e-Abstracts 2022
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Purpose :
Many children with sickle cell anemia (SCA) require blood transfusions, which carry risks and utilize a scarce resource globally, particularly in Africa. Realizing Effectiveness Across Continents with Hydroxyurea (REACH, NCT01966731) has documented the safety, feasibility, and benefits of hydroxyurea for children with SCA living in sub-Saharan Africa. In REACH, hydroxyurea escalated to maximum tolerated dose (MTD) significantly decreased vaso-occlusive events, malaria, and death; transfusions were decreased by ~70% over 30 months of treatment when compared to the 2-month screening period. Characterizing and investigating how hydroxyurea reduces transfusion needs in REACH could contribute to improved clinical understanding, better outcomes, a decreased transfusion burden, and preservation of the blood supply in these limited-resource settings.

Materials and Methods :
Transfusions were recorded prospectively in the REACH REDCap database. Transfusion rates and indications were compared during screening and treatment. Risk factors for receiving a transfusion were identified using time-varying predictors and landmark analysis. Univariate relationships were assessed using the Anderson-Gill model, plus multiple regression to estimate Hazard Ratios (HR) with 95% confidence intervals (CI).

Results :
A total of 635 children with HbSS from Angola, Democratic Republic of Congo, Kenya, and Uganda enrolled, and 606 started hydroxyurea treatment. During screening, 48 transfusions were administered to 43 children, and during the treatment phase 405 transfusions were administered to 214 children over a mean treatment time of 5.2 ± 1.3 years. The transfusion rate was 43.3 per 100 patient-years during screening, which decreased to 22.0 per 100 patient-years during the initial fixed dose treatment period (IRR = 0.50; 95%CI = 0.35-0.74, p<0.001 compared to screening) and then decreased further to 12.1 per 100 patient-years during the dose escalation period (IRR = 0.28; 95%CI = 0.21-0.39, p<0.001 compared to screening; IRR = 0.54; 95%CI = 0.43-0.73, p<0.001 compared to fixed-dose). For every 100 children treated for one year with hydroxyurea dose escalation, there were 31.4 fewer transfusions compared to the untreated screening period (number needed to treat = 3.2). Children with palpable splenomegaly at enrollment had a higher likelihood of receiving a transfusion during the trial (HR 1.98, 95% CI 1.59 to 2.47, p<0.0001). Age, gender, alpha thalassemia trait, and G6PD deficiency were not associated with differences in transfusion rates.

Conclusions :
Hydroxyurea significantly reduces blood transfusion administration in children with SCA in sub-Saharan Africa, especially when escalated to MTD. Overall, this demonstrates that treating children with SCA may not only improve individual patient outcomes through reduction in anemia, transfusion burden, and transfusion-associated complications including infections, but may also to help preserve the scarce blood supply for the benefit of the larger population. For example, applying similar outcomes to the population of Uganda predicts that about 30,000 transfusions per year are administered to patients with SCA and that with universal screening and treatment that 21,600 (72%) of these could be preventable, representing ~20% of all annual transfusions administered. The fact that treating SCA patients can benefit the entire population through reducing blood transfusion utilization reframes the importance of treating this population from an individual health decision to a public health strategy.

Purpose :
The use of Artificial Intelligence (AI) for personalized medicine has recently guided dramatic improvements in the diagnostic pathway of several diseases. The European Project GENOMED4ALL, aims at using European level data of patients affected by Multiple Myeloma, Myelodysplastic Syndromes and Sickle Cell Disease (SCD) to find correlation between genomics – and other omics data – with phenotypic manifestations and seize the opportunity of improving diagnostics through AI.
Silent Cerebral Infarcts (SCIs) are a significant determinant of morbidity since childhood in SCD. One of the aims of the SCD clinical case in GENOMED4ALL is the use of radiomics – quantitative method for the evaluation and interpretation of medical images- and AI firstly to develop an automatic and uniform identification and characterization of SCI through the analysis of cerebral MRI, secondly, to correlate imaging data with other types of omics data in order to predict risk of recurrence.

Materials and Methods :
The MRI protocol included 3D-T1, FLAIR and DWI sequences. Neuroradiological reports were cross checked for consistency.  A stepwise segmentation (identification of lesion volume), pre-processing and extraction of  MRIs  was performed utilizing different open-source software: Lesion Segmentation Tools and UNet for segmentation, Freesurfer for brain extraction. To optimize SCI identification, we have selected the best software for segmentation; freesurfer was substituted with a faster approach.

Results :
Six European SCD centers participated in this first phase of the Radiomics project with 501 MRIs: 225 were classified as abnormal by the local neuroradiologists due to presence of SCI, 275 were normal; 70% were pediatric MRIs. Different instruments were used in the 6 centers: Philips 1.5 T (n.2), Siemens 1.5 T and 3 T (n.2), GE 3T (n.2).
A stepwise procedure allowed the optimization of SCI identification, with distinction between SCI and background non clinically significant noise (i.e periventricular areas) or other white matter hyperintensities (i.e transient glial maturation).
As shown in Figure 1, to segment SCI in FLAIR MRI, we have used a pre-trained UNet [Li H, 2018]. Firstly, we have extracted the brain registering the MNI152 on the T1 image. Then we have applied the brain mask and a threshold taking only the largest component. The UNet performs the segmentation of hyperintensities. We have removed the regions surrounded by less than the 90% of White Matter or near the brain ventricles to remove non-SCI region. In the end, we have enlarged the remaining ones.
To date, segmentation on 303 exams from different centers showed very few false positive and false negatives highlighting the need to take into account different technical characteristics due to the various equipment used in the different centers.

Conclusions :
SCD is a systemic disorder with extreme phenotypic variability. Radiomics and AI offer the opportunity to seize the potential of big datasets analysis to optimize diagnostic in SCD. SCI can be detected automatically from different datasets. Four more European Centers will add their MRI in the second phase of the project, to increase variability and allow correlation of in detailed Radiomics data (lesion volume, lesion site, etc) with clinical-hematological characteristics and other omics data.

Figure 1 Stepwise Segmentation (identification and selection of lesion volume) workflow for automatic selection of all SCI. From left to right: Axial slices of the input scan, Slice after skull stripping to highlight only the brain parenchyma, Segmentatio

Purpose :
Etavopivat, an investigational, once-daily (QD), selective activator of erythrocyte pyruvate kinase (PKR) increases PKR activity, resulting in decreased 2,3-DPG and increased ATP in red blood cells (RBCs) of healthy volunteers and patients with sickle cell disease (SCD).^1,2 Here we report results of an open-label extension cohort from a Phase 1 study (NCT03815695) characterizing the safety and clinical activity of etavopivat at a maximal pharmacodynamic dose in patients with SCD.

Materials and Methods :
Fifteen patients were to receive etavopivat 400mg QD for 12 wks followed by a 4-wk follow-up. Assessments included safety, pharmacokinetics, pharmacodynamics, RBC health parameters, and systemic markers of SCD pathophysiology.

Results :
Fifteen patients were enrolled (age 32.3 ±10.1 years; HbSS/SC, n=13/2); 14 completed 12-wks of treatment; 1 discontinued treatment after ~2 wks. Etavopivat 400mg QD was generally well tolerated. Adverse events (AEs) reported during treatment and follow-up were commonly low grade (Gr) and consistent with patients’ SCD. Gr1-2 AEs in >2 patients (n, %) were sickle cell pain events (9, 60%), headache (5, 33%) and upper respiratory tract infection (3, 20%). The Gr3-4 AE in >1 patient was sickle cell vaso-occlusion (VOC; 4, 27%). On-treatment serious adverse events (SAEs; n=1 each) were Gr3 VOC post Gr3 COVID (not treatment-related) and Gr3 left-femoral deep vein thrombosis (possibly related, resulting in treatment discontinuation as stated above). SAEs (n=1 each) during the 4-wk follow-up were Gr3 acute chest syndrome + Gr3 VOC, Gr3 non-cardiac chest pain and Gr3 syncope (all unrelated).
Increases in ATP and decreases in 2,3-DPG were durable over 12 wks of etavopivat treatment. Etavopivat normalized HbS-oxygen affinity to that of HbA and improved measures of sickle RBC health, demonstrated by a reduction in RBC point of sickling and improved deformability (Figure 1) and hydration (Figure 2) (all P<0.01).
Etavopivat treatment over 12 wks resulted in a sustained significant increase in Hb compared with baseline (P<0.0001), with mean maximal increase of 1.5 (range, 0.7-2.3) g/dL. On-treatment increase in Hb >1g/dL was achieved in 11 (73%) patients, for whom the mean maximal Hb increase was 1.8 (1.2-2.3) g/dL. Absolute reticulocytes, indirect bilirubin and lactate dehydrogenase significantly decreased from baseline over 12 wks (all P<0.05), indicating decreased hemolysis and increased RBC lifespan. Several markers of disease activity significantly decreased from BL, including the inflammatory marker tumor necrosis factor-α, which decreased by 35% (P<0.001). Based on preliminary exploratory analysis with an aggregate duration of etavopivat exposure of 3.32 patient-years, the trend for VOC hospitalizations decreased: annualized historical and on-treatment VOC hospitalization rates were 0.93 and 0.30, respectively; the 1 on-treatment VOC hospitalization was COVID-related.

Conclusions :
Etavopivat 400mg QD for up to 12 wks demonstrated a tolerable safety profile with improvements in various markers of RBC health in patients with SCD. Rapid and sustained increases in Hb were associated with decreases in reticulocytes and hemolysis markers, supporting increased sickle RBC lifespan and improved anemia. Together, these results support further evaluation of etavopivat in the Phase 2/3 Hibiscus Study (NCT04624659) currently enrolling patients.
¹Kalfa et al, Blood 2019.
²Brown et al, Blood 2020.

Etavopivat improved sickle RBC PoS and deformability (EImax) during 12-weeks of dosing.

Etavopivat improved sickle RBC hydration (RBC Hb concentration [MCHC] and dense RBCs) during 12-weeks of dosing.

Purpose :
Vaso-occlusive crises (VOCs) are the hallmark of SCD and can lead to serious complications. P-selectin is a cell adhesion protein that plays a key role in the multicellular interactions that lead to VOCs. Crizanlizumab, a first-in-class humanized monoclonal antibody that blocks P-selectin, is approved in several regions to prevent/reduce VOCs for SCD patients aged ≥16 years. Since June 2018, patients in some countries have received crizanlizumab before health authority approval via a MAP (NCT03720626).

Materials and Methods :
The MAP was designed to provide access to crizanlizumab for patients with a serious or life-threatening disease (SCD) for which no comparable or satisfactory alternative to crizanlizumab was available in their country. Other eligibility criteria included: aged 16–70 years (18–70 years in Italy); history of VOCs as determined by the treating physician (including recurrent VOCs while taking preventative therapies eg hydroxyurea [HU]); and ineligibility for a crizanlizumab clinical trial. Patients’ disease burden during the baseline period was provided by the treating physician. This analysis describes the rate of VOCs and use of opioids for VOC-related pain relief 12 months before crizanlizumab initiation (baseline period) and after ≥12 months of crizanlizumab treatment in patients with SCD participating in the MAP, overall and stratified by genotype and prior HU use (in countries where publication of these data is allowed).

Results :
As of February 2022, 188 patients have received crizanlizumab in the MAP; 87 have been treated for ≥12 months (Brazil, n=79 [91%]; Italy, n=5 [6%]; Spain, n=2 [2%]; Israel, n=1 [1%]). Median (interquartile range [IQR]) age of the 87 patients was 33 (25–40) years, 57% were female, 45% were African American, 8% Caucasian, 20% Hispanic and 28% of ‘other’ ethnicity; 82% had an HbSS genotype. Prior HU use was reported for 41/56 (73%) patients with available data. During the baseline period, 85% (n=74/87) of patients were hospitalized for a total of 220 SCD-related complications.
Crizanlizumab led to a median (IQR) absolute reduction from baseline of –3.0 (–6.0 to –1.0) home-managed and –2.0 (–4.0 to 0) healthcare-managed VOCs after ≥12 months of treatment. Similar reductions in VOC rates post- versus pre-crizanlizumab were observed when stratifying the data by SCD genotype/prior HU use (Figure), although some groups only contain a small number of patients.
Opioids were taken for VOC-related pain relief by 95% of patients (n=83/87) at baseline and by 69% (n=60/87) in the 12 months after crizanlizumab treatment.
Adverse events were consistent with those reported in other crizanlizumab studies.

Conclusions :
Patients in the crizanlizumab MAP had significant disease burden at baseline, as evidenced by the high rate of home- and healthcare-managed VOCs, proportion of patients with SCD-related complications and use of opioids for VOC-related pain relief, despite many patients reporting prior HU use. Crizanlizumab led to clinically relevant reductions from baseline in the median annualized rates of home- and healthcare-managed VOCs and use of opioids in this real-world setting, consistent with results from SUSTAIN.

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Conclusions :

Purpose :
Sickle cell disease prevention and management programmes are traditionally based on 6 pillars: awareness, early detection, appropriate management, advocacy, research and training of health professionals. One of the main pitfalls is the lack of educational resources available to all healthcare professionals. Few studies quantify the training needs, but all conclude that it is necessary to deploy these activities at the preliminary levels of the healthcare system. In medical faculties and paramedical schools, little time is allocated to courses teaching about sickle cell disease. At the same time, between 2014 and 2019, average availability of 3G network coverage in sub-Saharan Africa increased from 42% to 73%, and from 4% to 44% for 4G. This massive development of information and communication technologies provides a major means of access to training for professionals working outside the capital cities, right down to the last mile.

Materials and Methods :
The e-sickle cell platform was created to address this issue. In order to offer content adapted to all healthcare professionals, a network of experts developed a training programme on all aspects of the disease with speakers from the North and the South, who are healthcare professionals in the field. This network guarantees the scientific independence of the training material. Low-tech tools have been selected: Dudal for receiving the courses (developed by RAFT), and a simple web platform for their distribution. The training is freely available on e-drepanocytose.com.

Results :
The tests carried out in early 2021 with health professionals in 4 countries did not reveal any major accessibility problems to the platform and evaluation in regard to the content was positive in terms of its form and content. Following the opening of the site to the public, 305 trainees were registered by 31st December 2021, at all levels of the health system. The study material has been used in mixed training courses by NGOs in the field (MSF in Niger, Santé Sud in Mauritania). Site content is updated regularly, taking into account feedback from users.

Conclusions :
The use of the platform must be extended beyond doctors based in capital cities who at present are the main users (33% of registered users), and access to the platform must be extended to healthcare workers in the most isolated areas. The implementation of MCQ-based assessment’s will provide for better mastery of the platform and also for evaluating its real impact on the trainees' practice.
(Project instigated, coordinated and funded by Fondation Pierre Fabre)

Bibliography (background)
de-Graft Aikins A et al. Tackling Africa's chronic disease burden: from the local to the global. Global Health. 2010 ; 6:5.
Guindo A et al. Le Centre de recherche et de lutte contre la drépanocytose de Bamako : histoire, bilan, défis et perspectives. Presse Med Form 2021; 2: 405–412.
Kafando E et al. Les syndromes drépanocytaires majeurs : une enquête anonyme auprès du corps médical au Burkina Faso. Med Trop. 2008 ; 68: 241-246.
Mbyia B et al. Sickle Cell Disease in the Democratic Republic of Congo: Assessing Physicians’ Knowledge and Practices. Trop Med Infect Dis. 2020 ; 5(3): 127.
 

Purpose :
Blood transfusion is an indispensable therapy in sickle cell disease, despite its multiple and often harmful complications. The objective of this study was to evaluate the complications of alloimmunization, infection and iron overload secondary to blood transfusion in sickle cell disease patients.

Materials and Methods :
This was a six-month cross-sectional case-control study conducted in the Clinical Hematology Department of Dakar (Senegal) on 253 patients followed for major sickle cell disease (153 polytransfused and 100 non-transfused patients). Transfusion practice (modalities, indications), post-transfusion complications (alloimmunization, iron overload, infections) and risk factors (sociodemographic, clinical, biological) for the occurrence of these complications were evaluated.  

Results :
The median age of the patients was 28.5 years (5-59). The sex ratio (M/F) was 0.86. Homozygous sickle cell disease was more frequent (95.3%). The median follow-up time was 9.1 years (2-26). Simple transfusion was performed in 142 patients, exchange transfusion in 29 patients; 22 patients were on a transfusion program. The indications for transfusion were dominated by acute anemia (57.06%) and prolonged CVO (14%). Red blood cell concentrates were administered in 143 patients (93.46%). The median number of units of blood received per patient was 10 units (2 - 48). The frequency of alloimmunization was 16% and the most frequent alloantibodies were anti-rhesus (34.19%) and anti-Kell (23.67%). The prevalence of hepatitis C in transfused subjects was 1.33% and 1% in non-transfused subjects (p=0.64). The prevalence of HBV was 2% in transfused patients and 3% in non-transfused patients (p=0.45). HIV antibodies were not found in either the transfused or the non-transfused patients. Twelve patients (7.84%) had post-transfusion iron overload (ferritin level >1000 ng/ml). The higher the transfusion, the higher the ferritin level (strong positive correlation, r = 0.8). The number of packed red blood cells transfused was the only risk factor for alloimmunization (p=0.03) and post-transfusion iron overload (p=0.023). The frequency of blood transfusion was not related to the transmission of infectious.

Conclusions :
The management of sickle cell disease often requires blood transfusion to correct anemia or prevent certain complications. Despite the progress made in transfusion safety, this therapy still remains a risk for the recipient.

Purpose :
Sickle cell anemia (SCA) increases childhood risk of neurocognitive impairment from severe anemia and vascular brain injury. We hypothesized that impaired neurocognitive function was more prevalent in Ugandan children with SCA than in their non-SCA siblings.

Materials and Methods :
In the BRAIN SAFE 1 cross-sectional study (Green NS, 2019), a sample of children ages 1-12 years were randomly selected from Mulago Hospital SCA clinic in Kampala. Their neurocognitive function was compared to that of unaffected siblings using age-appropriate tests:  Mullen Scales of Early Learning (Mullen) for children under age five and Kaufman Assessment Battery for Children, second edition (KABC-II) for ages 5-12. Raw scores were converted into age-adjusted Z-scores compared to established community controls; Z-scores -2 or lower were categorized as impaired neurocognitive function. Relationships between key variables and neurocognitive impairment (impaired vs. not impaired) were evaluated by logistic regression.

Results :
Overall, 369 participants were evaluated: 242 with SCA (mean age 5.44 ±2.9 years) and 127 non-SCA siblings (mean age 7.8± 3.3 years; p<0.0001). Mean hemoglobin was lower in SCA participants (7.3 ±1.02 g/dl vs. 12.1 ±1.45; p=0.0001); the groups were similar by sex (p=0.33). Neurocognitive impairment was present in 27 (11.2%) of children with SCA compared to 8 (6.3%) of non-SCA siblings. Children with SCA had lower median neurocognitive Z-scores compared to siblings (-0.62 vs. 0.30; p<0.0001) overall and for each age group tested: ages 1-4 years (-0.35 vs. 0.53, p=0.0001) and 5-12 (-0.88 vs. 0.25, p=0.0001) (Figure). SCA participants had higher odds of impairment (OR 1.88, 95%CI 0.83-4.27) compared to non-SCA; this relationship became stronger after accounting for age and caregiver education (secondary/tertiary school vs. primary/none) (aOR 5.07 (1.69-15.1)) (Table). After accounting for SCA status, increased age accounted for 50% increased risk of neurocognitive impairment (aOR 1.50, 95%CI 1.29-1.75). Overall, higher caregiver education modestly reduced the odds of neurocognitive impairment (OR 0.64 (0.23-1.72)). In participants with SCA, prior stroke or elevated TCD velocity each strongly predicted cognitive dysfunction (OR 6.60 (2.14-20.4) or 2.90 (1.09-7.74), respectively. These associations strengthened after accounting for age, hemoglobin and malnutrition (Table). Among siblings, age was significantly associated with impaired cognition (aOR 3.48 (1.00-12.1)), even after accounting for sex and hemoglobin (data not shown).

Conclusions :
In this Kampala-based sample and accounting for age, children with SCA had 4-fold greater odds of neurocognitive impairment compared to non-SCA siblings. For participants with SCA, major predictors of impairment were age, prior stroke and elevated TCD arterial velocity. These abnormalities were commonly found, translating into significant disease-associated morbidity and potential long-term disability. A treatment trial with hydroxyurea is underway to assess impact on neurocognitive function and reduction of stroke risk.

Supported by R21HD089791 and 3R21HD089791-02S (Idro, Green)
 

Children with SCA score lower on neurocognitive testing than their non-SCA siblings in both age groups sampled..

Table. Multivariable Regression Analyses evaluating Odds of Neurocognitive Dysfunction in Children Ages 1-12 years with SCA (n=242) Compared to Non-SCA Siblings (n=127).

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Desensitization procedure

Purpose :
Sickle cell disease (SCD) has a substantial emotional and physical burden on patients and their caregivers. Research on the impact of SCD on patient and caregiver quality of life (QOL) is limited, including on the health inequalities faced by different groups around the world. The limitations in research underscore the need for greater understanding of the challenges of living with SCD in different communities. The SHAPE survey aims to broaden the understanding of the global impact of SCD on patients living with the condition, on caregivers caring for those living with the condition, and on healthcare professionals (HCPs) who treat SCD. 

Materials and Methods :
The SHAPE survey comprised online, quantitative surveys of patients, caregivers, and HCPs conducted in 10 countries: France, Germany, UK, US, Canada, Brazil, Saudi Arabia, United Arab Emirates, Bahrain, and Oman (patients only). The surveys required participants to answer a range of closed-ended questions about their circumstances and experiences in order to build a robust and reliable dataset on which descriptive statistics were performed. This analysis focuses on findings from the patient and caregiver survey portion of the study. Patients were included if they were diagnosed with SCD by an HCP and were aged ≥12 years. Caregivers were included if they were caring for someone diagnosed with SCD and were aged ≥18 years. Informed consent was obtained from all participants, and all identifiable information was kept private and secure. The study protocol was reviewed and approved by an independent institutional review board.

Results :
A total of 919 patients and 207 caregivers completed the survey. The mean patient age was 32.3 years, and most patients and caregivers were female. Nearly all patients stated that reducing the risk of long-term complications, such as organ damage (93%) and hemolytic anemia (84%), was important to them (Figure). Caregivers felt that most areas of their lives—particularly their ability to attend and succeed at school or work (56%) and their own overall wellbeing (53%) and mental health (52%)—were impacted by caring for someone with SCD. The symptom experienced by patients with SCD that most impacted caregivers’ lives was fatigue/tiredness (49%), and 54% of caregivers reported a notable impact on their earning potential. Those caring for patients aged <18 years were more likely to have their ability to attend and succeed at school or work impacted than those caring for patients aged ≥18 years (61% vs 38%, respectively). A total of 46% of patients and 33% of caregivers reported that emergency room (ER) HCPs did not believe patients about their symptoms, and 48% of patients felt they were treated as a drug seeker by ER HCPs. Similarly, 54% of patients and 40% of caregivers reported ER HCPs provided poor care due to their lack of knowledge about SCD.

Conclusions :
The experience of a global group of patients with SCD shows that what most impacted caregivers’ lives was fatigue/tiredness and loss of earnings and this provides insight on the broad impacts of SCD and highlights areas that need greater support or improvement.

Figure: Percentage of Patients or Caregivers Who Agreed With or Endorsed the Following Statements

Purpose :
The spleen is a lymphoid organ that protects the body against blood infections and filters blood cells. Defective spleen function (hyposplenism) is caused by splenectomy or by immunological or hematological diseases. Counting vacuole-containing RBC (pocked RBC) by Differential Interference Contrast microscopy (DIC) is a robust marker of defective splenic function but it relies on the expertise of fewer and fewer specialized laboratory technicians, hence its rare application in routine medical use.

Materials and Methods :
We developed an automated method using a common fluorescent cytoplasmic marker, and imaging flow cytometry to quantify pocked RBC in 128 adult patients with Sickle Cell Disease (SCD). Spleen size was determined by 2 members of the team and scored as 4 categories: Splenomegaly, Normal, Small and Atrophic.
Six children with SCD who had partial splenectomy for hypersplenism were also included. Spleen were retrieved immediately after surgery, histology and electronic microscopy were performed. A venous blood sample was collected at the time of surgery and the splenic blood was retrieved from spleen fragments.

Results :
The proportion of RBC containing one or more fluorescent dots by IFC correlated with results of the conventional method, i.e., the operator-dependent quantification of pocked RBC by differential interference contrast microscopy. Proportions of pocked RBC in circulation correlated with spleen size, which varied widely from splenomegaly to atrophic spleen. Histology of post-splenectomy samples from 3 SCD children with normal, moderately, or markedly elevated pocked RBC counts, showed either an almost normal aspect with congestion, intense sickling, or widespread fibrosis, respectively. Electronic microscopy of the spleen from the child with normal pocked RBC counts showed persistent RBC filtration through inter-endothelial slits. Intrasplenic blood was enriched in RBC displaying a peripheral fluorescent dot, highly suggestive of ongoing intrasplenic expulsion of vacuoles (pitting). Operator-independent, fluorescent-based quantification of pocked RBC correlates with the reference method and confirms that spleen function is partially preserved in many adults with SCD.

Conclusions :
Because quantification of pocked-RBC correlates with the results of splenic scintigraphy, the new fluorescence-based counting method is a solid operator-independent quantitative surrogate of spleen function, is simple, and can be performed within hours on less than 20 µl of venous blood.
Some experts consider that adults with SCD are almost constantly asplenic with an atrophic spleen. In our study, patients with either splenomegaly, a normally-sized or small spleen had more variable counts, suggesting that spleen filtering function is often preserved in SCD, at least partially, even in adulthood.
In summary, this new fluorescence-based quantification of pocked-RBC, based on the spontaneous accumulation of the dye in vacuoles, correlates with the reference method, opening the way to a robust, operator-independent quantification of spleen function.  We show that spleen function is partially preserved in many adults with SCD. We also show that the fluorescent vacuoles accumulating in RBC from SCD patients are indeed expelled by the spleen-specific pitting process, as long as sinusal structures persist in the partially damaged SCD spleens.

Purpose :
Hydroxyurea is a highly effective disease-modifying treatment for sickle cell anemia (SCA) and designated an Essential Medicine by the WHO. Increasing access to hydroxyurea for SCA in low-resource settings is advocated, but the ideal approach to dosing and monitoring is debated. Escalation to maximum tolerated dose (MTD) achieves superior benefits without additional toxicities but requires incremental dose adjustments with serial monitoring. Pharmacokinetic (PK)-guided dosing uses a test dose and sparse blood sampling to model hydroxyurea metabolism and predict a personalized optimal dose, which should require fewer clinical visits, lab assessments, and dose adjustments. Currently PK-guided dosing requires expensive, complex analyses that are unavailable in low-resource settings. A simplified method for hydroxyurea PK analysis, coupled with a user-friendly dosing algorithm, could simplify dosing and monitoring.

Materials and Methods :
Concentrated stock solutions of reagents for chemical detection of serum hydroxyurea using high performance liquid chromatography (HPLC) were stored at -80C. On the day of analysis, hydroxyurea was serially diluted in human serum, then spiked with N-methylurea as an internal standard. After deproteination with trichloroacetic acid, a Fearon reaction to detect the urea moiety was performed with diacetylmonoxime, ferric chloride, sulfuric acid, and phosphoric acid. Thiosemicarbazide was added to minimize photolability of the colored product, which was analyzed using two commercial HPLC machines: (1) a standard benchtop Agilent HPLC system with a 449nm detector and 4.6mm x 250mm, 5 micron Zorbax Eclipse XDB-C18 column; and (2) a portable PolyLC SmartLife HPLC system with a 415nm detector and 4.6mm x 50mm, 3.5 micron Zorbax column XDB-C18 column (Figure 1). The mobile phases were acetonitrile and water. After validation in the US, the portable HPLC and chemicals were transported to Tanzania for repeat analyses.

Results :
A calibration curve was generated using the concentration of hydroxyurea dilutions ranging from 0mM to 1000mM, plotted against the hydroxyurea:N-methylurea ratio, and both HPLC systems yielded calibration curves with R²>0.99 (Figure 1). A separate stock of hydroxyurea was diluted to create known concentrations for analysis using the calibration curve, to confirm accuracy and precision within 10-20% of the known value. Both HPLC systems measured hydroxyurea concentrations with <10% variance from the prepared concentration. Paired analysis of samples on both machines yielded results with <15% variance. Serial measurements of a standard 100 mM concentration using the PolyLC system were precise with a 2.5% coefficient of variance. After transport to Tanzania, setup, and training, the SmartLife HPLC system was able to produce similar excellent hydroxyurea concentration calibration curves with R²>0.99.
 

Conclusions :
Improving access to hydroxyurea as an essential treatment for all children living with SCA requires an approach that eases financial and logistical barriers while optimizing safety and benefit, especially in low-resource settings. We modified a portable HPLC instrument to detect hydroxyurea, validated its precision and accuracy, and confirmed capacity building and knowledge transfer to a low-resource setting. Implementation of HPLC for measurement of serum hydroxyurea concentrations is now feasible in low resource settings using available laboratory infrastructure. PK-dosing of hydroxyurea may soon be possible, with initial treatment using a personalized optimal dose.
 

Agilent (unmodified) and SmartLife (modified) HPLC instruments for measuring serum hydroxyurea concentrations

Calibration curve of 0-1000 µM hydroxyurea measured with SmartLife PolyLC high performance liquid chromatography system demonstrating excellent R2 value in both the US (A) and Tanzania (B).

Purpose :
Sickle Cell Disease (SCD) is a major cause of morbidity and mortality worldwide, and affects more than 100,000 people in the US. Recurrent episodes of acute, vaso-occlusive pain crisis, a hallmark of the disease,  lead to chronic pain for many patients, though the mechanisms of this transition are poorly understood.  Although opioids remain the standard of care to treat SCD chronic pain,  their myriad adverse side effects (e.g., constipation, respiratory depression, abuse liability, dependence) as well as the fact that SCD chronic pain requires prolonged opioid treatment that results in tolerance, severely limit their therapeutic utility. Thus, a pressing need exists to identify effective non-opioid analgesic strategies to reduce SCD chronic pain. Humanized mouse models of SCD, such as the Berkeley (BERK) model, provide a useful tool to investigate disease pathophysiology and evaluate novel therapeutic strategies. Dorsal Root Ganglion (DRG) neurons are peripheral sensory neurons essential for the transmission of nociceptive afferents to the CNS. In rodent models of neuropathic pain, harvested DRG neurons show hyperexcitability. Inhibitors of the major degradative enzyme of 2-arachidonoylglycerol, monoacylglycerol lipase (MAGL), reduce nociceptive behavior in neuropathic and inflammatory preclinical models of pain through cannabinoid receptor-dependent and -independent mechanisms. As MAGL inhibitors have yet to be tested in BERK mice,  here we test whether this approach will ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice.

Materials and Methods :
Male and female HbSS-BERK (sickle) and HbAA-BERK (control) mice were used as subjects for these experiments. Nociceptive behaviors were assessed using the von Frey and Hot Plate tests. Motor-functional behavior was assessed using the Grip Strength and Nesting assays. Neuronal hyperexcitability was assessed using whole cell patch clamp electrophysiology of L4-S1 DRG  neurons.  Data were analyzed as two- and three-way ANOVAs followed by Tukey post-hoc analysis when appropriate (p < 0.05 considered significant).

Results :
HbSS-BERK mice displayed profound mechanical, thermal and deep tissue/musculoskeletal hypersensitivity. HbSS-BERK mice also exhibited deficits in nest-building behavior. Patch clamp studies revealed that DRG neurons harvested from HbSS-BERK mice displayed extremely hyperexcitability. MJN-110 dose-dependently reduced mechanical allodynia and thermal hyperalgesia, and ameliorated deficits in grip strength in HbSS-BERK mice. Importantly, seven days of daily injections of MJN-110 (5 mg/kg) continued to ameliorate the hyper-nociceptive phenotype of HbSS-BERK mice, which did not undergo tolerance. Moreover, DRG harvested from the MJN-treated HbSS-BERK mice showed similar DRG neuronal activity as seen in control mice.

Conclusions :
These findings validate that HbSS-BERK mice show hypersensitive responses to mechanical and heat stimuli, and exhibit motor-functional deficits in grip strength and nest building behavior. Additionally, this work demonstrates hyperexcitability of sensory neurons from the lower lumbar region of the spine of HbSS-BERK mice. Finally, the observations that MJN110 reduces these hyper-nociceptive behaviors and ameliorates neuronal hyperexcitability in HbSS-BERK mice suggest that MAGL inhibition represents a viable strategy to reduce chronic pain related to sickle cell disease.  

Purpose :
With no existing population-based national registry, there is a lack of comprehensive understanding of the sickle cell disease (SCD) population living in the United States. The Sickle Cell Data Collection Program (SCDC), funded by the Centers for Disease Control and Prevention (CDC), is addressing this need by developing state-level surveillance programs. SCDC currently operates in 11 states, maintaining identifiable individual-level data within each state. However, there is no data model to guide the harmonization of data collected for SCD public health surveillance. We describe an ongoing process for establishing and maintaining a common data model in the unique setting of public health surveillance and identify key elements for surveillance reporting on SCD.

Materials and Methods :
The core data sources common across SCDC states include newborn screening, vital records, and Medicaid claims.  However, many programs have additional data including electronic medical records (EMR), state all-payer data, and clinical cohorts. The SCDC Common Data Model will standardize key data elements by identifying data instruments, specific variables and formats. Key data instruments are shown in Figure 1.  We are developing  data hierarchy guides to standardize variables across multiple data sources. The creation of this Common Data Model is overseen by a committee from SCDC states with expertise in clinical care, claims data, epidemiology, database construction, and data analysis, with input from SCD champions. 

Results :
The SCDC has identified a core set of results that are feasible for most states to attain, are measured with reasonable accuracy, and have high public health and policy relevance. These Core Surveillance Data are reported annually by SCDC participant states and compiled by CDC.
Birth Information:  We report numbers of SCD births in 1-year and 5-year increments; by sex, race, ethnicity, and SCD genotype. Newborn screening records are the primary source, with state laboratory confirmatory testing used to identify SCD genotype.
Case Numbers: We estimate the number of prevalent cases across the state during a given date range and report by county, sex, and age group (<10, 10-19, 20-29, 30-39, 40-49, 50-59, 60+ years). The primary source for these tables is healthcare claims datasets linked with newborn screening and clinical case reports.  Cases are identified as possible, probable, or confirmed SCD using validated definitions.
Deaths: Deaths are reported by age (<20, 20-49, >=50 years) and stratified by sex.  State vital records are a core source of death information, followed in priority by Medicaid data, and clinical data when available.
Healthcare Utilization:  Core Surveillance Data reports acute care utilization including number of emergency visits, inpatient hospitalizations, hospital length of stay and visit’s payer type.  Results are reported by age group (<10, 10-19, 20-29, 30-39, 40-49, 50-59, 60+ years) and payer type (e.g., Medicaid, self-pay). 

Conclusions :
To standardized national SCD surveillance, we are implementing a common data model that will allow SCDC to move towards a distributed data network. In this report, we describe the process of developing and maintaining a common data model in this unique setting and identify key data elements for public health reporting on SCD.

Purpose :
Epidemiological modeling shows that universal screening of sickle cell disease (SCD) could save the lives of up to 9.8 million newborns with SCD by 2050. The World Health Organization (WHO) estimates that early diagnosis of SCD coupled with intervention programs would prevent 70% of existing SCD mortality. SCD newborn screening (NBS) performed in centralized laboratories has dramatically reduced SCD mortality in resource-rich countries. SCD NBS requires sensitive detection of low levels of certain hemoglobin (Hb) variants (i.e., sickle Hb, HbS) in the context of high levels of expression of other Hb variants (i.e., fetal Hb, HbF). The current centralized tests used for NBS for SCD are high performance liquid chromatography (HPLC) and isoelectric focusing. However, in SSA and central India, where >90% of annual SCD births occur, NBS programs have not been implemented universally due to the cost and logistical burden of laboratory diagnostic tests. As a result, there is a need for affordable, portable, easy-to-use and accurate point-of-care (POC) tests to facilitate decentralized Hb testing in low-resource settings for enabling nationwide NBS.

Materials and Methods :
We have leveraged the WHO listed Hb electrophoresis test and developed a POC electrophoresis microchip, Gazelle-Multispectral (Fig. 1A) that enables sensitive detection and identification of Hb types under both white and violet (410nm) light illumination. We hypothesized that the high absorbance of Hb at 410 nm wavelength would enhance the limit of detection and allow detection of low concentration Hb types o enable SCD screening in newborns. To test this hypothesis, we have conducted clinical testing over 441 subjects under the age of 6 months including 265 newborns with Hb variants including HbA (normal hemoglobin), HbF, HbS, and HbC (hemoglobin C variant) at Korle Bu Teaching Hospital, under IRB-approved protocol. 

Results :
Separated Hb variants are imaged under both white light illumination (Fig. 1B) and 410 nm illumination (Fig. 1C). The acquired data under white light illumination demonstrates the natural red color of hemoglobin and is used for test validation(Fig. 1B). The acquired data under 410 nm is used for sensitive and accurate identification and quantification of Hb variants (Fig. 1C&D), and to automatically generate report for result interpretation (Fig. 1E). Following the Standards for Reporting of Diagnostic Accuracy Studies guideline, 365 out of 441 tests were recognized as ‘Valid’. Gazelle-Multispectral determined Hb variant levels demonstrated high association with Person Correlation Coefficients of 0.97, 0.97, 0.93, and 0.95 for Hb A, Hb F, HbS, and HbC compared to HPLC. Gazelle-multispectral demonstrated accuracy of 96.8% in subjects of 0-3 days, and 96.9% in newborns. (Table 1).

Conclusions :
Gazelle-Multispectral imaging enables affordable and rapid identification of common Hb variants in newborns at the point-of-need. The internally integrated data analysis algorithm automatically reports Hb type identification and quantification results in an objective and easily understandable manner. Overall, Gazelle-Multispectral enables affordable, rapid, and accurate NBS for SCD at the POC in low resource settings where the prevalence of SCD is high.

Figure 1. Gazelle-Multispectral for screening hemoglobin variants in newborns. (A) The Gazelle-Multispectral perform real time imaging and data analysis tracking the Hb electrophoresis process under both white light illumination (B) and UV light illumina

Table 1: Summary of true positive, true negative, false positive, false negative, sensitivity, and specificity of the clinical testing among 294 subjects conducted at Korle Bu Teaching Hospital, Korle Bu, Ghana.

Purpose :
Sickle cell disease (SCD) is a group of inherited red cell disorders associated with substantial clinical and economic burden. Sub-Saharan Africa accounts for nearly 75% of the global burden of SCD. However, inequities in access to healthcare, including novel therapeutic options, absence of standard treatment guidelines, few dedicated SCD treatment centers and the high cost of treatment have made the management of SCD a challenging task in the region. The current study assessed treatment patterns and healthcare resource utilization (HCRU) and costs in patients with SCD using data from a national Private Medical Insurance database.

Materials and Methods :
Claims data from Ghana's Nationwide Medical Insurance Database were analyzed between January 1, 2015 and March 31, 2021. Patients with at least 12 months of follow-up data for ≥1 SCD diagnosis claim during the index period (July 1, 2015 to March 31, 2020) and continuous enrollment for at least one claim during the 6-months baseline period prior to index date and 12-months follow-up period post-index date, were included in the study. Healthcare resource utilization and associated costs for patients with SCD were assessed for outpatient and inpatient visits, medications, consumables (medical/surgical supplies) procedures (medical/surgical procedures, diagnostic investigations), services (consultation/hospital services) and other activities (administrative services) during the 12-month follow-up period.

Results :
Claims data for 2,863 identified patients with SCD (mean age: 20.1 ± 16.0 years; 43.9% males, 56.1% females) were considered. The cohort was stratified by age into two groups: pediatric (<16 years) and adult (≥16 years). The most common complications across all age groups included malaria, upper respiratory tract infection, sepsis, acute chest syndrome, and cellulitis. Most patients were prescribed non-steroidal anti-inflammatory drugs (75.6%), anti-infectives (52.2%), haematinics (27.9%), anti-malarials (26.5%), and opioid analgesics (9.7%), while standard-of-care hydroxyurea was prescribed to only 0.3% of patients. The mean time to first hospitalization was 129.9 ± 107.8 days, and the annual rate of hospitalization was 1.7 ± 1.4 during the 12-month follow-up period.
Outpatient claims were higher compared to inpatient claims (2.2 ± 2.4 vs. 1.7 ± 1.4). Most claims pertained to medications (1.9 ± 1.8) and services (2.2 ± 2.3). Costs incurred for inpatient claims were higher compared to outpatient claims (United States Dollar [USD] 261.4 ± 460.5 vs. 56.6 ± 60.4) (Figure 1) during the 12-month follow up period. This was mainly attributed to medications (USD 40.0 ± 117.2) and services (USD 43.3 ± 114.0) (Table 1).

Conclusions :
To our knowledge, this is the first cost-of-illness study conducted to evaluate HCRU for patients with SCD in Ghana. Our findings suggest that SCD has a substantial impact on healthcare costs in Ghana, consistent with the results of other studies evaluating the economic burden of SCD. Based on our results, use of hydroxyurea therapy as standard of care for SCD management was suboptimal for this cohort of patients. There is an opportunity to increase access to hydroxyurea and other emerging therapies to manage SCD and associated complications, and to reduce the economic burden of the disease.

Source for conversion of Cedi to USD currency: https://www.unitconverters.net/currency/ghs-to-usd.htm; Accessed on 21Feb2022 1 Cedi=0.1515234164.

Procedures examples: diagnostic investigations, surgical procedures. Services examples: specialist consultation, hospital services. Consumables examples: Oxygen cannula, dressings, syringes, needles, catheters, gloves etc

Purpose :
Ensuring consistent healthcare during transition remains challenging for SCD. The Sickle Cell Trevor Thompson Transition (ST3P-UP) study aimed to standardize the transition process for emerging adults with SCD while engaging the community as an equal partner. The ST3P-UP SCD HCT learning collaborative engaged 14 pediatric and adult paired sites (including community participation) using a standardized quality improvement (QI) process. Monthly virtual meetings provided coaching on clinical recommendations, QI methods, and practical implementation of Six Core Elements of HCT (6CE).

Materials and Methods :
Implementation of 6CE was measured across 14 clinical sites who collectively provide care for 1625 individuals with SCD aged 16-25. Each site comprised of both pediatric and adult clinics and a partner community-based organization (CBO). Clinical programs varied: 12 urban, 2 rural; 12 academic, 2 non-academic; 6 small, 8 large. The HCT Process Measurement Tool (HCT-PMT) assessed 6CE implementation adherence using iterative QI strategies at baseline (2018) and every 6 months thereafter through 42 months (2022). Pre and post results were compared for overall group and by type of practice. 

Results :
All 14 sites made substantial progress towards implementing a structured SCD HCT process over the past 42 months. Overall HCT-PMT scores increased over time from 19.4 at baseline to 61.5 at 12 months, 92.1 at 24 months, 96.8 at 36 months and 97.2 at 42 months. Pediatric site scores increased significantly from 24 (baseline) to 71 (12 months) to 96 (24 months) to 99 (36 and 42 months). Adult site scores also increased from 15 (baseline) to 52 (12 months) to 88 (24 months) to 95 (36 and 42 months). Pediatric sites scored higher at each scoring interval from baseline through 42 months versus adult sites. Both large and small sites demonstrated significant increase in PMT scores from baseline over the 42 months of the LC. Only one site – a large adult site – failed to achieve the collaborative goal of a PMT score >90. This site was unique in that they had a change in adult PI three times within the first 18 months of the collaborative however they continue to make progress towards their goal.

Conclusions :
All 14 pediatric and adult sites reported significant progress with adherence to a structured HCT process aligned with the 6CE within 42 months using quality improvement. Partnering with CBO’s facilitates sustainability and optimal patient engagement.

SCD Health Care Transition Process Measurement Tool Trend Over Time Ped & Adult

Overall Collaborative PMT Scores Over Time

Purpose :
Despite significant advances in the diagnosis and treatment of sickle cell disease (SCD), most of the >300,000 infants born with SCD will die before the age of 5 years, primarily due to the lack of timely and accurate diagnosis. In high-resource countries, early identification of SCD through newborn screening (NBS) is routine and subsequent delivery of preventive measures and comprehensive care is highly effective in reducing morbidity and early mortality. Despite the proven efficacy of NBS and successful pilot programs across Africa, large-scale implementation remains infeasible in many sub-Saharan countries due to inadequate financial, laboratory, and technical resources. The development and deployment of low-cost, rapid, user-friendly, and accurate diagnostic testing for SCD is imperative. Recently, several promising point-of-care (POC) diagnostics have been developed, but there have been few real-world studies that compare these tests head-to-head, particularly in the setting of NBS, where accurate diagnosis amidst high levels of fetal hemoglobin is critical. 

Materials and Methods :
We performed a prospective evaluation of two POC SCD diagnostics (Sickle SCAN and HemoTypeSC) at maternity centers and immunization centers in Luanda, Angola, with confirmation by isoelectric focusing (IEF). To evaluate the real-world feasibility and accuracy, with the guidance of the Angolan Ministry of Health, we planned to test 100 infants ≤6 months of age with each test at 10 different clinical sites (total of 2,000 infants tested). The study was designed to mimic a real-world setting with all POC testing performed and interpreted by local healthcare staff with basic instructions for the use of each test.

Results :
Of the 2,000 infants tested, 1958 results were included in the final analysis, with the exclusion of 42 tests that were not interpreted in the recommended time frame. Consistent with prior studies in Angola, the burden of sickle cell trait (HbAS, 21.9%) and sickle cell anemia (HbSS, 1.7%) was high. Tests were performed in maternity wards on the day of or following birth (63%) or at immunization centers through 6 months of life (37%). HemoTypeSC took an average of 15 minutes to obtain a result, was repeated 68 times (6.8%) due to invalid results, and was interpreted incorrectly only 4 times (0.4%). Sickle SCAN took an average of 7 minutes to obtain a result, was repeated 2 times (0.2%) due to invalid results, and interpreted incorrectly only 11 times (1.1%). The gold standard of IEF was imperfect with 5.5% of samples needing repeat testing at least once 12 samples with inconclusive results. Importantly, 92% of babies who were diagnosed and informed at the POC followed up in the SCD clinic, compared to the historical 56% who were successfully brought to care during the initial pilot NBS program in Luanda. 

Conclusions :
This study demonstrates the feasibility and accuracy of both HemoTypeSC and Sickle SCAN for diagnosing SCD in newborns in a Luanda, Angola. Given the complexities of testing in the newborn period at busy maternity hospitals, this study suggests that testing at immunization centers using novel POC diagnostics may be a more practical solution to early infant diagnosis programs for SCD. 

Purpose :
Endothelial cell (EC) activation and red blood cell (RBC) adhesion are central to both acute and chronic pathogenesis of sickle cell disease (SCD). Quantitatively, RBC-derived extracellular vesicles, REVs, are more abundant from SS RBCs compared with HbA-containing RBCs (AA RBCs). Sickle RBC-derived REVs (SS REVs) are known to transfer heme to endothelial cells and to promote endothelial activation through cell signaling and transcriptional regulation, triggering neutrophil adhesion to activated endothelium in vitro and vaso-occlusion in SS mice in vivo. Our objective is to test the hypothesis that SS REVs will trigger acute non-transcriptional regulation on human microvascular ECs. The adhesion characteristics of SS RBCs to ECs was used as a direct biomarker of endothelial activation.

Materials and Methods :
We examined the impact of SS REVs on microvascular EC activation and RBC adhesion using in vitro microfluidic assays. For in vitro studies, SS REVs were collected from pooled SS RBCs activated using calcium ionophore. Human pulmonary microvascular endothelial cells (HPMECs) were cultured in microfluidic channels under controlled shear stress and exposed to REVs at 37 ºC for 2 hours. Von Willebrand factor (vWF) display on REV-exposed HPMECs was quantified. Adhesion quantification was performed with and without the vWF protease ADAMTS13 at 1 dyne/cm2 shear stress, followed by a rinse step and quantification of adhered RBCs. In vivo, HbSS-Townes mice and a dorsal skin-fold chamber model was used to determine if ADAMTS13 would reduce microvascular stasis in response to hemin.
 

Results :
Compared with AA REVs, SS REVs promoted activation of human pulmonary microvascular endothelial cells (HPMEC) as reflected by increased vWF expression (Fig. 1A-C). Under microfluidic conditions, we found abnormal SS RBC adhesion to HPMECs that had been exposed to SS REVs (Fig. 1D). SS RBC adhesion was reduced by vWF cleaving protease ADAMTS13 to a level similar to HPMECs exposed to AA REVs (Fig. 1D). The RBC adhesion induced by SS REVs was variable, and was higher in SS RBCs from patients with increased markers of hemolysis (Lactate Dehydrogenase, LDH and reticulocyte count) or with a history of deep vein thrombosis (DVT, Fig. 2). Consistent with these observations, studies in SS mice with implanted dorsal skin-fold chambers found hemin-induced stasis was inhibited by ADAMTS13 (Fig. 1E).

Conclusions :
Our results emphasize the critical contribution made by REVs to the pathophysiology of SCD by triggering microvascular EC activation and abnormal RBC adhesion mediated by vWF. Additionally, results from both in vitro microfluidic RBC adhesion tests using patient samples and in vivo stasis tests using SS mice indicated the effect of ADAMTS13 in mitigating SS RBC adhesion and vaso-occlusion. These findings may help to better understand acute pathophysiological mechanism of SCD and thereby the development of new treatment strategies using vWF as a potential target.

Figure 1. SS RBCs generate higher levels of REVs which activate HPMECs, increase vWF expression and mediates enhanced adhesion of SS RBCs, which can be reduced by ADAMTS13. (A, B): HPMECs were treated with SS REVs (A) and AA REVs (B) for 2 hours in 37 °C

Figure 2. RBC adhesion to REV-activated HPMECs correlates with subject clinical phenotype including hemolytic and inflammatory biomarkers. (A): A subpopulation (Group 1, N = 6) with distinct hemolysis markers of lactate dehydrogenase (LDH) levels and abso

Purpose :
Hydroxyurea (HU) is a disease modifying oral therapy with demonstrated clinical efficacy and safety in patients with sickle cell disease (SCD) for over 30 years. In low- and middle-income countries (LMICs) where the disease burden is high, universal HU treatment is limited. Our aim for this systematic literature review was to summarise the available evidence conducted in low-resource settings on HU efficacy, safety and real-world effectiveness to demonstrate the need for wider access to HU for SCD patients in LMICs. The preliminary observations from clinical studies are presented here.

Materials and Methods :
Articles published in Embase, PubMed, OVID and Web of Science between January 2000–2022 were reviewed; studies assessing the impact of HU in patients with SCD were considered.

Results :
Out of 3709 screened articles, 124 unique studies were included (Figure) with 20 clinical trials. Majority of the studies are from sub-Saharan Africa (SSA) and Latin America. The initial dose of HU ranged from 10–25 mg/kg/day and the most common initial dose was 15–20 mg/kg/day; the maximum tolerated dose (MTD) was 25–35 mg/kg/day. Across various age groups, as anticipated, a significant increase in haemoglobin, foetal haemoglobin and mean corpuscular volume levels with HU at both fixed (20 mg/kg/day) and escalated dose (≤35 mg/kg/day). HU at doses 15–30 mg/kg/day reduced the need for transfusions during the treatment period. HU treatment initiation reduced VOC-related pain, and reduction was better with escalated (30 mg/kg/day) versus fixed dosing (20–25 mg/kg/day). Similar reductions across various age groups were observed for atherosclerotic events. Initial low-dose HU therapy of 10 mg/kg/day and a 12-month MTD of 24.1±6.0 mg/kg/day reduced the primary stroke risk. Secondary strokes were prevented with HU (3 years; initial dose: 15–20 mg/kg/day; MTD: 30–35 mg/kg/day), and long-term treatment (3.5–9.3 years; 30–40 mg/kg/day) prevented recurrent strokes. HU treatment showed a reduction in the rates of hospitalisation, and this was ?50% lower with escalated dose (29.0±3.5 mg/kg/day) when compared to fixed dose (19.3±1.7 mg/kg/day). Treatment with HU was also associated with significant reduction in malaria events and related hospitalisations. Significant reduction in the risk of mortality was observed after treatment with HU, assessed as events per 100 patients/person years, which can be attributed to fewer SCD–related clinical complications, infections, malaria and possibly transfusions. Various dosing of HU was well tolerated in children and adults, and the reported events of myelosuppression were mostly transient.

Conclusions :
Our preliminary observations suggest that clinical use of HU in low-resource settings is effective in improving haematological outcomes, reducing the risk of VOCs and primary and recurrent strokes, and reduction of malaria-related events. Need for transfusion, rates of hospitalisation and mortality were also reduced with HU treatment. These findings are in line with current consensus guidelines to initiate HU therapy to SCD patients starting as early as 9 months of age. We expect that the complete analysis of this systematic review will enable us to compile sufficient evidence for optimising the use of HU in LMICs.

Purpose :
Pulmonary Hypertension(PHTN) is a well-described, serious, and potentially life-threatening complication of hemolytic anaemia, including Sickle Cell Disease(SCD) and is associated with a significant increase in morbidity and mortality rate.In the paediatric population,however,death and morbidity is much less common and has not been well defined. In the past, a significant association of Pulmonary Hypertension with Tricuspid jet velocity and laboratory markers of hemolysis has been found. However there is paucity of data in this subject. Hence,it is important to study the prevalence of pulmonary hypertension and to identify the variables predictive of PHTN in this population that will benefit patients in early identification and appropriate intervention.

Materials and Methods :
A cross sectional study was conducted considering a total of 91 patients between 5-18 years, with SCD (homozygous) of either sex, diagnosed using HPLC attending outpatient clinic in their steady state for routine clinical care at a tertiary care hospital. Patients with history of acute illness or any SCD related complication within 4 weeks were excluded from the study.At the time of enrolment, detailed history, demographic data, age at diagnosis, details of blood transfusion and Hydroxyurea were taken.Detailed clinical examination of study subjects was recorded in structured data sheet .Investigations done were complete hemogram, reticulocyte count,LDH,liver function tests,renal function tests,pulse oximetry, chest radiograph, electrocardiogram and 2D echocardiogram.Pulmonary hypertension was defined as a peak TRV of at least 2.5 m/second equating to a pulmonary artery pressure of at least 30 mm Hg. All the data was validated and analyzed in statistical software STATA, version 10.1,2011. 

Results :
Total 91 patients (51 males, 40 females) between the age group of 5-18 years were enrolled in the study with mean age of 10.68 ± 4.03 years.Overall prevalence of pulmonary hypertension (PHT) was 11%. Mild PHT (TR Jet velocity 2.5 to 2.9m/second) was found in 8(8.79% ) patients and 2 (2.20% ) patients had moderate to severe PHT (TR jet velocity >3 m/second).The mean age of the patient with pulmonary hypertension was found out to be 14.7+-2.26 and there was significant association between pulmonary hypertension and increasing age(p value=0.0011).High reticulocyte count(p-value<0.0001),increased serum bilirubin(p value<0.0075),raised serum lactate dehydrogenase (LDH) (p value<0.0004) , high total leucocyte count(p-value<0.0001), lower platelet count (p-value 0.0018),low hemoglobin (p=0.0061), more frequent packed red cell transfusions (p value-0.0001) were significantly associated with elevated pulmonary artery pressures. On MLR analysis,only Age,Reticulocyte count and BT rate were found to be significant.In the study,there was no association of Hydroxyurea therapy with lesser prevalence of PHTN.Also,there was no significant association of gender or oxygen saturation between patients with and without PHTN.

Conclusions :
High pulmonary artery pressures do occur in children with SCD. This emphasizes the need for early screening of Pulmonary Hypertension by echocardiography for early detection and intervention in paediatric patients with SCD
 

Purpose :
One of the main treatments for the life threatening sickle cell disease (SCD) is the generic drug hydroxyurea, which reduces complications associated with SCD and therefore increases quality of life while prolonging life expectancy. Access to comprehensive disease management vary greatly between high- and low-resource countries especially for pharmacologic agents like hydroxyurea. This study therefore aims to examine how patients with SCD in Ghana,  access hydroxyurea treatment. Since a country’s supply chain management plays a crucial role in making health commodities available and thereby enabling access to pharmaceutical products, this paper also addresses two sub-questions:

• What is the status-quo of the supply chain of hydroxyurea in Ghana?
• What barriers can be identified that hinder SCD patients from accessing treatment with hydroxyurea in Ghana?

Materials and Methods :
We conducted qualitative in-depth virtual semi-structured interviews with 10 key stakeholders involved in the supply chain of health commodities, including but not restricted to hydroxyurea. The questions asked in the expert interviews were supported by a preparatory interview guide containing of semi-structured questions. The interview guide was reviewed by the study team ahead of the virtual interviews. The responses received were examined based on a content analysis and presented in a process map and a causal loop diagram.

Results :
Our results revealed a high fragmentation and decentralization of the supply chain of health commodities in Ghana that severely compromises the continuous availability of hydroxyurea in Ghana. The results also disclosed that there is currently no central procurement or distribution of hydroxyurea. Outside of the public sector, most patients have to pay for treatment out-of-pocket which is a great financial barrier that leaves many patients unable to afford the treatment. Furthermore, there is a lack of accurate data on SCD prevalence in Ghana, which makes effective forecasting and adequate health interventions challenging. Additionally, health facilities that offer hydroxyurea treatment are mainly located in urban areas, which are inaccessible to many patients living in rural areas.
The causal loop diagram depicted below (see Figure 1) summarizes the findings of our study and captures their complexity. It shows the factors that influence each other with regard to the accessibility of hydroxyurea treatment for patients with SCD in Ghana. The arrow between the factors shows the direction of influence and the sign next to the arrow represents the positive (+) or negative (–) influence a factor has on another factor.

Conclusions :
Based on our evaluation, this study identifies four main barriers that patients with SCD face to access treatment with hydroxyurea in Ghana. These barriers include the lack of accurate data on the number of patients with SCD in Ghana, the high financial hurdles to access treatment with hydroxyurea, the lack of treatment decentralization with hydroxyurea, and the limited availability of hydroxyurea. Digitalization was identified as a common denominator that could improve many of these barriers and therefore could support the wide-scale implementation of hydroxyurea access for SCD treatment.

Figure 1: Causal Loop Diagram (own representation)

Purpose :
HCP specialty and care setting may influence the care that SCD patients receive (Smeltzer et al. BMJ Open 2021). This analysis of SWAY reports potential differences in the treatment and management of SCD by HCPs, based on HCP specialty and care setting.

Materials and Methods :
SWAY was a cross-sectional survey of patients with SCD aged ≥6 years (N=2145) and HCPs (N=365) developed by international SCD experts, patient advocates and Novartis to assess patient and HCP experiences of SCD (conducted between April and October 2019). Eligible HCPs had qualified in their primary specialty by 2014 and were managing ≥10 SCD patients at the time of the survey (≥5 patients/HCP in Canada; ≥2 patients/HCP in the Netherlands). Data reflect only the experiences of the surveyed HCPs (recruited by Adelphi Real World Network).

Results :
HCPs were grouped by specialty (hematology, n=150; hematology–oncology, n=101; pediatric, n=31; general practitioner [GP], n=50; and other [internal medicine specialist, emergency department physician, physician assistant, nurse/nurse practitioner, other], n=33) and care setting (SCD center, n=46; university/teaching hospital, n=105; regional center/community hospital, n=57; private hospital/office, n=109; and other [health center and other], n=48). Acute (vaso-occlusive crisis [VOC]) pain was reported as one of the most common symptoms discussed with patients by most hematologists (82%), hematologist–oncologists (79%) and pediatricians (87%); reported as a discussed symptom by 54% of GPs and 42% of other HCPs (chronic pain was reported by 78% of GPs and 70% of other HCPs). Hydroxyurea (HU) – a treatment for VOCs – was initiated by 97% of hematologists, 88% of hematologist–oncologists and 90% of pediatricians versus 14% of GPs and 30% of other HCPs. More hematologists, hematologist–oncologists and pediatricians ranked reducing VOCs as a top treatment goal above reducing chronic pain levels; the opposite was true for GPs and other HCPs (Table). Exchange and standard blood transfusions were initiated by more HCPs at SCD centers and university/teaching hospitals than at other care settings (Table), although fewer HCPs at SCD centers versus other care settings reported iron overload as a complication patients experienced (SCD center, 26%; university/teaching hospital, 40%; regional center/community hospital, 47%; private hospital/office, 31% and other, 42%). HCPs at SCD centers were also more likely to provide information to patients in the form of leaflets or direct them to patient support groups (leaflets/patient support groups: SCD center, 70%/63%; university/teaching hospital, 60%/47%; regional center/community hospital, 49%/30%; private hospital/office, 59%/33%; other, 60%/33%).

Conclusions :
Hematologists, hematologist–oncologists and pediatricians in SWAY were more likely to discuss VOCs during consultation with their patients than GPs or other HCPs, a possible reason for why fewer GPs and other HCPs reported initiating HU than the other groups analyzed. Furthermore, HCPs at SCD centers appeared to be more likely to initiate certain SCD treatments than those at other care settings. Results from this analysis of SWAY support a multidisciplinary and collaborative approach to SCD care in order to optimize patient outcomes, with distinct but vital aspects of patients’ needs fulfilled by each HCP specialty and care setting.

Table. Top 10 therapies ever initiated to patients of any age, stratified by HCP specialty and care setting

Purpose :
Hematopoietic stem cell transplantation (HSCT) is the only curative therapy available for sickle cell disease (SCD). However, HSCT indications vary across countries, and HSCT mortality scores lack validation in SCD settings. Objectives: To identify how many adult patients followed at the SCD outpatient service of the Hospital das Clínicas de São Paulo meet at least one indication for HSCT, according to the Brazilian Ministry of Health (BMH) or to an international expert panel (Angelucci et al, 2014); to characterize the distribution of the hematopoietic cell transplantation comorbidity index (HCT-CI) and Charlson comorbidity index (CCI) scores, comparing them between transplantable and non-transplantable population to correlate with the severity of the disease. 
 

Materials and Methods :
Single-center, retrospective study. Using the set of HSCT indications from the Brazilian Ministry of Health (BMH) or Angelucci et al, we determined the frequency of patients with >1 indication. The concordance between the two sets was assessed using Cohen’s kappa coefficient. HCT-CI and CCI were used to assess risk of HSCT-related severity; median scores in transplantable and non-transplantable populations were compared using Mann-Whitney.

Results :
We included 219 SCD adults (143 SS ,47 SC and 29 Sβ); 138 were female;the median age was 38 (range,20-74) years. 33 patients had ischemic stroke, 2 had hemorrhagic stroke and 10 Moya-moya. 125 patients had acute chest syndrome and 210 had vaso-occlusive crisis (VOC). 11 patients had pulmonary arterial hypertension,73 avascular necrosis,35 priapism,65 proliferative retinopathy and 184 proteinuria. 103 patients were under hydroxyurea. 186 received blood transfusion; 72 were alloimmunized. 168 patients(77%)had at least one HSCT indication, 108(50%) by BMH and 166(76%) by Angelucci;60 met an indication only by Angelucci and 2 only by BMH.The overall agreement rate between the 2 sets was 0.71,with a Cohen’s kappa coefficient of 0.44. Considering only SS and Sβ0 patients,the Cohen’s kappa remains moderate (0.5). The main indication by both sets was alloimmunization,followed by CNS event(BMH) and recurrent VOC(Angelucci). Among age groups, the percentage of patients with >1 TCTH indication was:18-30 years:70%;31-40 years:85%; 41-50 years:71%;51-60 years:77%;+60 years:90%. Median HCT-CI in the transplantable population was 2(range,0-12) and 1(range,0-5) in the non-transplantable(p=0.004);median CCI was respectively 1(range,1-9) and 0(range,0-3)(p<0.001). 

Conclusions :
HSCT indications according to the BMH are stricter than those by Angelucci, explaining the moderate agreement on Cohen’s kappa coefficient. The rate of indications is higher than previously reported due to the higher rate of alloimmunization and older median age of our cohort, as HSCT indications increase over time. Median HCT-CI and CCI were significantly higher in the population with >1 HSCT indication, reflecting its higher severity. This study shows that indication of HSCT should be counterbalanced with the risk of mortality of SCD and HSCT.  Dynamic scores of mortality should be further validated to predict the best moment for HSCT in adults with SCD.

Purpose :
Sickle cell disease (SCD) is an inherited blood disorder caused by a single gene mutation that results in the production of abnormal hemoglobin, sickle hemoglobin (HbS). Under deoxygenation, HbS tends to polymerize, resulting in deformed, rigid, and fragile red blood cells (RBCs). These RBC alterations result in vaso-occlusion and hemolysis. The US FDA has recently approved voxelotor, an oxygen affinity modulating drug for the treatment of SCD. When voxelotor binds to HbS, it stabilizes HbS in an oxygenated configuration, which reduces HbS polymerization and RBC sickling [1]. However, the overall impact of voxelotor on RBC capacity for vaso-occlusion has not been examined. Here we use a microfluidic device, the OcclusionChip [2], to investigate the effect of voxelotor on the RBC capacity for occlusion in SCD.

Materials and Methods :
Venous blood samples were collected in EDTA from subjects with homozygous HbSS and healthy volunteers (HbAA) under an IRB-approved protocol. OcclusionChip devices were fabricated using soft lithography protocols [2]. RBCs were suspended in PBS at 20% hematocrit. To create Vox + samples, HbSS containing RBCs at 20% hematocrit were mixed with 67mg/ml voxelotor in 100% dimethyl sulfoxide (DMSO) to a final concentration of 600 µmol/L voxelotor and incubated at 37 C0 for 1 hour. RBCs were perfused through the OcclusionChip with a constant inlet pressure, followed by a PBS wash and microscopy. For hypoxia experiments, blood was deoxygenated in OcclusionChip (pO2 ~45 mmHg) [2]. The Occlusion Index (OI), representing the overall microcapillary network percentage occlusion, was quantified as previously described [2]. Data are reported as mean ± standard deviation (SD).

Results :
Using the OcclusionChip, we analyzed RBC-mediated microvascular occlusion for healthy (HbAA) and HbSS samples under normoxia and hypoxia in comparison to voxelotor treated HbSS samples under hypoxia (Figure 1A). There was no statistical difference between the OI values for HbAA samples under normoxic versus hypoxic conditions (P = 0.50). The OI values for HbSS samples were significantly elevated under hypoxia in comparison to normoxia (P = 0.007) (Figure 1B). However, when HbSS samples were treated with Voxelotor, the OI values significantly decreased under hypoxia by 52.6% ± 10.47% (mean ± SD, P = 0.010) (Figure 1B) but were still significantly greater than the OI of HbAA (P = 0.001) (Figure 1B).

Conclusions :
Our findings suggest that hypoxia alters the deformability of SCD RBCs but not healthy RBCs. This is likely due to HbS polymerization during deoxygenation. Under hypoxic conditions, HbSS RBCs treated with voxelotor exhibited significantly less occlusion in the Occlusion Chip, suggesting that voxelotor may reduce vasoocclusion in vivo. Voxelotor reduced OI to different degrees in RBCs from different individuals, indicating inter-patient variability in response to voxelotor for this aspect of SCD pathophysiology. Correlation between this in vitro test and in vivo response to voxelotor is warranted, as well as investigations with other novel SCD therapies, to validate the OI's utility as a functional indicator for evaluating the efficacy of novel therapies in preventing microcapillary occlusions.
[1] Elliott et al., NEJM, 2019,381:509-519
[2] Man et al., LabChip, 2020, 20, 2086-2099.

Figure 1. OcclusionChip: standardized microfluidic assessment of RBC-mediated microcapillary occlusion in Healthy (HbAA), SCD (HbSS), and SCD treated with voxelotor. (A) Occlusion Chip device for interrogation of RBC-mediated microcapillary occlusion. (B)

Purpose :
The degree of red cell phenotype correction, and therefore cure, in gene-based therapy and allogeneic hematopoietic stem cell transplant (allo-HSCT) with mixed donor chimerism (MDC), may not be captured by conventional laboratory tests like a complete blood count or hemoglobin profile.  We propose to define a cure as the achievement of at least the sickle cell trait level of red blood cell (RBC) deformability and sickling potential. RBC deformability is markedly abnormal in SCD; these abnormalities contribute to vaso-occlusion, ischemia, and SCD complications Even fully oxygenated, sickle RBCs are less deformable than those of HbAA or HbAS individuals; upon deoxygenation, deformability further declines. To determine if the rheology of blood from SCD patients post-cell-based therapy and post-HSCT with varying degrees of donor chimerism fall into the HbSS range of values, we functionally assessed the peripheral blood from a series of 7 post alloHCT SCD patients and one post-gene addition autologous hematopoietic stem cell transplant subject using oxygen gradient ektacytometry.

Materials and Methods :
Peripheral blood samples (EDTA) from6 SCD patients post-allo-HSCT, one matched sibling donor sample,  and a peripheral blood sample from an individual treated with Bluebird Bio’s Group C lentivirus-based gene addition were collected and immediately analyzed using oxygen gradient ektacytometry  (LORRCA). Oxygen gradient ektacytometry measures RBC deformability (elongation index, EI), under a range of pO2 (150-0 mmHg). EImax is the deformability of the oxygenated sickle RBC; EImin is the deformability of deoxygenated RBCs. The point of sickling (PoS) is the pO2 at which HbS polymerizes. Additional clinical data including Hb profile, donor chimerism, and symptoms were obtained via chart review. Reference ranges for genotypes were generated as described above using n=45 HbSS samples ages 2-21, on hydroxyurea, chronic transfusion, or untreated; n=14 HbAS, and n=43 HbAA.

Results :
6/7 allo-HSCT subjects exhibited normal or trait level rheology as measured by oxygen gradient ektacytometry, based on their donor genotype; one patient with 40% whole blood chimerism and an HbS less than 50% had EImin, EImax, and PoS values in the HbSS disease range. The gene addition treated subject had an HbS of 40%, yet their EImin, EI max and PoS also fell within the disease range. Both subjects with disease range RBC rheology reported SCD symptoms post-cell-based therapy, namely pain events. The other subjects did not report SCD symptomatology post-cell-based therapy.

Conclusions :
Oxygen gradient ektacytometry identified uncorrected, SCD level RBC dysfunction in two subjects post-cell-based therapies with conventional laboratory test values consistent with a cure. The current post-HCT evaluation depends on chimerism and hemoglobin profiles and would not detect the significant functional abnormalities visible by Lorrca with Oxygenscan biomarkers that indicate that Patient 2 is at risk for SCD related complications. Determination of a cure in gene-based therapies is an area of active research. Our results suggest that functional RBC analysis is needed for the management of post alloHCT SCD patients and may be even be more essential to assessing new gene-based therapy approaches to curing patients with SCD.

Oxygen gradient ektacytometry analysis of an allo-HSCT subject with 40% donor chimerism and their HbAA matched sibling donor (blue line).

Oxygen gradient ektacytometry analysis of a subject post gene addition based gene therapy, multiple measurements.

Purpose :
In Cameroon, pain management in children with sickle cell disease presenting at pediatric emergency services remains problematic due to the limited availability of morphine on the Cameroonian market. Although tramadol is not recommended by the WHO for the management of severe vaso-occlusive crises in children, this molecule is pharmacologically similar to morphine and remains the main analgesic used in pediatric emergency services in Cameroon.

Materials and Methods :
In order to compare the analgesic effect of oral morphine and injectable tramadol, we conducted a non-inferiority single-blinded randomized controlled clinical trial over a period of 2.5 months. We included in our study 45 patients. Patients included were sickle cell disease patients aged between 5 and 17 years who were admitted at the pediatric emergency department of the CME-FCB for vaso-occlusive crisis and had a pain intensity ³ 70/100 on the visual analogue scale (VAS). After obtaining informed consent from the parents, the patients were randomly divided into 2 groups, one receiving oral morphine 0.4 mg/kg/4h + injectable placebo/6 h and the other receiving injectable tramadol 1.5 mg/kg/6 h + placebo capsule/4h. Diclofenac in suspension (1 mg/kg/8 h) was prescribed as part of additional analgesia, in case pain intensity was ³ 5/10 on the VAS two hours after taking any of the treatments.

Results :
Forty-two (42) children completed the study. The male to female sex ratio was 1.3. The median age of the patients was 10.5 years (IQR 8.8-12.3). There was no significant difference between the medians of the initial pain intensity (p=0.184). Pain relief at the 30th minute was better for patients in the Tramadol group (p=0.019). Patients in the tramadol group used the additional analgesic twice as much as those in the morphine group (p=0.032). There was no significant difference between the median duration of analgesic treatment for morphine, 4.1 days (IQR 2.6-4.5) Vs 4.3 days (IQR 2.6-5.8), p=0.350 for tramadol respectively. The incidence of occurrence of pruritus was greater in the morphine group (p=0.008).

Conclusions :
The comparison of median pain intensity showed a difference in favour of tramadol at the 30th minute, a contrast that could be explained by the difference in bioavailability between oral morphine and injectable tramadol. Nonetheless, injectable tramadol is efficacious in the analgesic management of severe vaso-occlusive crises in children with sickle cell disease and may constitute, in combination with a non-steroidal anti-inflammatory drug (NSAID), an alternative to morphine in countries where the latter is still grossly unavailable.

Purpose :
Sickle cell anemia (SCA) is a monogenic disorder caused by a single point mutation in the beta-globin gene, however, phenotypic heterogeneity is commonly observed among the patients. Severity scores such as Tweel’s score were developed to differentiate SCA patients into various categories and identify severe patients who may need more clinical attention and management. However, most of these studies are conducted in Europeans and its utility has never been tested in Indian patients who are known to have a different clinical course and complications. We investigated the utility of the existing Tweel’s score comprising of 16 parameters in determining disease severity and further understand any differences to develop a severity score that can be applied for making informed management decisions

Materials and Methods :
One of the severity scores proposed by Tweel et al, reported for the SCA patients, uses multiple clinical and biochemical parameters to categorize the severity. Using this scoring system, we used data on clinical history, course and complications and various biochemical parameters from 171 Indian Sickle cell anemia patients recruited at Government Medical College and Hospital. The patients were identified as mild, moderate and severe based on the clinician’s assessment (DJ and SI). The patients were also scored using the 16 parameters included in this score and allocated into three categories; mild, moderate and severe. Correlation analysis was performed to evaluate the performance of the established scoring system with the clinical assessment in Indian patients. Further, various combinations of clinical course and biochemical parameters were used and correlation analysis was performed.

Results :
Approximately, 2/3rd of the patients (64.9%) identified as per clinicians’ assessments were correctly matched with those using Tweel’s scoring system, leaving one-third still uncategorized. Further, mild cases formed the majority of the concordant patients compromising the correct categorization of moderate and severe patients. Modified scores were generated by including in Tweel’s score, four clinical parameters as per the treating physician’s experience (MS1) and further, replacing the biochemical measures with clinical parameters based on clinical course and complications (MS2). Scores were recalculated for the Discovery group using two new scoring systems. Comparison of the modified scores with the standard Tweel’s score improved the disease severity prediction to 88.7% for severity scores having both clinical and biochemical parameters (MS1) and up to 92.3% using only clinical course and no biochemical parameters (MS2). Results were found to be robust even after designing and comparing several random sets of Discovery and validation groups from the 171 patients included in the study, as well as swapping the discovery group with the validation group for all random sets.

Conclusions :
We have developed a severity score for Indian sickle cell patients which could be used for better understanding and evaluation of the clinical course and hence disease management. This will help clinicians being able to evaluate sickle cell anemia patients and manage them accordingly.

Purpose :
Sickle cell disease (SCD) potentially causes numerous acute and chronic complications across the lifespan. The majority of patients with SCD live in sub-Saharan Africa and 1 in 50 Ghanaian newborns are affected. Foetal hemoglobin (Hb F) level is a known modulator of the severity of SCD. High levels of Hb F inhibit sickling and correlate with reduced disease morbidity and mortality. Hb F monitoring is also used to assess adherence and response to disease modifying (hydroxyurea) therapy. Standard hemoglobin electrophoretic techniques are unable to quantify Hb F. In resource-limited settings like Ghana, the use of high performance liquid chromatography (HPLC) for Hb F quantification is often challenging due to high cost and unavailability of laboratory equipment and skilled technicians. We report on the performance of a low-cost, point-of-care, microchip based cellulose acetate electrophoresis “Gazelle” compared to HPLC for Hb F quantification in Ghanaian children with SCD. GazelleTM is a fast (<8 minutes), easy-to-use test which can be performed by minimally trained personnel using only a finger-prick volume of blood.

Materials and Methods :
The study was conducted at the paediatric SCD Clinic in Korle Bu Teaching Hospital, Accra, Ghana.  Children (age ≥ 1 year to 16 years) known to have sickle cell disease who are on hydroxyurea therapy, and followed at the paediatric sickle cell clinic were enrolled in the study. The study is still ongoing.

Results :
A total of 110 children were included in the analysis. Gazelle showed a correlation of 0.93 when compared to HPLC (Figure 1).

Conclusions :
Haemoglobin variants are quantified by Gazelle offering the ability to monitor disease modifying therapy in SCD such as hydroxyurea, where quantification of HbF levels is the key to assessing treatment adherence and determining response to therapy. Gazelle has the potential to be utilized as a POC test for quantification of foetal haemoglobin (Hb F) levels in monitoring hydroxyurea therapy for children with SCD.

Hb Quantification Performance of Gazelle compared to HPLC

Purpose :
For long sickle cell and thalassaemia were thought to be childhood diseases. It was believed that there was no future for these kids but to suffer the consequences of inheriting the genes and so their future was unplanned.
I am Shifneez, 39 years old sickle beta-thalassaemic from Maldives. My parents were told I will not live up to be 14. I grew up as a very active student in school, graduated in teaching and worked at the very school I studied at.

Materials and Methods :
I had my first sickle crisis at the age of 14. After about 5 years of not knowing what sickle cell can do, I was introduced to this drug called Hydroxyurea (HU) in 2003. After I got married and was ready to have a family, I was advised by my physicians to avoid becoming pregnant while taking HU. Understandably, they told me about the potential harm to the fetus. I was left with a real dilemma: Take a drug with unknown effects on fetal development or give up a helpful, necessary medication that has kept me healthy since I started it.
 

Results :
Many SCD patients are not willing to take a chance and go through pregnancy, let alone take the risk of continuing pregnancy with HU.  hence, there is not a lot of clinical evidence with pregnancy while taking HU. My determination to start a family, despite being aware of the risks associated, past 17 years, I have had 3 pregnancies each experience different with regard to HU therapy. Today, I am a mother of 2. I had my last pregnancy and lactation while taking HU.

Conclusions :
HU, the only disease-modifying therapy approved for SCD, has continuously shown to improve the quality of life of adults and children with sickle cell.  I hope the story of my personal journey can break the barrier and open the possibility for others like me to reach their milestones and dreams.
 

Growth Chart of kids with and without exposure to Hydroxyurea during pregnancy and lactation

Experience: ferritin level through Hydroxyurea therapy Vs Blood transfusion

Purpose :
Patients with sickle cell disease (SCD) are at risk for developing chronic vascular complications. The presence and the severity of these complications are not related to the severity of the vaso-occlusive disease and their pathophysiology is not completely elucidated. Circulating endothelial activation markers and their interaction may contribute to the pathophysiology of these vascular complications. In the present study, we hypothesized that plasma levels of endothelial activation markers: Circulating Endothelial Cells (CEC), E-Selectin, Progenitor’s Endothelial cells (PEC) and circulating macrovesicles (MVs), could be evaluated to characterize chronic vascular complications related to endothelial dysfunction in SCD patients. We explored the predictive value of these markers in the occurrence of disease-related microvascular injury of the eye (retinopathy), kidney (nephropathy) and skin (chronic active ulcers). Finally, we analyzed the relevance of these markers in the degree of severity of SCD and in the presence of associated thromboembolic disease (deep venous, peripheral or pulmonary thromboembolic disease).

Materials and Methods :
Fifty patients with SCD aged 18 years or more were enrolled in this study at steady state. We investigated the relationship between plasma endothelial activation markers (CEC, E-Selectin, EPC, circulating MVs) and SCD-related microvascular complications in 3 different territories, retinopathy, nephropathy and leg ulcer.

Results :
Sixteen patients (32%) had SCD related retinal, renal and/or cutaneous vasculopathy. Retinopathy and nephropathy were found in 9 out of 50 and 6 out of 50 patients respectively. Three patients had leg ulcer. SCD patients with vasculopathy were older than patients without vasculopathy [37.5 (26.5-41) vs. 27.5 (21–33) years, P = 0.039]. No differences were found between patients with or without vasculopathy for sex ratio, sickle cell genotype, thromboembolic event, severe disease or hydroxyurea treatment.
Among the markers of endothelial activation studied, only a significantly higher level of E-selectin was found in SCD patients with microvasculopathy (p=0.015). In subgroup analysis, patients with proliferative sickle cell retinopathy presented higher levels of E-Selectin compared to patients without retinal damage (p< 0.001). Our cohort and at steady state, whatever vasculopathy or disease severity, did not expressed high count of CEC and EPC that are markers of endothelial injury and repair. High levels of circulating Erythrocyte-MVs and Platelet-MVs were found in our SCD patients. In contrast, numbers of Leucocyte-MVs and Endothelial-MVs, respectively, were most often within the normal range. We did not shown any significant differences in MVs levels between vasculopathy and not vasculopathy SCD patients.
In patients with severe vaso-occlusive disease, E-selectin level was significantly higher although other endothelial markers count were not different from SCD patients without severe disease. Patients with severe vaso-occlusive disease were significantly younger, had higher level of HbF, they more often had history of deep venous embolism and mostly treated by hydroxycarbamide.
 

Conclusions :
Our study provides evidence that circulating E-Selectin is associated with sickle cell retinopathy. Its may be considered as an accurate biological marker for endothelial involvement in SCD retinopathy. Further studies will be required to determine whether the E-Selectin could be used as an early biomarker of retinopathy sickle cell development, or even become a therapeutic target.

Purpose :
Sickle cell anemia (SCA) is characterized by a myriad of acute and chronic complications.  These complications negatively impact the ability to achieve educational and occupational goals making this group a particularly vulnerable population to socioeconomic challenges (PMID 30540112).  In the general population, indices of socioeconomic distress are associated with poor nutrition and kidney disease risk (PMID 25573510). The association of socioeconomic distress with nutritional status and kidney function in people with SCA is unknown.

Materials and Methods :
We conducted a cross-sectional study to describe the burden of socioeconomic distress and its impact on nutritional status and kidney function in an SCA cohort (n=332) treated at an urban academic institution.  The distressed community index (DCI), a composite of socioeconomic indicators that estimate the economic well-being of a community, was determined for each individual using the following website: https://eig.org/dci/interactive-map.  A higher DCI score indicates a more distressed community. The associations of DCI with measures of nutritional status (serum albumin, body mass index [BMI], vitamin D 25-OH) and kidney function (estimated glomerular filtration rate [eGFR]; albuminuria; serum bicarbonate, potassium, and erythropoietin levels), were determined using linear or logistic regression.  The analyses were adjusted for age, sex, and hydroxyurea use for nutritional status as well as for APOL1 G1 and G2 risk status for kidney function. The beta coefficients provided are based on increments of 10 for DCI.  Median and interquartile ranges (IQR) are provided.

Results :
The median age of the cohort was 31 years (IQR, 24 – 41 years), 55% were female, and 53% were on hydroxyurea therapy.  The median DCI in this cohort was 87 (IQR, 61 – 96) with 61% of SCA patients living in a distressed tier (DCI≥ 80) and only 5% living in a prosperous tier (DCI< 20).  The DCI tiers for SCA patients trended towards more socioeconomically distressed communities compared to the general African American population (P=0.1) (Figure 1A).  A higher DCI, reflecting an increase in socioeconomic distress, was associated with a trend for a lower odds ratio (OR) of being on hydroxyurea therapy (OR 0.93, 95% CI: 0.86 – 1.01; P=0.08).
An increasing DCI was significantly associated with lower serum albumin (β= -0.02; P=0.016) (Figure 1B), BMI (β= -0.01; P=0.045), and vitamin D 25-OH levels (β= -0.7; P=0.001) (Figure 1C).  A higher DCI was associated with a trend for a lower eGFR (β= -0.6; P=0.09) (Figure 1D) while no association was observed with urine albumin concentration (P=0.8).  Lower serum bicarbonate (β= -0.1; P=0.02) and lower erythropoietin (β= -0.9; P=0.1) levels, but not serum potassium concentration (P=0.9), were also associated with higher DCI.

Conclusions :
In conclusion, we demonstrate that a high proportion of patients with SCA treated at an urban academic institution live in distressed socioeconomic communities.  Furthermore, higher distressed community indices are associated with lower measures of nutritional status and with some markers of impaired kidney function.  Future studies investigating the effects of socioeconomic distress on access to healthy foods and kidney disease risk may guide public health strategies to improve health disparities in the SCA population.

Figure 1

Purpose :
Individuals with sickle cell disease have many options for mobile applications. Everything from monitoring pain, nutrition, exercise, and more.  This stakeholder-driven mobile application is a self-management tool that was developed to assist young adults with sickle cell disease with access to information. The aim was to develop a stakeholder-driven mobile application to assist persons with sickle cell disease with a tool for self-management, and access to information from trusted resources. The purpose of the LWSC® App is to provide the user with a tool to track sickle cell episodes within the context of daily living with access to quality, trusted information available through the LWSC® App. It is well documented that mobile applications have utility and the concept is widely accepted by persons living with sickle cell disease.

Materials and Methods :
The Living Well with Sickle Cell® App (LWSC®App) is a component of the Living Well with Sickle Cell® Initiative focusing on improving the lives of children and adults with sickle cell disease(SCD). The design is a collaboration with g youth and young adults with sickle cell disease to improve their overall wellness. The LWSC®App design research was conducted over a two-year period, engaging 3 groups of participants in 2 sets of focus groups; youth ages 13-17 and their parents, young adults aged 18-26, and a survey conducted through our social media networks. The design of the LWSC®App is the result of our commitment to remain stakeholder-driven and patient-focused throughout the process.  Pain crises may be caused or influenced by a variety of factors including overall wellness, mood, and hydration. With the mobile application (LWSC®App) patients can log pain, mood, and hydration levels.  This information can help avoid certain behaviors and adapt to stay healthier, live longer, leading to more time between crises. Many persons with sickle cell disease suffer from memory issues and chronic fatigue. The calendar tool helps manage doctor’s appointments, medication and improve adherence to treatment regimens.

Results :
LWSC®App analytic dashboard is used to understand what features are used the most and use this 
information to make quality improvements. Users range from persons living with sickle cell to parents, students, and medical professionals with 95% of users reporting that they are satisfied or very satisfied with the  LWSC® App information and ease of use. Currently, the mood, hydration, and pain logs are used the most, respectively. In year one, post-beta testing, there were 37 users from two countries; the United States and Canada.  In year two, after developing promotional videos and launching a social media campaign through a private Facebook Group, Sickle Cell Mom's Rock, the number of users has grown to nearly 100 from four countries. 

Conclusions :
The LWSC®App is a disease self-management tool, available at no cost, yet, has tremendous value to persons living with sickle cell disease and their families, and the network of medical professionals providing quality care across the lifespan.  This powerful mobile application can be downloaded for both android and IOS.  It was designed with stakeholder input and remains stakeholder-driven. 

App Short

Purpose :


Materials and Methods :


Results :


Conclusions :

Specific total force production in SCDCON and SCDGBT before and after the 8-week period. Each point represents a single measurement and lines are linking consecutive points. p-values are mentioned on the top of the figure.

Metabolic indices in SCDCON and SCDGBT before and after the 8-week period.

Purpose :
Sickle cell disease (SCD) is a hereditary and chronic life-threatening disorder, characterized by haemolytic anaemia. Increased 2,3-diphosphoglycerate (2,3-DPG) concentrations, along with decreased oxygen affinity of hemoglobin, may be related to the variability of clinical outcomes in SCD. Furthermore, genomic health data holds promise to improve the prediction of disease severity in SCD. Based on the integration of genomics, metabolomics and clinical data from 1000 SCD patients, to be included in 2022, GenoMED4all aims to develop Artificial Intelligence (AI) based deep learning algorithms to improve the prediction of disease severity and phenotype in SCD. This study aims to correlate non-quantitative metabolomics data obtained from dried blood spots (DBS), one of the inputs for GenoMED4all, to quantitative measurement of 2,3-DPG. Aiming to improve the potential of non-quantitative metabolomics from DBS to asses 2,3-DPG.

Materials and Methods :
In snap frozen blood samples from 37 SCD patients and 29 healthy controls, 2,3-DPG was quantified by liquid chromatography mass spectrometry. 2,3-DPG was also detected in DBS from the same subjects by direct infusion high resolution mass spectrometry (DIHRMS). The oxygen tension at 50% Hb saturation (p50) was determined using a Hemox Analyzer (TCS). Statistical analysis were performed by Spearman’s correlation coefficients (SPSS v26.0.0.1) and Mann Whitney testing (GraphPad Prism v9.3.0).

Results :
After correcting for Hb, 2,3-DPG concentrations were higher in SCD patients than in controls (p<0.001) and Z-scores for 2,3-DPG, as assessed by DIHRMS, were similar in patients and controls. The Z-scores positively correlated with 2,3-DPG concentrations (Fig1A, 0.353, p=0.004). Because of the anaemia in SCD, red blood cells (RBCs) and plasma make up lower and higher volumes in the blood, respectively, compared to healthy controls. This affects the ratio of RBC and plasma metabolites on the DBS. DIHRMS detects a wide range of RBC and plasma metabolites, whereas the quantitative measurement is restricted to measuring 2,3-DPG of RBCs. To correct for those differences between methods, we applied a correction factor to the DIHRMS data using (1/Ht)*(1-Ht/1), correcting for the RBC volume (1/Ht) and the plasma volume (1-Ht/1). This resulted in a trend towards higher Z-scores in patients than controls (p=0.0597). Moreover, the positive correlation with 2,3-DPG concentrations increased to 0.526 (Fig1B, p<0.001). As 2,3-DPG affects the oxygen affinity of Hb, all measurements were correlated to p50. Expectedly, 2,3-DPG concentrations positively correlated with p50 (0.842, p<0.001). After applying the correction factor to the DIHRMS data, p50 correlations increased from 0.361 (p=0.003) to 0.529 (p<0.001).

Conclusions :
Strongest correlation between quantitative and non-quantitative methods for 2,3-DPG detection were observed after correcting for both RBC and plasma volumes in non-quantitative metabolomics. After correction, DIHRMS can be used to assess 2,3-DPG concentrations in DBS. This translation of non-quantitative to quantitative metabolomics for 2,3-DPG and potentially other RBC metabolites adds significant value to the use of DIHRMS, as DIHRMS is far more efficient in obtaining extensive information about the total metabolome than quantitative methods. The large population size and the AI based deep learning algorithms of GenoMED4all will enable to evaluate the potential of non-quantitative metabolomics in SCD. 
Funding: Horizon2020, GenoMED4all(https://genomed4all.eu/), Agios Pharmaceuticals Inc., ERN-EuroBloodNet


 

Fig 1. correlations between non-quantitative (Z-score) and quantitative 2,3-DPG detection A) non-quantitative and quantitative 2,3-DPG detection B) non-quantitative detection corrected for RBC and plasma volume and quantitative 2,3-DPG detection

Purpose :
La rate est un organe lymphoïde secondaire ayant pour fonction la filtration du sang et elle joue un rôle dans l’immunité innée et acquise.
La drépanocytose est une hémoglobinopathie très répandue dans le monde avec près de 300.000 nouveau cas chaque année en Afrique sub-saharienne où la mortalité reste élevée.
La littérature affirme que la rate dans la drépanocytose subit une fibrose et atrophie avant l’âge de 5 ans donnant lieu à une asplénie fonctionnelle. Par contre, certaines études en Afrique Sub-saharienne décrivent une splénomégalie persistante ; ce qui peut suggérer un mécanisme physiopathologique différent de l’asplénie et/ou l’influence des facteurs environnementaux comme la malaria.
Peu d’études sont décrites sur la structure de la rate chez des sujets drépanocytaires vivant en milieu tropical. Nous nous proposons dans cette étude de décrire les aspects anatomo-histologiques et immuno-histochimiques de la rate chez des sujets drépanocytaires congolais comparés à un groupe témoin non drépanocytaire.
 

Materials and Methods :
Il s’agit d’une étude transversale descriptive et analytique, effectuée sur des pièces de rates recensées à Kinshasa, en RDC sur une période de 20 ans (2001-2021).
Tous les spécimens ont été fixés dans le formol à 10%, déshydratés et éclaircis, enrobés dans la paraffine, coupés au microtome et colorés à l’HE et au Rouge Sirius, marqués par les Ac anti CD8, anti CD34, anti CD68, anti AML.
Notre échantillon global comportait 43 rates dont 32 de sujets homozygotes SS et 11 témoins de rates de sujets traumatisés dont un sujet AA et 10 autres dont le profil hémoglobinique n’était pas précisé.
 

Results :
Chez les drépanocytaires, le volume de la rate était plus élevé (valeur moyenne : 20,8 x 12,2 x 7,4 cm et âge moyen de10 ans), la densité et la taille des follicules lymphoïdes étaient réduites (densité : 23,9 vs 45,8/10 ch, p<0,001 ; taille : 49,5 x 34,9 vs 65,1 x 46,2 µm ; p=0,001) avec rarement la présence de la zone marginale. Les corps de Gamna-Gandy étaient présents (86,2% vs 13,8%) et la fibrose plus intense. La densité des sinusoïdes CD8+ était faible tandis que la densité des micro-vaisseaux CD34+ était plus élevée. La densité des macrophages CD68+ était élevée tandis que la densité des cellules myoïdes AML+ était faible (CD68+ élevé chez 71,4% de drépanocytaires vs 57,2% chez les témoins; AML+ : 33,3% vs 65,4%) (Fig.1).
Nos résultats contrastent avec ceux raportés dans une étude similaire réalisée au nord (Tab.1).
 

Conclusions :
Nos données montrent qu’en Afrique sub-saharienne, chez les drépanocytaires, la pulpe rouge reste fonctionnelle alors que la pulpe blanche devient peu fonctionnelle comme démontré par la faible densité et la dimension réduite des follicules.
Nous notons des différences avec les observations faites chez les drépanocytaires vivant en milieu non tropical ; ce qui suggère l’impact de l’environnement qui nécessiterait des études ultérieures plus approfondies.
 

Tab.1 : Comparaison des paramètres immuno-histochimiques entre notre étude et celle réalisée en Italie par Pizzi M. et al. en 2017.

Figure 1 : Aspects anatomiques, histologiques et immuno-histochimiques de la rate drépanocytaire: a : grosse rate d’un drépanocytaire b : GR falciformés dans une rate congestive chez un drépanocytaire c et d : densité et taille des follicules lymphoïdes r

Purpose :
Sickle cell disease (SCD), the most frequent genetic disease worldwide, results from a unique point mutation leading to the synthesis of the abnormal hemoglobin S (HbS). Under hypoxic condition, HbS polymerization causes a mechanical distortion of red blood cells (RBCs) called sickling, which is accompanied by a decrease of their deformability and their life span.
SCD is characterized by a wide variability of clinical expression and a complex pathophysiology including vaso-occlusive processes, intravascular hemolysis as well as pro-inflammation, pro-coagulant and vaso-constrictive states.
Recently, the abnormal mitochondria retention in mature RBCs has been documented in small series of SS patients but the biological consequences of this abnormality have not been fully documented and not been documented in other sickle cell syndromes. In the present study, we analyzed mature RBC mitochondria retention in both SS and SC patients, the second most frequent sickle cell syndrome, taking into account the biological/clinical features associated with these abnormal RBCs.
 

Materials and Methods :
Eighty-nine adult SCD patients were included: 35 SS patients treated with hydroxyurea (HU), 15 SS patients not treated with HU and 39 SC patients. All of them were at steady-state and regularly followed by the sickle center of Guadeloupe. Twenty-one Guadeloupean adult controls (AA) were also included. Mature RBCs containing mitochondria were identified by flow cytometry using MitoTracker® probe as well as thiazole orange and anti-CD71 antibody to discriminate total and stress reticulocytes respectively. Correlations between the percentage of mature mitochondria+-RBCs and the following parameters were then analyzed: RBC deformability (Ektacytometry), reticulocytes (both total and CD71+) counts and levels of hemoglobin, total bilirubin, aminotransferase aspartate and lactate dehydrogenase levels using routine procedures. The impact of splenic function was evaluated using Howell-Jolly bodies, assessed by classic May-Grünwald Giemsa smears, as a marker of splenic dysfunction. We also analyzed the relationships between mitochondria+-RBCs and HU treatment as well as the rates of hyper vaso-occlusive (painful vaso-occlusion, acute chest syndrome, osteonecrosis) and hyper hemolytic (micro/macro-albuminuria, leg ulcer) complications.
 

Results :
Abnormal mitochondria+ RBCs were detected not only in SS but also in SC patients, although at a less extend. In both sickle cell syndromes, positive correlations with mitochondria+ RBCs were detected with hemolysis markers (percentage of total and stress reticulocytes). In addition, a negative relationship with hemoglobin level was detected in SS patients. A negative correlation was also detected between these abnormal mitochondria+ RBCs and RBC deformability. In contrast, no association between the presence of this RBC sub-population and clinical manifestations, hydroxyurea treatment or Howell-Jolly bodies was detected.
 

Conclusions :
In the present report, we demonstrated that abnormal retention of mitochondria in mature RBCs occurred not only in SS but also in SC patients. We also presented evidence that this phenotype could be related to hemolysis rate.
Further studies are warranted to reveal the molecular mechanisms involved.
 

Purpose :
Sickle cell nephropathy is a major complication of sickle cell disease and microalbuminuria is an early predictor of renal damage. Hence it is important to study the prevalence of renal dysfunction and its associated clinical, biochemical, and nutritional factors, benefitting patients in early identification and appropriate intervention thus, preventing the renal complications and associated morbidity.

Materials and Methods :
A cross sectional study was conducted considering a total of 163 patients between 5-20 years, with SCD (homozygous) of either sex, diagnosed using HPLC attending outpatient clinic in their steady state for routine clinical care at a tertiary care hospital. Patients with history of blood transfusion within 2 weeks or in acute crisis or febrile illness were excluded from the study. Random spot urine sample was used for estimation of microalbuminuria by immunoturbidimetry method. Demographic details, clinical, biochemical, and nutritional parameters of each were recorded, analysed and compared. Microalbuminuria was said to be present if urine albumin creatinine ratio was between 30-300 mg/gm urine creatinine and value more than 300 mg/gm of urine creatinine was labelled as macroalbuminuria. A positive screening test was repeated twice with an early morning urine sample before labelling patient as having microalbuminuria. eGFR estimation was done using modified schwartz formula.

Results :
Out of total 163 patients (5-20 years), majority (32.52%) were in the age group of 6.1-9 years followed by 5-6 years (31.29%). Male female ratio was 1.3:1. Mean BMI was 14.67 ± 2.32. Mean VOC episodes and mean number of blood transfusions were 3.25 ± 2.91 and 3.23 ± 4.39 respectively. 3 patients had macroalbuminuria (1.84%), microalbuminuria was found in 24 patients (14.72%) while 83.44% patients were normal. 71.8% patients had normal eGFR, 18.4% had hyperfiltration while 9.8% had mild decrease in eGFR. The mean age was significantly higher in children with microalbuminuria than in those without microalbuminuria (11.69 ± 3.86 vs 8.55 ± 3.59 years; P=0.001), youngest patient being 6-year-old. Amongst positive patients, 66.67% were males and 33.33% were females. However, no significant correlation was found between gender and microalbuminuria (P= 0.293). 70.8% of positive patients had ≥3 episodes of vaso-occlusive crisis during their lifetime (P= 0.028) which was found to be statistically significant. Blood pressure taken at first visit were within normal limits for all the patients. 75% patients were already on hydroxyurea at the time of enrolment. 16.67% patients with microalbuminuria had hyperfiltration, 12.5% had mild decrease in eGFR while 70.83% had normal eGFR. No significant correlation was found between patient’s eGFR, nutritional status, blood transfusion frequencies, acute febrile illness episodes or any laboratory parameters with microalbuminuria.

Conclusions :
Age and frequent episodes of vaso-occlusive crises were found to be major factors associated with occurrence of microalbuminuria. These results confirm the need for early screening of microalbuminuria in patients with sickle cell disease and will be helpful in designing target strategy for early identification and introduction of appropriate timely intervention (drugs like ACE inhibitors) so that long term renal complications can be prevented.

Purpose :
Patients with sickle cell disease (SCD) are prone to dys- or autoimmune disorders, such as autoimmune hepatitis (AIH), sclerosing cholangitis, or ulcerative colitis. Sclerosing cholangitis can develop in young children with an unusually severe course due to associated vaso-occlusion and ischemia. Liver transplantation may be indicated early but is a challenging procedure in SCD, with uncertain outcomes (Hurtova Liver Transpl 2011, Duvoux [abstract] JFHOD 2021). Hematopoietic stem cell transplantation (HSCT) can cure SCD, although conditioning may induce hepatic complications. Herein we report a series of 6 children with SCD and autoimmune disorders, 3 for whom liver or digestive symptoms resolved after successful HSCT, and 3 for whom cirrhosis precluded HSCT conditioning.
 

Materials and Methods :
Of the 6 children (5 girls), 5 were HbSS and 1 was HbSC. Dysimmune symptoms appeared at a median age of 4.5 (3-12) years and were colitis (n=2), cerebral vasculitis (n=1), and AIH (n=3). In 4/6 patients, liver disease was an overlap AIH-sclerosing cholangitis, while 2/6 patients had either isolated AIH or sclerosing cholangitis. Liver biopsy showed cirrhosis in 3 patients. Median age at last follow-up (FU) was 13.5 (8-17) years.
 

Results :
The 3 patients without cirrhosis, including 2 with sclerosing cholangitis and dilated bile ducts, underwent HSCT with a geno-identical donor, at 6, 11.5 and 16 years old. Autoimmune disease was controlled with steroids, azathioprine, infliximab or cyclophosphamide with plasma exchanges and rituximab. Conditioning included hepatotoxic drugs busulfan (n=2) and treosulfan (n=1), with antilymphocyte globulins, fludarabine (n=3) and thiotepa (n=1). Transient hepatitis developed in 1 patient and moderate graft-vs-host disease (GVHD) in 2, which resolved after adapting immunosuppression treatment. HSCT was uneventful in the third patient. With a FU of 1 to 2.5 years, all 3 patients had no digestive or liver symptoms, normal or mildly elevated liver enzymes and unchanged liver imaging.
The 3 patients with cirrhosis upon first visit, at ages 7, 8.5, 13 years old, were on regular exchange transfusions at last FU. One patient developed recurrent infectious cholangitis and received a liver transplant at age 10, with many complications; at 7 years of FU, sclerosing cholangitis relapsed and re-transplantation was discussed. Another had moderate portal hypertension, bilirubin levels of 100-200 µM, and prothrombin time 60%, on steroids. The third patient was on tacrolimus, had presented 2 episodes of reversible liver failure, bilirubin levels of 100 µM and prothrombin time 45% at last FU and had developed myasthenia.
 

Conclusions :
Autoimmune liver disease in children with SCD may rapidly lead to end-stage liver disease. Upon diagnosis, these disorders should be considered as severe complications of SCD and prompt early discussion of HSCT. HSCT should be performed before conditioning with hepatotoxic drugs is not feasible. If a geno-identical donor is not available, alternative donors could be considered (haplo-identical, unrelated). If the liver disease is not stabilized after HSCT, liver transplantation could be performed with a standard risk, in a patient rid of SCD. If liver transplantation must be performed before HSCT, HSCT could be discussed rapidly afterwards to avoid possible relapse of SCD-related liver disease and dysimmune liver disease.
 
 

Purpose :
Studying stress erythropoiesis (SE) in sickle cell anemia (SCA) is difficult because of the rigor in accessing bone marrow samples. However, the characterization of circulating cell populations reflecting SE is feasible and critical, especially in the perspectives of gene therapy.

Materials and Methods :
Clinical and laboratory data were collected from 49 subjects with SCA (SS-genotype) under chronic transfusion (CT, n = 15), hydroxyurea therapy (HC, n = 20), or under both treatments (CT/HC, n = 14) at the Blood Center of Ribeirão Preto - Brazil. The mean age was 35.22 y/o (20-64), and 53.06% were males. Six patients from each group, under steady-state, and five healthy donors (HD group) were selected for downstream analysis of PBMCs. Briefly, 5 mL of blood were collected in EDTA-evacuated tubes and processed immediately with Ficoll-Paque PLUS (Cytiva). 106 live cells were stained with anti-CD235a, -CD123, -CD36, and -CD34 (stemness and erythroid maturation markers) BD Biosciences antibodies and analyzed by FACS in a BD FACSCanto II. FACS analysis was conducted in the FlowJo software v.11, using conventional gating strategies and dimensionality reduction machine learning algorithms. Complete blood and reticulocyte count, liver function, hemoglobin profiling, and FACS data were plotted for statistical comparison of mean ranks using Kruskal-Wallis, Dunn’s multiple comparisons, or Spearman's rank-order correlation tests. Data analysis was performed in statistical software GraphPad Prism v.6.0 and open-source statistical programming language R v.4.0.2, using packages ‘Hmisc’ and ‘corrplot’ for correlation analysis and visualization of results. Significance levels were considered when p<0.05 in which ‘*’, ‘**’, ‘***’ and '****' indicate p<0.05; p<0.01, p<0.001, and p<0.0001, respectively.

Results :
Subjects under HC had overall higher median Hb, HCt, MCV, MCH, HBS, and HBF while CT groups presented higher RDW. The number of nucleated RBCs (nRBC) was similar among groups and the ratio of RBCHb/RetHb increased under HC treatment. Also, levels of plasma-free hemoglobin (calculated based on total and cellular Hb) were lower in HC. Reticulocytosis was higher in CT and hemolysis parameters appeared discretely lower in HC (Table 1). FACS analysis revealed that patients had higher circulating CD34+ cell counts. Levels of CD235+ cells inside the PBMC compartment were higher in HC patients. Also, CD34+CD235a+ population counts were significantly higher in HC than in CT and CT/HC, representing more than 15% of the circulating CD34+ compartment in some patients. Up to 60% of the CD34+CD235a+ were also CD36+ (Figure 1 A). In contrast, CD34+CD123+ were increased among CT and CT/HC groups. CD235a+ and CD34+CD235a+ populations positively correlated with HbF levels in the HC group, whilst its dose did not associate with any blood count or hemoglobin parameters (Figure 1 B). Interestingly, the same population counts positively correlated with age among non-HC takers. 

Conclusions :
These results reaffirm the capacity of HC treatment to improve hematological parameters in SCA. Notwithstanding, HC presented increased levels of SE cell populations than the CT groups, reflecting different mechanisms of SE onset between CT and HC treated patients. Financial Support: CAPES, CTC/FAPESP-(2013/08135-2); INCTC-(465539/2014-9).

Complete blood count and hemolysis biomarkers of HC and CT / HC groups compared with CT, calculated using Kruskal-Wallis test. ‘*’, ‘**’, ‘***’ and '****' indicate p?0.05; p?0.01, p?0.001, and p?0.0001, respectively.

A) Levels of CD34+ and CD34+CD235a+ cell populations. B) Correlogram of stress erythropoiesis cell FACS data versus hematological parameters of hydroxyurea treatment groups. Levels of stress cells are positively correlated with levels of HbF.

Purpose :
Red blood cell (RBC) deformability is a biomarker to assess the clinical status and response to therapy in sickle cell disease (SCD). RBC deformability has been measured by ektacytometry for decades, using shear or osmolar stress. However, ektacytometry averages the RBC population, and cannot report small fractions of abnormal RBCs. A single cell-based, functional RBC deformability measurement provides essential information on the presence of unmodified cell populations that can perpetuate disease complications after the implementation of drug or cell-based therapies. There are two goals in this study: to determine the reproducibility of values generated by the standardized OcclusionChip assay and commercially available Oxygen Gradient Ektacytometry (LORRCA); and to determine if the OcclusionChip assay could complement Ektacytometry for detection of abnormal RBCs in small fractions.

Materials and Methods :
OcclusionChip microfluidic assay design includes a gradient of capillary network-inspired micropillar arrays embedded into the microfluidic channel forming microcapillaries from 20 μm down to 4 μm. This mimics the non-uniform, continuously changing capillaries in the capillary bed, retaining RBCs with significantly decreased deformability in the upstream array with coarser openings, and those with modestly decreased deformability in the downstream array with finer openings. Micropillar arrays were coupled with two 60-μm side passageways mimicking capillary bed arteriovenous anastomoses. This helps regulate blood flow; when an upstream portion of the array is obstructed, incoming RBCs can flow into the microfluidic anastomosis and re-enter the array downstream. To test the relative merits of the OcclusionChip and Ektacytometry, we measured the elongation index (EI by Oxygen Gradient Ektacytometry) and occlusion index (OI by OcclusionChip) of blood samples containing gradient concentrations of glutaraldehyde-stiffened RBCs. We also tested clinical blood samples obtained from subjects with SCD using oxygen gradient ektacytometry, which measures the EI at a range of pO2 level (159 mmHg to ~5 mmHg), and the hypoxic OcclusionChip assay, which measures OI at physiologic hypoxic pO2 level (45 mmHg).

Results :
We tested glutaraldehyde-stiffened RBCs for up to 1% volume fraction; ektacytometry detected no significant change in Elongation Index (EI), while the OcclusionChip showed significant differences in OI (Figure 1). OcclusionChip detected a significant increase in OI in RBCs from an individual with sickle cell trait (SCT) and from a subject with SCD who received allogeneic hematopoietic stem cell transplant (HSCT), as the sample was taken from normoxic (pO2:159 mmHg) to physiologic hypoxic (pO2:45 mmHg) conditions (Figure 2). Oxygen gradient ektacytometry detected no difference in EI for SCT or HSCT.

Conclusions :
The OcclusionChip assay is more sensitive to alterations in RBC deformability in SCD, particularly when %hemoglobin S (HbS) is small, thus able to complement Ektacytometry for a more accurate assessment of patient clinical status. These results suggest that the single cell-based OcclusionChip enables the detection of HbS-related RBC abnormalities in SCT and SCD, particularly when the HbS level is low. We conclude that the OcclusionChip is complementary to the population-based Ektacytometry assays and provides additional sensitivity and capacity to detect small but clinically significant populations of abnormal RBC.

Figure 1. The OcclusionChip enables the detection of small-fraction glutaraldehyde-stiffened RBCs in mixtures with normal RBCs. (A) Elongation Index (EI) by ektacytometry. (B) Occlusion Index (OI) results on the same RBC mixtures.

Figure 2. OcclusionChip enables detection of HbS-related RBC abnormalities under hypoxia in SCT and SCD. Shown are (A) Elongation Index (EI) results by oxygen gradient ektacytometry and (B) Occlusion Index (OI) results by the OcclusionChip.

Purpose :
Crizanlizumab (ADAKVEO®, Novartis, Switzerland) a monoclonal P-selectin blocking antibody designed to disrupt the blood cells and endothelium interactions in sickle cell disease (SCD) patients, which leads to reduction of vaso-occlusive crises, the major manifestation of the disease. We report the novel microfluidic platform (endothelium-on-a-chip), to test the effects of Crizanlizumab on the adhesion of red blood cells (RBCs) to perfusion-cultured, acutely and chronically activated human endothelial cells (ECs).

Materials and Methods :
Whole blood samples collected from SCD subjects (n=21), 20 HbSS and 1 HbSC in EDTA and sodium citrate. RBCs were isolated via centrifugation from whole blood and resuspended in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10 mM of HEPES. Human umbilical vein endothelial cells (HUVECs; Lonza, Morristown, NJ, USA) were cultured within the microfluidic platform channels at 15 dyne/cm2 for at least 48 hours prior to experiments. To mimic chronic pre-activation in SCD HUVECs were pretreated for 4 hours with heme (40 µM), TNFα (20 ng/ml) or 50% plasma of SCD patients in basal media, +/- 100 µg/ml Crizanlizumab followed by injection of blood samples through the microfluidic channels. For acute EC activation, blood samples were supplemented with 40 µM heme +/- 100 µg/ml Crizanlizumab and injected through the microfluidic channels for 15 minutes. Thereafter, non-adherent RBCs were rinsed via washing solution with or without Crizanlizumab and the remaining RBCs were quantified based on previous published methods. Paired t-test was used to calculate statistical significance.

Results :
The inhibitory effects of Crizanlizumab on blood cell adhesion to ECs was linked to the type and duration of EC activation (fig. 1). Crizanlizumab slightly reduced RBC adhesion to 4-hours heme activated ECs (1170±413vs1671±522 p>0.05). Reduction of RBC adhesion due to Crizanlizumab treatment was significant in 15-minutes heme activated ECs (135±40 vs 1513±617, p≤0.05). The effect of Crizanlizumab on decreasing RBC adhesion was significant when ECs were pre-activated by TNF-α (4404±1393 vs 2016±609, p≤0.05) or subjects’ autologous plasma for 4 hours followed by a 15-minute heme activation (5876±2579 vs 2397±1381p≤0.05).

Conclusions :
In accordance with the label of Crizanlizumab to reduce VOC, application of microfluidic platform associated with Crizanlizumab use displayed a reduced RBC adhesion to acutely heme-activated EC. Effects were preserved in presence or absence of chronic TNFα or autologous plasma pre-activation. Endothelium-on-a-chip microfluidic platform has been shown as reliable in monitoring patient response to anti-adhesive therapies in SCD.

Effects of Crizanlizumab on RBC adhesion levels to HUVECs activated for (A) 4-hours with heme (B) 15-minutes with heme (C) 4-hours with TNF-? and 15-minutes with heme, (D) 4-hours with plasma and 15-minutes with heme. Error bars represent the standard err

Purpose :
Sickle cell disease (SCD) has a global incidence of approximately 300,000/yr. About 100000 individuals live with SCD in the US. The hallmark of SCD is anemia, recurrent acute painful Vaso occlusive episodes (VOE’s) and progressive multi-organ involvement.  VOE’s begin in infancy (dactylitis) and increase in frequency with age. Prompt administration of short-term full agonist opioids, combined with Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) is the mainstay of VOE treatment. However, a subset of patients will develop chronic pain (CP) beginning early in adolescence. Long term opioid therapy has been used to treat SCD CP due to a lack of available alternatives. LTOT for CP has been proven to have poor efficacy and high toxicity and is associated with reduced quality of life. Buprenorphine is a partial opioid agonist, which physiologically exerts a ‘ceiling effect’ with less opioid related toxicity and thus is a safer option for patients who experience poor efficacy and increased toxicity from LTOT.

Materials and Methods :
From 12/2020 to 02/2022, 10 patients met program eligibility criteria: SCD diagnosis, debilitating CP, receiving >90 morphine equivalents daily, (both outpatient and inpatient totals) with poor analgesic response. Each patient underwent a comprehensive evaluation of SCD status and other comorbidities, a psychological evaluation and participated in several education sessions with their caregiver prior to enrollment. Buprenorphine was initiated using a micro induction regimen that comprised of transdermal Buprenorphine 10mcg/hour or 20 mcg/hour on day 1 and adding buccal Buprenorphine/Naloxone starting on day # 2 or 3 and titrating upwards to individualized requirements. Full opioid agonists were slowly weaned down. Symptoms of withdrawal were monitored using the clinical Opioid withdrawal scale (COWS).  

Results :
Patient were between ages between 12-19 years, with median of 18 years. 7 were Female.  All 10 patients were successfully transitioned off LTOT onto a Buprenorphine formulation and none experienced withdrawal symptoms. Of the 10 patients, 7 remain on Buprenorphine beyond 3 months post induction with reductions in both acute care utilization and inpatient hospitalization.
Utilization data: 6-months pre buprenorphine: Emergency room (ER) visits ranged from 3-20 with a mean of 9 encounters. Number of inpatient admissions were from 3-10 with a mean of 5.7 and length of stay between 3-19.3 days with a mean of 8.75 days. In the 3-6 months post Buprenorphine: number of ED encounters ranges from 1-7 with a mean of 2.2; the number or admissions range from 0-5 with a mean of 1.6 and length of stay ranged between 0-15 days with a mean of 4.85 days. 

Conclusions :
Pediatric and young patients with SCD on high dose LTOT can be transitioned successfully to buprenorphine in the inpatient setting, using a modified micro induction titration regimen. A system wide multidisciplinary approach to include nursing, emergency department, pharmacy, psychologists and psychiatrists and an education dissemination plan is needed. Both patients and parents need to be ready for an alternative in order to achieve long term success. With the increasing reports of the use of Buprenorphine for CP in SCD, large prospective multi-center clinical trials are warranted.

Purpose :
Sickle cell disease (SCD) is a chronic life threatening disorder, caused by the presence of structurally abnormal adult hemoglobin S (HbS). Under low oxygen saturation, HbS forms hemoglobin polymers that deform the red blood cell structure, referred to as ‘sickling’. Sickled erythrocytes result in hemolytic anemia and recurrent vaso-occlusive crisis, which lead to long-term morbidity and early death. The patient specific pO2 at which sickling starts (PoS) along with RBC deformability at normoxia (EImax) and upon deoxygenation (EImin) can be measured by oxygen gradient ektacytometry (Laser Optical Rotational Red Cell Analyzer (LoRRca)). In the GenoMed4ALL project, oxygen gradient ektacytometry data will be integrated with genomics, metabolomics and clinical data of 1000 SCD patients, allowing better characterization of SCD and development of Artificial Intelligence (AI) algorithms for personalized medicine.

Materials and Methods :
SCD patients with SS, Sβo, SC and Sβ+ genotypes, older than 1yo at steady state with no transfusion in the last 3 months were analyzed by oxygen gradient ektacytometry. All samples were processed in duplicates. Derived parameters were correlated with genotypes, and for severe SCD (SS, Sβo) with hydroxyurea options (HU-, HU+) and HbF. An Anova analysis has been carried out to test the relation  of the parameters among groups. The post-hoc test Scheffe was used to test which groups are different two by two. 

Results :
49 samples were analyzed: 32 SS, 2 Sβo, 12 SC and 3 Sβ+. Mean and standard deviation values for PoS, EImax and EImin are shown in Table1. PoS and EImin allowed patients’ clustering in 2 groups according to genotype: SS PoS: 34.34±5.0 Elmin: 0.23±0.1 vs Sβo, SC and Sβ+ PoS: 24.62±3.7 and Elmin: 0.34±0.1. EImin showed statistically significant Scheffe test value in discriminating SS vs Sßo and SS vs SC, while PoS was statistically significant only in discriminating SS vs SC.
31 samples from severe SCD patients (SS, Sβo) were further analyzed for correlation with HU (7 HU-, 24 HU+) and HbF. Mean and SD values are shown in Table1. The differences in mean values for PoS, EImax and EImin between HU- and HU+ were of -27%, 54% and 135% respectively. The best correlation between Lorrca parameters and HbF was found in group of patients without treatment (HU-). EImin showed the highest correlation being 93% in HU- and 81% in HU+ group.

Conclusions :
Our results demonstrate the value of oxygen gradient ektacytometry for  SCD patients characterization. PoS and Elmin were able to distinguish between SS and other genotypes, regardless of HU treatment. In addition, PoS, EImax and EImin all showed differences between HU+ and HU- groups, being Elmin the most valuable one. Elmin also showed the highest correlation with HbF. It is highly likely that PoS could be impacted by other variables: 2,3 DPG, pH, genetic variants or metabolomics. Pooling data of SCD patients from at least 9 EU centers in the GenoMed4LL project is needed to improve results robustness. Standardization of Oxygenscan data generation should be ensured for data comparison and development AI algorithms for personalized medicine. 

Purpose :
Sickle cell disease (SCD) is a public health problem in the Democratic Republic of Congo. While reference sickle cell centers have been implemented in capital cities of African countries and have proven to be beneficial for SCD patients. In the Democratic Republic of Congo, they have never been set up in remote areas for families with low or very low sources of income.

Materials and Methods :
A cohort of 143 children with SCD aged 10 years old (IQR (interquartile range): 6–15 years) (sex ratio male/female = 1.3) were clinically followed for 12 months without any specific intervention aside from the management of acute events, and then for 12 months with a monthly medical visit, biological follow-up, and chemoprophylaxis (folic acid/penicillin), adequate fluids and malaria prevention.

Results :
The median age of patients at the diagnosis of SCD was 2 years (IQR: 1–5). The implementation of standardized and regular follow-ups in a new sickle cell reference center in a remote city showed an increase in the annual mean hemoglobin level from 50 to 70 g/L (p = 0.001), and a decrease in the lymphocyte count and spleen size (p < 0.001). A significant decrease (p < 0.001) in the average annual number of hospitalizations and episodes of vaso-occlusive crises, blood transfusions, infections, and acute chest syndromes were also observed.

Conclusions :
The creation of a sickle cell reference center and the regular follow-up of children with sickle cell disease are possible and applicable in the context of a remote city of an African country, and represent simple and accessible measures that can reduce the morbimortality of children with sickle cell disease.

The city of Mbujimayi in the Democratic Republic of the Congo

Purpose :
Hydroxyurea (HU), also known as hydroxycarbamide has been commonly used as a therapeutic strategy in Sickle Cell Disease (SCD) since 19841. HU has been recognized as efficient in SCD patients with an acceptable toxicity2. From a clinical point of view, HU has been shown to reduce painful episodes, hospitalizations, blood transfusions, acute chest syndrome and mortality3,4. These beneficial roles of HU are mediated by the increase of fetal hemoglobin (HbF) production and the decrease of abnormal hemoglobin (HbS) levelin erythrocytes. HU can then reduce the polymerization-sickling cascade thereby causing a decrease in the number of circulating sickled red blood cells5. HU also induces physiological changes such as an increase in both number and size of erythrocytes. It can also decreases hemolysis6. HU is also known as a nitric oxide (NO) donor which can directly affect the microvascular function. Although microvasculature is known to be remodeled in SCD (capillary rarefaction and enlargement8), the corresponding effects of HU have been scarcely reported. Given that HU improves hemorheologic and hemodynamic functions in SCD patients, one can hypothesize that HU may be beneficial for skeletal muscle microvasculature.

Materials and Methods :
Gastrocnemius muscles of 11 HbSS mice and 12 HbSS HU-treated mice (Townes model) were used to perform histochemical analyses. Morphometric analyses of microvasculature were performed by staining the endothelial cells with CD31 antibody. Microvascular network analysis included Capillary Outer Diameter (COD), Perimeter (CP), Surface Area (CSA), and Density (CD, number of capillaries per mm2) as well as the capillaries to muscle fibers ratio for a given muscle area (C/F). ANOVA and Tukeys post-hoc tests were used to compare data of HbSS and HbSS HU-treated mice. Significance was accepted when p≤ 0.05..

Results :
HbSS HU-treated mice displayed significantly higher COD, CP and CSA. than control HbSS mice (p = .033, p = .013, and p = .005 respectively, Figure 1). On the other hand, CD and C/F did not differ between HbSS and HbSS HU-treated mice (Figure 2).

Conclusions :
Given that HU is a NO donor on the one hand and that NO can induce hyperemia on the other hand, one can hypothesize that that the widening of capillaries might be related to hyperemia related to HU-treatment. As supporting evidences, Bosman et al 9 reported that hyperemia induced capillary distension/enlargement while Kano et al.10 observed a reduced capillary luminal diameter in response to a reduced blood flow

Figure 1: Capillary outer diameter (COD), area (CA) and perimeter (CP) in HbSS and HbSS HU-treated mice.

Figure 2: CD and C/F Indices of microvascular network in HbSS and HbSS HU-treated mice

Purpose :
Data regarding red blood cell (RBC) alloimmunization in sickle cell disease (SCD) patients are scarce in the Democratic Republic of the Congo (DRC) which is the third most affected country in the world by sickle cell disease. We aimed to determine the prevalence of red blood cell alloimmunization and alloantibody specificity in SCD patients in Kisangani (DRC).

Materials and Methods :
We conducted a multi-hospital, multi-site based cross-sectional study among 125 SCD patients at Kisangani and 136 at the Centre Hospitalier Régional de la Citadelle (CHR) of Liège (Belgium). Data were collected using a survey form. Diagnostic con?rmation of SCD was made by high-performance liquid chromatography coupled to mass spectrometry. Alloantibodies were screened using the agglutination technique on gel cards and their specificity determined using 11 and/or 16 cell panels. Statistical analyses were carried out using SPSS.  

Results :
The prevalence of RBC alloimmunization was 9.6% for samples collected at Kisangani versus 22.8% (including historical antibodies) at the CHR and increased to 10% at Kisangani versus 29.3% at the CHR when considering only patients transfused in the last five years prior to the study. At Kisangani as well as at CHR, the median age of alloimmunized patients was higher than that of non-alloimmunized patients, 15.5 years (IQR:4.8-19.8) vs 10 (IQR: 6.5-17) and 24 years (IQR:14-31) vs 17(IQR:12-24), respectively. The median number of blood units was higher in both Kisangani and CHR immunized patients compared to non-immunized patients, 8(IQR:5-11) vs 5(IQR:3-13) and 41(IQR:6-93) vs 6.5(3-37) respectively. At Kisangani(N=14), the most frequent antibodies were anti-D (28.6%), anti-C (21.4%), anti-M (14.3%,) and anti-N (14.3%) versus anti-E (13.6%), anti-S (13.6%), and anti-Lea (11.4%) at the CHR(N=44).

Conclusions :
Red blood cell alloimmunization is a common complication of transfusion therapy in SCD patients in the DRC and is mostly directed against the RH system. In a low income context in the DRC, blood transfusion with a minimum ABO, D, C, E antigen matching could significantly reduce the incidence of this complication.

Table 1. Comparison of characteristics of alloimmunized patients

Fig.1. Distribution of identified alloantibodies according to blood group systems(Kisangani, N=14; CHR, N=44)

Purpose :
Sickle cell disease (SCD) alters the hemoglobin molecule, modifying the properties of red blood cells (RBC) and causing their destruction in the circulation, leading to multiple complications. The increased adhesive properties and reduced deformability of sickle RBC make a major contribution to the vaso-occlusive processes that characterize the disease. Piezo1 is a mechanosensitive membrane protein that is activated by changes in vascular flow, triggering calcium (Ca2+) influx into cells. We aimed to identify a role for Piezo1 in altering the adhesive properties of sickle RBC and their propensity for sickling, and the contribution of Ca2+ influx and Gardos Channel (GC) activation to these alterations.

Materials and Methods :
Peripheral blood was collected, with informed consent, from patients with HbSS SCD, cared for at the Hospices Civils de Lyon and Institut d’Hématologie et d’Oncologie Pédiatrique de Lyon, France, and at the Hematology Center, University of Campinas, Brazil. Piezo1 activation on separated RBCs was achieved by incubation of cells with Yoda1 (0.1-10µM), a Piezo1 agonist. RBC membrane protein expression and Ca2+ influx were observed by flow cytometry, microfluidic adhesion assays were used for examining the adhesive properties of cells and RBC deformability was monitored using ektacytometry under oxygen gradient. Channel activity under Piezo1 activation was monitored using change in membrane potential method (CCCP).

Results :
Oxygen gradient ektacytometry demonstrated that activation of Piezo1 on sickle RBC reduces cellular deformability and increases the sickling propensity of sickle RBC during deoxygenation. In contrast, inhibition of Piezo1 with GsMTX4 increased the deformability of sickle RBC and decreased their sickling propensity. Adhesion assays indicated that activation of Piezo1 was associated with a significant increase in the ability of sickle RBC to adhere to laminin under flow conditions. While flow cytometry demonstrated a clear effect of Piezo1 activation by Yoda1 on phosphatidylserine exposure on sickle RBC, accompanied by Ca2+ influx, no alteration in the expression of the laminin-binding BCAM adhesion molecule was observed on these cells. However, co-incubation of cells with a BCAM function-neutralizing antibody molecule abolished the Yoda1-dependent increase in sickle RBC adhesion, indicating a role for BCAM adhesion molecule activation (rather than expression) in this mechanism. Importantly, membrane potential measurement showed that Piezo1 stimulation triggers pronounced membrane hyperpolarization linked to GC activation, a phenomenon exacerbated by low oxygen partial pressure; furthermore, the increase in RBC sickling propensity following Piezo1 activation occurred in a GC-dependent manner (as demonstrated by co-incubation with TRAM34, a GC inhibitor).

Conclusions :
In conclusion, pharmacological activation of Piezo1 on the sickle RBC surface significantly modulates the function and properties of these cells, suggesting that this mechanosensor may represent a potential therapeutic target molecule in SCD.

Purpose :
Previous studies have identified sleep-related metrics as important players in patient wellbeing, with links between sleep disturbances to both acute and chronic pain, as well as quality of life (QoL), overall physical health, functional disability, and clinical depression in Sickle Cell Disease (SCD).
This work therefore aimed to improve patient QoL and overall outcomes through the identification of key underlying factors in patients with SCD. This focused on generating real-world evidence that describes the potential impact of live sleep biometrics upon patient QoL, driving understanding of causal factors and potential preventative best practice at a patient self-management and clinical level.

Materials and Methods :
An FDA approved, CE marked wearable monitoring device (Withings ScanWatch) was provided to 85 patients and worn over a 12-month period, automatically recording key biometrics including sleep quality and heart rate. This was supplemented by manual patient self-reported EQ-5D scores, entered via a patient-reported outcomes (PRO) portal. Analysis was conducted within a final cohort of 54 patients (64%) who had both recorded sleep data and EQ-5D data.
Patients were stratified into low, average, and high categories regarding their averages across several key sleep metrics, and where these averages fell within the calculated cohort percentiles. A comparison of the average EQ-5D score was then compared across patients between the three categories for each sleep metric, in order to identify any measures with potentially statistically significant impacts upon resulting patient QoL.

Results :
While no statistically significant differences were seen between the three categories in terms of their EQ-5D score across the measures ‘total sleep duration’, ‘sleep heart rate’, ‘number of wake ups per night’, ‘time spent awake’, ‘time to sleep’, and ‘time to wake up’, a potential link was seen between EQ-5D scores and ‘deep sleep’ levels.
Indeed, analysis of the link between patients' average percentage of deep sleep (as a proportion of total sleep duration) and EQ-5D score found that patients within the lower range (0-39%) had statistically significantly higher average EQ-5D scores than those in the high ranges (50-83%) of deep sleep (p = 0.030), as shown in Image 1 below.

Conclusions :
Our data identified an early association between the amount of deep sleep patients with SCD have and their self-reported EQ-5D scores, as an indicator of QoL, suggesting longer periods of deep sleep each night may be linked to lower patient-perceived wellbeing.
Moreover, previous publications have indicated an optimal deep sleep percentage between 13-23% of total sleep duration, our data suggesting that a significant proportion of this patient cohort exceeded the accepted levels of “healthy” deep sleep (96%, 52/54).
Further investigation is required to explore any potential causal relationship, in order to establish whether poorer QoL may see patients enter longer periods of deep sleep, or whether this widespread excess in deep sleep itself exerts an impact upon patient QoL in SCD.
Future Steps

• Expansion to a larger cohort of patients, with analysis for breakdown between different patient subgroups, including genotype, age, and gender.
• Exploration of any additional links to measures such as healthcare utilisation, comorbidities, or pathology results.

Correlation between deep sleep percentage and EQ-5D score (left), with associated statistical significance analysis between patients within the low vs. high deep sleep categories (right).

Purpose :
Sickle cell disease (SCD) combines, in its homozygous form, chronic hemolytic anemia, vasoocclusive complications and susceptibility to infections. It is well known that the combination of pregnancy and SCD promotes the occurrence of complications that are sometimes fatal for the mother and/or the fetus. The objective of the current study was to compare pregnancy outcomes among women with SCD with those of no-SCD women in our country.
 

Materials and Methods :
It was a case-control study carried out in the obstetrics and gynecology departments of the 4 main hospitals in the city. It was conducted over a period of 24 months (2017 – 2019). The case group was made up of pregnant women with SS homozygous SCD (n=65). The control group was made up of non-SCD gestants (Hb AA), two controls for one case. Excluded were women whose delivery time was less than 28 weeks of amenorrhea (because in our country, the resuscitation of children born before 28 weeks of gestation is extremely delicate), women admitted to the expulsion phase, or women who gave birth outside the centers mentioned above. The parameters analyzed were as follows:
(i) Maternal : age, complications linked to SCD ;
(ii) Obstetric : gestational age (prematurity before 37 weeks of gestation), mode of delivery, postpartum complications ;
(iii) Fetal : birth weight (low birth weight = birth weight <2500 g), complications requiring admission to the intensive care unit, mortality.
The chi-square test was used for the comparison of the proportions, the Student t-test for that of the means, and the Mann–Whitney for that of the medians. The odds ratio with the 95% confidence interval was calculated to assess the association between two variables. The p value of the probability was considered significant for a value < 0.05. Logistic regression was applied in order to identify the associated factors having a great power and to eliminate those of confusion.
 

Results :
The average age was 27 years for SCD women and 31 years for non-SCD women. The average gestational age at delivery was 35 weeks for SCD women and 38 weeks for non-SCD women. From the logistic regression analysis using the comparison group as the reference group, there was excessive risk in SCD compared to non-SCD of infection (29.3% vs. 4.6%, OR = 21.7, 95% CI [7.6–62.7]; p = 0.001), cesarean (63% vs. 35.4%, OR = 3.1, 95% CI [1.6–5.7]; p = 0.001), prematurity (75.4% vs. 30.8%, OR = 8, 95% CI [3.0–23.2]; p = 0.001), low birth weight (52.3% vs. 16.1%, OR = 4.7, 95% CI [2.4–9.4]; p = 0.001), neonatal requiring admission to the intensive care unit (40.3% vs. 17.5%, OR = 3.2, 95% CI [1.6–6.3]; p = 0.01), and neonatal death (21.5% vs. 4.8%, OR = 4.3, 95% CI [1.5–12.2]; p = 0.01).
 

Conclusions :
Maternal and fetal complications are still significantly higher in pregnant sickle cell patients. A close supervisor of these pregnancies with a multidisciplinary approach between hematologist, obstetrician, and pediatrician is essential. Information, education and communication sessions for sickle cell women are essential to minimize these risks.
 

Purpose :
Blood transfusions are a scarce resource globally and particularly in sub-Saharan Africa. Without disease-modifying therapy, in sub-Saharan Africa sickle cell anemia (SCA) has excess morbidity, mortality, and healthcare resource utilization, specifically requiring a high reliance on blood transfusions. Hydroxyurea is a safe medication for the treatment of SCA that can decrease rates of blood transfusions. The widespread use of hydroxyurea in sub-Saharan Africa is limited in part, however, by logistical challenges in determining an individual patient’s optimal dose. Pharmacokinetic (PK) dosing of hydroxyurea may streamline the dosing process, maximizing the clinical benefits and minimizing toxicities, while reducing the time and resources needed to determine the optimal dose. 
We hypothesize that hydroxyurea treatment will be associated with a 50% reduction in blood transfusion rate in children with SCA, compared to pre-treatment utilization, and that determining a PK-based hydroxyurea starting dose will be feasible in 80% of participants.

Materials and Methods :
To test this hypothesis, we will conduct the Alternative Dosing and Prevention of Transfusions (ADAPT) trial, a prospective cohort study of children with SCA in Jinja, Uganda (Figure). Transfusion rates and indications will be prospectively recorded during a 3-month pre-treatment period and compared to a 12-month treatment period. Every participant will undergo baseline hydroxyurea PK testing using timed serum collections based on our published sparse-sampling method and HPLC analysis. If generated, the participant will start on their PK-guided dose; when PK testing is not feasible, the participant will be started on 20 mg/kg/day per Ugandan guidelines. Regardless of starting dose (PK or default), all participants will undergo the same laboratory monitoring schedule with dose escalation and adjustments as necessary. Hydroxyurea will be purchased from a local commercial source using 500mg capsules.

Results :
The primary endpoint will be incidence rate ratio of transfusions per 100-patient years during hydroxyurea treatment as compared with the pre-treatment period. Based on sample size analysis, a total of 100 children with SCA (HbSS genotype), aged 1 to 10 years, will be enrolled in ADAPT.  A student’s t-test will be performed to compare the rate of transfusions per 100 patient-years prior to and during hydroxyurea treatment. The secondary endpoints include feasibility of determining a PK-based starting dose and the rates of transfusions, clinical events, dose-limiting toxicities, and dose adjustments in participants on PK-based versus default start dose.

Conclusions :
Overall, the potential for hydroxyurea to reduce the rate of blood transfusions in children with SCA will have important individual and public health implications. Determining the feasibility and accuracy of PK-dosing of hydroxyurea could simplify the safe and effective treatment of SCA. ADAPT is a collaborative research partnership that will provide important data toward increasing the use of hydroxyurea in Uganda and potentially across sub-Saharan Africa.

Figure: Schema of the ADAPT trial design

Purpose :
Successful, full chimerism hematopoietic stem cell transplant (HSCT) from a matched sibling donor is curative for sickle cell disease (SCD). Current cell therapy trials of less established strategies typically have reduction in pain events as their primary endpoint, which is important, but not equivalent to a cure. Therapies with curative intent need to also be comprehensively assessed using analytical assays that provide detailed and robust functional assessment of red blood bell (RBC) properties. We hypothesize that microfluidic BioChip Assays can complement clinical assessments of efficacy in curative therapies. To test this, we assessed RBC adhesion using SCD BioChip A and RBC deformability using SCD BioChip D, before and after HSCT.

Materials and Methods :
Venous blood samples were collected in EDTA tubes from a patient with SCD at University Hospitals, Cleveland Medical Center. The samples were tested with the SCD Biochip A and D at multiple time points between 2016 and 2022. SCD BioChip A devices  were fabricated using lamination processes and were functionalized with Intercellular Adhesion Molecule 1 (ICAM-1) and Laminin (LN). Whole blood was injected into the ICAM-1 and LN-immobilized microchannels. An inverted microscope and microscopy camera were used to obtain high-resolution images of the whole channel for processing (Adobe Photoshop, San Jose, CA) and quantification of adhered RBCs per unit area (32 mm2). Adhesion Indices for ICAM-1 and LN were calculated. SCD BioChip D devices were fabricated using standard soft lithography protocols. RBCs were isolated from whole blood, re-suspended in PBS at 20% hematocrit, and passed through the device with a constant inlet pressure. Following a wash step, the microchannel was imaged, processed and Occlusion Index (OI) was quantified

Results :
The ICAM-1 and LN RBC Adhesion Indices are shown in Figure 1a and 1b. Occlusion Index is shown in Figure 1c. The green rectangle represents the normal range value for the HbAA controls as previously established. The patient had high Adhesion indices in both ICAM-1 and LN before HSCT, and decrease in Adhesion Indices to the normal range was observed after HSCT. The LN Adhesion Index was still within the normal range one year after HSCT, while the ICAM-1 Adhesion Index increased. Regarding the OI, the patient had high levels before HSCT which decreased 5 months after transplant but increased at 1-year follow-up. The number of hospital visits related to pain crisis were elevated before HSCT; after HSCT, the patient did not have any pain related visits for 5 months, but between 6 and 12 months from HSCT, the patient experienced new pain related visits.

Conclusions :
The Adhesion Index for ICAM-1 and LN and OI were abnormal in the patient with SCD pre-transplant, and decreased to normal levels in the first 5 months post-transplant with no pain related visits recorded in that period. Adhesion and Occlusion indices increased at 1-year post transplant as the number of pain related visits recorded in that period. These results suggest that SCD BioChip microfluidic devices could be an effective platform to estimate the RBCs functional properties to validate the efficacy of experimental cell-based therapies in SCD patients.

. a) RBCs Adhesion Index before and after the HSCT to Laminin a) and ICAM1 b). Occlusion index before and after HSCT c) Number of pain related visit before and after HSCT d).

Purpose :
In Guadeloupe, a French West Indies Island of 379,710 inhabitants of which about 90% are of Afro-Caribbean origin, sickle cell disease (SCD) is the most common genetic disease. The Delta variant SARS-CoV2 4th wave was particularly deadly for the general population of Guadeloupe between July and October 2021 with 822 deaths attributed to COVID.
We report here characteristics and prognosis of SCD adult patients infected by Delta variant during 4th wave.

Materials and Methods :
This retrospective study was conducted at the Reference Center for SCD located in University Hospital of Guadeloupe.

Results :
Thirty-six SCD adult patients had symptomatic COVID-19 infection confirmed by nasal RT-PCR, with a sex ratio F/M of 2.6. None of them was vaccinated. The median age (IQR) was 43 years (27.0-50.0), 49 years (36.8-64.5) in SC patients, and 27.00 years (21.00-47.0) in SS patients (p=0.0007). Eighteen (50%) were SC, 15 (41.7%) were SS, and 3 (8.3%) were Sβ+-thalassemia patients. Body mass index (BMI) > 30 was observed only in SC patients (n=8). Nine patients had arterial hypertension, 13 had nephropathy with renal failure in one; three had mellitus diabetes. Three women were pregnant. Eleven SS, 1 SC, and 1 Sβ+-thalassemia were under Hydroxyurea treatment. Four SC patients had regular phlebotomies and 2 SS patients were on transfusion exchange program.
Fifteen (41.7%) patients were hospitalized; among them, 4 had Dexamethasone, 4 had monoclonal specific therapy by casirivimab plus indevimab combination, 10 had transfusion and 1 transfusion exchange. Eight patients need oxygen supplementary. Among 21 (58.7%) outpatients, none had corticoid therapy and 3 received specific monoclonal therapy. Eleven needed oxygen support.
Four (11.1%) patients died, all of them were SC patients. A 33-week pregnant 29-year-old woman was hospitalized with a vaso-occlusive crisis and developed pulmonary and neurologic symptoms with multivisceral failure despite high corticoid pulses, transfusion therapy, and delivery. An overweight 66-year-old woman with arterial hypertension and nephropathy died at home before medical management. A 48-year-old woman exhibiting obesity (BMI>45), arterial hypertension, asthma, nephropathy, and obstructive-sleep-apnea-syndrome (OSAS), was hospitalized for respiratory symptoms and died despite exchange transfusion, high corticoid pulses and tocilizumab. A 66-year-old man exhibiting obesity (BMI>35), arterial hypertension, diabetes, chronic renal failure and OSAS was hospitalized for worsening anemia and respiratory symptoms and died of probable myocardial infarction despite aggressive medical management.
We compared the 4 deceased patients with the 32 survivors. Only median BMI and obesity were significantly different (p=0.009 and p=0.03 respectively). No difference were detected in terms of age, sex, genotype or other co-morbidity. Our numbers are too small to detect prognostic factors for death in the group SC patients.
 

Conclusions :
In this first Caribbean cohort of SCD patients infected with the delta variant, the SC genotype is over-represented (50%) whereas it represents only 36% of SCD patients followed in our referral center. However, our limited series does not allow us to conclude that SC genotype is a risk factor for mortality in COVID-19 infection. Further studies are needed to understand whether characteristics of this genotype or co-morbidities explain the severity.
 

Purpose :
Hydroxycarbamide (HU) is used sickle-cell disease (SCD) after having showed to induce fetal haemoglobin (HbF)[1]. Although this was an old molecule, few pharmacokinetics-pharmacodynamics (PK/PD) models were developed on HU.

[1] Platt OS. Hydroxyurea for the treatment of sickle cell anemia. N Engl J Med. 2008; 358 (13): 1362-69. 10.1056/NEJMct0708272.


 

Materials and Methods :
The aim of this analysis was to develop retrospectively, a population PK (popPK) and PK/PD models to patients receiving both HU formulations (capsules or tablets), in order to:
Describe the exposure-efficacy relationships between HU and biomarkers (HbF% and mean corpuscular volume (MCV))
Compare PK/PD parameters, HbF% and MCV, obtained with the results observed in Paule et al[1],  
Simulate HbF% and MCV levels according to two regimens (7/7 vs 5/7 days) for 14 weeks at 1000 mg daily dose.
We combine three PK datasets from renal study[1], bioequivalence study[2]and retrospective PKPD data from patients followed in hospital Mondor.
A popPK model for HU was established. The selected popPK model was used in PK/PD modelling, consisting of two independent PK/PD models respectively for HbF% and MCV. Indirect PK/PD models were tested for both relations since drug impact on each marker was observed after few weeks of treatment.

[1] Clinical study report, "Evaluation of the impact of renal function on the pharmacokinetics of hydroxyurea (siklos®) in normal-renal, hyperfiltrating and renal failure sickle cell disease patients (DARH)". Final version, 02-February-2018.
[2] Clinical study report, "a phase I pharmacokinetics study of hydroxyurea 1,000 mg coated breakable tablets and hydroxyurea 500 mg capsules in paediatric, adolescent and adult patient treated for sickle cell syndrome". Final version, 15 april 2005.
 

[1] Paule I, Sassi H, Habibi A, Pham KP, Bachir D, Galactéros F, Girard P, Hulin A, Tod M. Population pharmacokinetics and pharmacodynamics of hydroxyurea in sickle cell anemia patients, a basis for optimizing the dosing regimen. Orphanet Journal of Rare Diseases. 2011.
 

Results :
The popPK model was confirmed as a linear model with a 2-compartment parameterized in terms of CL/F, V2/F, Q/F, V3/F and ka, allometric scale with exponents of 0.75 and 1 for clearances and volumes of distribution, respectively was integrated. For a bodyweight of 70 kg, clearance was 12.1 L/h with a variability of 63.9%, V2/F and V3/F were respectively 48.1 and 31.1 L. Population parameters were very closed to observed CL/F and V/F obtained in the study published by Paule et al2 ..

 

Conclusions :
Precision of the model was acceptable and individual prediction of HbF% levels were good (Figure 1).
For MCV, HU is assumed to reduce kout (inhibition of the elimination of the response) (Figure 2).
Regarding simulations, MCV reached a plateau around 3 months of HU and HbF% continue to increase over 14 weeks (Figure 3).
Indeed, the increase of HbF% and MCV levels was more marked than using the regimen with 2 days of HU interruption (especially in patients having the highest values). The daily regimen seemed to be more appropriate regarding both responses.
Both HbF% and MCV are good biomarkers for evaluation of SCD.
 

Figure 1 pcVPC of level of HbF% vs. time after treatment initiation (left: lin-lin scale; right: lin-log scale) - Figure 2 pcVPC of level of MCV vs. time after treatment initiation (left: lin-lin scale; right: lin-log scale)

Figure 3 SIMULATED HBF% AND MCV LEVELS USING PKPD MODEL, CONSIDERING 2 DOSING REGIMENS (left: HBF%; right: MCV)

Purpose :
India shoulders the second highest burden of sickle cell disease (SCD) and around 44000 new-borns with SCD are borne in India. SCD in India is localized to certain geographical areas in central India (from South-Eastern Gujarat to South-Western Odisha) and southern India (Eastern Ghats and Nilgiri Hills). Further, the disease is more prevalent in certain indigenous groups (tribes). Prior knowledge of disease prevalence in different geographical areas and tribal groups may help in designing locally appropriate policies and implementation plans for control and management of SCD. The aim of this study is to map out the geographical and ethnic distribution of SCD in Madhya Pradesh and Chhattisgarh states of India.

Materials and Methods :
In the present study, retrospective data from previously published in journals, books and conference proceedings were retrieved. Prevalence of SCD in different districts of Madhya Pradesh and Chhattisgarh state were recorded. Similarly, disease prevalence in different tribes of Madhya Pradesh and Chhattisgarh state.

Results :
In Madhya Pradesh state sickle cell disease is commonly found in southern part of the state and 22 districts have high burden of sickle cell disease. The majorly affected tribes are Gond, Baiga, Bharia, Panika and Pradhan. Among schedule caste community, sickle cell is more prevalent in Jharia and Mehra of certain areas. The prevalence of sickle cell trait within the community varies from one place to another (Figure 1). In Chhattisgarh, the SCD is common in central and southern parts and barring few districts. Twelve districts in Chhattisgarh falls into sickle belt of the state. The sickle cell trait prevalence was more than 10 percent in Abhujmaria, Gond, Halba tribes and Ghasia, Mahar and Ganda schedule caste communities of Chhattisgarh. In other backward class community, it is more prevalent in Agariha, Teli, Kurmi, Kumhar community of certain area (Figure 2). 

Conclusions :
The study mapped out the distribution of sickle cell disease in multi-ethnic population of Madhya Pradesh and Chhattisgarh State of India. This will help in planning the universal screening strategy for prevention of SCD.

Ethinic distrubition of SCT in Madhya Pradesh

Ethnic distribution of SCT in Chhattisgarh

Purpose :
Red blood cells transfusion is a mainstay in the management of sickle cell disease (SCD). However, this medical procedure is not without risk, particularly in patients with SCD. Indeed, they are particularly at risk of post-transfusion alloimmunization for several reasons. This complication will lead to an increased risk of additional immunizations, delayed hemolytic reactions and reduced chances of finding compatible blood products. The objective of this work is to evaluate, through a retrospective study, the effectiveness of the transfusion policy adopted at the regional hospital center of the Citadelle in Liège (Belgium).

Materials and Methods :
We studied clinical and immunohematological data in SCD population. Our cohort consisted of 164 patients followed regularly at the hospital's SCD center. The different sickle cell syndromes were represented (SS, SC, S/β0, S/β+).

Results :
Seventy-four percent of our cohort was transfused (n=121), 91% (n=110) via our hospital blood bank. Our results reveal a post-transfusion alloimmunization rate of 19%. Antibodies directed against the RH system are predominant, with a rate of 38% (n=17), followed by the MNS system with a rate of 29% (n=13), the Lewis system with a rate of 18% (n=8), the Kell system with a rate of 7% (n=3), and finally the Duffy and Kidd systems, each with a rate of 4% (n=2) (Figure 1).
Our study shows that advanced age, single transfusion, presence of an alloantibody, inflammatory background and an acute event are all risk factors for alloimmunization. We could not find any significant difference concerning gender, the presence of an autoantibody or splenectomy.
RH genotyping has been performed in 87 patients. The distribution of the different RHD gene types found is shown in Figure 2. Phenotype has been changed from D-positive to partial D on the basis of this analysis for 35% of the patients, i.e. to be considered as D-negative for transfusion purposes. Among these patients, 2 had developed an anti-RHD antibody before the genotyping was performed. In 2017, prophylactic RH genotyping was implemented for all SCD patients. No anti-D was found since then.

Conclusions :
The significantly higher alloimmunization rate found in our SCD population is similar to rates reported in other centers meeting the guidelines. The major explanation probably lies in the difference in the distribution of erythrocyte antigens between African and Caucasian populations. The risk is particularly elevated for Rh system. That is why, in addition to the extended blood group phenotyping, prophylactic RH genotyping was introduced in our lab for all SCD patients since 2017. We have not detected any alloimmunization against antigens of this system since then.
Alloimmunization risk factors highlighted in our study are consistent with the literature.
In conclusion, the precautionary measures used in our center appear to be effective in minimizing the risk of post-transfusion alloimmunization in patients with SCD. However, improvement in this field still possible, including optimization of donor-recipient matches, better control of chronic inflammation associated with the disease and better understanding of genetic polymorphisms influencing the immune response.

Figure 1 - Distribution of specific alloantibodies according to erythrocyte antigenic systems.

Figure 2 – Distribution of RHD genes identified by genotyping.

Purpose :
Leg ulcers are the most common skin complication in sickle cell patients. Though it is associated with great morbidity and cost of care, research is rather sparse on this condition. Through this study, we wished to determine the factors associated with leg ulcers in sickle cell patients in two hospitals in Douala.
 

Materials and Methods :
This was a retrospective cross-sectional study carried out over a period of 05 years (January 01, 2015 to December 31, 2019). This study was carried out at two sites: the outpatient hematology department of the General Hospital Douala and Sickle cell care center at the Laquintinie hospital Douala. We included all sickle cell patients aged 10 years and above. We excluded patients with incomplete files. Data on sociodemographic characteristics and clinical parameters were collected. Analysis was done using SPSS version 25.0 software. Qualitative variables were compared using Pearson's chi-square test. Multivariate analysis was done using logistic regression to determine the factors associated with leg ulcers. Significance level was set at p <0.05.

Results :
Out of 620 sickle cell patients, 41 (6.6%) had a leg ulcer. The median age of patients with a leg ulcer was 28 years (IQR 22-33). The male to female sex ratio was 1.15. Leg ulcers were traumatic in 70.7% of cases. The leg ulcer was unique in 65.85% (n=27) and located next to the malleolus in 26 (63.34%) of patients. Sociodemographic characteristics significantly associated with leg ulcers were the age groups 30-39 years [OR=9.3 (3.1-27.3), p <0.001], 20-29 years [6.3 (2.3-17.1), p <0.001] and a history of leg ulcer [2.7 (1.1-6.4), p=0.020]. For clinical characteristics, pulmonary hypertension [3.5 (1.7-6.7), p<0.001] and aseptic osteonecrosis of the femoral head [1.5 (0.01-2.25), p=0.020] were significantly associated with leg ulcers.

Conclusions :
In the pathogenesis of leg ulcer, a triad of mechanical obstruction of the microcirculation, hémolysis-vascular dysfunction syndrome and venous incompetence has been incriminated. in our study, one out of fifteen patients with sickle cell disease in Douala has a leg ulcer. Factors associated with leg ulcers were age between 20-39 years, pulmonary hypertension, aseptic osteonecrosis of the femoral head and history of leg ulcer. These factors are probably the result of chronic hyperhemolysis in our population. 

Purpose :
A substantial number of patients with sickle cell disease (SCD) have HbSC disease, especially those living in West Africa. HbSC may cause less early morbidity and mortality than sickle cell anemia (HbSS/Sβ0), but individuals with HbSC disease suffer from the same complications and have reduced quality of life. Retrospective data suggest some benefits of hydroxyurea treatment for people living with HbSC disease, but rigorous prospective evidence is lacking regarding the optimal dose, the effects on clinical and laboratory parameters, the safety of long-term treatment, and the best study endpoints to consider for a definitive Phase III trial of hydroxyurea therapy for HbSC disease. Therefore, we designed a clinical trial to achieve the following aims: 1) to measure the toxicities of hydroxyurea treatment on laboratory parameters; 2) to assess the effects of hydroxyurea treatment on a variety of sickle-related clinical and laboratory parameters in a large cohort of children and adults with HbSC disease; and 3) to identify study endpoints that are suitable for a future Phase III trial of patients with HbSC disease receiving hydroxyurea therapy.

Materials and Methods :
Prospective Identification of Variables as Outcomes for Treatment (PIVOT) is a double-blinded, randomized, placebo-controlled phase II trial of hydroxyurea. Children and adults with HbSC disease, ages 5 to 50 years, and receiving care at Korle Bu Teaching Hospital in Accra, Ghana will be eligible. Participants will undergo initial screening for 2 months and then will be randomized to placebo or hydroxyurea (donated by Addmedica, Inc, Paris) with approximately 120 participants in each arm. Randomization will be stratified by age and sex to ensure that an equal number of children and adults with balanced baseline characteristics are enrolled in each arm.

Results :
Participants will first be treated with hydroxyurea or placebo for 12 months. Hydroxyurea will be prescribed initially at 20±5 mg/kg/day with an opportunity to escalate the dose twice over the first 6 months if appropriate laboratory parameters are met. If safety of hydroxyurea is confirmed, participants will be able to continue in a subsequent open label treatment phase for up to 48 months. Laboratory safety measures will include assessment of treatment-related cytopenias and change in whole blood viscosity. Primary outcomes of clinical efficacy will be the rate of SCD-related acute complications, and secondary outcomes will include change in hemoglobin and change in chronic organ function with measurement of intracranial arterial blood flow by transcranial Doppler ultrasound, near infrared spectrometry for oxygen utilization, spleen size and function by ultrasonography, renal assessment of proteinuria and kidney volume, and retinal imaging to assess for damage. Exploratory outcomes will include change in functional RBC analysis using a Laser Optical Rotational Red Cell Analyzer (Lorrca) and change in patient reported outcomes using the PROMIS questionnaire and 6-minute walk test.

Conclusions :
PIVOT is scheduled to begin enrollment by May 2022. For children and adults with HbSC disease PIVOT will determine the safety of hydroxyurea and identify measurable changes in a variety of laboratory and clinical parameters, suitable for future prospective testing in a definitive Phase III clinical trial.

Purpose :
Sickle cell disease (SCD) is a serious public health problem worldwide. Currently, hydroxyurea (HU) is the most widely used drug in SCD as it promotes a reduction in severity of sickle cell events, improves hematological parameters and increases the quality and expectancy of life. Despite the evident benefits of HU, there is significant inter-individual variability in pharmacological response and genetic factors seem to be associated. The aim of this study was evaluate the individual variability of pharmacological response to treatment with HU, analyzing pharmacogenetic markers and hematological parameters of patients with not treated SCD and after treatment.

Materials and Methods :
185 patients with SCD treatment (n=93) and without treatment (n=92) with HU followed at Fundação Hemominas of Governador Valadares/Minas Gerais (MG)- Brazil were evaluated. The mean levels of hemoglobin (Hb), hematocrit, reticulocytes, global leukometry and fetal hemoglobin (HbF) were assessed before and after treatment. Patients were genotyped by real-time PCR (qPCR) for the polymorphisms G>T (rs1799983) and T>C (rs2070744) on the endothelial nitric oxide synthase (eNOS) gene, C>T (rs17599586) of arginase type 1 (ARG1) gene, A>C (rs766432) and G>A (rs4671393) of the B-cell lymphoma/leukemia 11A (BCL11A) gene, G>A (rs9960464) of the Urea Transporter (UTA) gene and A>G (rs2182008) of the Fms-related tyrosine kinase 1 (FLT1). The Ethics Committee has approved this study.

Results :
The average age of patients was 15.8±11.3 years, among which 54% were men. Genotypic and allele frequencies for polymorphisms in the gene of  eNOS (rs1799983: G=0.76; T=0.26/rs2070744: T=0.65;C=0.35), ARG1 (C=0.86; T=0.14), BCL11A (rs4671393: G=0.76; A=0.24/ rs766432: A=0.76;C=0.24), UTA (G=0.61; A=0.39), and FLT1 (rs2182008: G=0.92; A=0.08) were in  Hardy-Weinberg equilibrium and were similar to those found in other populations. In the group of patients treated with HU, those with the GT genotype for the polymorphism in eNOS gene had higher baseline Hb values when compared to GG patients (p=0.033). When patients were grouped according to HU response profile in “responders” (HbF ≥ 20%) or “non-responders” (HbF < 20%), it was found that those with the AC and CC genotypes in the BCL11A and GA gene in the UTA gene responded more effectively to drug treatment than patients homozygous for the most frequent allele. No significant influences were found related to other polymorphisms or the response to HU after analyses by logistic regression.

Conclusions :
This study is pioneer in describing the frequencies of 7 polymorphisms in 5 candidate genes in a population of individuals with SCD treated with HU in the state of MG. The findings suggest that patients with the GT genotype in the eNOS gene (rs1799983) have higher Hb values and that the polymorphisms in the BCL11A (rs766432) and UTA seem to affect the hematological response to HU. Support: FAPEMIG (APQ-02608-14, PPSUS/APQ-03560-13) and UFJF.

Purpose :
The convergence of innovative biotechnologies is propelling the field of gene therapy with consequential implications for treating and curing genetic and infectious diseases globally. Although dozens of gene therapies for a broad range of disease areas are expected to receive regulatory approval worldwide over the next decade, patients are unlikely to receive equitable access to these medical breakthroughs. In particular, the sophisticated infrastructure required to deliver gene therapies pose critical challenges for low and middle-income countries (LMICs) seeking to integrate gene therapies into resource-constrained health systems. Without critical foresight and targeted investments across LMICs, gene therapies will perpetuate global health inequity.

Materials and Methods :
Using gene therapies for sickle cell disease and human immunodeficiency virus (HIV) in sub-Saharan Africa as use cases, this project examined the necessary infrastructure required for effectively and sustainably delivering gene therapies in low-resource settings. Data was obtained through a series of interviews with expert stakeholders from sub-Saharan Africa, Europe, and the US. Interviewees represented multiple sectors including hospitals and community clinics, gene therapy companies, manufacturing, patient advocacy and community engagement, technical training, and global health priority setting. Interviews were supplemented with an extensive literature review.

Results :
An analysis of stakeholder interviews revealed the need for core infrastructure across seven thematic areas: research; engagement and education; facilities and manufacturing; information systems, workforce, regulation; and finance. Although assessed individually, these domains are interdependent, highlighting the need to invest and co-develop across all areas simultaneously and synergistically.

Conclusions :
Building the requisite infrastructure for delivering gene therapies is a multi-decade endeavour. This long horizon should not discourage immediate action; rather, it should be acknowledged and appreciated as an opportunity for strategic preparation assuring future success.

By establishing and maintaining infrastructure across several thematic areas, countries can accelerate the development and delivery of gene therapies. Throughout this process, key steps should be taken to improve outcomes:

• Engage communities early and often: The process of providing gene therapies must be patient-centred. Involving communities in the design and implementation process will lead to effective therapies that are acceptable and accessible.
• Leverage existing infrastructure: Maximizing the impact of limited resources and removing redundancy requires integrated services, multi-use facilities, and increased coordination.
• Collaborate internationally: While countries in SSA build research capacity, promote private sector innovation and commercialization, and update regulatory frameworks, international collaboration will facilitate the exchange of scientific equipment, knowledge and training, and policy.
• Improve and adapt iteratively: The success of gene therapy hinges on iterative cycles of feedback between research and clinical deployment. Infrastructure should support the ability of scientific and societal findings to inform healthcare practices and regulations.

While this study focuses on gene therapy delivery and its requisite infrastructure, considerations of health systems strengthening should remain at the forefront. The delivery of gene therapies should expand to align with health priorities set by countries (e.g., attention to non-communicable diseases). A narrow focus on gene therapies can create vertical systems of planning, management, and monitoring and evaluation that are not suited for other health areas and do not maximize the use of limited resources.

Purpose :
This paper reports on empirical research which was conducted in England in 2018-2020 before the COVID-19 pandemic. It involved trying to understand barriers and enablers to employment for people who have sickle cell disorder (SCD).
 

Materials and Methods :
The project worked with people with SCD, and two of their voluntary organisations, the Sickle Cell Society and OSCAR Sandwell as partners. It used a method of ‘democratic co-production' and people with SCD led two focus group discussions to formulate and critique questions to ask about work and to share their understandings of what barriers and enablers to employment had been in their experiences. Then forty-seven individual interviews were conducted with people with SCD incorporating feedback from those focus groups 

Results :
We found that work was about more than just employment for people with SCD who had to engage in self-management of their conditions and were contributing a lot to their families and communities. There were considerable barriers to access employment and once in employment people could face various forms of discriminations. However, reasonable adjustments for peope with SCD in the workplace were relatively simple and inexpensive to put into practice. While people with SCD have a right to accomodations in the workplace they did not often understand legislation or how that affected things like disclosure or their rights. 

Conclusions :
We development an employment policy guide to aid employers and employees in the workplace in England. Post-COVID-19 we have also thought about how to apply this to flexible work and working from home. 

Purpose :
India has been identified as having the 2nd most number of births with sickle cell anemia in the world, after Africa. Apart from biomarkers, environmental factors play an important role in the clinical severity. As there are no such studies from India,current study attempts to address the effects of environmental factors in children with SCD.

Materials and Methods :
Prospective observational study was conducted in 122 consecutively recruited hospitalized children of 1-12 years of age with SCD diagnosed by HPLC,living within a 10km radius of the tertiary care centre.Convenience sampling method was used.This a hospital based study conducted from 1st January 2020 to 31st December 2021.The severity of morbidity were identified by duration of hospital stay,number of blood transfusions,infections & VOCs and correlated with various environmental factors like temperature,rainfall,humidity,windspeed & particulate matters on the day of admission.

Results :
The study was conducted in Jan 2020-Dec 2021.A total of 212 admissions from 122 subjects were included, 88 females(41.51%) & 124 males(58.49%).Of this,35.85% of children were under 5 years & 64.15% of children above 5 years.The causes of admission included vasoocclusive crisis(54.25%),severe anemia  requiring blood transfusions(41.98%) & infections(34.91%).
Various environmental factors recorded during this period were as follows, maximum temp 32.85( 22-46.8),min temp 21.46(0-30.8), relative humidity 78.41(20-100),wind speed 5.98( 0-28km/hr),rainfall 4.89(0-100.3), atmospheric NO2 levels 27.29(0-95ug/m3),SO2 7.06(0-39ug/m3) & Ozone 41.76(0-148ug/m3).
We noted an increase in the admissions as the relative humidity increases.Both extremes of temperature & relative humidity were the 3 important factors in causing VOCs & infections across 3 different seasons(p=0.0001).Among air pollutants, rising NO2 levels were significantly related to VOCs(p=0.0002).Rainfall had a strong correlation with infections(p=0.0035).All the environmental factors, except ozone were found significantly correlated with VOC & severe anemia while Infection was significantly related to all the environmental factors except wind speed,NO2 & Ozone.

Conclusions :
Studying the effect of environmental factors in SCD is important,as it allow patients and families to be given accurate information on how to reduce the risk of acute complications, because they can be manipulated relatively easy with simple advices,unlike genetic factors.However,further long term studies need to be conducted, to assess the effects on chronic complications and to drive public health policies.

Purpose :
Diverse clinical variability among sickle cell disease (SCD) patients opposes crises prediction, health monitoring and streamlined management. Thus, an unmet need for objective biomarkers prevails. Exosomes are extra-cellular nano-vesicles (~50-150 nm), enriched in bioactive lipids, proteins, mRNAs and miRNAs, released by cells. They transport molecular cargo to nearby/distant cells to affect-regulate biological processes. Recent studies by Khalyfa et al assessed the plasma exosome content, their sources and transcriptomics signature as predictive marker in SCD children with acute chest syndrome. However, the small sample sizes (32 and 33 individuals, respectively) may not capture the clinical variability?Thus, we aim to screen large population (~150 patients) with good follow-up available at Regional Blood Center, Ribeirão Preto using omics - proteomics and transcriptomics - of plasma-derived exosomes to identify biomarkers in SCD. However, the grand challenges to this expansive undertaking are: 1) establishing a R3 (reliable, robust and reproducible) exosome extraction protocol, 2) performing high-throughput mass-spectrometry and next generation sequencing, and 3) establishing a reliable multi-level comparative bioinformatics platform to analyze the omics data. Here we present our approach with an international collaborative team to resolve these challenges and preliminary results.

Materials and Methods :
 Exosomes from plasma from steady state SCD patients and healthy donors were extracted using ultracentrifugation. Exosome characterization involved size-concentration estimate by Nano Tracking Analysis (NTA); western-blot for exosome surface marker CD81, CD63, and CD9, and HSP70 and ALIX as internal controls; shape confirmation by transmission electron microscopy. Interestingly, the size and concentrations were different (n=4 each): size: 96.05+/-29.71 nm (healthy) and 65.23+/-21.7 nm (sickle), and concentrations: 51.3+/-10.3e9 (healthy) and 107+/-77.5e9 (sickle) particles/ml. Reverse-phase LC-MS/MS was done using an Orbitrap Fusion mass-spectrometer at Butantan Institute. From the bioinformatics pipeline established at the University of Nebraska-Lincoln, we were able to extract expression of ~2000 proteins per sample from 8 SCD, 2 healthy and 1 mast cell culture samples (mast cells, taken as control, excrete exosomes in physiologic state). We identified 25 significantly down-regulated proteins in SCD samples (vs. healthy) using t-test with equal variance (p<0.05) – which include blood proteins, complement proteins and immunoglobins. 

Results :
We identified expression of selected sno-RNAs and miRNAs: RNU 44, miR15a, miR361, miR132, miR16, miR125 and miR181 by qRT-PCR in SCD exosomes. While we are still in process of getting RNA-seq analysis, and the proteomics data are preliminary, these validations are pre-requisites for establishing exosome-based omics-pipeline for biomarker discovery for health monitoring of SCD patients, crises prediction and assessing response-to-therapy.

Conclusions :
NA

Purpose :
Introduction: Sickle cell disease management is a public health problem in developing countries. According to the World Health Organization (WHO), more than 300,000 of the 500,000 annual births with sickle cell gene, in the world live in Africa, particulary  in the sub-Saharan region. The risk of infant mortality is 50% to 80% in the lack of quick and appropriate care. In Senegal, the gene frequency is estimated at 10%, with 0.5% of annual births of major forms, (1700 newborns per year). In order to improve the management of this disease, Senegal State and Monaco Principality have co-financed a dedicated structure: Ambulatory Care Unit for Children and Adolescents with Sickle Cell Disease (USAD), in the National Children Hospital Albert Royer in Dakar, as part of a project with a partnership agreement. The main missions are care, training and research.

Materials and Methods :
Methods: In order to share the results of the first four years, we used data from the project's activity reports and the Hospital's Medical Information Service.

Results :
Results: between 2017 and 2020, 14899 consultations were carried out, on average: 3700/year, including 4604 Emergencies, on average 1151/year and 1062 new cases, on average: 265/year. During the same period, 743 long-term hospitalizations were carried out, an average of 186/year. The number of daily hospitalizations was 2686, an average of 672/year. In 2020, according to SIM statistics, the management of sickle cell disease occupied the 1st place in specialized consultations at the hospital, and was the 1st reason for hospitalization in the two main general pediatrics Units (Unit M and Unit K).  About the laboratory, 4099 complete blood counts were performed with 3063 Hb electrophoresis and 797 thick drops. Concerning the training of human resources, 8 paediatricians were certified in the management of sickle cell disease. In addition, 64 doctors and 82 nurses received capacity building. Advanced training courses were organized for 6 nurses and a laboratory technician. Associative activities made it possible to organize 28 talks with 2,831 people made aware of sickle cell disease. Three communications plan development workshops were organized with 60 people as well as training in social communication for 90 members of the association with the production of 1,800 awareness leaflets on sickle cell disease. 

Conclusions :
Discussion and Conclusion: Sickle cell disease is a neglected disease that can be functionally or life-threatening. Its management requires a national and international commitment. The State of Senegal and the Monegasque Cooperation have contributed to improving the diagnosis and supported. However, the main challenges remain the sustainability and the implementation of neonatal screening, as part of a national program to fight against sickle cell disease. 
Keywords: sickle cell disease, management, cooperation.

Purpose :
Among various Hemoglobin (Hb) disorders, approximately 1.5% of the world population carry β-thalassemia, of which 40,000 newborns are affected each year. β-Thalassemia is caused by reduced (β+) or absent (β0) synthesis of the β-globin chains of hemoglobin. Three clinical and hematological conditions are recognized, including the disease states of β-thalassemia intermedia and β-thalassemia major, and the less severe or asymptomatic β-thalassemia carrier state (β-thalassemia trait). Early screening and timely diagnosis are required in β-thalassemia patients for prevention and management of later clinical complications. High performance liquid chromatography (HPLC) is the major laboratory test available for screening of β-thalassemia carriers. In countries where β-thalassemia is most prevalent, diagnosis and screening of the condition is a challenge due to the high cost and logistical aspects of the existing laboratory diagnostic tests. Therefore, there is a need for an affordable and easy-to-use point-of-care (POC) test to facilitate decentralized hemoglobin testing in low-resource settings. Here, we present the first POC diagnostic test for β-thalassemia, Gazelle. Gazelle enables detection, accurate identification, and quantification of Hb types under ultraviolet (UV 410nm) illumination.

Materials and Methods :
In this study, 368 subjects, in the age range of 4 – 63 years, were tested at Apple Diagnostics lab in Ghatkopar, Mumbai, India, under a local IRB-approved study protocol. Additionally, 32 blood samples were prepared to match expected untreated β-thalassemia intermediate and β-thalassemia major samples. In total, 400 tests were conducted in this study.

Results :
As defined by the Standards for Reporting Diagnostic Accuracy (STARD), 394 out of 400 (98.5%) tests were valid tests. Gazelle correctly identified all subjects with β-thalassemia major and β-thalassemia intermedia, as well as β-thalassemia trait. 6 normal subjects were identified as β-thalassemia trait by Gazelle. Sensitivity was 100% for all β-thalassemia tested. Specificity was 100% for disease vs. normal and disease vs. trait, and 98.3% for trait vs. normal (Table 1).

Conclusions :
Gazelle provides hemoglobin type identification and quantitative results of relative hemoglobin percentages for a broad range of hemoglobin variants including Hb A, Hb S, Hb F, and Hb A2, enabling accurate detection of β-thalassemia carriers. Gazelle has significant advantages over laboratory tests, including timely results (in <8 min), automatic interpretation, digital storage and printed reports. Gazelle is a versatile point-of-care system that enables affordable, accurate, decentralized hemoglobin testing in resource-limited settings.

Table 1: Gazelle’s diagnostic accuracy for beta-thalassemia major, intermedia, and trait

Purpose :
In sickle cell disease (SCD), a single β-globin gene mutation results in sickle hemoglobin (HbS) that polymerizes upon deoxygenation, causing red blood cells (RBCs) to sickle leading to various complications. In thalassemia, RBCs have an imbalance in the ratio of α/β globin chains and aggregated, unpaired globin chains increase metabolic demands. These stresses result in ineffective erythropoiesis and shorten RBC lifespan, leading to chronic anemia. The resultant anemias, exacerbated by impaired RBC health, are associated with lower ATP levels than in healthy RBCs. Supportive care and agents like hydroxyurea are used most in treating SCD, with some patients on regular transfusions. Regular or episodic transfusions, with their own set of complications, are the mainstay of treatment for thalassemia.
Etavopivat, an investigational, once-daily, selective, erythrocyte pyruvate kinase (PKR) activator increases ATP and decreases 2,3-DPG.^1,2 In a Phase 1 study, etavopivat 300-600 mg once daily in patients with SCD not regularly transfused was well tolerated, improved hematologic markers, decreased hemolysis and improved markers of RBC health.^1,2 Etavopivat 200 and 400 mg once daily (doses predicted to provide desired pharmacodynamic response profiles) are being evaluated in a Phase 2/3 study of patients with SCD not on chronic transfusions (The Hibiscus Study, NCT04624659). We describe the design of a Phase 2, open-label, multicenter study (NCT04987489) evaluating efficacy and safety of etavopivat in patients with SCD on chronic transfusions (Cohort A), transfusion-dependent thalassemia (Cohort B) and non–transfusion-dependent thalassemia (Cohort C).

Materials and Methods :
Key eligibility criteria are outlined in Figure 1. Patients will receive etavopivat 400 mg once daily for 48 wks (Figure 2). Patients will provide written informed consent.
Transfusions received during the study (every ~3-5 wks) will be recorded and include Hb values before and ≥15 min after transfusion, transfusion dates, number of RBC units, volume of packed RBCs and hematocrit of the transfused unit (if available). If a patient has an increase ≥1.0 g/dL in pre-transfusion Hb vs baseline, the investigator may delay transfusion 1 wk or reduce the number of RBC units transfused. RBC exchange transfusions may also be performed in patients with SCD.
Primary, secondary and exploratory endpoints are outlined in Figure 2. The following additional endpoints will be assessed in all cohorts: change from baseline in quality of life (using SF-36 and PROMIS); change from baseline in serum ferritin levels at 12, 24 and 48 wks; liver iron at 48 wks; 2,3-DPG and ATP; pharmacokinetics; and safety. Primary endpoints will be analyzed using a 1-sided test at α=0.025.

Results :
Results are not yet available for this trial in progress. Planned enrollment includes ≤20 patients (aged 12-65 y) in each of the 3 cohorts (Figure 2).

Conclusions :
Etavopivat is a novel, investigational, once-daily, selective PKR activator with potential to improve RBC health and lifespan. This Phase 2 study will assess the safety of etavopivat and its impact on Hb levels and transfusion burden in patients (aged 12-65 y) with SCD or thalassemia.
¹Brown et al, Blood 2021.
²Kalfa et al, Blood 2021.

Key eligibility criteria for patients 12–65 y of age.

Study Design.

Purpose :
Nicaragua is a developing country located in Central America. The country has a multiethnic population, with Mestizos, American Indian, and black being the most predominant ethnic groups. Generally, there is a lack of national screening programs as well as a research center dedicated to the study of hemoglobinopathies. Commonly, solubility is used as screening test. Other Diagnostic methods such as electrophoresis or HPLC are restricted to a very small number of patient due to their high cost and availability. Therefore, Sickle cell disease (SCD) is diagnosed on the basis of the patient’s clinical examination, history, and routine hematologic analysis. Altogether, the deficient facilities, expertise, and economical limitations contribute to the unknown prevalence of this pathology. Recently, great efforts have been made to carry out screening studies in different parts of the country. Overall more than 800 were tested by using cellulose acetate electrophoresis (CAE).  The aims of this study are to estimate the prevalence of homozygotes and heterozygotes as well as analyze the accuracy of the clinical diagnostic. Additionally, to foreseen the SCD distribution statistical analysis will be performed

Materials and Methods :
Hemoglobin lysate from peripheral blood was obtained and cellulose acetate electrophoresis was carried out to define the hemoglobin phenotype and to confirm the clinical diagnostic. Descriptive statistics and probabilistic graphical model (Markov influence diagrams) were used to calculate the SCD prevalence and to foresee SCD distribution, respectively.

Results :
In the first group, 150 blood samples from patients with a clinical diagnosis of SCD were analyzed over a 2-year-long study period. Sixty-five patients (43%) with the hemoglobin phenotype of SCD were confirmed. However, 85 patients (57%) had an SCT or normal phenotype. In the second group, 450 blood samples from healthy individuals were screened, 21 (4.6%) were positive for SCT, and the other 429 (95.4%) were normal

Conclusions :
Although significant advances have been made in the diagnostic and prevention of SCD, in low-income countries such as Nicaragua, there is a great need for resources including newborn screening and counseling programs, specialized and equipped laboratories, and training for healthcare professionals. In this context, epidemiological studies, for instance, the one here presented are vital to assess the current status of the disease and can provide guidance for future clinical care of the patient with SCD

Purpose :
Sickle Cell Anaemia (SCA) is an inherited autosomal and lethal blood disorder caused by a mutation in the HBB gene that promotes haemoglobin (Hb) polymerization and consequent sickling of red blood cells (RBCs) in hypoxia. Regardless of being a monogenic disease, SCA has a remarkably high clinical heterogeneity in its phenotypic expression. Several factors have been shown to modulate the clinical manifestations of SCA, namely genetic markers such as α-thalassaemia and β-globin cluster haplotypes, that can modulate biological parameters like the degree of haemolytic anaemia or the levels of foetal haemoglobin (HbF).
 

Materials and Methods :
Our study incorporates 112 patients of all ages and genders that are followed in the SCA consultation at Clínica Girassol and obstetrics consultation at Maternidade Lucrécia Paim, in Luanda. All patients or their legal caretakers, in the case of minor patients, signed an informed consent form. Clinical, biochemical, and haematological data were collected, as well as the patient's clinical history including the age at diagnosis, the number of hospitalizations, blood transfusions and strokes, among others Additionally, samples of peripherical blood were used for genetic analysis. All samples were genotyped for the HbS mutation by PCR-RFLP to confirm the SCA diagnosis. Moreover, four SNPs were genotyped in the β-cluster to determine the HbS haplotype by RT-PCR (rs968857, rs10128556) and PCR-RFLP (rs28440105, rs3834466). The presence of the 3.7kb deletion of the α-globin gene was determined by Gap-PCR

Results :
A total of 112 SCA patients, including 76 females (67,9%) and 32 males (28,6%), were studied with ages ranging from 1 to 67 years old (mean of 18,8±12,1). All the samples were homozygous for the HbS mutation. The observed frequency of homozygous for 3.7kb α-thalassaemia deletion was 14%, with an allelic frequency of 35% in this sample. The data was consistent with the Hardy-Weinberg equilibrium (χ2=0.823, p=0.364) with the wild-type homozygous genotype being 44% and the heterozygous genotype being 42%. Most of the patients have the CAR/CAR haplotype (86 patients, 76,8%), and all the patients have at least one CAR allele. We observed an apparent differential survival of SCA patients that co-inherit the α-thalassaemia deletion, and with the homozygous CAR haplotype. We observed apparent higher mortality for homozygous with the normal alpha gene and for heterozygous, as well as an increase in 3.7kb α-thalassaemia deletion frequency in the individuals above ten years old. This effect was more evident when comparing the under-10 and over-10 years classes. Moreover, a clear decrease in the frequency of CAR homozygotes was observed in older age classes. This differential survival should be taken into account during patient management since the first ages of diagnosis. Analysing the haematological data, we observed also that the presence of the α-thalassaemia deletion reflects on the red blood cell count (p-value < 0.001) and on the haemoglobin level (p-value = 0.029).
 

Conclusions :
We confirm the positive impact of co-inheritance of 3.7kb α-thalassaemia deletion in SCA patients, particularly in the clinical severity and survival. The genetic polymorphisms studied can be powerful prognostic markers.
 

Purpose :
Intravascular hemolysis in sickle cell anemia (SCA) leads to release of heme into the circulation and oxidative stress predisposing to vaso occlusive crisis (VOC). Heme is metabolized by the Heme Oxygenase (HO) enzyme resulting in decrease in oxidative stress. The HO-1 expression is altered by HMOX-1 gene promoter polymorphism. We investigated the role of three HMOX-1 gene polymorphisms [two single nucleotide polymorphisms (SNP), 19(G>C)(rs2071747) and -413A>T(rs2071746), and one dinucleotide (GT)n length polymorphisms (rs3074372)] on VOC in SCA patients of eastern India.

 

Materials and Methods :
A total of 170 SCA cases (80 mild and 90 severe phenotypes) and 101 healthy controls were considered for this study. Those without incidence of a single pain episode or VOC episode were considered as ‘mild phenotype’ of SCA while those who had three or more acute pain episodes in the last twelve months were considered as ‘severe phenotype’. Genotyping of HMOX-1 (19 G>C) polymorphism was done by ARMS-PCR. Whereas, -413A>T polymorphism and (GT)n repeats were done by DNA sequencing.  In HMOX-1 gene promoter, the (GT)n repeats were categorized into three types: first, (GT)n≤27 is noted as ‘Small (S)’ repeats; second, (GT)n=28-32 as ‘Medium (M)’ repeats and third, (GT)n≥33 as ‘Large (L)’ repeats. We categorized repeats into two groups: group-I contains S/S and S/M; whereas, group-II contains L/L, L/M, M/M, and S/L. Genotypes and allele frequency were calculated and compared between cases and controls using SNPstarts. Fisher’s exact test, odds ratio (ORs) and 95% confidence intervals (95% CIs) were calculated by using GraphPad Prism v5.0. The p-value of <0.05 was considered statistically significant.

Results :
On analysis of HMOX-1 (19G>C) polymorphism (rs2071747) between the mild and severe phenotype of VOC, no significant (p>0.05) variation was found in the allele and genotype distribution. Whereas, in HMOX-1 (-413 A>T) polymorphism, a significantly higher mutant allele (T) (p=0.0185) and genotype were found [AA vs TT (p=0.011) and AA vs AT+TT (p=0.02)] in severe phenotype. However, there is no difference among AA vs AT (p=0.56). On (GT)n repeat analysis, it was found that small repeats were associated with the mild phenotype and large repeats with that of severe phenotype in SCA {Gp-I vs Gp-II [p=0.0014; OR(95%CI)- 3.143 (1.538-6.422)]}. The difference was significant when comparison of severe phenotype SCA was done with controls for (GT)n repeats with larger repeats being commoner in severe phenotype.

Conclusions :
No difference was noted between mild and severe SCA phenotypes HMOX-1 (19G>C) polymorphism. The prevalence of the small repeat (GT)n was found to be significantly higher in the mild SCA phenotypes and controls. Higher incidence of mutant allele and genotype of HMOX-1(-413T) polymorphism and larger (GT)n repeats in the promoter region of the HMOX-1 gene were associated with the severe phenotypes. Both have a negative effect on the HO-1 enzyme activity leading to increased VOC in severe SCA- phenotype. The capacity of HO-1 enzyme activity appears to be overwhelmed by heme-induced oxidative stress in severe phenotypes and the affected patients suffer more VOC compared to mild SCA phenotypes and controls.

Purpose :
Sickle cell disease (SCD) is a heritable blood disorder caused by a point mutation in the β-globin gene. Polymerization of deoxygenated mutated hemoglobin is the major molecular process in SCD pathogenesis that leads to red blood cell sickling and hemolysis. In addition to the major complications of SCD—hemolytic anemia, inflammation, and vascular occlusion—patients often experience secondary complications such as stroke, organ damage, shortened lifespan, and painful and debilitating skin ulcers. The prevalence of leg ulcers among people with SCD is likely underestimated, given the lack of registries and large prospective studies examining this complication. Current estimates of leg ulcers vary widely by geographic region: 43% in Brazil, 30% in Jamaica, 27% in Nigeria, 19% in Ghana, 13% in Sierra Leone, 8% in Saudi Arabia, and 1%-5% in the US. Among Americans with SCD, an estimated 14%-18% may develop leg ulcers. Voxelotor, a sickle hemoglobin polymerization inhibitor, is approved in the US for the treatment of SCD in adults and pediatric patients aged ≥4 years and in the EU for the treatment of hemolytic anemia due to SCD in adults and pediatric patients aged ≥12 years as a monotherapy or in combination with hydroxycarbamide. In a post hoc analysis of voxelotor-treated patients from the phase 3 HOPE study (NCT03036813), leg ulcers resolved within 24 weeks in 10 out of 14 patients, and leg ulcers improved or resolved in 13 out of 14 patients by week 72.

Materials and Methods :
RESOLVE is an ongoing phase 3, randomized, double-blind, placebo-controlled, multicenter trial investigating the efficacy of voxelotor with standard of care (SOC) in the resolution of leg ulcers in patients with SCD. Target enrollment is 80 patients from Nigeria, Kenya, and Brazil with a confirmed diagnosis of SCD (HbSS or HbSβ0 genotype), aged ≥12 years, and with ≥1 cutaneous ulcer on the lower extremity (leg, ankle, or dorsum of foot) that meets the following criteria: ≥2 weeks’ and <6 months’ duration at screening and >2 cm2 in area before randomization. After a 2-week run-in period, participants are randomized 1:1 to receive once-daily oral voxelotor 1500 mg or placebo in addition to SOC for 12 weeks. After the randomized treatment period, all participants receive open-label voxelotor 1500 mg plus SOC for 12 weeks (Figure). 

Results :
The primary objective of the study is to assess the efficacy of voxelotor plus SOC compared with placebo plus SOC on leg ulcer healing, measured by the proportion of patients with resolution of target ulcer(s) in each treatment group by week 12. Key secondary endpoints include days to resolution of target ulcer(s) up to week 12, change from baseline in total surface area of target ulcer(s) at week 12, and incidence of new ulcers by week 12.

Conclusions :
Results from this study will further guide clinicians and patients regarding the clinical use of voxelotor for the treatment of leg ulcers in patients with SCD. Participants will have the option to enroll in a separate open-label extension study after the 24-week treatment period.

Figure. RESOLVE Study Design, Population, and Timeline

Purpose :
A higher Neutrophil-Lymphocyte ratio (NLR) has been associated with poor prognosis in many diseases. Most Africans are neutropenic due to the duffy antigen null status. The duffy antigen null status however, has been associated with a higher risk of organ damage in people living with sickle cell disease (SCD). We therefore sought to determine the effect of the proportion of these blood cells to one another on SCD prognosis in Nigeria.

Materials and Methods :
One hundred and thirty (130) sickle cell disease patients in steady state and 117 healthy age- and sex-matched controls were recruited from a tertiary institution in Nigeria. All the participants gave informed consents and blood samples were taken for Full Blood Count and phenotyping by High Performance Liquid Chromatography. Demographic and clinical details were obtained by questionnaire administration. Neutrophils make up over 90% of granulocytes, hence the granulocyte-lymphocyte ratio was determined from the available absolute counts of granulocytes and lymphocytes. A subset of 110 patients who were not on hydroxyurea was used for the analysis. The patients were divided into 3 age groups (< 7years, 8 – 17years and >17years). Data analysis was done using IBM SPSS version 23.

Results :
The mean absolute lymphocyte counts (ALC) and absolute granulocyte counts (AGC) in SCD patients (5.142 and 5.295 x103/µL respectively) were higher than in controls (2.952 and 2.171 x 103/µL respectively) (p<0.001). ALC and AGC decreased with age (p<0.001, p=0.02 respectively) in the SCD patients. A similar trend in ALC was observed in the control group while there was no significant difference in the AGC of children <7years and adults (>17years) (Fig 1). The mean granulocyte-lymphocyte ratio (GLR) in SCD patients increased from 1.01 in children <8years to 1.40 in adults. For the control group, the GLR decreased from 0.75 in children  <8years to 0.66 in children 8–17years old and an increase to 0.95 in adults (Fig 2). In the SCD patients, GLR was positively correlated with RBC (rs=0.351, p<0.001), HGB (rs=0.256, p=0.004), HCT (rs=0.295, p<0.001) and HbA2 (rs=0.293, p<0.001). HbA2 was positively correlated with RBC (rs=0.201, p=0.028) while HbF was negatively correlated with RBC (rs=-0.257, p=0.006) and showed no correlation with the GLR (rs=-0.008, p=0.930). ALC however had a negative correlation with RBC (rs=-0.395, p<0.001), HGB (rs=-0.452, p<0.001) and HCT (rs=-0.395, p<0.001). AGC also had a negative correlation with HGB (rs=-0.245, p=0.006) and HCT (rs=-0.179, p=0.047). These relationships were not observed in the control group except for a positive correlation between HGB and GLR (rs=0.273, p=0.003) and a negative correlation between HGB and ALC (rs=-0.295, p=0.001). GLR was negatively associated with the frequency of painful crisis in the last one year (p<0.001). 

Conclusions :
AGC and ALC decreased with age in our patients with an increase in the GLR. A positive correlation of GLR with RBC, HGB, HCT and HbA2 infers a protective role of a higher GLR/NLR to RBC survival in our patients. A higher GLR may be associated with better clinical outcomes, and neutropenia due to the duffy antigen null status may exacerbate the course of SCD in blacks.

Fig 1: The mean absolute counts of granulocyte and lymphocytes in both cases and controls.

Fig 2: The percentage of granulocytes and lymphocytes in both cases and controls

Purpose :
Defective spleen function (hyposplenism) affects subjects with sickle cell disease (SCD) and is associated with complications. Red blood cell (RBC)-related markers are very accurate to assess spleen function. Vacuole-containing RBC (pocked RBC) are observed in high proportions (10 to 70%) of hyposplenic subjects whereas they do not exceed 5% of RBC in healthy subjects. Vacuoles in pocked RBC are considered by many as remnants of erythropoiesis-related processes.

Materials and Methods :
Patients with SCD who benefit from an exchange transfusion program, have not only their own HbSS RBC but also transfused HbAA RBC in circulation. In vitro sickling “Emmel test” discriminates HbSS RBC, that sickle, from HbAA RBC, that do not.
We perfused a mixture of HbSS RBC and HbAA RBC (from exchange transfusion left-overs) into human spleens ex-vivo. We then analyzed spleen sections and quantified vacuoles in RBC containing hemoglobin polymers or not. 

Results :
Using Emmel test, we observed vacuoles in HbSS RBC but also in transfused HbAA RBC. Pocked HbAA RBC were more frequent in transfused SCD patients than in healthy subjects (25.1±4.41% vs. 2.8±2.1%).
Into human spleen ex-vivo, we observed a high rate of vacuoles in either polymerized and non-polymerized RBC (29.8±3.7% vs. 17.9±5.8%). In our experimental set-up, HbAA RBC is deducted as the absence of polymerization.

Conclusions :
Taken together, these results strongly suggest that vacuoles are created de novo in transfused HbAA RBC.Counting vacuoles in RBC labeled with anti-HbS and anti-HbA antibody is ongoing to validate these observations. When the spleen is dysfunctional, these pocked RBC stay in circulation hence their validity as a spleen function marker. De novo appearance of vacuoles in transfused HbAA RBC likely impacts the kinetics of reappearance of pocked RBC in circulation following regular or exchange transfusion to hyposplenic subjects, and opens interesting prospects on cell processes involved in their genesis.

Purpose :
In sub-Saharan Africa, universal sickle cell disease-newborn screening (SCD-NBS) programs are nonexistent due to high costs associated with conventional laboratory technologies, competing for national healthcare priorities, and budgetary constraints. However, the recent emergence of lateral-flow immunoassay microtechnology point-of-care tests (POCT) such the SickleSCAN™ and HemoTypeSC™ present exciting opportunities to change the status quo and potentially revolutionize healthcare for children as best practice for early diagnosis of SCD.

Materials and Methods :
We perform a matrix comparison of diagnostic accuracy in field conditions for the SickleSCAN™ and HemoTypeSC™ tests. We assessed the performance characteristics of SickleSCAN™ and HemoTypeSC™ by evaluating children with positive Emmel test for HbSS, HbSC, and HbAS; and children, including newborns, without known coagulopathy in a rural district in Sierra Leone.

Results :
A total of seventy-four (33 females/41 males) children from three participating community health centers were analyzed by both methods. The ages ranged from twenty-four hours old to seventeen years old. HemoTypeSC™ and SickleSCAN™ correctly identified hemoglobinopathy phenotypes in 26 children with positive Emmel test as follows: twenty HbSS and four HbAS. Two positive Emmel test results (HbAS) were identified as HbAC and HbAA by the two tests indicating that the two tests have excellent sensitivity and specificity for the identification of the HbS pattern, consistent with SCD. Of the 74 samples that had two SCD POCT scores, there was no discordant result between the HemoTypeSC™ and the SickleSCAN™. Results for both tests were as follows: HbAA 36 (46.8%), HbAS 17 (22.1%), HbSC 1 (1.3%), and HbSS (26.0%). No newborns were identified with sickle cell disease (HbSS). The two tests identified seven newborns with sickle cell trait (HbAS). Twenty-one newborns had the normal HbAA genotype. Overall, HemoTypeSC™ and SickleSCAN™ exhibited sensitivity and specificity of 100% and 100% respectively for all possible phenotypes (HbAA, HbAS, and HbSS) detected.
 

Conclusions :
The HemoTypeSC and SickleSCAN tests demonstrated 100% concordance for identifying homozygote healthy people, sickle cell trait, and disease patients in this study. Our matrix comparison study highlighted the potential use of POCTs as both first?tier and second-tier screening and confirmation tests when presumptive SCD or trait is indicated. This is an acceptable practice for SCD screening as the current consensus is to use the same method on the same sample if no second?tier screening method is available and to confirm the screening result with a second method on a second sample to make a diagnosis. We, therefore, conclude that POCTs are suitable for population and newborn screening for the HbS phenotype in limited-resource settings where the disease burden is greatest and conventional laboratory methods are nonexistent.
 

Purpose :
A child’s growth reflects it nutritional and pubertal status and it is proven that nutrition is a factor affecting pubertal development. Sickle cell patients often have slowed growth and delayed pubertal development. This work aims to assess the nutritional and/or pubertal profile in children with sickle cell diseases (SS) admitted at the CEMECO center.

Materials and Methods :
This was a cross-sectional study and study participant under 16 years were randomly selected from the health center database having about 6497 cases and enrolled in the study.The participants were divides into two groups based on the electrophoresis of hemoglobin: Sickle cell disease including 103 cases of SS (homozygote) and 18 cases of SC, S, β-Thalassemia and SE (heterozygous). While the group of non-sickle cell participants includes 87 (AA and AS)

Results :
We included 208 children among them121 sickle cell disease patients and 87 non sickle cell diseasechildren. with a sex ratio M/F was about1.02. The meanage of sickle cell patients was 8.7±4.4 years while that of non-sickle cell patients was 9.5 ± years. The family income evaluated according to the MICS, was similar between the two groups (P= 0.123).

Evaluation of nutritional status using the weight / height score-z, revealed that in children under five years of age with sickle cell patients, was lower than that of non-sickle cell patients(p=). However, the difference was not significative (P = 0.155).

The height/age ratio express as Z score showed a significative difference between sickle cell patients (1.1 Z score)while non-sickle cell patients (P =0.000). 

Underweight evaluated by weight/age z-score showed that the in-sickle cell patients were -0.91 and -0.12 in non-sickle cell patients (P=0.014). However, the BMI evaluation did not show any significant difference between sickle and non-sickle cell patients P=0.188.

The proportion of delayed puberty in sickle cell patients were (4.8%) compared to 6.3% in non-in sickle cell patients. puberty. Uni-variate analysis showed an association between sickle cell disease and testicular development (P=0.046).

Conclusions :
The height/age ratio expressed as Z score result is similar to those found by Sharon E. Cox et al. in Tanzania in 2011. Contrary to the report by Al Saqladi et al, a difference was found in the weight/age z-score between the body mass index in control children compared to sickle cell children. These results are different from those found by Shongo et al. in 2015 that were statistically significant with a P >0.05 (21). This difference may be justified by the fact that our sample was larger than theirs.

In some, this study highlighted growth retardation and slowed testicular development in children with sickle cell disease.

Purpose :
La drépanocytose représente 1/260 naissances en Guadeloupe. L’insuffisance rénale est l’une de ses complications, son diagnostic repose sur le calcul du débit de la filtration glomérulaire (DFG). La créatinine sérique (CrS) est basse chez ces patients à cause de l’hémolyse et l’inflammation et augmente au cours du développement précoce de l’hypertrophie glomérulaire. La CysC est un polypeptide cationique non glycosylé, synthétisée à taux constant au niveau des lysosomes des cellules nucléées. Son taux ne varie pas avec l’âge, le poids, la taille et le sexe. Elle est utilisée pour l’évaluation du DFG chez les enfants drépanocytaires.

Materials and Methods :
Dans cette étude rétrospective au CHU de la Guadeloupe, la CysC est dosé par la technique PENIA sur BNII et la CrS par la méthode Jaffé. Nous avons comparés le DFG calculé par la formule à base de CrS (Schwartz) et le DFG calculé par les formules à base de CysC (Zappitelli, Hoek, Filler et Larsson). L’hyperfiltration est définis par un DFG ? 140 ml/min ou un taux de CrS < 30 pour les enfants de 4 à 12 ans et CrS < 50 µmol/l pour les enfants de 12 à 19 ans, ou un taux CysC < 0,53 mg/l. Nous avons utilisé Epi info, test t, test Chi 2 et le test de Pearson pour les corrélations.

Results :
40 enfants drépanocytaires de 6 à 17 ans sont inclus, 22 garçons et 18 filles, 25 sont de génotype SS, 12 S/C et 3 S/beta+thal. 76 % des patients âgés <12 ans, ont une CrS < 30 µmol/l et seulement 24% des patients ont CysC < 0,53 mg/l (p=0,0035). Alors que 95 % patients >12 ans ont CrS < 50 µmol/l  contre 16 % CysC < 0,53 mg/l (p<0,0001).
Le taux de  la CysC n’est pas corrélé à la CrS (r=0,298), Par contre une corrélation modérée existe entre la CysC et la clairance mesurée après recueil des urines (r=0,5201).
97.5 % des patients sont en hyperfiltration avec CrS =21,8 (5,8) et CysC <0,53 vs CrS =32,2 µmol/l (11,3) et CysC= 0,67 mg/l (0,08) pour les patients avec filtration normal (p<0,0001). Ce pourcentage baisse à 72% si on utilise la formule de Filler, 60% Larsson, 40% Zappitelli, et 30% Hoek (p<0,001). Une protéinurie (PTU) significative est présente chez 3 patients avec CysC 0,75 (0,11) vs 0,61 mg/l (0,11) chez les patients sans PTU (p=0,040)

Conclusions :
La CysC est un meilleur marqueur pour l’évaluation du DFG et un indicateur précoce de l’hyperfiltration glomérulaire. La formules de Hoek semble appropriée pour le calcul du DFG du patient en état basal et détecter l’hyperfiltration.
Une cohorte plus large est souhaitable chez des enfants drépanocytaires sans micro-albuminurie, pour consolider ces données sur cet indicateur de l’altération de la fonction rénale. Le coût du test de la CysC reste plus cher que CrS.

Purpose :
Sickle cell disease (SCD) is one of the most common hemoglobinopathy, affecting more than 300,000 infants each year globally and 1 in 365 Black or African American births in the United States.  SCD is associated with various potential complications, including, but not limited to pain, acute chest syndrome, splenic sequestration, and neurological insults, among others. Patients with SCD who have a history of silent or overt cerebral infarcts or strokes are at risk for cognitive deficits. There is also evidence to suggest SCD carries risk for cognitive deficits even in the absence of silent or overt strokes, and factors associated with such deficits are not well understood. Sickle cell disease (SCD) is one of the most common hemoglobinopathy, affecting more than 300,000 infants each year globally and 1 in 365 Black or African American births in the United States.  SCD is associated with various potential complications, including, but not limited to pain, acute chest syndrome, splenic sequestration, and neurological insults, among others. Patients with SCD who have a history of silent or overt cerebral infarcts or strokes are at risk for cognitive deficits. There is also evidence to suggest SCD carries risk for cognitive deficits even in the absence of silent or overt strokes, and factors associated with such deficits are not well understood. 

Materials and Methods :
The study was approved by Children’s National Hospital Institutional Review Board. Sixty-nine study participants (7-16 years old) completed the Wechsler Intelligence Test for Children Fifth Edition (WISC-V), where we collected Full Scale Intelligence Quotient (FSIQ), working memory, processing speed, and verbal comprehension. Socioeconomic factor data were collected from the Census and Maryland Department of Education, based on the participants' postal code.

Results :
Overall, there were no significant correlations between socioeconomic factors and cognitive function (weak correlations ranging from r = 0.1 to r = 0.21, ps > .05). The highest correlation was 0.21 between processing speed and the number of libraries within a zip code, and this was not statistically significant (p > .05).

Conclusions :
These data suggest that community-level socioeconomic factors do not appear to relate to an individual’s cognitive functioning. That said, socioeconomic factors on an individual or familial basis may have an impact on cognitive function in children and adolescents with SCD, and further exploration is needed. Particular attention to cognitive functioning and literacy-based resources may help to elucidate the non-significant though larger correlation found in our data. Limitations of our study include small population size, and limited data on socioeconomic factors. In the future, a larger scale study with person-specific socioeconomic data rather than community level data could potentially describe relationships further.

Purpose :
Introduction and Aim: Sickle cell anemia, an inherited blood disorder has now become the concern of global health due to its wide presence in sub-Saharan Africa, parts of the Middle East and some areas of the Indian subcontinent. The tribal population of India, as per 2011 census, is 10.43 crore, constituting 8.6% of the total population. Despite of the highest population of indigenous people in India, there is a very little and scattered knowledge about the actual burden and frequency distribution pattern of the disease in this underprivileged group of the society. In state Chhattisgarh, where 30 % of the total population is contributed by indigenous people, there are limited studies with large scan. The objective of this study is to find out the genetic burden and distribution of the sickle gene in tribal and non tribal communities of State Chhattisgarh.

Materials and Methods :
Methods: A large scale population based screening program was conducted during the year 2007 to 2017 under the State Sickle Cell Screening Program of Chhattisgarh, in which a total of 17,18,909 people in 11 districts along with the special camps in 6 districts was screened using sodium dithionite turbidity test (DTT) after taking informed consent. Identified 2,09,460 positive cases were subjected to confirmation by hemoglobin electrophoresis. The samples not apparently identified using electrophoresis and Sickle homozygous (SS) were further confirmed by HPLC.

Results :
Results: The data indicated the presence of the sickle gene in ~10 % while two sickle gene (homozygous; SS) in ~0.47 % of total screened population of Chhattisgarh. It was observed that the prevalence of the sickle gene in tribal group is highest in Gond, Halba and Kanwar tribes. Among non-tribals the prevalence highest occurrence in Teli followed by Ravat and Kurmi  in OBC while in SC category, Satnami, Ganda, Mahar and Ghasiya communities showed the highest occurrence. Brahmin and Rajput communities showed the highest numbers among general social group. 
 

Conclusions :
Conclusion: This was one of the largest population based study, which showed high prevalence of sickle gene in non tribal group as compared to tribal group in Chhattisgarh. The study has not only provided information on the disease burden and distribution pattern of the sickle gene among tribal and non-tribal communities of the State Chhattisgarh, but also contributed a lot in creating social awareness about the disease. Now, the people are voluntarily coming forward for their screening and adopting required management/ treatment strategies such as lifestyle management, pre/post marriage counseling and hydroxyurea therapy. The information can be used to link to their natural history and to develop better measures for management, control and effective eradication of the disease. 

Purpose :
Sickle cell disease (SCD) is a monosomic genetic disorder in which affected individuals might have a worse outcome in respiratory diseases such as covid-19 due to its induced hypoxemia sickling.

Materials and Methods :
We described a group of SCD pediatric patients from a hematologic service that seeked medical care in an emergency department (ED) in the period of March 2020 to November 2021, in a tertiary hospital in Brazil that were tested for covid-19. We described clinical and laboratory characteristics: age, gender, SCD genotype, comorbidities, and ambulatorial treatments. We gathered information regarding clinical presentation and also laboratory findings for all participants, which included: hemoglobin, mean corpuscle volume (MCV), leucocytes, neutrophiles, lymphocytes, platelets count, total, direct and indirect bilirubin. We compared laboratory data from the last routine ambulatory visit and ED admission for all participants. We compared two groups of patients: those with positive polymerase chain reaction (PCR) for Sars-Cov-2 and the negative PCR  group. We described treatments given to positive PCR individuals and their outcomes: hospitalization, intensive care unit (ICU) admission, death, venous thromboembolism, pediatric acute distress syndrome (PARDS), multisystem inflammatory syndrome in children (MIS-C), acute chest syndrome (ACS), vaso-occlusive crisis (VOC) and discharge before 24 hours of admission.

Results :
Twenty-nine individuals were included on our criteria and fifty-three admissions were analyzed. Five patients that have positive PCR had 40% of ICU admissions, but no death, venous thromboembolism, PARDS or MIS-C were registered. Median age for all patients at admission was 11 years old while positive patients had a median of 16 years old. The majority was male and had genotype SS. Laboratory findings at admission day at ED and a basal laboratory data did not show statistical significant differences, except for neutrophil count and direct bilirubin for all individuals and in the negative PCR group of participants. 
The main manifestations presented at ED were fever, followed by respiratory symptoms, hypoxia and VOC. During hospitalization antibiotics, oseltamivir, blood transfusion and enoxaparin were utilized as therapy and patients did not receive specific medications for covid-19 infection.

Conclusions :
We expected that Sars-Cov-2 infection would have a worse effect in SCD patients due to their comorbidities and their predisposition to worse outcomes in respiratory infections. That hypothesis though is not confirmed neither from this study nor other authors.
 

Table 1 Characteristics of participants with SCD at ED who tested PCR positive and PCR negative for SARS-CoV-2

Table 2 Comparison of laboratory values of participants tested PCR positive and negative for SARS-CoV-2 between last routine visit and admission in the ED

Purpose :
Occurrence of sickle cell gene in Odisha state is known since 1952. It is very widespread among the people of western districts of the state. A great deal of literature on distribution, clinical presentation and haematological parameters of sickle cell disease cases have been documented.  Due to varied morbidities the sickle cell disorder patients regularly visit the health centres frequently. The gene frequencies in certain caste groups have been reported to be very high and probably due to various social and traditional beliefs.  Further most of the patients were from low income groups and rigid cultural tradition. Keeping in view this study was performed to explore some of the socio-economic aspects of such debilitating health problem in the state.

Materials and Methods :
The various haemoglobinopathy cases including sickle cell disorder cases reporting to Government Medical college hospitals (Burla and Berhampur) and district headquarters hospital, Balasore, Sishu Bavan hospital, Bhubaneswar, representing the different part of the state were taken for the study. Relevant data pertaining to the study were collected by applying questionnaire method from either of the parents of 100 haemoglobinopathy cases from Balasore, 98 cases from Bhubaneswar, 100 cases from Burla and 90 cases from Berhampur.

Results :
Majority of sickle cell disorder cases were from western and southern districts of the state. However such cases were also reported from the eastern and northern districts of the state also. Nineteen percent (19%) of parents of Beta thalassaemia patients had consanguineous marriage, where as it was 10% among the parent of sickle cell patients. Because of the disease a good number of children were not attending school (Beta Thalassaemia-24%, Sickle cell disease-28%) and school dropouts were 3% and 5% respectively. Forty two (42%) percentage of sickle cell patients were having blood group O+ and the blood groups O+ and B+ were equally (38%) present among the studied thalassaemia patients. More than eighty percent of parents’ income of sickle cell disorder patients was less than Rs.4000/- per month and only 2% of such parents’ income exceeds the income range of above Rs.10000/- per month. Father’s of the studied sickle cell patients were not well educated, 34% ware illiterate and 45% received primary education. Similarly Forty percent (40%) mothers were illiterate and 43% were having primary level of education.

Conclusions :
The sickle cell disease patients were reported from all the regions of the Odisha state and majority were from western part of the state. Contrary to the belief very few numbers of consanguineous marriages were observed among the parents of sickle cell disease cases. The parents of sickle cell disease cases were facing heavy financial burden for proper clinical management. The literacy levels of the parents were also very inadequate. The need of blood transfusion particularly of the B+ and O+ blood groups is also very high.  It is highly desirable to initiate social intervention and economic support for these patients and their parents by the government as well as non government organisations of the state.

Purpose :
Sickle cell disease (SCD) is a genetic blood disorder that affects the shape and transport of red blood cells (RBCs) in blood vessels, leading to various clinical complications. The pharmacological reactivation of Fetal Hemoglobin (HbF) is considered to be a viable therapeutic method in SCD. In this regard, hydroxyurea (HU), a powerful ribonucleotide reductase inhibitor, is being employed as a HbF-inducing pharmaceutical. However, its cytotoxicity, carcinogenic potential and variable effects limit its use. Thus, a major challenge today is to identify new agents, with high HbF-inducing activity, low cytotoxicity, and available in low- and middle-income countries, such as natural compounds. Quercetin, a natural flavonoid, has been identified as a potential HbF inducer. The main aim of this work was to evaluate Quercetin role in the reactivation of fetal hemoglobin (HbF) by analyzing the expression of globin and HbF regulatory/silencing genes.
 

Materials and Methods :
Gene expression was studied in K562 cells previously exposed for 24 hours to two concentrations of quercetin (0.2 and 20 mM) dissolved in DMSO and 25 μg/ml hydroxyurea (HU) as a positive control. The exposed cells and controls were collected, and cell viability and proliferation parameters were evaluated microscopically.  Variation in gene expression after CPMLE exposition was quantified from the total RNA isolated from cultured cells, using quantitative Real Time PCR. The studied genes were α, β and γ-globin genes, as well as the HbF regulators genes MYB, KLF1, BCL11A and BGLT3, and GAPDH as reference.
 

Results :
The proliferation rates were calculated as the ratio between the value at 24h and the initial number of cells (1X105 cells/well). The results for the quercetin concentrations of 0,2 and 20 mM were of 1,95 and 0,967, respectively, while for vehicle (DMSO) the value was 2,40. The percentages obtained for the viability, as assessed by trypan blue staining, were of 95,18% in vehicle, 91,40% and 88.99% for the quercetin concentrations of 0,2 and 20 mM, respectively. Altogether, these results indicate that quercetin slightly affects the proliferation and viability of the K562 cell line at both concentrations, although without cytotoxic effects. Transcriptional analysis demonstrated that both concentrations inhibit BCL11A, MYB, KLF1 and HBB gene expression levels; and increase the expression of HBG and BGLT3. The effect of quercetin on BCL11A gene expression was similar to the effect of HU, however the expression of MYB, KLF1, HBB and BGLT3 genes differed between the two molecules. In addition, the 0.2 mM concentration induced an overexpression of the HBA gene, similar to the effect of HU.
 

Conclusions :
The results presented are preliminary, however it is possible to observe that quercetin can modulate the expression of HbF, thus potentially constituting an effective alternative treatment of SCD.
Acknowledgments - This project was supported by Instituto Politécnico de Lisboa under the grant IDI&CA-IPL/2021/EpiCa/ESTeSL and partially supported by FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020). The authors are grateful to Fernando Nunes for kindly providing the Carica Papaya leaves.
 
 

Purpose :
In high-income countries (HICs), advances in sickle cell disease (SCD) care have led to >95% survival of children to age 18 years.  In low-and-middle income countries (LMICs), like Brazil, survival is approaching 80% by age 18.  With higher survival rates, a growing number of young adults need to be prepared (i.e., build health literacy, improve self-efficacy and gain transition skills), transferred, and integrated into adult care.  Implementation of evidence-based health care transition (HCT) practices can address this need.  Despite the increasing SCD pediatric survival in LMIC, no systematic evidence-based HCT practices are in place.  While guidelines to support HCT in HICs exist, the readiness for implementation of HCT practices is unknown in LMICs, therefore implementation cannot be appropriately planned or their environment prepared.  

Materials and Methods :
To address the need for implementation of HCT in Brazil, we plan to undertake a 3-Aim process: Aim 1) Select SCD-specific evidence-based practices for HCT from HICs, Aim 2) Identify barriers and facilitators to implement HCT for SCD in 2 Brazilian institutions (Instituto Estadual de Hematologia, HEMORIO, Rio de Janeiro and Universidade Federal de Sao Paulo, UNIFESP, Sao Paulo), and Aim 3) Adapt evidence-based practices for HCT to the Brazilian institutions.  We will begin by examining the contextual factors that influence the successful implementation of HCT practices through surveys to providers, patients, and clinic leadership, with complementary focus groups with a selected sample (Table 1).  To investigate the barriers and facilitators, we will conduct a QUANTITATIVE->qualitative mixed-methods evaluation, guided by the Exploration, Preparation, Implementation and Sustainment (EPIS) framework (Figure 1).  In addition to examining the context of each site, we will perform a literature synthesis and investigate the mechanisms of action (i.e., what works and how it works) of HCT practices in a HIC program (St. Jude Children’s Research Hospital, Memphis, TN).  Using implementation and mechanism mapping, we will define the core components and the adaptable steps required to build capacity and expertise to implement HCT practices in Brazil.  Additional focus groups with patients, providers, and clinic leaders at each Brazilian hospital will examine the fit of the adapted approach to their organization.  To complement the mechanism mapping process, we will collect data to map the implementation steps and cost for implementation using the Stages of Implementation Completion (Chamberlain P, et al. ImplementSci.2011;6:116.) measure and its associated cost mapping tool, the Cost of Implementing New Strategies to track the entire implementation process.

Results :
Our research and implementation teams have been assembled and data collection is underway. Ongoing results will be presented. 

Conclusions :
Evidence-based HCT services are needed to address the higher survival rates among young adult populations with SCD in LMICs.  Our project will be the first to rigorously evaluate the readiness for implementation of HCT in LMIC and to design a context-specific HCT tool for Brazil.  Finally, documentation of milestones of pre-implementation will allow us to track and compare the effectiveness and cost of future HCT implementation strategies, ensuring the optimal implementation and sustainability of HCT programs in LMICs.

Figure 1. EPIS as the guiding framework for implementing HCT for SCD in LMICs. We will identify Evidence-based practices (EBPs) for HCT in SCD, assess determinants of implementation in Brazil and begin adapting HCT EBPs for their context. (Aarons GA, et a

Table 1. Mapping of study measures according to the EPIS constructs.

Purpose :
Sickle cell disease (SCD) is one of the known autosomal recessive hereditary disorders of hemoglobin known to associate with serious complications and responsible for recurrent hospitalization, considerable morbidity and mortality in the affected individuals. Despite of being a monogenic autosomal recessive genetic disease, it is one of the most heterogeneous diseases known. In the face of high prevalence and heterogeneity in the area, there are limited studies addressing the hematological pattern of sickle cell disease for proper therapeutic management and treatment. Therefore, the study aims t­o establish a baseline data of hematological values in pediatric age group (1 - 14 years) and adults (> 14 years) and to establish an interrelationship between red cell indices with hemoglobin subtypes in sickle cell disease (SCD) in Chhattisgarh, India.

Materials and Methods :
Following the preliminary screening by sickle solubility test, blood samples of positive suspected cases were collected in EDTA tubes for hemoglobin fractionation by cation exchange high performance liquid chromatography (HPLC) and hematological indices by the automated cell counter.

Results :
The results indicated that out of total 4674 sickle cell disease cases, 3338 (71.42 %) cases were of the pediatric group while 1336 (28.58 %) adult cases. Surprisingly, SCD subjects showed high fetal hemoglobin (average 18.68 %) with no significant difference in both the age groups. However, a significant difference was observed in red cell indices with moderate total hemoglobin (average Hb 9.5 %) and red cell indices in both the groups. Furthermore, the result of correlation analysis indicated a significant negative correlation between fetal hemoglobin and RBC, MCHC and RDW; Hb A2 showed significant negative correlation with MCHC while positive with RDW, further Hb S showed a significant positive correlation with MCHC in both the age groups. 

Conclusions :
The single most striking observation to emerge from the data is that the sickle cell disease subjects from Chhattisgarh exhibit mild phenotypes with the presence of high fetal hemoglobin with moderate to normal total hemoglobin and red cell indices. It is expected that the study will act as a database to characterize the hematological indices which can be taken into consideration when dealing with SCD patients for therapeutic management.

Purpose :
The management of sickle cell disease (SCD) can be challenging, requiring different therapeutic approaches because SCD could induce multiple complications, precipitating multi-organ failure. Red blood cell (RBC) transfusion is well known as one of the disease-modifying therapies available for long-term management of this condition. It is supported by multiple randomised clinical trials for the prevention of disease complications such as stroke in adults and children with SCD. RBCs can be delivered via three methods: Simple transfusion(ST), manual exchange(ME) and automated red blood cell exchange(aRBCX). In a simple transfusion, patients receive additional units of blood. The purpose is to raise the haemoglobin (Hb) to a steady state and maintain the oxygen-carrying capacity of the blood. Manual exchange transfusion removes the patient’s sickle-shaped RBCs and replaces them with healthy ones — lowering the concentration of sickled cells without increasing blood viscosity. This is performed using repeated alternating isovolumetric phlebotomy and blood transfusion. Automated red blood cell exchange transfusion involves removing sickled RBCs from the patient and rapid replacement with healthy RBCs while maintaining isovolemia. An advantage of aRBCX is avoiding or minimising costly iron chelation therapy to treat iron overload.
As Sickle Cell disease (SCD) management is a critical cost driver to health systems, this research intended to compare the cost-effectiveness of different transfusion modalities. We wanted to compare the following transfusion types: Automated red blood cell exchange (aRBCX), Top-up transfusion and Manual red blood cell exchange (mRBCX). More specifically, we wanted to perform this evaluation in Oman as 0.2% of the Omani population suffers from this disease, which is the most frequent genetic disorder with the highest incidence.
 

Materials and Methods :
Variables that drive costs have been collected to populate a de Novo cost model. We collected data from the Royal Hospital and Sultan Qaboos University Hospital. The most significant cost drivers which we took into consideration were: disease incidence, cost blood transfusion, medical equipment, disposables, complications, chelation therapy, hospitalisation, lives lost.
 

Results :
Our results reflect a 5-year overview demonstrating the costs and benefits experienced by healthcare providers. When considering the cost of treatment, consequence, complications, lives lost, life years lost, aRBCX is dominant over the transfusion modalities.
 Yearly intervention cost per patient per year in Omani-Rial: [Top-up 1933.5;Manual 3101.5;aRBCX 5857.5].The yearly cost of treatment choice related cost per patient per year in Omani-Rial:[12128.88; 6233.41; 789.88].Cost of disease complications per patient per year in Omani-Rial:[12706;12864; 11346].Cost of lives lost per patient per year in Omani-Rial:[1315;1288;1102). The total yearly cost per patient per year in Omani-Rial is estimated:[28084;23487.5;19095]
 

Conclusions :
aRBCX is 47% more cost-effective when compared to Top-up transfusion. aRBCX is 23% more cost-effective when compared to mRBCX.
aRBCX promotes reduction in overall resources, disease burden, bed retention and the need for chelation therapy when compared to top-up transfusion and manual exchange transfusion. Cost savings may lead to better adoption. We recommend researchers rerun these calculations on an individual hospital basis.

Purpose :
Introduction: Hemoglobinopathies are genetic disorders resulting from heritable mutations through changes in amino acid sequence, structure, behaviour, production rate, and/or its stability.  Although more than one thousand hemoglobin mutations have been reported globally, only a few are common and clinically significant. In Central India, no systemic study has been carried out to either map the common hemoglobin variants or to identify rare mutations. In the present studies, efforts have been made to identify hemoglobin mutants among tribals and non tribals of Central India to address the paucity of data in assisting the care givers and administrators in developing early therapeutic interventions to reduce the morbidity and mortality of the disease

Materials and Methods :
Material and Methods:   In Tribal dominated Madhya Pradesh, no public health facilities or screening programs exists. Community screening including field tests and High performance Liquid chromatography tests were carried out to identify all haemoglobin disorders. 

Results :
Results: Mapping of the entire state for prevalence of sickle hemoglobin, beta thalassaemia and G6PD deficiency among unscreened Scheduled tribes and Scheduled caste populations revealed Sickle hemoglobin (HbS) was mainly distributed over 32 districts (comprising of 240 blocks/ taluks) with prevalence varying from 3 to 35%. Moreover, several uncommon mutations were detected for first time among these ethnic populations and belonged to both classes of beta and alpha chain variants. These mutations altered the clinical manifestations when co-inherited with other common hemoglobin disorders.

Conclusions :
Conclusions:   A systemic study to identify uncommon hemoglobin mutations and major hemoglobinopathies among scheduled caste and scheduled tribe populations need to be undertaken to reduce the morbidities and associated mortalities.

Purpose :
A child’s growth is a reflection of it nutritional and pubertal status and its proven that nutrition is a factor affecting pubertal development. Sickle cell patients often have slowed growth and delayed pubertal development. This work aims to assess the nutritional and/or pubertal profile in children with sickle cell diseases (SS) admitted at the CEMECO center.

Materials and Methods :
This was a cross-sectional study and study participant under 16 years were randomly selected from the health center database having about 6497 cases and enrolled in the study.The participants were divides into two groups based on the electrophoresis of hemoglobin: Sickle cell disease including 103 cases of SS (homozygote) and 18 cases of SC, S, β-Thalassemia and SE (heterozygous). While the group of non-sickle cell participants includes 87 (AA and AS).

Results :
We included 208 children among them121 sickle cell disease patients and 87 non sickle cell diseasechildren. with a sex ratio M/F was about1.02. The meanage of sickle cell patients was 8.7±4.4 years while that of non-sickle cell patients was 9.5 ± years. The family income evaluated according to the MICS, was similar between the two groups (P= 0.123).
Evaluation of nutritional status revealed that in children under five years of age, the weight / height index z-score of sickle cell patients (-0.82 ± 1.1 [-3.01-1.62]) was lower than that of non-sickle cell patients ( -0.29 ± 1.4Zscore { -2.91-2.86]). However, the difference was not significative (P = 0.155).The height/age ratio express as Z score showed a significative difference between sickle cell patients (1.1 Z score)while non-sickle cell patients ( -0.2 Z score) (P =0.000).  Underweight evaluated by weight/age z-score showed that the in-sickle cell patients were -0.91 and -0.12 in non-sickle cell patients (P=0.014). However, the BMI evaluation did not show any significant difference between sickle and non-sickle cell patients P=0.188.
The proportion of delayed puberty were 4 (4.8%) in sickle cell patients compared to 6.3% in non-in sickle cell patients. puberty. Uni-variate analysis showed an association between sickle cell disease and testicular development (P=0.046).
 

Conclusions :
The results of our study highlighted growth retardation and slowed testicular development in children with sickle cell disease.

Purpose :
Hemoglobin SC disease is the second most common genotype of sickle cell disease (SCD), and is characterized by erythrocyte dehydration, intra erythrocyte crystal formation and increased blood viscosity. Although it is considered less severe than SS and SB-0 genotypes, it may be associated to potential severe comorbidities, as avascular necrosis, retinopathy, vaso-occlusive crisis (VOC) and acute chest syndrome (ACS). These complications can cause low quality of life and justify surveillance and timely intervention. Evidence-based recommendations for its clinical management are lacking, with much of the treatment extrapolated from sickle cell anemia. This study describes the clinical profile of pediatric patients with hemoglobinopathy SC treated with hydroxyurea in a reference service in Brazil, outcomes and HU- related toxicity.

Materials and Methods :
Retrospective single-center study that reviewed all children with hemoglobin SC disease treated with hydroxyurea in a tertiary hospital in Brazil, between 2009 and 2020, for at least 12 months by evaluating the laboratory profile, the number of episodes of VOC and ACS and the drug-related toxicity. 

Results :
Eight patients have met the inclusion and exclusion criteria and were included in the study. There was a significant reduction in the number of episodes of vaso-occlusive crisis and acute chest syndrome  after 12 months of treatment compared to the previous 12 months. Median age of start of HU treatment was 13 y. Six patients had neutropenia as medication toxicity, but only 2 of them failed to reach the maximally tolerated dose. One patient had transient thrombocytopenia. There were no other cases of significant toxicity.

Conclusions :
The use of hydroxyurea in the study group of children with hemoglobinopathy SC was shown to be safe and associated to a lower incidence of episodes of vaso-occlusive crisis and acute chest syndrome with minimal toxicity. 

Table 1. Clinical and demographic data of 8 children with hemoglobinopathy SC treated with HU between 2009 and 2020 in a tertiary hospital in Brazil

Table 2. Comparison between laboratorial data before treatment, six months, and 12 months after treatment with HU in children with hemoglobinopathy SC

Purpose :
Although nearly 80% of all sickle cell disease (SCD) births occur in sub-Saharan Africa (SSA) and nearly 90% of children diagnosed with SCD will die from the condition, efforts to improve health outcomes in this context have been riddled with implementation limitations. Aside from the geo-political and economic challenges that have historically and currently undermine the capacity to deliver healthcare particularly in Sierra Leone, poor understanding of cultural context has also been implicated. To increase knowledge of the cultural elements which could improve SCD interventions, this study aimed to describe cultural factors which influence SCD related health seeking behavior in Kono District, Sierra Leone.

Materials and Methods :
Ethnography was used to explore the cultural influences of SCD care. Participant observation and semi-structured interviews revealed cultural values, beliefs, and practices of professional and family caregivers as well as those with SCD. Participants included persons with SCD, professional and familial caregivers, and community informants. Field observations were conducted in participant homes and healthcare facilities. Semi-structured interviews were conducted with 27 participants. Constant comparative analysis was used to generate codes, categories, and themes that explained cultural influences on health-seeking behavior in this community.

Results :
Participants explained recognizing overt symptoms, then seeking family and community support and advice (cultural values) on how to treat these symptoms based on supernatural, traditional, and/or biomedical beliefs about disease (cultural beliefs, Figure 1). This process led to defining the illness and making a treatment decision according to one’s belief system. Participants described illness as: 1. a community and family burden that led to family division and loss of economic productivity or 2. an opportunity for increased support and collectivism. In describing their health seeking behaviors, participants described a process of trial and error when accessing care in traditional and biomedical practices. Participants then explained returning to family and community support systems to gather new ideas for symptom treatment informed by their cultural beliefs following poor care experiences or after returning to their communities is worse health. Several participants attributed their poor health care experiences to a lack of knowledge about SCD and its treatment. Many participants living with SCD and their family caregivers recounted years of seeking treatment in this manner until receiving a diagnosis. All participants interviewed suggested the path which integrated aspects of traditional, spiritual, and biomedical practices has been the most successful (The Hybrid Way).

Conclusions :
These findings provide an opportunity for researchers and clinicians to operate within the cultural systems of this community rather than outside of it. Belief and value systems were linked to specific cultural practices aimed at providing healing for those with SCD. Many cultural practices led to interactions with leaders within a belief system; these leaders may or may not have had exposure to SCD treatment and management. Therefore, the development of educational and training interventions for religious leaders, traditional medicine practitioners, and medical personnel is imperative. To accomplish this, implementation science community-based research techniques are suggested to ensure sustainability of such interventions and to meet the needs of the community. 

Patterns of Health Seeking Behavior for SCD Care

Purpose :
Microparticles (MPs) are submicron vesicles derived from the plasma membrane of their parent cells. MPs have acquired a biological and clinical interest and are recognized as entities able to modulate many biological functions. Literature suggests that they may have a role in the pathophysiology and downstream manifestations of sickle cell disease (SCD). However, in SCD most of these MPs were derived by eryptosis, the programmed death of red blood cells.
This present study aims to determine the cellular biomarkers to implement new and innovative methods of the preventive diagnosis of SCD acute complications. We propose to study the mechanisms involved in the triggering of eryptosis and to quantify microparticles derived from platelets, erythrocytes and endothelium of homozygous sickle cell patients.

Materials and Methods :
Homozygous SCD patients and healthy donors were sampled for hematological and cellular assays. The exploration of eryptosis was performed by a flow cytometry by determining the viability parameters of red blood cells: the exposure of PS to the GR surface by annexin V labeling, the determination of intracellular calcium concentration by Fluo3-am labeling, quantification of ROS by CM-H2DCFDA labeling and determination of ceramides by specific labeling.
The exploration of MPs was performed by evaluating the number of circulating microparticles as well as their cellular origin by triple labeling with Annexin V / Ac anti CD41 for platelets MPs/ Ac anti CD235a for erythrocytes MPs.

Results :
The outcome of our study indicated that Eryptosis in sickle cell patients is triggered essentially by high Ca2 + entry and narrowing of red blood cells. However, the pathway of ceramides and ERO can also considered a stimulating factor of eryptosis. Eryptosis ensures healthy erythrocyte quantity in circulation whereas excessive eryptosis is the cause of acute anemia and may contribute to vaso-occlusive crisis in sickle cell patients.
For MPs study, we confirmed that microparticles derived from platelets and erythrocytes clearly increased in SCD patients, with predominance of the number of erythrocyte microparticles. This suggests that erythrocytes MPs may contribute to thrombotic risk in sickle cell patients.

Conclusions :
Our research describes promising biomarkers of the preventive diagnosis of acute complication in sickle cell disease. These findings allow considering the eryptosis and circulating MPs as potential biomarkers for implementation a new preventive diagnosis method.

Purpose :
Sickle cell disease is the most widespread hereditary genetic disease in the world, particularly in black populations [1]. In Niger, there were 20% sickle cell trait and 1.8% were homozygous SS [2]. Subjects with major sickle cell disease may have serious clinical manifestations whose evolution will lead to numerous hepatobiliary and splenic complications variable in their severity and temporality.
The objective of this work is to analyze the frequency and the characteristics of the hépatobiliary and splenic complications, in order to prevent them and to facilitate their taking up.

Materials and Methods :
We led a retrospective and transverse study in the National Reference Center of sickle cell disease (CNRD) of Niamey between 2014 to 2021. The regularly followed patients were identified on the basis of medical file. On the whole 3000 files with a request of abdomino-pelvic ultrasound and hepatic enzymes had been consulted. Patients of less than 3, those of more than 50 years old and those whose medical file was incomplete are not included.

Results :
Approximately 4,8% patients (145 patients) presented complications to abdomino-pelvic ultrasound. The sex ratio (M/F) was of 1,15. The Average Age is 14,13 with extremes ranging from 4 to 49 years. The age bracket <10 years was more frequent (52,41%), followed by the one of 10 to 20 (27,01%). The sickle cell anemia phenotype consisted of 84,8% SS (123 cases), 14,5% of SC (21 cases) and 0,7% of Sβ° thalassaemia (1cases). The circumstances of the discovery of the disease are dominated by the splenomegaly and the vaso-occlusive crisis respectively in 22,8% and 15,2%. The hepatic enzyme aspartate amino transferase (ASAT) was high in 72,6% of the patients.
The most frequent complications were hepatomegaly alone (HPM) (60,7%), followed by the hepato- splenomegaly in 17,3% and splenomegaly alone (SPM) in 9,66% of the cases. The vesicular lithiasis was found in 12,4% of the cases.
The average age of patients with hepatomegaly and splenomegaly was 36,5 years old. The average age of the of onset of the lithiasis alone was 21,85 years old. Those with splenomegaly alone had an average of age younger 10,64. The average age of onset of the 3 associated complications hepatomegaly+ splenomegaly+ Lithiasis was 18,8 years old. Hyperbilirubinemia is higher among patients presenting hepatomegaly + Lithiasis, it is the same as for the hepatic enzymes. Indeed 73.79% of patients were hospitalized either because of vaso-occlusive crisis (35.42%) or anemia (21.53%). All the patients had received medical care with antibiotics, antimalarials, antispasmodics, analgesics and parenteral rehydration. Three patients had undergone cholecystectomy.

Conclusions :
Sickle cell disease is a serious public health problem in black african populations and major clinical findings despite anemia are liver and splenic complications which must been looked out and followed because of their morbidity and invalidity keeping home young pupils compromising their scholar education. A great carefull follow up should be done for these kind of patients.

Purpose :
Background: In patients with sickle cell disease (SCD), pain results in varied physical and emotional consequences. Insufficient information about underlying mechanisms affecting pain remains a barrier to addressing SCD pain. Multiple biological and psychological factors (i.e., environmental stress) known to contribute to other pain conditions are understudied in SCD where the median survival (38 years for men and 42 years for women) is lower than the general population. Emotional stress has been shown to be a trigger of pain in patients with SCD. Genetics plays an important but still not fully-understood role in the variation of pain. The rs10877969 SNP in the Arginine vasopressin receptor 1A gene (AVPR1A) is associated with aspects of acute pain and stress-related to pain, but it is unknown whether the genotype frequency differs between those younger or older than the median survival age for SCD. Our aim was to determine the association between AVPR1A genotype with stress and age in adults with SCD who had chronic pain.

Materials and Methods :
Materials and methods: In a cross-sectional study, 169 participants with SCD and chronic pain (100% African descent; 62% female, mean age 36.4±11.6 years [younger adults n =110, 18-39 years and older adults n=59, ≥40 years]), we used the Perceived Stress Questionnaire (PSQ). As a part of a larger study, we genotyped samples using the Axiom Precision Medicine Research Array. We used the Michigan Imputation server to examine our single nucleotide polymorphism (SNP) of interest, rs10877969. The SNP was evaluated as the imputed score was R2>0.8. We used ANOVA to compare stress by genotype and age (younger and older adults).

Results :
Results:  The rs10877969 genotype frequency for the overall sample was: CC 47 (28%), CT 72 (43%), and TT 50 (30%). The genotype frequency was similar for older adults vs. younger adults: CC 16 (27%) vs. 31 (28%), CT 24 (41%) vs. 48 (44%), and TT 19 (32%) vs. 31 (28%). Mean stress scores were not significantly different by genotype: CC mean = 0.39 ± 0.16, CT mean = 0.41 ± 0.19, and TT mean = 0.36 ± 0.16, p=0.22. Mean stress scores also were not significantly different by genotype for younger adults: CC mean = 0.39 ± 0.15, CT mean = 0.44 ± 0.19, and TT mean = 0.36 ± 0.14, p=0.09 or older adults: CC mean = 0.37 ± 0.19, CT mean = 0.34 ± 0.16, TT mean = 0.34 ± 0.19, p=0.90. Mean stress scores were significantly lower (p<0.05) for the older adults (mean = 0.35 ± 0.18) than the younger adults (mean = 0.41 ± 0.17).

Conclusions :
Discussion-Conclusion: Younger adults had higher average stress than older adults. In contrast to prior research, in this study there was not an association between genotype and stress. Previously, the stand-out result was that individuals with SCD and CC genotype cited stress much less than the other two genotypes. The current result has a very different pattern. Further research is needed to examine stress and the rs10877969 genotype using other indicators of stress in this population.

Purpose :
Social determinants of health (SDOHs) are the conditions in which people are born into and surrounded by throughout their life. Generally, these include economic stability, education, social and community context, health and health care and housing/ neighborhood characteristics. In the pediatric population, it is these factors that are thought to influence health, well being and development. Additionally, patients with sickle cell disease face significant complications from their chronic disease and racial disparities, which may contribute to effects of adverse SDOH. Prior studies have suggested the importance of implementing a universal screening to identify adverse SDOHs in order to provide necessary resources.

Materials and Methods :
In this prospective, quality improvement initiative, we administered a universal screener for social determinants of health in our pediatric sickle cell clinic. The paper questionnaire included screening about housing and food insecurity, transportation difficulties and education concerns.  The aim of the study was to determine feasibility of administering screening in pediatric sickle cell clinic and to better understand the socio-economic needs of our sickle cell disease population.

Results :
Data was collected between August 2020 to May 2021, during which time 226 screening questionnaires were completed. With each PDSA cycle, percentage of patients screened increased over time and by May 2021, we achieved about a 35% screening rate. When we looked at specific needs, housing insecurities and transportation difficulties were reported the most, at 39.6% and 44.4% respectively. This data allowed us to identify and provide additional resources for patients.

Conclusions :
The clinical complications of sickle cell disease place extensive burdens on patients. These strains are additionally exacerbated by significant adverse SDOHs that these patients often face. It is feasible to implement a universal screening tool in a pediatric sickle cell clinic, to identify these adverse events. This information can be used to provide appropriate resources and follow up for these patients. We hope to look at this data in more detail to evaluate the success of providing additional resources.

Monthly run chart to compare percentage of patients surveyed and percentage of patients previously surveyed in the month

Purpose :
Priapism is a frequent vaso-occlusive complication of sickle cell disease, the major risk of which is the impairment of erectile function. The objective of this study was to evaluate the sexuality of SS sickle cell patients with a history of priapism.

Materials and Methods :
This was a case-control study of adult SS sickle cell patients. The occurrence of priapism and the nature of the priapism were investigated. Patients were divided into three groups: Group 1 (no priapism), Group 2 (intermittent priapism) and Group 3 (acute priapism). The sexuality of the patients was studied using the IIEF-15 questionnaire. The scores were compared between the three groups to look for an impact of priapism on their sexual life.

Results :
We interviewed 191 SS sickle cell patients. The mean age was 27.1 +/- 7.1 years. Priapism was observed in 43.5%. Only 77 patients were eligible for the IIEF15 questionnaire. Groups 1 and 2 had significantly better results than group 3 on EF and FO scores. There was no significant difference in EF and FO scores between groups 1 and 2. There was no significant difference between the three groups for SD, IS, OS scores. The impairment of erectile function in group 2 was related to the age of the first and last episode of priapism. The impairment of erectile function in group 3 was related to the duration of evolution. 

Conclusions :
This study shows that acute or intermittent priapism is responsible for impaired erectile function in adult SS sickle cell patients. Associated prognostic factors were prolonged duration of priapism, age of first and last episode of priapism.

Purpose :
Sickle cell disease (SCD) is a hemoglobin disorder characterized by chronic hemolysis, inflammation, and oxidative stress. Requirements of vitamin B-6 are thought to be elevated in individuals with SCD due to increased red blood cell (RBC) turnover. Vitamin B-6 also has in vitro anti-sickling properties and is thought to reduce oxidative stress as a coenzyme in the glutathione antioxidant defense system. The aim of this study was to determine the status and dietary intake of vitamin B-6 in Canadian children with SCD.

Materials and Methods :
Fasted plasma samples were collected at baseline from individuals with SCD in British Columbia, Canada participating in a folic acid supplementation trial (ClinicalTrials.gov: NCT04011345). Samples were analyzed for plasma B-6 concentrations using high-performance liquid chromatography. Marginal vitamin B-6 status was defined as plasma pyridoxal 5’-phosphate (PLP) concentrations of 20-30 nmol/L, while deficiency was defined as PLP <20 nmol/L. Dietary vitamin B-6 intake data was collected and analyzed from two non-consecutive dietary recalls using the Automated Self-Administered 24-hour (ASA24) Dietary Assessment Tool-Canada 2018 version. Medication use was collected from the medical chart. Supplement use was self-reported by questionnaire.

Results :
A total of 18 individuals (67% female; SCD type: HbSS n=16, HbSβ0-thal n=2) with a median (IQR) age of 10 (8, 14) years were included. The majority (89%) of participants were prescribed hydroxyurea (median (IQR) dose: 22.0 (19.5, 25.5) mg/kg/d)). Median (IQR) PLP concentrations were 30.2 (23.9, 51.9) nmol/L. A total of 33% of participants (n=6) had marginal vitamin B-6 status, and 17% (n=3) were deficient. Median (IQR) intake of vitamin B-6 from diet and supplements was 1.1 (0.8, 2.0) mg/d. Four participants (22%) did not meet the age- and sex-specific Recommended Dietary Allowance (RDA; Average daily intake sufficient to meet the requirement of ~97% of healthy individuals) for vitamin B-6, and 3 of them (17%) did not meet the Estimated Average Requirement (EAR; Average daily intake sufficient to meet the requirement of 50% of healthy individuals). There was no significant correlation between dietary intake of vitamin B-6 and PLP or total vitamin B-6 concentrations (r=0.12 and r=-0.07, respectively).

Conclusions :
Despite most participants having sufficient estimated dietary B-6 intakes, half had marginal or deficient vitamin B-6 status. Further research which investigates the estimated needs and utilization of vitamin B-6 in individuals with SCD is warranted.
(Funding: Canadian Institutes of Health Research (CIHR))

Purpose :
Thalassemia alfa (Alpha-Thal) and sickle cell disease (SCD) are hemoglobinopathies that can be inherited concomitantly. The scientific literature suggests an improvement in the clinical picture of SCD when these conditions coexist, due to the greater potential for deformation of red blood cells and lower rates of hemolysis, positively impacting complications and transfusion needs. The aim of this study was to evaluate, in a population of patients with SCD, the frequency of deletions for the alpha-Thal genes and whether this characteristic resulted in a significantly lower number of transfusions.

Materials and Methods :
138 medical records of children born between 1998 and 2007, with SCD registered at Fundação Hemominas Juiz de Fora - Brazil, from August 1998 to July 2018 were evaluated. The inclusion criteria for the analysis were patients with active follow-up, diagnostic test through neonatal screening standardized (High Performance Liquid Chromatography- HPLC and Isoelectric Focusing Electrophoresis - IFE) by Núcleo de Ação e Pesquisa em Apoio Diagnóstico of Universidade Federal de Minas Gerais and the exclusion criteria were diagnosis through another methodology, loss of follow-up and death. The variables analyzed were the presence or absence of the deletion for Alpha-Thal(3,7) and the number of transfusions in the observational period using the Qui-Square test (SPSS 15 ®). Study approved by the Research Ethics Committee.

Results :
10 patients died before the exams, there was a loss of follow-up in 11 patients due to the transfer or abandonment of treatment and 5 patients did not attend the testing for Alpha-Thal(3,7)  in the period for collection. In the total sample, 60.9% were diagnosed with HbSS genotype, 28.3% HbSC, 6.5% HbSBeta0, 3.6% HbSBeta+ and 0.7% HbSD. 4.27% of the patients had two deletions, against 25.64% with one deletion and 65.81% without Alpha-Thal(3,7). Regarding polytransfused patients (10 or more transfusions), 22.1% of patients had no deletions, compared to 16.67% of those with a deletion and 0% of patients with two deletions for Alpha-Thal(3,7) (p = 0.036).

Conclusions :
It was observed a lower number of patients undergoing hypertransfusion and a lesser need for transfusions among those with deletions of Alpha-Thal(3,7) genes when comparing them to non-carriers, strengthening the role of Alpha Thalassemia coexistence as a protective factor in the SCD.

Purpose :
Infection a factor triggering sickle cell crises, a frequent cause of hospitalization as well as a factor of poor prognosis in sickle cell patients. The objective is to contribute to improving the management of sickle cell disease in Guinea by determining their infectious profile.

Materials and Methods :
This was a descriptive and analytical prospective study lasting 08 months from September 01 to April 30, 2021 carried out at the University Hospital of Conakry. We included in this study all patients hospitalized for sickle cell disease in our departments with an infectious syndrome, who had carried out the infectious assessment.

Results :
out of 68 sickle cell patients included, 62 had an infection, i.e. 91.17%, mean patient age was 26.17±11.97 years,the female sex wasslightlyrepresented i.e. 51.61%,and a female to male sex ratio of 1.06.
The most common reasons for consultation were osteo-articular pain 96.77% followed by fever 95.16%.
The most common type of infection in our study was parasitic 93.55%, followed by bacterial 30.65%. The germs found were: Escherichia coli (68.75%), Salmonella typhi (31.25%), staphylococcus (50.00%), Koch bacillus 0.14%, candida albicans (66.66%) Plasmodium (87.10 %), Ascaris Eggs (46.15%), Amoeba Cyst (23.07%), Hookworm (11.54%), HBV (7.01%), HIV (5.26% ), Taénia (7.69%), Gardenerella vaginalis (33.33%) and Neisseria Gonorhée (25.00%). The management of infections was made according to the etiology found: antiparasitics (Artesunate, Artemether/Lumefantrine, Albendazole, praziquantel), antibiotics (Ceftriaxone‚ metronidazole, amoxicillin-clavulanic acid, ciprofloxacin), antifungals (Nystatin, Fluconazole , Miconazole and antiretrovirals (Emtricitabine, Lamuvidine and Tenofovir).evolution after treatment was favorable in88.71% of cases and we noted 4.48% transfer, 3.23% discharge against medical advice and 3.23% death.

Conclusions :
Infection is a common cause of hospitalization in sickle cell patients. Bone and joint pain and Fever were the most frequent clinical presentations, the types of infections were bacterial, parasitic, viral and fungal. The treatment of infections was made of antibacterials, antiparasitics, antivirals and antifungals.

Purpose :
Objective: To describe the morbidity and mortality of sickle cell disease patients in the Hematology Department of the Ignace Deen National Hospital in Conakry.

Materials and Methods :
This was a retrospective descriptive and analytical study that took place over a period of  7 years from January 1, 2013 to December 31, 2019. The study population consisted of records of sickle cell patients hospitalized during the study period.

Results :
150 records of hospitalized sickle cell patients were selected with 67.33% male and a sex ratio of 2.06. Their ages ranged from 15 to 65 years, with a mean of 29.17 years. The age group under 25 years represented 45.33%.  Students accounted for 42%.
The main reasons for hospitalization were anemic syndrome (93.33%), osteoarticular pain (67.33%), infectious syndrome (91.33%), hemolytic crises (32.67%) and acute chest syndrome (28.66%).
 Sickle cell disease-related mortality was 5.33%.
 The major causes of death in hospitalized sickle cell patients were severe anemia (100.0%) and infection (50.0%).

Conclusions :
It emerges from our study that the morbi-mortality of sickle cell disease is non-negligible, marked by acute and chronic complications including severe anemia and infections.
Early diagnosis and regular monitoring of patients with adequate care would minimize them.

Purpose :
Over 200 000 babies are born with sickle cell disease (SCD) annually in sub-Saharan Africa. In many African nations, however, 50% to 90% of children with SCD will not survive until their fifth birthday and will die due to susceptibility to malaria, sepsis, anemia, and infection without being diagnosed with SCD. The incidence of SCD and trait in newborns in Sierra Leone is unknown. Early diagnosis of SCD and treatment can greatly improve patient outcomes. The purpose of this study was to assess the feasibility of a Nurse Champion sickle cell disease point of care testing (SCD-POCT) model in community healthcare centers using a novel microtechnology diagnostic device.
 

Materials and Methods :
This cross-sectional study was conducted at a regional government hospital in a rural district in Sierra Leone. Immediate, free screening was offered to newborns over 24 hours old delivered at the maternity ward.  We used a validated, low-cost, point-of-care, lateral flow immunoassay (SickleSCAN®) device to screen for SCD and trait.  The screening tests were conducted by 12 nurse-midwives. A brief questionnaire was used to record the test evaluators’ experience with the device.
 

Results :
Fifty-two (30 males/22 females) newborns screened within 24-48 hours of birth over four weeks. Two newborns were identified with sickle cell disease (HbSS). The test identified 13 newborns with sickle cell trait (HbAS). Thirty-seven newborns had the normal HbAA genotype. The heterozygous HbSC genotype was not observed in the study cohort. 12 nurse-midwives evaluators rated the acceptability and feasibility of screening for SCD at the bedside as very good. None of the evaluators deemed the device to be inappropriate for SCD screening.
 

Conclusions :
Our study demonstrates a practical method of instituting SCD screening in maternity wards with nurses and midwives as frontline implementers. Our study is a “real world” confirmation that the SickleSCAN device produces reliable results; that nurses are uniquely positioned to educate and advocate for newborn screening, and serves as a “proof of concept” for service delivery, particularly in settings where access is limited and point-of-care testing is less well studied.
 

Purpose :
Ethnic minorities in the UK have a 3-5 times higher risk than white British patients of developing diabetes II, however not all of the HbA1c tests, which are widely used to screen for Type II diabetes, are unsuitable for some ethnic minority groups, such as where haemoglobin variants such as sickle haemoglobin (HbS) are common. Sickle cell trait (SCT) is found in up to 25% of ethnic groups of African, Middle Eastern and southern European heritage and there are 250,000 people with SCT in the UK. This paper will explore if the current HbA1c testing can illuminate mechanisms of structural racism in UK healthcare.

Materials and Methods :
Methods of HbA1c testing and evidence of haemoglobin variant interference with different HbA1c testing systems were examined through literature searching and the USA National Glycohemoglobin Standardization Program database. A policy analysis and literature search explored potential mechanisms of structural racism in the case of HbA1c testing and finally a case study of a patient diagnosed with SCT through a HbA1c test was used to explore ethical dilemmas and possible information needs subsequent to incidental finding of SCT through HbA1c screening.

Results :
Five methods of testing and over 200 different kinds of machines used to assay HbA1c were found. Only 16% of all systems have been evaluated using robust methods, 57% of those evaluated showed an interference with at least one haemoglobin variant, 51% for HbS. Some systems gave false positives, some false negatives. The extent of use HbA1c machines that are inaccurate for SCT patients in the UK is unknown. No national or international guidance on HbA1c screening for haemoglobin variants was found. An inquiry into sickle cell care, ‘Nobody’s Listening’, found sub-standard care for SCD, low awareness of sickle cell among healthcare professionals and inadequate training and inadequate investment in sickle cell care. Various ethical dilemmas arise through an incidental finding of SCT through an HbA1c test. These include an unanticipated incidental diagnosis of SCT without prior informed consent, communicating and understanding incidence of potential symptoms associated with SCT, not being given timely access to genetic counselling services, how results are appropriately delivered to patients or carers and dealing with stigma and racism that may result as having a SCT status revealed.

Conclusions :
Screening for diabetes in the UK, and potentially globally, is fraught with lack of information regarding suitability of HbA1c testing systems for patients with haemoglobin variants, therefore potentially providing mechanisms for structural racism in health care for different ethnic groups. There is a need to conduct a thorough assessment of which HbA1c testing systems are in use and if these systems are able to accurately screen for diabetes in patients who may have haemoglobin variants such as HbS. If any systems in use are not able to accurately screen for Type II diabetes in patients with SCT alternative routes for screening should be made available so to not exacerbate health inequalities in ethnic minority groups in the UK. How and when SCT status results are communicated with patients should be carefully considered.

Purpose :
Melioidosis and its germ Burkholderia pseudomallei are increasingly reported on the African continent and particularly in Central Africa. It is called "Great Mimicker" because it produces a wide range of clinical characteristics that would be found in patients living with major sickle cell syndrome. Few cases have been reported in Africa. But In DRC no cases have been previously described in SCD patients.

Materials and Methods :
We describe here 3 clinical cases presenting this very rare association between melioidosis and sickle cell disease.

Results :
Three homozygous SS children residing in Lubumbashi in the Democratic Republic of Congo were concerned.
One patient presented with sepsis as a clinical form of the disease. All 3 had presented a pulmonary form, 2 pneumonia and 1 pleuropneumonia. The 1rst one patient with spesis was treated specifically with infusion ceftazidime for 21 days followed by oral cotrimoxazole for 2 months after the diagnosis of melioidosis. Regular clinical improvement was observed with treatment although fever did not decrease until the 17th day after admission. The patient was discharged on the 22nd day. The patient was seen again in consultation three months after his discharge from the hospital. He was asymptomatic and the clinical examination was unremarkable. For the other two cases, diagnosis was confirmed in postmortem. Globally, the death rate was 66.67%.

Conclusions :
These cases are, to our knowledge, the first cases of melioidosis reported in sickle cell patients. This association requires further researches to establish whether, like beta-thalassemia major, sickle cell anemia can be considered as a risk factor for melioidosis. A screening of cases of melioidosis in SCD cohorts should allow a best comprehension of a such association.

Purpose :


Materials and Methods :


Results :


Conclusions :

Fig 1:The morphology of sickle cells that were incubated under hypoxic conditions in the absence or presence of various concentrations of Sailin-HbS after 4hrs observed under 40x magnification.

Fig 2 : Clinical profile of sickle cell patients administered with Sailin-HbS.

Purpose :
The first marketing approval of hydroxyurea in SCD was granted in US in 1998 in adults. 10 years after EMA gave a marketing approval in SCD in children from 2 years old. In 2017 in US HU was approved in children. However, in the majority of African countries, in Brazil or in India where the SCD population is important hydroxyurea is not approved in SCD, but only in myeloproliferative disorders and in adults only. Is there an interest in patients and clinicians to have hydroxyurea products dedicated to the SCD?
 

Materials and Methods :
Escort HU (European Sickle Cell Disease Cohort – Hydroxyurea) is a non-interventional prospective cohort study conducted in patients treated with HU according to current clinical practice with SCD. The main objective was to refine the safety profile of hydroxycarbamide, but clinical effectiveness were evaluated by recording painful crises lasting more than 48 hours, episodes of ACS, number of hospitalisations related to SCD, and biological parameters were also regularly recorded according to the frequency of the hospital visits.
 

Results :
At inclusion, 926 (48.7%) patients had been previously treated off the label with HU (as HU was only approved for myeloproliferative disorders at the time), with mean duration of HU treatment of 5.74 ± 4.98 years. The mean age of this subgroup was 27.60 ± 14.81 years compared to 19.75 ± 15.79 years of the patients never been treated with HU before enrolment
As showed in the table 1, hemoglobin level remains unchanged during the first two years but the HbF% increased in the HU-pretreated subgroup. In the “HU-naïve group”, despite lower baseline Hb level et HbF% at baseline, similar outcome were obtained after 12 or 24 months. In terms of clinical outcomes, after 1 year of treatment the number of vaso-occlusive crises (VOC), acute chest syndrome (ACS) or hospitalizations decreased dramatically (table 2) in both group. The median duration of follow-up in the cohort was 45 months (0-128). 123.7 ± 62.7 months. In term of safety, neutropenia, thrombocytopenia and dry skin were the most frequent HU- related adverse events reported but with comparable cumulated incidence between the HU-pretreated subgroup and HU-naïve subgroup (Table 3).
 

Conclusions :
In real life setting, a significant improvement of the vaso-occlusive symptoms was observed even in patients previously treated with HU for a long time. It was observed an improvement of compliance and consequently of effectiveness as suggested by the lower MCV than expected, at baseline in patients previously treated with HU. Paule and al (2011) demonstrated that daily regimen of HU could be superior in term of efficacy to weekly regimen. The fine tuning of the daily dose possible with HU tablets might be another reason of clinical optimization. Even hydroxyurea is available for SCD patients, dedicated marketing approvals of the drug for SCD patients seem support the compliance of the patients materialized by better clinical benefits. These data may encourage Health authorities and pharmaceutical companies to develop and to approve drugs in the indication and so to avoid off-label uses.
 

Table 1 Biological parameters in the subgroups after 12 and 24 months Table 2 : Number of VOC, ACS and hospitalizations after 1 year of HU treatment in the cohort compared to the previous year before enrolment

Table 3: Most Frequent Adverse Events Sorted by Relative Risk by Previous HU Treatment Other Than HU tablets

Purpose :
Sickle cell disease (SCD) is one of the most common human genetic disorders, which is caused by a single point mutation (Glu6Val) on the HBB gene. Currently, one of the treatments for this global health problem involves induction of fetal hemoglobin (HbF). There are some drugs on the market that pharmacologically induce HbF, namely Hydroxyurea (HU), however, their safety concerns and the expensive cost in low- and middle-income countries limit their use. In this context, it is essential to study novel fetal hemoglobin-inducing compounds that have fewer adverse effects and are widely available, such as natural compounds. Therefore, the main aim of this work was to evaluate the effects of Carica Papaya methanolic leaf extracts (CPMLE) in HbF reactivation.
 

Materials and Methods :
In order to achieve these goals, gene expression studies on globin and HbF regulators/silencers genes were performed using three biological replicates in K562 cells. The cells were exposed for 24 hours to three concentrations of CPMLE (0,5; 50 and 100 μg/ml), and to 25 μg/ml of HU, which was used as a positive control. Exposed cells and controls were harvested, and parameters such as cell proliferation and cell viability were microscopically evaluated. Variation in gene expression after CPMLE exposition was quantified from total RNA by quantitative Real Time PCR. The studied genes were α, β and γ-globin genes, as well as the HbF regulators genes MYB, KLF1, BCL11A and BGLT3, and GAPDH was used as a reference gene.
 

Results :
The proliferation rates were calculated as the ratio between the value at 24h and the initial number of cells (1X105 cells/well). The results for the CPMLE concentrations of 0,5; 50 and 100 μg/ml were of 2,12; 2,48 and 2,15, respectively, while for control (cells non-treated) the value was 2,35. The percentages obtained for the viability, as assessed by trypan blue staining, were of 90,02% in control cells, 94,33%; 89,22% and 84,42% for the CPMLE concentrations of 0,5; 50 and 100 μg/ml, respectively. Altogether, these results indicate that some concentrations of CPMLE affect the proliferation and viability of K562 cells, although no cytotoxic effects were observed. Transcriptional analysis demonstrated that all CPMLE concentrations repress BCL11A expression and increase expression of HBA, HBB, MYB and KLF1, relative to control cells, which is in line with the HU results observed for each gene. Additionally, an overexpression of γ-globin and BGLT3 genes was observed upon incubation with 0,5 μg/ml of CPMLE. Thus, the results observed for the BGLT3 gene where distinct between CPMLE and HU. For the other CPMLE concentrations, there was a reduction in the expression of the same genes.
 

Conclusions :
Overall, this preliminary study suggests that CPMLE can modulate the expression of HbF and regulator genes, thus potentially constituting an effective approach for treatment of SCD.
Acknowledgments- This project was supported by Instituto Politécnico de Lisboa under the grant IDI&CA-IPL/2021/EpiCa/ESTeSL and partially supported by FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020). The authors are grateful to Fernando Nunes for kindly providing the Carica Papaya leaves.
 

Purpose :
Glucose is the single most essential component of the red blood cells (RBCs), being its sole energy provider and therefore is implicated in the energy metabolomics of the RBCs. This in turn play a crucial role in RBC lifespan, which in Sickle Cell Disease (SCD) patients is reduced to about 20 days from the usual average 120 days. This motivated us to investigate the process of glucose consumption and metabolism in red blood cells of Sickle Cell Patient of Odisha.

Materials and Methods :
Blood samples of homozygous SCD patients (HbSS) were collected randomly with informed consent from different parts of Odisha state depending on the availability and accessibility of the subjects. The samples were studied for their hematological and biochemical parameters including levels of glucose. The red blood cells of SCD patients aged and sex matched normal (HbAA) as well as sickle cell traits (HbAS) were separated and incubated for different time intervals to measure the glucose consumption and Pyruvate and Lactate formation.  Along with this the membrane stability of the cells were estimated and time dependent glucose uptake was also carried out. Identification of the haemoglobin variant was done by performing sickling test, haemoglobin electrophoresis and HPLC Hb. Variant analysis. All the biochemical estimations were done following the standard methods.

Results :
In the red blood cells of SCD cases (HbSS), the levels of glucose consumption were noted to be the higher than the sickle cell trait (HbAS) cases and control/normal (HbAA) samples. Concurrently, formation of blood Pyruvate and Lactate showed higher rate in comparison to HBAA and HbAS red cells indicating raised metabolic turnover in the sickle RBCs. In the time dependent study, the red cells of HbSS patients showed higher rate of glucose consumption compared to trait and normal cells. This data also correlates with the higher hemolytic features of the RBCs showing lower membrane stability with increased metabolism.

Conclusions :
The sickle cell mutation causes much more damage to the life of RBCs than mere morphological sickling of cells. The energy consumption plays a vital role in the life span, shape, stability and overall biochemistry of the RBCs, thus being a critical factor of RBC metabolism, which in turn determines the overall blood-based health of the SCD patients. Understanding and pin-pointing the key factors of energy metabolism thus holds many potential and promising factors in controlling the sickling, and indeed overall health of patients in case of sickle cell disease.

Purpose :
The frequency of priapism is very high in adult sickle cell patients. In Africa, the assessment of the level of knowledge of priapism in sickle cell patients showed that priapism was poorly known. The objective of this study was to assess the knowledge, prevalence and clinical features of priapism in adult sickle cell patients in Senegal.

Materials and Methods :
This was a seven-month descriptive study of 219 male patients with sickle cell disease (SS, SC, S-beta-thalassemia). SS sickle cell patients were in the majority. A questionnaire was drawn up with a 20-minute interview with each patient to assess his or her knowledge and the clinical features of priapism.   

Results :
The mean age of the patients was 27.1 years (18-54) (SD=7.1). Twenty-three patients (10.5%) were aware of priapism (p=0.004). The prevalence of priapism in sickle cell patients was 41.5%. SS sickle cell patients were at higher risk (91.2%). Eighty-five patients (93.4%) had at least one episode of intermittent priapism and 24 patients (26.4%) had acute priapism. The mean age of onset of the first episode of priapism was 20.4 years +/- 6.1 years. The majority of patients (62.5%) had consulted within the first 24 hours. The last episode of priapism in 50.5% of patients was more than 6 months ago. Priapism occurred at night during sleep in 92.3% of patients. Fifty-eight patients (63.7%) did not have any factors favouring the occurrence of priapism. 

Conclusions :
Priapism is a frequent serious vaso-occlusive complication in sickle cell disease. We show in this study, a high prevalence, the lack of knowledge of priapism and the clinical particularities of priapism in sickle cell disease in Senegal. 

Purpose :
The purpose of this qualitative study was to explore the academic and emotion of secondary students with sickle cell disease school experiences as their parents recalled them. Educators are responsible for ensuring access to quality education for all students. Students with disabilities have a legal right to a free appropriate public education under Section 504 and the Individualized Education Program (IEP).

The chronic illness this research will focus on is children living with sickle cell disease. Sickle cell disease is an inherited blood disorder that causes severe pain episodes when sickled blood cells get stuck in blood vessels. To explore these experiences, the researcher employed a qualitative phenomenological approach to inquiry. The researcher also employed a social constructivism approach.

Materials and Methods :
The study purpose was to investigate students’ academic and emotional school lived experiences through interviews and a focus group. The research method involved using the data from interviews, artifacts, and the focus group to analyze the parents’ experiences, background stories, and perspectives.

The research method also included reviewing some parents’ artifacts, such as attendance records, grades, IEPs and 504 plans, and psychological evaluations to understand their educational attainment, absenteeism, and achievement. One participant shared the school attendance policy with the researcher as an additional artifact to provide additional insight into the phenomenon.

Results :
. The four themes that emerged were: (a) the need for a consistent approach by school staff, with awareness and understanding in an inclusive setting, (b) challenges faced by children with sickle cell disease in a school setting, (c) parents need for empowerment, and (d) need for adaptability in school culture and practices.

The findings, results, and interpretations from this qualitative, phenomenological study support the need for increased awareness and support for secondary students with sickle cell disease as it relates to student achievement, emotion, and psychosocial challenges.

Conclusions :
Recommendation 1: Effective collaboration between schools and medical/psychosocial team
Recommendation 2: Provide Targeted Professional Development
Recommendation 3: Create Personalized Learning Options for Some Students with Sickle Cell Disease
Recommendation 4: Promote Parent and Student Empowerment
1. Listen to mother’s story
2. Parent, Student and School Team Engagement Council
3. Parent Support Group
4. Review Hospital Homebound policy

Themes and Subthemes in results

Purpose :
 
Sickle cell disease (SCD) is associated with a high frequency of chronic kidney disease (CKD), which constitutes an independent risk factor for death in this population. In term of epidemiology SCD-associated nephropathy (SCAN) is a growing matter of concern because chronic renal failure affects from 12% to 16% of adult patients^1-3. Thus, the early diagnosis of SCAN together with the recognition of the associated clinical and biologic risk factors are of major interest to make it possible to initiate kidney-protective therapy at early stages of renal impairment and to impact the outcome of the disease. Albuminuria is a relevant biomarker for the detection of early glomerular damage in patients with SCD. Several observational studies, especially in the adult population, have provided evidence that hydroxycarbamide (HU)would be beneficial in SCD patients with impaired glomerular function^4-5

[i] Falk RJ, Scheinman J, Phillips G, et al. Prevalence and pathologic features of sickle cell nephropathy and response to inhibition of angiotensin-converting enzyme. N Engl J Med. 1992 Apr 2;326(14):910-5.
2.Powars DR, Chan LS, Hiti A, Ramicone E, Johnson C. Outcome of sickle cell anemia: a 4-decade observational study of 1056 patients. Medicine (Baltimore). 2005 Nov;84(6):363-76.
3.Yeruva SL, Paul Y, Oneal P, Nouraie M. Renal Failure in Sickle Cell Disease: Prevalence, Predictors of Disease, Mortality and Effect on Length of Hospital Stay. Hemoglobin. 2016 Sep;40(5):295-9.
 4Bartolucci P, Habibi A, Stehle T, et al. Six Months of Hydroxyurea Reduces Albuminuria in Patients with Sickle Cell Disease. J Am Soc Nephrol. 2015 Nov 19. Eng.
5Laurin LP, Nachman PH, Desai PC, Ataga KI, Derebail VK. Hydroxyurea is associated with lower prevalence of albuminuria in adults with sickle cell disease. Nephrol Dial Transplant. 2014 Jun;29(6):1211-8.

Materials and Methods :
 The SIKAMIC trial evaluates the effect of six-month of hydroxycarbamide compared to placebo on albuminuria. It is a Phase-IIb, international, multicentric, randomized, double blind placebo-controlled trial in adult patients with HbSS or HbSβ0 SCD and ACR between 3 and 100 mg/mmol.

 

Results :
Initially it was planned to enroll 120 patients (60 patients for each study group) in 21 French centres.  The recruitment started in August 2019 but1,5 years later only 9 patients were randomized. Because most of the patients with renal impairment in France were often treated with HU or converting-enzyme inhibitors which was not the case in Africa. Consequently 3 African centres were selected to participate in the study.

 

Conclusions :
 Important points must be considered when extension to Africa is planned:
Regulatory processes are longer than in Europe,
Support with local CRO experimented in the conduct of trials in Africa is recommended,
Local features such as unavailability of some exams, long distance from home to clinical centre, more dedicated time for information of patients must be managed in term of finance and training.In Sikamic trial, from the submission of amendment at the end of 2020 to the recruitment of the first African patient nine months were required. On March 2022, 28 patients were randomized (20 in France, 8 in Africa). The recruitment is still ongoing.

Purpose :
Management of Sickle cell disease in India the is evolving. The pediatric fatal disease is shifting to adult chronic organ disease. Sickle hepatopathy is an umbrella term which covers hepatic sickle crisis, acute hepatic sequestration crisis although uncommon but manageable. Acute sickle cell intrahepatic cholestasis (SCIC) is a rare phenomenon, difficult to treat and has very high mortality. The pathogenesis of Acute sickle cell intrahepatic cholestasis involves sickling within the hepatic sinusoids leading to vascular stasis, hypoxia, and ballooning of the hepatocytes, resulting in intracanalicular cholestasis. We describe a difficult case of Acute sickle cell intrahepatic cholestasis which has a subacute course, challenges faced, and lessons learnt from managing it.
 
 

Materials and Methods :
A 14-year-old girl with sickle cell anemia who was non-compliant with hydroxyurea treatment presented to us with complaints of pain in the right side of abdomen, drowsiness, lethargy, nausea and loss of appetite. She was transfused twice at another center before reaching our center. On examination, she was disoriented, febrile, icteric, severely pale with active bleeding from gums and mouth and moderate hepatosplenomegaly. Hemoglobin was 7.2 grams/ dl with reticulocyte count of 2%, with neutrophilic leukocytosis and normal platelet count. Hemoglobin S was 42%. Serum bilirubin was 68 mg/dl with direct fraction of 65.3mg/dl. SGPT and SGOT were 263 and 202 IU/dl respectively and prothrombin time was raised with INR of 1.79. Viral hepatitis A, B, C and E were ruled out. Bacterial infections were ruled out.
The possibility of Acute sickle cell intrahepatic cholestasis was considered, and the child was managed with partial exchange transfusion till Hemoglobin S was brought to <10%. She also received fresh frozen plasma in view of raised prothrombin time and active bleeding. 

Results :
She did not improve until 14 days after admission.Gradually after 20 days she started talking and eating. However, liver enzymes and bilirubin normalized only after 40 days. She was discharged in stable condition and is continued on follow up. 

Conclusions :
Sickle cell hepatopathy is an umbrella diagnosis for all the varied causes of hepatic dysfunction ranging from sickling crisis to transfusion transmitted viral infections. In our case, viral hepatitis was ruled out conclusively.A very rare condition  Acute sickle cell intrahepatic cholestasis was considered in view of the clinical presentation serum bilirubin was extremely high and mildly elevated transaminases. However. Hepatic sequestration was unlikely based on the hemoglobin level and reticulocyte count. Differentiating the varied causes in resource constrained centers can be often a daunting task and multipronged management needs to be initiated at the earliest to ensure a positive outcome.
 

Purpose :
Parental knowledge about SCD has a direct effect on reducing mortality and complications related to Sickle cell disease. Families of children affected by SCD face several hardships caused by raising a child afflicted with this disease. However, parental knowledge about SCD and challenges faced by families of children with SCD have not been assessed in Rwanda. The aim of this study was to explore the knowledge on sickle cell disease among parents of children affected by SCD and to identify their experiences while caring for affected children.

Materials and Methods :
A qualitative method using thematic analysis was used. Data collection was done using semi-structured interviews with thirteen parents from one referral hospital with Pediatric hematology clinic in Rwanda.

Results :
Thirteen caregivers including eleven mothers and two fathers participated in the study. Two broad themes were identified: knowledge and understanding of SCD, and experiences and hardships. The majority of parents had some knowledge of SCD but most of them had no knowledge of inheritance pattern of SCD. Prior to the diagnosis being made, all parents had never heard of SCD and they had misconceptions about the condition of their children. Parents face several challenges including persistent psychosocial distress and practical hardships caused by SCD. This study also revealed coping strategies used by parents.

Conclusions :
This study showed that parents had some knowledge of SCD with gaps of inheritance pattern. Families of children affected by SCD face several burdens including nutrition for the affected child, poverty and persistent emotional distress. Raising awareness of SCD among the public, availing premarital screening program for high risk people and providing social support for affected families would help to mitigate the impact of this disease.

Purpose :
La drépanocytose est la maladie hématologique héréditaire la plus répandue dans le monde. Les études actuelles démontrent que les effets multiformes de la drépanocytose sur la santé mentale d'une personne et la douleur chronique associée à cette maladie ont un impact négatif sur la qualité de vie psychologique et physique. En plus, c’est un véritable fardeau pour les familles et pour l’économie nationale, du fait des couts élevés des analyses biologiques, des médicaments et des hospitalisations.
L’objectif de cette étude est de déterminer l’impact social, économique et psychologique de la drépanocytose chez les enfants suivis à la Clinique Amis des enfants à Bukavu.
 

Materials and Methods :
Notre étude est descriptive et transversale menée sur une série de 32 enfants suivis à la Clinique Amis des enfants à Bukavu à l’Est de la RDC. Les données ont été recueillies par un questionnaire d’enquête adressé aux parents ou tuteurs des enfants malades après consentement éclairé, saisies en utilisant le logiciel Microsoft Word 2014 et analysées par les logiciels Epi info version 7.2 et Excel 2014.
 

Results :
En ce qui concerne le profil socio démographique des enfants concernés, le sexe féminin est le plus concerné dans 62.50% de cas et la tranche d’âge la plus intéressée est comprise entre 11-15 ans. La plupart des enfants vivent en milieu urbain, plus dans la commune d’Ibanda soit 68.74%. En ce qui concerne le profil de leurs parents, 50 % des pères de ces enfants sont ouvriers, 56.25% des mères sont ménagères et 75% des parents de ces enfants vivent en couple.   En ce qui concerne le profil social, les pères s’occupent de leurs enfants malades et ces derniers vivent normalement en contact dans la société.
En ce qui concerne l’impact psychologique, 67.75% des enfants ont comme réaction des pleurs face aux crises drépanocytaires, seulement 15.63% sont soit isolés, déprimés ou stressés au quotidien en dehors des crises et environ 53.12% des parents se questionnent sur la survenue de la drépanocytose chez leurs enfants.  
En ce qui concerne l’impact économique, environ 43.75% des dépenses mensuelles pour les soins de ces enfants en termes d’argent est compris entre 50-100$ ; alors que le revenu mensuel d’environ 40.63% de ces parents est compris entre 10-50$.
 

Conclusions :
La drépanocytose a un impact social et psychologique considérable chez les enfants suivis à la Clinique Amis des enfants à Bukavu. Le niveau économique des familles des enfants concernés est précaire alors que le coût de soins reste plus élevé. Une implication de l’Etat ainsi qu’un soutien psychosocial sont indispensable pour alléger le fardeau des familles concernées
 

Purpose :
* Suneeta Patra1,  Anju Bhandari2, P.K.Patra3
 *1 Govt. N.P.G. College of Science , Raipur, patrasuneeta@yahoo.com,
Corresponding and presenting author
  2Bhilai Mahila Mahavidyalaya , Bhilai, bhandarianju786@gmail.com,
 3Chandulal Chandrakar Memorial Government Medical College Durg Chhattisgarh
 
ABSTRACT
Introduction : - Sickle cell anaemia also known as drepanocytosis , is a genetic blood related disease in which red blood cells form an cresent ( sickle ) or abnormal shape. This genetic disease is due to a change in the beta globin chain of haemoglobin glutamic acid ( a polar amino acid) is replaced by valine ( a non – polar) amino acid . This genetical change reduces the binding of haemoglobin for oxygen. At the low oxygen level the mutant haemoglobin , known as sickle haemoglobin (Hbss) . Medicinal plants have been used in Indian folk medicine in the remedy of sickle cell anaemia by inhibiting sickling . This work was therefore aimed at screening the antisickling study of the aqueous extract of stems of T. Cardifolia and fruits of T.bellirica used in the Indian herbal medicine.
 Objective :- The present study investigates the preliminary screening of secondary metabolites                               of aqueous extracts of T. Cardifolia( stems) and T.bellirica(fruits) and invitro antisickling test (sickling reversal test).

Materials and Methods :
Materials and methods :- Standard methods of  phytochemical analysis of T. Cardifolia( stems) and T.bellirica(fruits) and  sickling reversal test ( invitro antisickling test), stems of  T. Cardifolia  ,fruits of  T.bellirica and Hbss blood samples etc.

Results :
Results -Screening of secondary metabolites (bioactive molecules) and sickling reversal test were carried out using standard methods. The phyto-chemical  screening indicated the presence of secondary metabolites (bioactive molecules) viz -  alkaloids,flavonoids,phenols, saponins, steroids and cardiac glycosides etc. The results of antisickling reversal test that  T. cardifolia( stems) and T. bellirica (fruits) exhibited potential antisickling property.

Conclusions :
Conclusion: - The percentage of sickle cells of untreated Hbss was statistically significant higher than treated Hbss. The percentage  of sickle cells decreased on the addition of aqueous extract of T. cardifolia stems and  T. bellirica fruits on induced Hbss blood, which indicates the reversal of of Hbss blood to normal biconcave shape due to the presence of bioactive molecules, which have sickling reversal ( anti-sickling property) and anti haemolytic properties .

Purpose :
Sannipatika Panduroga, described in Classical Ayurveda Scriptures resembles the clinical features of Sickle cell Diseases (SCD), which results from substitution of a single amino acid, Glutamic Acid to Valine at 6th position on the β globin polypeptide chain, The resulting condition from homozygous (HbSS) and heterozygous (HbAS) states of the mutation are a cause of debilitating health conditions of millions across the globe including in India. One remedial approach as per Ayurveda for such condition, is application of processed stem parts of Tinospora cordifolia (called Guduchi in Ayurvedic term) in powdered starch form known as Guduchi Satwa.  This study includes clinical application of Guduchi Satwa on SCD patients and the clinical improvements observed thereafter.

Materials and Methods :
A clinical study was conducted on 30 SCD patients (15 patients each in Group-A and Group-B) registered at the OPD and IPD of Govt. Ayurvedic College and Hospital, Balangir, Odisha. The Subjective Parameters of study included Panduta (Pallor of the skin), Durvalata (Weakness), Sadana (Fatigue), Angamarda (Body ache), Jwara (Fever), Swasa (Dyspnoea), Aruchi (Anorexia), Gourava (Heaviness), Chhardi (Vomiting), Mala-Mutra and Netraswetata (pale colouration of urine, stool and eye), while Objective parameters as CBC, ESR Sickling test and Hb Electrophoresis were included for the clinical study. Group-A patients were treated with Herbo-Mineral Compound (500mg), which contains parts of silver, calcium, pearl paste, coral derivative among others; and Group-B patients with Guduchi Satwa (500 mg) twice daily in empty stomach with honey orally for 30 days respectively. The patients were assessed at 10 days interval up to 30 days in order to find the efficacy of both the trial drugs. 

Results :
There were 65.15% and 40.85% of improvement in signs and symptoms and 25% & 17.65% of increase in the percentage level of Hb in the patients of Group-A and Group-B respectively. The statistically significant (P<0.05) result was revealed in both Group-A and Group-B but improvement was noticed more in Group-A.

Conclusions :
The overall study revealed that the application of Guduchi Satwa has  Substantial and Noticeable improvement in the SCD specific parameters and over all health condition of the test subjects. Herbo-Mineral Compound i.e., Group-A showed better   than Guduchi satwa i.e, Group-B.­ The composition of Herbo-Mineral Compound helped more to develop the body immunity as well as maintain the hematological parameters rather than single Guduchi satwa. No side effect was noticed during clinical trial in both groups.
The compound is natural, chemical free and easily affordable compared to synthetic drugs, which holds a very promising future for prophylactic relief to SCD patients in the long-run.

Purpose :
Thalassemia remains a public health problem in Tunisia. The difficulties in setting up a prevention program can be explained in part by negative social representations and the general public's ignorance of this disease.
This study examined the health literacy (HL) of Tunisian secondary school students regarding thalassemia as well as their emotional and behavioral attitudes towards this disease and preventive measures.

Materials and Methods :
This is a quantitative study; a cross-sectional survey was conducted among Tunisian youths (n = 182; male 23,3 %, female 76,7 %). Data collection took place in two public secondary schools.
Students aged 17 to 19 completed a paper and pencil questionnaire and described their knowledge level about Thalassemia, their affective and behavioral attitudes as well as their estimates of hereditary transmission risks and usefulness of prevention measures. Statistical software (SPSS, v.25.0) was used to analyze the data.

Results :
The results indicate low HL scores concerning Thalassemia. Lack of knowledge and confusion about the cause, symptoms, and diagnosis of Thalassemia is a common finding at all school levels surveyed.
48,9 % has not identified the hereditary nature of diseases and confuse it with dietary iron anemia 30,7%.
Although 70,5% of respondents recognized severe anemia as a symptom however, 57,4% did not link jaundice to thalassemia, almost 30% said this disease causes rash, fever, dry cough and sore throat.
The results show low levels in relation with assessment of the hereditary transmission risks on the one hand and the prenuptial screening usefulness on the other hand. 24,4% do not consider it necessary to inform their partner of their carrier status. 45% do not see the benefit of genetic counseling before marriage and prenatal diagnosis even if the family of one of the two partners has a history with disease.

Conclusions :
Primary prevention of thalassemia depends on a comprehensive strategy of promotion and health education for Tunisian’s youth. This survey reveals the difficulties of youths to consider the risks of hereditary transmission of thalassemia and to understand the importance of screening and genetic counseling. These results can be explained by the deficit of communication about disease severity and the lack of adequate educational content in Life Sciences programs.

Purpose :
L’ostéonécrose aseptique de la tête fémorale est une complication chronique invalidante de la drépanocytose. Ses données chez l’enfant africain sont rares. Ce travail a eu pour objectifs d’en déterminer la prévalence, en décrire les caractéristiques et en identifier les déterminants dans notre contexte d’exercice.
 

Materials and Methods :
La première partie de l’étude était descriptive, menée sur une période de 4 ans au centre national de référence de la drépanocytose sur des enfants drépanocytaires homozygotes atteints d’ostéonécrose aseptique de la tête fémorale. Les variables étudiées étaient épidémiologiques (âge et sexe), cliniques et paracliniques. Le score fonctionnel de Postel Merle-d’Aubigné a été utilisé pour évaluer l’état fonctionnel des hanches et la classification d’Arlet et Ficat pour la stadification radiographique.
Dans un second temps, une étude cas-témoins a comparé ces enfants drépanocytaires homozygotes atteints d’ostéonécrose de la tête fémorale à des témoins appariés par âge afin d’identifier les facteurs prédictifs. Les variables étudiées étaient épidémiologiques, cliniques (complications aiguës), paracliniques (hémogramme inter-critique) et thérapeutiques (qualité du suivi). Les tests statistiques de khi2 et l’Odds ratio ont été utilisés pour la comparaison des variables, avec un seuil de significativité p ?0,05 et un intervalle de confiance IC >95 %.
 

Results :
L’âge moyen des enfants drépanocytaires était de 11,51±2,17 ans avec des extrêmes de 8 et 17 ans.
La prévalence de l’ostéonécrose aseptique de la tête fémorale était de 2,15 %. Près de la moitié avait moins de 11 ans (45,16%). Au diagnostic, l’atteinte était bilatérale chez 10/31 cas, la douleur de la hanche était constante et associée à une boiterie dans 83,87 %. Une inégalité de longueur des membres inférieurs était objectivée chez 21 patients soit 67,74 % des cas. La fonction de la hanche était bonne, passable et médiocre respectivement dans 41,94 %, 54,84% et 3,22 %. Un enfant présentait deux hanches ankylosées. Au plan radiologique, les stades I, II, III et IV d’Arlet et Ficat étaient retrouvés respectivement dans 21,95%, 19,51%, 31,71% et 26,83% des cas.
Les 31 cas ont été comparés à 62 témoins Le sex-ratio était de 1,38 pour les cas et 0,93 pour les témoins. Un suivi médical irrégulier (OR=4,87), une fréquence annuelle de crises vaso-occlusives (CVO) supérieure à 3 (OR=3,10), un nombre annuel d’hospitalisations pour CVO supérieur à 3 (OR=17,60) et un nombre de transfusions sanguines supérieur à 3 (OR=2,82) constituaient des facteurs prédictifs de survenue de l’ONATF. Le sexe, le nombre de leucocytes en phase inter-critique ou encore la concentration corpusculaire moyenne en hémoglobine étaient non prédictifs.
 

Conclusions :
La prévalence de l’ostéonécrose aseptique de la tête fémorale est l’une des plus élevées d’Afrique noire. L’affection touche des enfants très jeunes. Le diagnostic est fréquemment tardif au stade d’impotence fonctionnelle. Il est impératif de mettre en place une stratégie nationale pour un diagnostic et une prise en charge précoces de la drépanocytose afin de réduire le risque de survenue de cette complication grave, source inéluctable d’handicap en l’absence de prise en charge.
 

Purpose :
Les AVC représentent une complication majeure au cours de la drépanocytose caractérisée par une lourde mortalité et morbidité. Notre objectif était de décrire les circonstances de découverte, les facteurs de risque et le profil évolutif sous traitement des AVC chez les drépanocytaires.

Materials and Methods :
il s’agissait d’une étude rétrospective, analytique et descriptive portant sur des patients suivis sur une période de 8 ans allant de décembre 2012 à Octobre 2020. Les données recueillies concernaient les paramètres sociodémographiques, cliniques, paracliniques, les facteurs favorisants ainsi que les aspects thérapeutiques et évolutifs pour chaque patient.

Results :
Pendant la période d’étude nous avons recensé 22 patients ayant présenté un ou plusieurs épisodes d’AVC dans une population de 2634 drépanocytaires soit une prévalence de 8,3 pour 1000. L’âge moyen au diagnostic était de 23 ans avec un sex ratio à 1,45. Tous les patients étaient drépanocytaires homozygotes. Le symptôme le plus représentatif était les céphalées retrouvées chez 12 patients soit 54,6% de la population ; suivies du déficit moteur hémi corporel objectivé chez 11 patients (50%). Les principaux facteurs favorisants retrouvés étaient : nombre de CVO / année > 3 , au moins un épisode de STA / année, taux d’hémoglobine S en moyenne de 94,06 %, hyperleucocytose moyenne de 15,63 G/L,  hématocrite en moyenne de 28,13 %,  thrombocytose avec une moyenne de 657 G/L , taux de LDH élevé et une hyper bilirubinémie à prédominance non conjuguée. La TDM cérébrale et/ou l’IRM avait(ent) permis de confirmer le diagnostic et de distinguer la forme ischémique (77,3% des épisodes d’AVC) de la forme hémorragique (22,7% des épisodes d’AVC). Le territoire sylvien était le plus touché, retrouvé dans 19 épisodes d’AVC (79,16%) dont 2 récidives. A la phase aiguë, tous les patients ayant un AVC hémorragique avaient bénéficié d’un traitement antalgique, d’une oxygénation aux lunettes ainsi qu’une transfusion de concentré(s) de globules rouges. Pour les AVC ischémiques, seuls 10 patients soit 58,8% avaient été transfusés en concentrés de globules rouges ; 13 patients (59,09%) avaient bénéficié d’un échange transfusionnel dans l’immédiat ; 8 patients (47%) avaient bénéficié d’un traitement anti thrombotique. L’évolution à court terme (<1 mois) était marquée par une aggravation du tableau clinique chez 7 patients (31,8%), un état clinique stationnaire chez 8 patients (36,4%). On avait noté une régression du tableau clinique chez 7 patients (31,8%). Le traitement à long terme était fait d’un programme mensuel d’échange transfusionnel effectué chez 12 patients (54,54%) ; 10 patients (45,45%) avaient bénéficié d’une rééducation fonctionnelle. L’évolution à long terme était surtout marquée par la survenue de récidives chez 2 patients (9%) ; de séquelles neurologiques chez 5 patients (22,72%) et de décès chez 8 patients (5 AVCI et 3 AVCH).

Conclusions :
Cette étude montre que les AVC au cours de la drépanocytose sont plus ischémiques que hémorragiques . Les perturbations des paramètres de la NFS et du bilan d’hémolyse ont été retrouvés comme principaux facteurs favorisants des AVC. L’échange transfusionnel constitue un acte majeur dans la prévention et la prise en charge de cette complication.

Purpose :
La drépanocytose (anémie falciforme) est l'une des maladies hématologiques héréditaires les plus courantes chez l'homme. Une évaluation précise et fiable de l’hémoglobine en biologie délocalisée peut être essentielle pour le diagnostic précoce et l’éventuel traitement immédiat des anémies, notamment dans les pays en voie de développement sans laboratoire de référence à proximité. Le sang des patients drépanocytaires présente des propriétés rhéologiques particulières. Il n’existe à notre connaissance qu’une publication sur l’étude des performances d’un lecteur HemoCue® chez des patients drépanocytaires
Le but de notre étude était d’évaluer les performances de deux lecteurs aux principes différents, StatStrip (Nova Biomedical®) et Hb 201+ (HemoCue®), en les comparant à la méthode de référence du laboratoire central (XN10, Sysmex®) sur le sang de patients drépanocytaires.

Materials and Methods :
Les mesures ont été réalisées à partir de sang veineux prélevé sur tube EDTA de 57 sujets drépanocytaires au CHU de Toulouse, âgés de 2 à 60 ans, avec un taux d’hémoglobine compris entre 5,4 et 12,6 g/dL et un taux d’hémoglobine S entre 13 et 95%.

Results :
La moyenne (écart-type) des différences de taux d’hémoglobine comparativement au Sysmex® était de 0,3 g/dL (0,2) pour l’Hb 201+ et de 0,3 g/dL (0,9) pour le StatStrip. Toutes les valeurs obtenues par l’Hb201+ et 75 % des mesures du StatStrip étaient à moins de 1 g/dL de la méthode de référence. Les coefficients de corrélation de Pearson étaient respectivement de 0,99 et de 0,68. Les figures montrent les 2 diagrammes de Bland-Altman ainsi que les droites de régression linéaire.

Conclusions :
Les résultats de l’Hb 201+ sont très bien corrélés au XN10 et présentent juste un léger biais de surestimation. Le StatStrip montre des résultats moins précis, mais cela est inhérent à la technique et pas spécifique des sangs de patients drépanocytaires (données non publiées). Il ne semble donc pas y avoir d’interférence de l’hémoglobine S ou des drépanocytes lors de la mesure de l’hémoglobine totale. Mais les erreurs pré-analytiques sont des déterminants essentiels concernant l’utilisation des dispositifs en biologie délocalisée. Il reste donc à évaluer un plus grand nombre de patients notamment à partir de sang capillaire. 
 

Droite de régression et diagramme de Bland-Altman de l'automate Hb 201+ HemoCue® pour le dosage de l’hémoglobine de 57 patients drépanocytaires, en comparaison avec le XN 10 de Sysmex®

Droite de régression et diagramme de Bland-Altman de l'automate StatStrip (Nova Biomedical®) pour le dosage de l’hémoglobine de 57 patients drépanocytaires, en comparaison avec le XN 10 de Sysmex®

Purpose :
Le syndrome thoracique aigu (STA) est une complication pulmonaire de la drépanocytose, qui peut engager rapidement le pronostic vital en l’absence de prise en charge adéquate. Il y a peu d’études portant sur les relations entre COVID 19 et drépanocytose, notamment sur leurs localisations pulmonaires. La description des cas de comorbidités entre ces deux affections permettrait de réduire les incertitudes par rapport à leur diagnostic et leur prise en charge.

Materials and Methods :
Nous rapportons 4 cas de STA à COVID 19 survenus chez des patients drépanocytaires homozygotes, suivis au
niveau du service d’Hématologie clinique du CNTS.

Results :
Sur une cohorte de 2704 patients drépanocytaires suivis au CNTS, 28 patients ont été admis en hospitalisation continue durant lanpériode d’étude dont 4 patients pour un STA sur COVID 19. Il s’agissait de 2 hommes et de 2 femmes. L’âge était compris entre 17 et 27 ans. Ils étaient tous drépanocytaire SS et suivis au service d’hématologie clinique du CNTS depuis 7 ans en moyenne. Les motifs d’hospitalisation étaient essentiellement faits de douleurs osseuses diffuses évoquant une crise vaso-occlusive, plus ou moins associées à une toux, une douleur thoracique, une dyspnée dans un contexte fébrile. Il y’avait trois patients qui saturaient en deçà de 80 % à l’air ambiant et un qui avait une saturation normale. L’examen de
l’appareil pleuropulmonaire avait retrouvé chez deux des patients un syndrome de condensation pulmonaire alors qu’il était normal chez les deux autres. Trois patients présentaient une hémolyse aigue faite d’un syndrome anémique associé à un ictère de type hémolytique. La radiographie du thorax avait permis de confirmer le STA chez les 4 patients en montrant des infiltrats parenchymateux récents. Dans le cadre de la recherche étiologique du STA, tous les quatre patients
avaient bénéficié du test rapide antigénique du COVID 19 qui était positif. Deux patients ont réalisé un scanner thoracique qui avait permis d’évaluer la sévérité des lésions. Sur le plan thérapeutique tous les patients avaient bénéficié d’une oxygénation par voie nasale, d’une antibiothérapie, d’un traitement rhéologique, d’une anticoagulation à dose préventive ainsi qu’un traitement antalgique. Tous les quatre patients ont été transfusés : transfusion simple de concentrés de
globules rouges chez trois patients et échange transfusionnel chez un patient. L’évolution était marquée par l’apparition d’une coagulopathie intravasculaire disséminée chez un patient et une aggravation de la symptomatologie respiratoire chez un autre patient entrainant ainsi leur décès. Les deux autres patients ont eu une bonne amélioration avec un amendement de la symptomatologie et un test PCR de contrôle revenu négatif après une semaine de traitement.

Conclusions :
Les manifestations cliniques du STA chez les drépanocytaires sont similaires à celles du COVID 19 et leur
association doit toujours été évoquée, et le dépistage du SARS-COV 2 réalisé chez tout drépanocytaire avec une crise vaso-occlusive. C’est une comorbidité qui expose à des complications qui mettent en jeu le pronostic vital, d’où l’importance d’une hospitalisation et d’une prise en charge adéquate.

Purpose :
La drépanocytose est une maladie chronique, héréditaire, invalidante et douloureuse qui cause le problème aux : Malades, Familles, Communautés, Nations.
PROBLEME PSYCHO-SOCIAL : Une maladie exigeant une prise en charge multisectorielle: Famille, Personnels soignant, Communauté, Psychologues, Sociologues, Anthropologues, Organismes nationaux et internationaux, Etat.
Depuis longtemps, les drépanocytaires sont marginalisés dans nos sociétés africaines en disant que la survie des drépanocytaires ne dépasse pas 5 ans. Ce qui décourage les familles de poursuivre les soins comme si elles sèment dans le désert.
PROBLEME ECONOMIQUE : La RDC étant un pays en développement, les soins médicaux sont pris en charge par la famille. Avoir un drépanocytaire dans une famille est un véritable fardeau suite aux multiples consultations,
hospitalisations, bilans et soins réalisés. Cela nécessite un minimum de connaissance de cette maladie afin de bien adhérer au traitement pour diminuer les dépenses.

Materials and Methods :
Il s’agit d’une analyse retrospective des carnets de suivi des drépanocytaires de la colombe (Association de lutte contre la drépanocytose) qui ont consultés ELISABETH MEDICAL CENTER à la période de Janvier 2019 à Décembre 2021 dont un échantillon de 123 drépanocytaires homozygotes SS confirmés a été retenu.

Results :
Sur les 123 drépanocytaires suivi à la période de 2019 à 2021, le sexe féminin domine 51% contre 49% du sexe masculin ; la tranche d’âge de 6-10 ans est régulièrement suivie à 17% ; 42% sont suivi pour douleur articulaire modérée de localisation osseuse, 25% pour fièvre et 11% sont cliniquement stables ; 9% sont sous hydroxy-urée ; la complication majeure est l’osteonécrose aseptique de la tête fémorale à 11% suivi du décès à 7% dont 4% des patients de plus de 15 ans décédés en 2020 pendant le confinements.

Conclusions :
En raison de l’allongement de l’espérance de vie des drépanocytaires, les unités de prise en charge devrait être mieux équipées pour le diagnostic et la prise en charge précoce des complications aiguës de la maladie. C'est ainsi qu'un engagement ferme de tous est nécessaire dans la lutte contre la drépanocytose.

Purpose :
The Globin Research Network for Data and Discovery, GRNDaD (2016), registry and its recently initiated partner, Collaborating to Understand Sickle Cell Disease Internationally, CoUSIn (2021), were developed by providers at sickle cell centers in order to collect prospective data on the modern cohort of people living with SCD. The goal of these registries is to have the tools to describe the natural history of the disease and ensure the provision of guideline-based care. Neither foundations nor government-supported registries have, to date, been able to supply a comprehensive prospective detailed and patient-centered registry like GRNDaD or CoUSIn. 

Materials and Methods :
GRNDaD, comprising over 300 individual data elements, collects data from 12 sites at present, using de-identified data, and relying on a central IRB and annual updates (Table 1, N-1319, Fall 2019). The prospective REDCap database has been flexibly amenable to the addition of more highly detailed data sheets, encompassing queries on end stage renal disease, pregnancy, and thrombophilia.

Results :
To date, 12 sites in the US are fully functional, with 8 additional sites on-boarding. GRNDaD serves as the companion registry for the National Alliance of Sickle Cell Centers, NASCC, and is expected to grow briskly in the next 24-36 months. Most patients have sickle cell anemia (HbSS or HbSb0, Table 1), many have contributed patient reported outcome (PRO) surveys, and half of all entrants are adolescent/young adults (percent of total, Figure 1). As of February 2022, 2160 people with SCD have consented for data entry. There is a robust range of treatments in GRNDaD; of 2160 subjects, 53% of subjects with HgbSS or SB0thalassemia were on hydroxyurea, 12% of all subjects were on chronic transfusions, 2.4% were on L-glutamine, 2.2%were on crizanlizumab, and 2.0% were on Voxelotor.  We estimate that >100 patients are likely to be added each year for recently-approved novel therapies Crizanlizumab and Voxelotor. Of note, the older adult with SCA had a depressed reticulocyte count and a trend towards a higher creatinine (not shown). 
Finally, the GRNDaD data dictionary has been shared with investigators in the UK, who have developed an identical parallel database, CoUSIn, which will serve as a platform for comparisons going forward of prospectively collected data between SCD populations that are cared for in 2 entirely distinct settings.

Conclusions :
Multi-site prospective registries designed to better understand the contemporary person living with SCD are crucially important, for tracking, and analysis of guideline-based care delivery in this disease. With funding from several sources, these registries will continue to grow and have plans for sustainability well into the future.

Shown are sites, genotype distribution, and patient reported outcome survey attainment in GRNDaD, as of Fall 2019.

Shown is the age distribution for the contemporary SCD population (%), in GRNDaD, as of February 2022.

Purpose :
Sickle cell disease (SCD) is a devastating red blood cell (RBC) disorder in which polymerization of sickle hemoglobin (HbS) upon deoxygenation results in poorly deformable sickled RBCs with a shortened lifespan. 2,3-Diphosphyglycerate (2,3-DPG), a glycolytic intermediate in RBCs, promotes deoxygenation by lowering hemoglobin (Hb)-oxygen affinity. Mitapivat (AG-348) is an oral allosteric activator of pyruvate kinase (PK), a key enzyme in RBC glycolysis generating adenosine triphosphate (ATP) and reducing 2,3-DPG levels. Here, we report follow-up data of the safety and efficacy of mitapivat treatment in subjects with SCD enrolled in the currently ongoing ESTIMATE study (www.trialregister.nl NL8517; EudraCT 2019-003438-18).

Materials and Methods :
The ESTIMATE study is a phase 2, investigator initiated, open-label study. Subjects ≥16 years with SCD (HbSS, HbS/β⁰ or HbS/β⁺), 1-10 vaso-occlusive crises (VOCs) in the prior year and/or prior SCD-related complications, a Hb level >6.1 g/dL and ≤11.1 g/dL, and not receiving chronic transfusions are eligible. In the 8-week mitapivat dose finding period, initial dosing is 20 mg twice daily escalated to max 100 mg twice daily, and followed by ≤52-week fixed dose extension period (FDEP). Primary endpoints are safety, based on adverse events (AEs), and efficacy of mitapivat treatment based on RBC sickling (Point of Sickling (PoS), oxygen gradient ektacytometry). Key secondary endpoints are changes in Hb level, markers of hemolysis, ATP and 2,3-DPG levels, and Hb-oxygen affinity (p50, Hemox Analyzer).

Results :
Until Jan. 2022, nine subjects received mitapivat. Baseline characteristics: median age 22 years (range 16-59), 6/9 (67%) female, and 6/9 (67%) used hydroxyurea. 7/9 (78%) had HbSS, 1/9 (11%) HbS/β⁰, and 1/9 (11%) HbS/β⁺. One subject with a non-treatment related serious AE (SAE) of a urinary tract infection (grade 4), was lost to follow-up shortly after first dosing. No other SAEs or treatment-emergent AEs (TEAEs) grade ≥3 occurred. The remaining eight subjects had a median treatment duration of 38 weeks (range 11-60). 6/8 (75%) took 100 mg mitapivat twice daily in the FDEP, and 2/8 (25%) took 50 mg mitapivat twice daily after a single dose reduction because of transaminase increase. The most common TEAEs (n>2 subjects): ALT or AST increase (both 5/9, 56%; all grade 1), headache (4/9, 44%; grade 1-2). Three VOCs occurred: one related to excessive alcohol consumption with no need for hospitalization, and two in subjects with documented noncompliance the week before hospitalization. Mean annual VOC rate and SCD-related hospital admission days were, respectively, 1.5±1.3 and 5.9±7.1 days at baseline, and reduced to 0.5±0.7 and 1.6±3.1 days when weighting cases by follow-up duration (p=0.021 and p=0.134, respectively). Table 1 summarizes mean changes in different endpoint parameters of scheduled visits in the FDEP versus baseline. Hb level significantly increased, accompanied by a decrease in markers of hemolysis (absolute reticulocyte count, total bilirubin, and lactate dehydrogenase). Mean PoS decreased, and p50, 2,3-DPG level and ATP/2,3-DPG ratio significantly improved.

Conclusions :
Treatment with mitapivat for up to 60 weeks in subjects with SCD showed no treatment related TEAEs grade ≥3. Improvements in anemia, markers of hemolysis, oxygen affinity, 2,3-DPG level and ATP/2,3-DPG ratio were seen. Follow-up data will be collected.

Table 1

Purpose :
Sickle cell disease (SCD) is one of the most prevalent genetic disorders, affecting around 20 to 25 million individuals throughout the world. In Sub-Saharan Africa, where it is more prevalent, it can contribute up to 80% of under-5 mortality. Clinical manifestations of SCD are very heterogeneous and the intestinal microbiome has recently been reported to be crucial in the modulation of inflammation, cell adhesion and induction of aged neutrophils, which are key interveners of recurrent vaso-occlusive crises. Since gut bacteria can regulate aged neutrophils, defects in either the integrity of the intestinal walls or a chronic disequilibrium of the microbiota are very likely to emerge in SCD patients. Moreover, it has been suggested that Hydroxyurea (HU), the most common treatment for SCD, shows a multimodal action and may reduce microbiome dysbiosis and aged neutrophils. In this context, we aimed to understand how SCD and HU treatment modulates the microbiome and if these changes could be related with disease severity.
 

Materials and Methods :
In order to characterize the gut microbiome of an SCD pediatric population, which consisted of Angolan children before and after 6 months of continuous HU treatment, a total of 66 stool samples were collected in tubes with a preservative solution. Then, the metagenomic DNA was extracted, quantified and the bacterial 16SRNA gene for the V3-V4 regions was sequenced by NGS. Microbiome taxonomic profiling analysis was performed with the EzBioCloud pipeline and differences between the two groups were assessed with the Statistical Analysis of Metagenomic Profiles (STAMP) software, using Welch’s t-test.
 

Results :
Significant associations were observed in alpha-diversity between the two groups, with higher values for the children naïve for HU in several parameters, namely in OTU species count (p<0.001), phylogenetic diversity (p=0.004) and microbial richness (p<0.001), which was calculated by the ACE, Chao1 and Jackknife indices. We also noticed that children after HU treatment had higher proportions of some bacteria associated with health, including Blautia luti, Roseburia inulinivorans, Lactobacillus rogosae and Faecalibacterium, when compared to the beginning of the study. Additionally, the proportion of Firmicutes phylum was significantly lower before HU.
 

Conclusions :
This was the first study to report gut microbiome changes before and after HU treatment in SCD children. Overall, our findings provide a rationale for further research about gut microbiota dysbiosis in this population. Determining the effect of specific bacteria will give evidence for the correction of microbiota composition and supplementation with beneficial strains in order to reduce gut microbiota-driven inflammation, which could ultimately mitigate disease severity. This work was supported by FCT/Aga Khan (project nº330842553) and FCT/MCTES (UIDB/05608/2020 and UIDP/05608/2020) –H&TRC.
 

Purpose :
Sickle cell disease (SCD) is a monogenetic disorder with a highly complex pathophysiology. There is an unmet need for robust reproducible biomarkers that can assess red blood cell (RBC) function and predict disease severity and complications.

Materials and Methods :
In this study, we investigated the association between oxygen gradient ektacytometry-derived biomarkers, and blood viscosity with incidence of major (acute) SCD-related complications. Oxygen gradient ektacytometry measures RBC deformability continuously while the sample is gradually deoxygenated and subsequently reoxygenated, and identifies the oxygen tension at which sickling occurs. We examined associations between the occurrence of acute chest syndrome, cerebral infarction and vaso-occlusive crisis (VOC) and known biomarkers such as fetal hemoglobin, blood viscosity as well as exploratory oxygen gradient ektacytometry-derived biomarkers in an adult cohort of 50 individuals with SCD (HbSS or HbS/βo-thalassemia) and a pediatric cohort consisting of 177 children with SCD (HbSS or HbS/βo-thalassemia). A substantial number of subjects were on hydroxyurea therapy (64% of adults and 89% of children). Subjects that received a blood transfusion less than three months prior to measurements were excluded from the study. A logistic regression analysis was performed; odds ratios were adjusted for age and hydroxyurea therapy.

Results :
In the adult cohort, for every 10 mmHg increase in Point of Sickling (PoS, the pO2 tension where RBCs start to sickle, reflecting sickling tendency) the likelihood of >1 acute complication increased; the adjusted odds ratio (aOR) was 3.00 (p=0.015). For every 0.1 increase in EImax (reflecting RBC deformability at normoxia), the aOR was 0.33 (p=0.035, Table 1). In the pediatric cohort, for every 10 mmHg increase in PoS, the likelihood of >1 acute complication increased; the aOR was 1.65 (p=0.006). For every 0.1 increase in EImin (reflecting RBC deformability at hypoxia), the aOR was 0.50 (p=0.007). Fetal hemoglobin and blood viscosity levels were not associated with likelihood of multiple acute complications. However, fetal hemoglobin was associated with reduced likelihood of VOC in adults (aOR of 0.32 for every 10% increment, p=0.010) but not in children (aOR of 0.68, p=0.231, data not shown). In the adult cohort higher EImax was associated with reduced likelihood of VOC (aOR 0.31, p=0.029). There was a trend found for an association between higher PoS and greater likelihood of VOC (aOR 2.22, p=0.050), and no association for EImin. In the pediatric cohort only EImin was associated with VOC (aOR 0.68, p=0.036).

Conclusions :
These findings indicate that oxygen gradient ektacytometry generates novel clinically relevant biomarkers and provide a rationale for further development of these biomarkers in the evaluation of novel therapies, as part of clinical care, or clinical trial endpoints. In particular, in assessment of treatment strategies that do not target HbF induction, such as pyruvate kinase activators, voxelotor and l-glutamin, oxygen gradient ektacytometry can generate relevant biomarkers.

Purpose :
Sickle cell disease (SCD) is a monogenetic red blood cell (RBC) disorder that is characterized by hemolytic anemia and vaso-occlusive crises. Among the many factors that contribute to disease pathophysiology is stiffening and sickling of RBCs, which is the direct result of the polymerization of abnormal hemoglobin S. Sickling is modulated by glycolytic intermediates such as 2,3-diphosphoglycerate (2,3-DPG) and ATP. Previously we showed that RBC pyruvate kinase (PKR), the key regulatory enzyme of glycolysis, is impaired in SCD and that ex-vivo treatment with mitapivat, an allosteric activator of PKR, increased enzymatic activity and thermostability, reduced 2,3-DPG levels, decreased p50, and subsequently reduced sickling (Rab et al, Blood 2021). Currently, mitapivat is being investigated in phase 1, phase 2, and phase 2/3 trials in patients with SCD (#NCT04000165, EudraCT#2019-003438-18, and NCT05031780). Recently, AG-946, a novel PK activator, has been developed. Here we investigate the pharmacodynamic effects of AG-946 in ex vivo treatment of RBC from SCD patients in comparison with mitapivat. 

Materials and Methods :
Buffy coat depleted whole blood obtained from five SCD patients was incubated for 20-24 hours in absence or presence of mitapivat (100mM) or AG-946 (1mM, 5mM, 50mM). After ex-vivo treatment, enzymatic activities of PKR and PKR-thermostability were measured. Glycolytic intermediates ATP and 2,3-DPG were measured using LC-MS/MS. Hemoglobin oxygen affinity (p50) was measured and RBC sickling was analyzed with oxygen gradient ektacytometry, a method that characterizes individual sickling behavior. Individual tendency to sickle is reflected by Point-of-Sickling (PoS) that indicates the specific pO₂ at which RBCs start to sickle during deoxygenation under shear stress. 

Results :
PKR activity was increased compared to vehicle (DMSO) to a similar extent in the presence of mitapivat or AG-946 (Figure 1A). In addition, PKR thermostability was significantly increased compared to vehicle (mean 22%, SD 6%) in samples treated with mitapivat 100 mM (mean 78%, SD 11%), as well as AG-946 5mM (mean 66%, SD 23%), and AG-946 50mM (mean 95%, SD 17%, Figure 1B). After incubation with mitapivat or AG-946, 2,3-DPG decreased (Figure 1C), which was further illustrated by the improved ATP/2,3-DPG ratio (Figure 1D). Accordingly, p50 decreased significantly after incubation with mitapivat 100mM (mean 95%, SD 2%), as well as AG-946 1mM (mean 96%, SD 2%), AG-946 5mM (mean 94%, SD 2%), and AG-946 50mM (mean 95%, SD 3%, Figure 1E). The improved metabolic status and p50 was accompanied by a decreased PoS in RBCs treated with mitapivat or AG-946, indicating reduced RBC sickling tendency (Figure 1F).

Conclusions :
Ex vivo treatment of SCD RBCs with the novel PK activator AG-946 activates and stabilizes PKR, decreases 2,3-DPG levels, improves the ATP/2,3-DPG ratio, improves p50 and lowers the PoS. These beneficial effects are similar to ex-vivo treatment with mitapivat but, importantly, are obtained at much lower concentrations. Taken together, these results are the first in an ex-vivo model to demonstrate that the novel PK activator AG-946 has a similar favorable pharmacodynamic profile to mitapivat with enhanced PKR-stabilizing properties and, hence, might represent a potential novel therapeutic option in addition to mitapivat for the treatment of SCD.

Pharmacodynamic effects of AG-946 in ex vivo treatment of red blood cells (RBC) from sickle cell disease (SCD) patients in comparison with mitapivat. Buffy coat depleted whole blood was incubated for 20-24 hrs in absence or presence of mitapivat (100 ?M,

Purpose :
Sickle cell disease (SCD) is a major contributor to child morbidity and mortality. In Sub-Saharan Africa (SSA) approximately 90% of the children born annually with SCD will die before their fifth birthday, often undiagnosed. The absence of early diagnosis through laboratory-based newborn screening (NBS) programs is a major barrier to the implementation of timely preventive measures in SSA. Emergent, novel, inexpensive, point-of-care tests (POCTs) with validated analytic characteristics and field-tested performance comparable to laboratory methods for identifying SCD present a unique opportunity for implementation of localized, community-based newborn NBS programs in primary health centers (PHCs). Nurses comprise the single largest category of available health workers in SSA. Therefore, we describe a Nurse Champion protocol to provide (1) genetic counseling education, (2) perform sickle cell disease and carrier screening with POCTs, (3) and prompt linkage to care in PHCs.
 

Materials and Methods :
Our protocol delineates a type-2 hybrid implementation-effectiveness design for the evaluation of feasibility and acceptability of the Nurse Champion model protocol in limited-resource settings. The model’s components are provider capacity education, care support tools, care coordination, and an expert-driven process of professional peer oversight of the nurses. We use the Consolidated Framework for Implementation Research (CFIR) to guide comprehensive pre-implementation evaluation and adaptation of the Nurse Champion protocol. The CFIR provides a structure for understanding barriers and facilitators to successful protocol implementation in a specific setting and enhancing fit with the local context. We combine the CFIR with Implementation Mapping, a protocol that guides the design of multi-level health promotion interventions and implementation strategies. Implementation Mapping posits that the primary and secondary outcomes of implementation and innovation effectiveness depend on logic modeling. We add the Learning Evaluation Model (LEM) to the protocol to establish feedback loops for evaluation and adapt the protocol to contextual changes.
 

Results :
Nurse champion models, while evidence-informed, have not been tried in PHCs in SSA for SCD diagnosis. Therefore, we need to know whether nurses can diagnose SCD using POCTs and what kind of training and technical assistance they would require? What characteristics of a SCD POCT intervention will support (a) identification of babies with SCD at birth; (b) broad adoption across primary care settings (including rural and urban); (c) implementation by nurses with moderate levels of training and expertise; (d) replicable effects, and (e) at a reasonable cost? Our protocol outlines multimodal steps to address these questions.
 

Conclusions :
The Nurse Champion protocol showcases the wide variety of nursing roles in the early diagnosis of a genetic disease such as SCD with POC diagnostics.  The World Health Organization recommends task shifting of clinical roles as a workforce strategy to improve access to services and reduce disparities in healthcare. Task-shifting and upskilling are innovative strategies for enhancing nurses' knowledge inventory and skills to manage health conditions or coordinate health service delivery. A burgeoning literature supports the effectiveness of nurse-led interventions to improve access to preventive health services.
 

Purpose :
Haemoglobinopathies, including sickle cell disease (SCD) and thalassaemia syndromes, represent the commonest monogenic diseases in the world. Although their pathogenicity is well established, the diverse clinical manifestations and the varying degree of severity are less understood and are thought to be governed, in part, by genetic modifiers. Despite the identification and characterisation of a few genetic modifiers by previous studies, these are as yet insufficient to guide treatment recommendations or stratify patients reliably. Larger, multi-ethnic studies are needed to identify and validate further disease modifiers that can be used for patient stratification and personalised treatment. There is a growing need for deeper insight into the availability of novel targeted therapies and potentially curative options like gene therapy in both SCD and thalassemia. 
The International Haemoglobinopathy Research Network (INHERENT) is a recently established network with the aim of investigating the role of genetic modifiers in haemoglobinopathies, through a large-scale, multi-ethnic genome-wide association study (GWAS).

Materials and Methods :
INHERENT brings together nine existing international or regional consortia in the field of haemoglobinopathies, namely ITHANET, RADeep, ARISE, SPARCO, SADaCC, REDAC, the HVP Global Globin Network, the International Health Repository, and the ClinGen Haemoglobinopathy VCEP. The activities of INHERENT are currently divided into five working groups, as follows: clinical, genotyping, data management and analysis, ethics, and knowledge translation. Participation in INHERENT is open for any group that can submit a minimum number of samples with their core phenotypic description. INHERENT membership is international and interdisciplinary and, currently, includes over 200 experts from over 105 organisations, spanning 43 countries worldwide. INHERENT aims to recruit over 30,000 hemoglobinopathy patients, which is larger than any previous GWAS in the field.

Results :
A survey amongst 67 of the participating centres revealed that INHERENT has the potential to recruit over 73 500 patients, of which 53755 are SCD, 15691 are β thalassemia (62% transfusion-dependent), 4179 (α thalassemia). A sample size of 10000 individuals can detect associations down to MAF=0.05 with an OR=1.5 in binary phenotypes assuming a case/control ratio of 0.3 and p-value of 5x10^-8. For survival endpoints, 5000 individuals will be sufficient to detect an HR of 1.5 and MAF of 0.05, if we assume an event rate of 30%.

Conclusions :
We demonstrate that the current membership of INHERENT has the potential to reach this sample size target. The large increase in the sample size and the diversity in the studied populations will enable novel discoveries and expand knowledge on haemoglobinopathy genetics, thus paving the way for advancing the science of personalised diagnosis and treatment.

INHERENT patient distribution

Power calculations

Purpose :
Few studies have described in depth the burden of sickle cell anemia (SCA) related morbidity and mortality in children in Sub Saharan Africa, the continent with the highest burden of SCA. The goal of this study was to describe the morbidity and mortality in children with SCA

Materials and Methods :
We enrolled 248 children aged 1-5 years with SCA who participated in the Zinc for Infection Prevention in Sickle cell anaemia (ZIPS) clinical trial and followed them for 1-year (Clinicaltrials.gov, NCT03528434) at Jinja Regional Referral Hospital, Uganda. Children were evaluated for all infections, SCA related complications and deaths during the study period by taking standard history and diagnostic work-up. All children received standard maintenance care for SCA, which included daily folic acid, penicillin prophylaxis, monthly sulphadoxine-pyrimethamine for malaria prophylaxis, and an insecticide treated mosquito net for malaria prevention. We initiated hydroxyurea to children meeting Uganda Ministry of Health SCA hydroxyurea treatment criteria at the time of the study, which included: > 5 pain crises, stroke, and baseline Hb<6g/dL, and history of or new acute chest syndrome (ACS). Infections were defined as severe or invasive if they met strict clinical and laboratory criteria

Results :
Between 2018 and 2019, 248 children with SCA were enrolled with a mean (SD) age of 2.8 (1.1) years. The mean (SD) baseline white cell count, hemoglobin level and fetal hemoglobin % were 19.2 (8.4) X109/L, 8.2 (1.3) g/dL and 13.7 (8.0) %, respectively. A total of 117 (47.2%) children were initiated on hydroxyurea at some point in the study. There were 1513 sick visits over 12 months, giving an incidence of 6.4 sick visits per year. Of these, 467 visits required hospital admission. A total of 121 children had more than 1 sick visit requiring hospitalisation. The mean (SD) hospital stay was 3.0 (1.8) days. The incidence of any severe or invasive and clinical infections was 276 and 516 infections per 100-person year respectively. Sepsis, malaria, gastroenteritis, and pharyngitis/tonsillitis were the most common forms of severe or invasive infections, while upper respiratory tract infections were the most common clinically defined infections. Vaso-occlusive crises (VOC) and severe anaemia requiring blood transfusion were the most common SCA related complications, with incidences of 160 and 135.7 events per 100 child years. Two children had a stroke over the 12 months study follow up. A total of 9 children died, and the likely cause of death was pneumonia/lower respiratory tract infection with accompanying respiratory failure in five children, sepsis with accompanying heart failure in one child, severe malaria in one child, and an unspecified cause in two children. The incidence of infections and SCA related complications reduced significantly in children initiated on hydroxyurea (5.2 vs. 2.0 infections per child per year and 12.0 vs.1 complication before and after hydroxyurea therapy, P<0.001 for both) 
 

Conclusions :
There is high burden of SCA related morbidity and mortality in young children with SCA in Uganda. There is need to implement wider use of disease modifying therapies and research to understand risk factors for the high morbidity and mortality.

Purpose :
In 2006, WHO declared sickle cell disease (SCD) as a global public health priority and recommended the development of Reference Centers in the sub-Saharan African region where the disease affects 1 to 2% of births. In Mali, the birth rate was estimated at 714,000 per year and the incidence of sickle cell anemia (homozygous SS + S/β-thalassemia + SC), at 0.8% of live births.  To begin to adress the issue of reducing the burden of the SCD in Mali, a Sickle Cell Research and Control Center was established in Bamako in 2010. This presentation, reports the experience of the first decade of operation of this Center.

Materials and Methods :
Through the circumstances of its creation, the principle of operation and the results acquired by the Center in ten years of functioning, we make an analysis on the possible future orientations to optimize the management of sickle cell disease in sub-Saharan Africa by the Reference Centers.

Results :
The « Centre de Recherche et de Lutte contre la Drépanocytose (CRLD) » was established as a result of coordinated efforts by the Government of Mali, SCD patients association, and a consortium of partners led by Pierre Fabre foundation. The assigned objectives were: to support access to quality care with equity, to develop medical research on sickle cell disease, to train medical personnel on sickle cell disease, to develop education, information and communication on the disease, to promote national and international collaboration (South-South, North-South). In order to ensure an equitable access to care, the center provides treatment with no consideration of the financial status of the family. Since March 2010, when the Center began its activities, 956 patients from the first year to 10745 sickle cell patients in 2019 have been recruited and are being provided with regular follow-up and access to treatment.  Sickle cell mortality assessed in 2016 was estimated at 0.035 per 100 person-years (0.040 per 100 person-years before 5 years). In terms of research, 44 research protocol have been conducted and 26 scientific papers were published. Within the framework of an university degree, the Center has trained 126 physicians from 12 French-speaking countries in East and West Africa, Madagascar and Haiti. More than 300 communications relays were trained.

Conclusions :
If the growing increase in patients recruitment has shown the validity of the creation of the Center with satisfactory results in the support of specific care, the results of some of its asigned objectives have remained below expectations. We propose a new approach for the management of sickle cell disease in sub-Saharan Africa that follows a pyramid-schape of healthcare services.

Purpose :
Sickle cell disease (SCD) is the most commonly inherited monogenic hemoglobinopathy and results from a mutation in the β-globin gene (HbS), leading to red blood cell (RBC) sickling, vaso-occlusive episodes (VOE), and organ damage. Chronic hemolysis, inflammation, and repeated red blood cell transfusions in SCD can disrupt iron homeostasis. Patients who receive multiple blood transfusions develop iron-overload, and another subpopulation of SCD patients manifest iron deficiency. SCD mice exhibit increased iron absorption via upregulation of the intestinal transcription factor hypoxia inducible factor-2a(HIF-2a) leading to the development of iron overload. Disruption of intestinal HIF-2a or limiting iron intake by dietary iron restriction in SCD mice, significantly reduced systemic iron overload, lowered cellular sickle hemoglobin as reflected by reduced RBC mean corpuscular hemoglobin concentration (MCHC), and consequently improved hemolysis and ameliorated anemia. Understanding iron homeostasis is therefore critical to SCD pathogenesis and holds the promise for identifying novel approaches to treat SCD.

Materials and Methods :
SCD mice were generated by transplanting bone marrow cells from Berkeley SCD (Tg[Hu-miniLCRα1GγAγδβS]Hba−/− Hbb−/−) mice into 8-week old, lethally-irradiated C57BL/6J mice. Immediately after transplantation, SCD mice were fed either a regular-iron diet (Ctrl) containing 185 parts per million (ppm) iron or an otherwise identical iron-restricted diet (IRD) containing 3 ppm iron. VOE and organ damage analysis were done 3-4 months after treatments. 

Results :
IRD resulted in a reduced organ iron concentration (Fig-1A) in SCD compared to Ctrl group, confirming that dietary iron restriction ameliorates organ iron deposition in SCD mice. SCD mice treated with IRD exhibited reduced MCHC (Fig-1B) and serum indirect bilirubin(IDBILC) (Fig-1C) indicating a decrease in cellular sickle hemoglobin and hemolysis. Furthermore, IRD reduced the fraction of aged neutrophils by >50% in SCD mice (Fig-1D), which corresponded with improved VOE parameters. Specifically, intravital microscopy (IVM) demonstrated elevated mean centerline RBC velocity and blood flow rate and decreased leukocyte adhesion in IRD compared to Ctrl mice (Fig-1E-G). Importantly, IRD led to prolonged survival after TNF-a challenge in SCD mice (Fig-1H). IRD resulted in a dramatic reduction in lung leukocyte infiltration, indicating significantly reduced lung inflammation in SCD mice (Fig-2A). Compared to Ctrl liver samples, IRD resulted in significantly less liver necrosis, leukocyte infiltration and fibrosis (Fig-2B,C), decreased serum liver enzymes (ALT, AST; Fig-2D), and reduced levels of total and direct bilirubin (TBILC, DBILC; Fig-2D).We further hypothesized that, in addition to reducing iron concentration in organs, IRD ameliorates organ damage by preventing translocation of microbial compounds.IRD inhibits translocation of FITC-Dextran by 5-fold compared to Ctrl suggesting functionally improved gut permeability (Fig-2E). We detected significantly decreased TLR2 ligands in serum of IRD mice (Fig-2F) suggesting ligands from gram-positive bacteria (TLR2 agonists) may play a role in triggering SCD complications.

Conclusions :
Our study demonstrates for the first time that dietary iron restriction reduces VOE severity and alleviates chronic organ damage in SCD mice. Novel approaches that target iron availability/absorption in the gut, the gut microbiome and aged neutrophil crosstalk and gut barrier integrity, provide an important area of further investigation with the potential of improving hemolysis, VOE, and organ damage in SCD patients. 

(A)The bone-marrow/spleen/liver Non-heme iron content. (B)MCHC, (C)IDBILC,(D)aged neutrophils percentage in Ctrl/IRD SCD mice. IVM parameters (E) (Vrbc) (F) blood flow rate (J) the number of adherent leukocytes and (J) survival rate after TNF-a injection

(A)Leukocyte infiltration in lung and (B)liver tissue. (C)Liver necrosis, leukocyte infiltration, and fibrosis. (D)Liver ALT and AST, TBILC, and DBILC from sera. (E)serum FITC-Dextran and (F) TLR2 ligands activity in Ctrl/IRD SCD mice

Purpose :
Many paediatric studies describe the association between selected biological parameters and increased incidence of sickle cell disease (SCD) complications. However, blood levels of these biological parameters change over time (figure 1), during growth and as the switch from foetal haemoglobin (Hb) to adult HbS is completed. In addition, blood levels may vary depending on the occurrence of complications, or during treatment. The aim of our study was to assess the impact of i) the methods used for biological parameters collection and use in cohort studies in the literature, ii) the statistical method used in subsequent analysis iii) the overall effect on the association between those biological parameters and clinical SCD outcomes in the paediatric context.
 

Materials and Methods :
First, a literature search was performed to review how the biological parameters were collected and used in statistical analyses in published cohort studies in SCD. The second step was to test those statistical analyses on our cohort dataset of SCD patients in order to evaluate the impact of the methodological strategy on the results for this given cohort. In particular, we investigated the impact of statistical strategies on the relationship between biological parameters and macro-cerebral vasculopathy (CV) in our paediatric cohort of SCD patients followed at the Robert-Debré University Hospital.
 

Results :
In 16 cohort studies, biological parameters were collected either once or at different time-points during follow-up. The identified methods in the statistical analysis were: (1) one baseline value per patient (2) last known value; (3) mean of all values; (4) modelling of all values in a two-stage approach, which take into account the course of laboratory parameters over time. Applying these four different statistical methods to our cohort, the results and interpretation of the association between biological parameters and CV showed marked difference depending on the method used (figure 1). Overall, the final number of significant parameters can differ from 1 to 4, depending on the statistical method. Furthermore, one biological parameter can be found significant with a method and not with another (figure 2).  In fact, using the Cox model encompassing linear multivariable modeling of longitudinal quantitative variables, on both clinical and biological parameters in our cohort, we identified some new independent protective or risk factors for CV. Years per upper-airway obstruction or bronchial obstruction and reticulocyte count were risk factors whereas foetal Hb level was protective. Specific treatment for upper or lower-airway obstruction and indirect targeting of foetal haemoglobin and reticulocyte count by hydroxycarbamide could potentially reduce the risk of CV.
 

Conclusions :
This study shows that different methods on the same dataset can lead to different conclusions as to the prognostic value of the laboratory parameters predicting the morbidity of SCD. It illustrates how important it is to apply appropriate statistical methods. In order to take into account longitudinal changes of biological parameters, appropriate statistical analyses in cohort studies should be considered.
 

Figure 1. Evolution of fetal haemoglobin level and reticulocyte count over time. Biological parameters were recorded at least 3 weeks distant from any clinical event or any intensification (hydroxycarbamide, transfusion program, or haematopoietic stem cel

Figure 2. Illustration of results obtained according to the four methods used to study the relationship between the biological parameters and the occurrence of cerebral vasculopathy. Each panel represents one of the four methods used in multivariable anal