ALBERT

Description: Production of abnormal hemoglobin (HbS) in sickle-cell disease (SCD) results in its polymerization in deoxygenated conditions and in sickled-RBC formation. Dense RBCs (DRBCs), defined as density 〉1.11 and characterized by increased rigidity, viscosity and HbS concentration (main polymerization factor), are absent in normal AA subjects, but present at percentages that vary from 1 SCD patient to another but remain stable throughout adulthood for each patient. Polymerized, but not nonpolymerized, HbS has reduced affinity for oxygen, demonstrated by the rightward shift of the oxygen-dissociation curve, leading to disturbances in oxygen transport. We recently described a correlation between %DRBCs and some clinical SCD manifestations. Notably, some SCD patients have unexplained, very low oxygen saturation (SpO2), without heart or lung dysfunctions. %DRBC variability within SCD patients could be the main pathophysiological explanation of those manifestations. This study was undertaken to determine whether a link exists between the %DRBCs and Hb affinity for oxygen, and to look for a potential clinical implication for SCD patients. 92 patients (44.6 ± 7.7 years; 51 women and 41 men) were included in the study. Blood samples were obtained at steady state to measure hemorheological and hematological parameters. Using a Percoll-gradient fractionation method, total RBCs were separated into non-DRBCs (NDRBC) (d1.11) fractions. The %DRBCs was determined using the phthalate-gradient method. P50 in venous blood gases was measured with a radiometry analyzer. Oxygen-affinity curves of Hb dissociation and association in RBC fractions were obtained with dual wavelength spectrophotometry. All patients had a 6-minute walking test (6MWT) and 10 of them (38.1 ± 6.1 years; 6 men and 4 women) had done so before and after 〉6 months (〉6M) on hydroxyurea (HU). Times 75th centile), were analyzed for the times

Description: Introduction: We recently proposed that B-cell depletion in immune thrombocytopenia (ITP) promotes the generation of long-lived plasma cells in the spleen, some of them being auto-reactive; but it remained possible that this observation was related to ITP itself rather than to B-cell depletion. Primary warm autoimmune hemolytic anemia (wAIHA) is a rare disease characterized by IgG auto-antibodies directed against antigens at the surface of red blood cells (RBCs) antigens, leading to their accelerated destruction. The use of B-cell depletion in wAIHA leads to 60 % to 70% of overall response at one-year and beyond. Nevertheless, 30-40 % of patients may resist to rituximab and then require a splenectomy. Nothing is known about antibody secreting cells (ASC) in the spleen of wAIHA patients, who have previously been treated or not with rituximab. In this study, we analyzed at the single cell level the splenic ASC from patients with chronic and active wAIHA, previously treated or not with rituximab (RTX), and we compared them with splenic ASC from ITP patients, and with splenic and bone marrow plasma cells from healthy donors (HD). Methods: We took advantage of the different therapeutic outcomes to analyze the splenic B-cell compartment of wAIHA patients, not previously treated with RTX (n=6), or after failure RTX treatment (n=3).Splenic tissues from organ donors and bone marrow from cardiovascular thoracotomy were used as controls. Blood samples from wAIHA (n=19), and (HD) (n=8) enrolled in this study were obtained after giving written informed consent in accordance with the Declaration of Helsinki. Results: We observed by flow cytometry and microscopy that the spleen from wAIHA patients who received less than 3 months of steroid therapy was the site of a B-cell response characterized by the presence of Bcl6+ germinal-center (GC) B-cells. Furthemore, splenic ASC secreted anti-red blood cell IgG in vitro. In line with this observation, we observed in the peripheral blood from patients with a newly diagnosed wAIHA (n=11), that short-lived IgG plasmablasts were increased compared with HD (n=8) (Mean 4.2 ± 0.84 % vs 0.99 ± 0.19% of CD19+ cells, p〈 0.01). Moreover, for patients receiving long term steroid therapy (〉 6 months) the plasmablast response was suppressed in the peripheral blood (Mean: 0.68 ± 0.2 % of CD19+ cells, n=8) and the splenic GC B-cell reaction was impaired (n=3). We conclude that short-lived IgG ASC result from an over-activity of GC reactions in wAIHA. We then analyzed the spleen of 3 patients who failed to respond to RTX, and observed a residual population of CD19+B-cells (median: 0.9% of lymphoid cells), including non-proliferative memory B-cells and plasma cells (PC). A fraction of these residual PC secreted anti-red blood cells IgG in vitro, thus accounting for the faillure of the B-cell depletion therapy. By using a single cell multiplex quantitative RT-PCR (Fluidigm dynamic arrays), we showed that such RTX-resistant plasma cells display a long-lived transcriptional program, which differs from PC from untreated wAIHA patients or HD, as well as from plasmablasts. Interestingly, the gene expression profile of wAIHA long-lived plasma cells segregated with long-lived PC previously observed in the spleen of ITP patients treated with rituximab. By a principal component analysis, we observed a gradient of maturation from plasmablasts to bone marrow plasma cells in which PC from RTX-treated spleens segregated close to bone marrow PC. We also observed that the cytokine BAFF was increased in the supernatants of spleen cell cultures from wAIHA patients treated with rituximab compared with controls (p〈 0.05), suggesting, in keeping with our previous report in ITP, a role for BAFF in the differentiation of short-lived plasma cells into long-lived plasma cells. Conclusion: The presence of splenic long-lived autoreactive PC in wAIHA may explain why some patients cannot achieve a response after RTX. Our results show that, the B-cell depletion induced by rituximab itself, as opposed to the underlying auto-immune condition, promotes a suitable environment for the maturation of auto-immune long-lived plasma cells in the spleen. Targeting specifically some factors such as BAFF right after rituximab injection could be an interesting therapeutic option in the future. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: Fetal hemoglobin (HbF) expression is a major modulator of sickle cell disease (SCD) severity by decreasing the HbS polymerization. However, the distribution of HbF in red blood cells (RBC) is heterogeneous in SCD patients. In the hypothesis of an HbF "threshold" in RBCs for inhibiting the HbS polymerization, accurate measurement of the HbF content in each red blood cell (HbF/RBC) is mandatory. To this purpose we developed a new and accurate method allowing the direct measurement of HbF content per RBC. Thanks to it, as a proof of concept, we analyzed HbF distribution and content in RBC from SCD patients before and after 6 months of treatment by hydroxyurea (HU). To determine if a threshold of HbF/RBC could modulate SCD, we analyzed the associations between the %RBC reaching different thresholds of HbF (in picograms), and biological parameters and the incidence of severe VOC. Methods: 14 SCD (βS/βS or βS/β0) patients were included to study HbF distribution during HU for a period of 6 months. RBCs were collected during each outpatient visit (Week 0, Week 2, Week 4, Week 12, and ≥ Week 24). HbF content was measured in RBCs using an anti-Human-HbF antibody by flow cytometry. Normalized RBC fluorescence intensity was then converted in picograms of HbF/RBC by using the linear association between mean HbF content and mean RBC fluorescence obtained from subjects presenting homogeneous HbF distribution (patients with hereditary persistence of HbF (HPFH), or β-Thalassemia or δβ-Thalassemia). Quantitative analysis were performed before and during HU treatment to characterize the response by comparing percentages of RBC classes based on different ranges of HbF/RBC during HU treatment. We therefore analyzed the associations between HbF/RBC thresholds (%RBC containing at least 2, 4, 6, 8, 10 or 20 pg HbF) and biological parameters before and ≥ 6 months of HU treatment. Finally HbF/RBC thresholds at Week 0 were compared to the incidence of hospitalized VOC within 3 years before W0, and HbF/RBC thresholds at Week 24 were compared to the incidence of hospitalized VOC within 3 years after W24 at a stable dose. Results: After 6 months of HU, mean %HbF, assessed by HPLC, raised from 6.16% (±3.5) to 15.2% (±8.7) (mean ± standard deviation). Quantitative analysis of HbF/RBC revealed a statistically significant decrease between D0 and ≥M6 of 24% of RBCs containing less than 2 pg (p = 0.0015) and a 2-fold increase of RBCs containing between 2 and 4 pg (p = 0.0025) (Friedman test). For biological parameters we observed an increase in mean %HbF, MCV and MCH and a decrease in RBC count significantly associated (p