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Background Sickle cell disease (SCD) children are frequent travellers to countries where yellow fever (YF) is endemic, but there are no data regarding the safety and immunogenicity of the vaccine in such children treated with hydroxyurea (HU). The main objective of this study was to compare the tolerance and immune response to YF vaccination in SCD children treated or not with HU. Method SCD children Results Among the 52 SCD children vaccinated against YF, 17 (33%) were treated with HU. Only mild adverse events, mainly fever and local reaction, were observed in the HU group with a similar frequency in the non-HU group (57 and 35%, respectively, P = 0.30). YF antibody titres were measured in 15/17 patients in the HU group and 23/35 patients in the non-HU group after a median of 6.0 months (3.5–8.5) following vaccination. The geometric mean of YF antibody titre was similar in both groups. A protective antibody level was observed in 85% of the children in the HU group vs 100% in the non-HU group (P = 0.14), suggesting a lower effectiveness of the vaccine in patients on HU similarly to what has been described in patients on immune suppressive therapy for other vaccines. Conclusion YF vaccination seems to be safe and efficient in SCD children treated with HU. Considering the potential risk of severe complications in cases of YF while travelling in Africa for those patients, the benefit-to-risk ratio argues for YF vaccination in all SCD children. Control of a protective antibody titre may also be useful to ascertain an adequate response in those treated with HU.

Spleen dysfunction and susceptibility to pneumococcal infection is a well known feature in homozygous sickle cell disease (HbSS), whilst to date splenic function in hemoglobin SC disease (HbSC) has been poorly investigated. The aim of this study was to analyze spleen function in children with HbSC disease using a high-throughput validated method (1) and to examine if the current recommendations regarding pneumococcal risk are appropriate in this population. Spleen function was evaluated using a flow cytometry quantification of red blood cells (RBCs) with Howell-Jolly bodies (HJBs), in a cross-sectional study of patients at steady state during an outpatient visit in an expert center. Quantification of HJB-RBCs was performed in children with HbSC disease aged < 10 years and compared to children with HbSS disease or healthy children of the same age groups, or splenectomized children. Additional exploratory analysis was performed according to age (under or above the age of 5 years old) and treatment group (hydroxyurea). The median (Q1-Q3) HJB-RBCs count was 16 (11-28.25) /100.000 RBCs in 40 HbSC children (Figure 1). This result was not statistically different from the control group of 22 healthy children (p=0.96) nor in subgroups < or ≥ 5 years old, indicating that children with HbSC under 10 years have a preserved splenic function. Expectedly, the HJB-RBCs counts differed significantly from splenectomized children (419 (296-489)/100.000 RBCs, n=15, p By contrast, among the 53 HbSS children, the median HJB-RBCs count was 134 (29-216) /100.000 RBCs, differing significantly from HbSC children (p The result of this study suggests that spleen function in children under 10 years old with HbSC is not altered. The routine administration of prophylactic penicillin to young children with SC disease may therefore be questioned. Similarly, fever in children with HbSC under 3 years old may not require parenteral antibiotics as it is generally currently recommended by analogy to children with HbSS. Functional or anatomical asplenia in children with HbSC is delayed compared to those with HbSS at least after the first decade of life. Future large cohort studies using similar methodology will allow better evaluation of the pneumococcal risk in adolescents and adults with Hb SC disease. Bibliography (1) El Hoss S, Dussiot M, Renaud O, Brousse V, El Nemer W. A novel non-invasive method to measure splenic filtration function in humans. Haematologica. oct 2018;103(10):e436-9. Figure 1 Figure 1. Disclosures El Nemer: Hemanext: Consultancy.

Sickle cell disease (SCD) is a severe hemoglobinopathy due to abnormal hemoglobin S (HbS). One of the most serious complications of SCD is cerebral vasculopathy (CV) leading to ischemic stroke in 8% of homozygous SS children without prevention strategy. Typical CV in SCD is a stenosis of one or more intracranial or cervical arteries, including internal carotid arteries, anterior cerebral arteries and middle cerebral arteries. However, several studies have reported the occurrence of stroke in SCD children in the absence of typical cerebral vasculopathy. The aim of the study is to investigate the prevalence of stroke without CV in SCD children, and to compare them to "classical" stroke (i.e in a context of typical CV) in terms of suspected etiologies, triggering events, treatment and risk of recurrence. In a large cohort of about 1500 SCD children living in Paris area, France, we recorded all the strokes occurring between 2007 and 2020, excluding venous thrombosis and PRESS Syndrome. These children were followed in a university hospital, and all benefited from early screening for cerebral vasculopathy and an adapted stroke prevention program. We considered as a "typical" stroke any new ischemic lesion of the cerebral parenchyma associated with an acute neurological syndrome occurring in the territory of a pre-existing and/or non-regressive stenosis. 25 strokes occurred during the study period. 12 of them (48%) did not met the definition of a "typical" stroke related to sickle cell CV. The children with "atypical" stroke were older (9 years old +/- 4.6, vs 6.5 years old +/- 4.3 in the typical group, p = 0.0086) and less frequently of SS genotype (33% non-SS vs 8% non-SS in the typical group). They had lower leukocyte count (11.3 G/L +/- 4.6, vs 15.7 G/L +/- 2.3 in the typical group, p=0. 04) and higher hemoglobin level at the time of the stroke (11 g/dl +/- 3 vs 7.4 g/dl +/- 1.3 in the typical group, p= 0.027). 17% of atypical strokes had posterior ischemic lesions, 33% had anterior lesions and 17% had multiple systematized lesions, in counting junctional lesions. We also found 33% of ischemic lesions of the cerebellum. Considering a potential trigger of the stroke, 58% of atypical events were hospitalized in an anesthesia or intensive care unit at the time of the stroke or less than 7 days before, compared to 8% of children with a "classic" stroke (p= 0.011). The etiologies adopted by clinicians and radiologists for the atypical stroke were reversible cerebral vasoconstriction syndrome (RCVS) (Figure 1), cerebral fat embolism, hyperviscosity and vasculitis in systemic inflammatory context. The evolution in the atypical group was more favorable in terms of recurrence (0% within 2 years vs 42 % in the typical group, p= 0.045), although only 33% (3 children) of atypical strokes were still under exchange transfusion program 24 months after the stroke vs 92% in the group of typical stroke. Overall, in a cohort of SCD children with efficient stroke prevention program, atypical strokes account for nearly half of all acute ischemic neurological events, related to hyperviscosity, RVCS or inflammatory vasculitis. Physicians must be aware of the potential triggers and of the context in which such events occur. Nevertheless, stroke without CV may not require long-term transfusion program contrary to stroke with CV, given the very low risk of recurrence we highlighted. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Sickle cell disease (SCD) is a severe hemoglobinopathy due to abnormal hemoglobin S (HbS). Although red blood cell dysfunction is at the core of the SCD pathophysiology, several studies have highlighted the important role of inflammatory cells like neutrophils. One of the most serious complications of SCD is cerebral vasculopathy (CV), due to the occlusion of one or more intracranial or cervical arteries. In 1998, the STOP study demonstrated that monthly blood transfusions could reduce the risk of stroke by 90% in children with CV. However, there is large heterogeneity in the evolution of CV under chronic transfusion, sometimes requiring exchange transfusion (ET) program for years without succeeding in healing the CV. The aim of the study is to investigate the impact of long-term transfusion program on neutrophil dysfunction, in order to understand if persistent inflammation could contribute to the non-healing of CV despite HbS permanently below 40%. In SCD children undergoing ET program for at least 1 year, we analysed i)the phenotype of neutrophils with 8 markers of activation/adhesion/ageing, ii)the plasmatic levels of elastase, witnessing the NETose activity of neutrophils, and iii)the ex-vivo adhesion of neutrophils on activated endothelial cells. One hundred and two SCD children with an ET transfusion program for at least 6 months because of CV were included in the study. ET session, carried out every 5 weeks and most of the time by erythrapheresis, reached their biological objectives with a mean HbS rate after ET session of 14.1%, and 35.4% before the next ET session, which means that these patients globally live at an average HbS level of 24% for at least 1 year. We managed to limit iron overload with a mean ferritinemia of 207 µg/L in the whole cohort. Despite these satisfactory results in terms of HbS reduction, the efficiency in curing the CV was modest in accordance with the previously described efficiency of ET program in SCD children: after a mean ET program duration of 4.4 years only 22% of them had an improvement of their CV since the beginning of the ET program, while 60% of them had a stagnation of their CV, and 18% of them worsened their vascular lesions. Considering inflammatory parameters, the patients had persistence of high leukocytosis and high neutrophils count (respective mean of 9810 G/L and 5742 G/L), significantly not different of neutrophils count before inclusion in the ET program. In a random subgroup of 20 patients, we analysed neutrophils phenotype, NETose and endothelial adhesion and compared them to healthy controls and SCD children without ET, treated or not with Hydroxyurea (HU). Overall, we observed as expected an activated, aged and adherent profile of neutrophils from untreated SCD children compared to healthy controls, characterized by an overexpression of CD18/CD11b (p=0,03), CD18/CD11a (p=0,02), CD162 (p=0,01), CD66a (p=0,01) and the ageing markers CD184 high/CD62Llow (p=0,04) as well as a higher plasmatic level of elastase (p=0. 01) and higher adhesion of neutrophils to endothelial cells. All these parameters were alleviated in SCD patients treated with HU. In SCD patient undergoing ET program, we found a similar profile of activated neutrophils to that of untreated SCD patients with a similar expression of activation molecules, high level of elastase and the same increase of neutrophils adhesion to endothelial cells compared to controls, witnessing a persistence of chronic inflammation despites years of ET. Overall, our study highlights that the replacement of sickle red blood cells, even for years, is not sufficient to reverse the deleterious inflammatory phenotype of neutrophils. Given the major role of inflammation in endothelial dysfunction, these could contribute to the persistence of CV in a majority of patients despite efficient ET programs. This raises the question of systematically combining ET program with anti-inflammatory treatment such as HU or P-selectin inhibitors in children with CV. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Le phenotype erythrocytaire Emm- a ete decrit en 1973 mais reste l’un des derniers groupes sanguins de base genetique inconnue (antigene de prevalence elevee de la serie 901). Il avait ete propose qu’Emm serait porte par une proteine ancree a la membrane du globule rouge (GR) par un glycosylphosphatidylinositol (GPI) car l’expression de cet antigene est fortement reduite dans les GRs de patients atteints d’hemoglobinurie paroxystique nocturne (HPN). Cette maladie est causee par des mutations somatiques du gene PIGA, implique dans la synthese du GPI. En utilisant une approche genomique basee sur le sequencage d’exome chez 3 rarissimes individus Emm- (dont le cas index), nous avons identifie des mutations homozygotes sur un gene commun implique dans la synthese du GPI, PIGG. En utilisant l’approche Crispr/Cas-9, nous avons developpe des lignees cellulaires deficientes pour plusieurs genes PIGs impliques dans la synthese et le trafic des proteines ancrees au GPI. L’analyse de ces cellules par cytometrie en flux et western blot en utilisant un anticorps anti-Emm a montre que Emm est porte par un GPI libre et que l’epitope est forme par le 2e et 3e ethanolamine du squelette du GPI. Nous avons montre que l’anticorps anti-Emm detecte parfaitement les defauts de synthese du GPI, causes par des mutations germinales dans les genes PIGs et responsables de pathologies neurologiques severes. Il est a noter que le cas index etait atteint d’un deficit intellectuel et que l’absence de PIGG semble donc etre associee a des troubles du developpement du systeme nerveux central. Ce travail a permis d’etablir Emm comme un nouveau systeme de groupe sanguin chez l’Homme (n°42, ISBT 2020) ( Fig. 1 ).

BACKGROUND: There is a variability of the results in the published prognostic sickle cell disease (SCD) studies. The aim of this article is to assess and demonstrate how the methods used for analysis of laboratory parameters impact the prognostic study results in children with sickle cell disease. METHODS: Firstly, a systematic review of published studies was conducted to identify the methods used in statistical analyses on laboratory parameters. Secondly, four different identified analysis were successively applied on laboratory data collected prospectively in our cohort of newborns with SCD in order to evaluate the association between laboratory parameters and cerebral vasculopathy, depending on the analysis strategy. RESULTS: Thirty-one studies were included in the systematic review in which hemoglobin and reticulocyte counts were the most frequently collected parameters. Through this review, we noted four different statistical strategies. In almost one half of the cohort studies, laboratory parameter was collected only one time per patient. When several values were available, only one statistical analysis considered the time variation. When we applied these four different statistical strategies to our cohort of SCD children, we highlighted that the conclusion on prognostic value of parameters (either as protective or as risk factor) depends on the analysis strategies applied. CONCLUSION: The laboratory parameter prognostic value depends on the analysis strategy chosen for its analysis. It raises fundamental questions about methodological approaches that concern clinician researchers. Identifying prognostic factors is currently essential for better understanding of SCD. Taking into account longitudinal data and standardization of analyses in prognostic cohort studies should allow drawing valuable and reproducible conclusions.

BACKGROUND Chronic exchange transfusion is effective for primary and secondary prevention of stroke in children with sickle cell anemia (SCA). Erythrocytapheresis is recognized to be the most efficient approach; however, it is not widely implemented and is not suitable for all patients. The aim of our study was to compare automated exchange transfusion (AET) with our manual method of exchange transfusion and, in particular, to evaluate the efficacy, safety, and cost of our manual method. STUDY DESIGN AND METHODS Thirty-nine SCA children with stroke and/or abnormal findings on transcranial Doppler were included in the study. We retrospectively analyzed 1353 exchange sessions, including 333 sessions of AET and 1020 sessions of manual exchange transfusion (MET). RESULTS Both methods were well tolerated. The median decrease in hemoglobin (Hb)S per session was 21.5% with AET and 18.8% with our manual method (p

Nitrous oxide (N2O) is widely used as an anesthetic or an analgesic. N2O prolonged and recurrent administration is known to affect vitamin B12 metabolism with subsequent clinical consequences. We report herein the case of a 13-year-old girl with sickle cell disease exhibiting severe neurological and biochemical signs of functional vitamin B12 deficiency due to prolonged and repeated exposure to N2O. This was an incentive to prospectively investigate functional vitamin B12 deficiency in patients affected by sickle cell disease regularly exposed to N2O. We measured plasma concentrations of vitamin B12, total homocysteine, methionine and methylmalonic acid in 39 patients with sickle cell disease between 2015 and 2016. No patients developed neurological symptoms related to N2O administration but 19 patients (49%) had biochemical abnormalities suggesting mildly disturbed vitamin B12 metabolism e.g. decreased B12 vitamin, hypomethioninemia, or slightly increased methylmalonic acid or homocysteine. The clinical case highlight the potential severe deleterious effects of N2O over exposure on B12 vitamin metabolism in particular in patients affected with sickle cell disease. Conversely, when used without excess even repeatedly, there seem to be no overt clinically relevant abnormalities in vitamin B12 metabolism as observed on the cohort of 39 sickle cell disease affected patients., Highlights • Nitrous oxide (N2O) overexposure has a deleterious impact on vitamin B12 metabolism. • Regular use of N2O in sickle cell disease patients can induce minor abnormalities in vitamin B12 metabolism. • N2O administration should be carefully monitored and quantified as any drug. • Functional vitamin B12 deficiency should be investigated prior to N2O administration in at-risk patients.

Tuberculosis (TB) and sickle cell anaemia (SCA) may affect the same population of patients, particularly in Africa but also in high-TB incidence areas in developed countries. However, few data are available from children with SCA who develop TB. The aim of this study was to describe the clinical features and outcome of TB diagnosed in children with SCA. We conducted a retrospective, descriptive study in three referral centre of Sickle Cell Disease in Paris, France. We included 11 patients with SCA who develop TB. The median age at TB diagnosis was 11 years [7.5-14.5]. Two patients were asymptomatic and nine patients were symptomatic. Six patients had pulmonary TB (pulmonary, pleural and mediastinal lesions). Five patients had extrapulmonary TB (osteoarticular TB, hepatic TB, cervical and mediastinal TB). Mycobacterium tuberculosis was isolated in four of the 11 cases. All patients recovered after a median of 6 months of anti-TB treatment. The localisation of TB and outcome after treatment in our SCA patients were similar to the one observed in an age-and sex-matched control group of non-SCA patient with TB.despite the low number of patients included in our study, SCA does not seem to be a risk factor for severe TB. What is Known: • Tuberculosis (TB) remains a global health problem particularly in developing countries, and Sickle cell anaemia (SCA) is currently one of the most common genetic diseases in the world that mainly affects African populations. • Very few data are available on TB in SCA patients. What is New: • The features of TB in children with SCA seem to be comparable to those expected in general population, with favourable outcomes in response to standard treatment. • Monitoring the dosage of anti-TB treatments could be of interest because of the possible impact of SCA on drug metabolism.