Red Blood Cell Alloimmunization Research Articles

Alloimmunization to Erythrocyte Antigens in Patients Receiving Multiple Blood Transfusions

Background and Objectives: The risk of red blood cell (RBC) alloimmunization is always a concern for multi-transfused patients. Various factors have been found to be associated with alloimmunization. Alloimmunization rates were reported from 4% to 50%, 1.9% to 13% and 1.27% to 13.1% in thalassemic, onco-hematology and renal patients respectively. Transfusion of extended phenotype-matched blood was found to reduce rate of alloimmunization. This study investigated the various risk factors associated with RBC alloimmunization and the impact of transfusing phenotype-matched PRBC to multi-transfused patients. Methods: The retrospective observational study included 4350 multi-transfused patients of hemoglobinopathies, onco-hematological diseases and chronic renal failure (CRF). Pre-transfusion testing that included ABO and Rh(D) typing, antibody screening and crossmatching were performed following departmental standard operating procedure (SOP). Rh (C, c, E, e) and Kell (K) antigen phenotyping of blood donors as well as patients were performed by recommended techniques. Statistical analysis was done using the SPSS statistical package. Results: The mean age of multi-transfused patients was 41.4 years with a female preponderance. A total of 122 alloantibodies with various specificities were found in the 4350 patients (2.8%). Anti-E was the most frequent alloantibody (27.87%) followed by anti-c (11.48%). Frequency of alloimmunization in patients with hemoglobinopathies receiving phenotype-matched blood was 1.89% which was significantly lower than those receiving phenotype-unmatched blood (p=0.0019). Gender, age, splenomegaly status, number of PRBC unit transfused, phenotype-matched status, and transfusion duration in years were independent risk factors of alloimmunization. Conclusion: Alloimmunization to red blood antigens challenge the proper management of multi-transfused patients. Based on the present results we suggest considering antibody screening as an important tool of pre-transfusion testing to ensure safe blood transfusion. It will be prudent to adopt a transfusion policy that provides at least the Rh-Kell phenotype-matched blood to minimize red cell alloimmunization among multi-transfused patients.

HLA and HNA Antibodies Are More Prevalent in Multiparous Women with Red Blood Cell Alloimmunization

Background: Leukocyte antibodies against HLA class I, class II and human neutrophil antigens (HNA) are formed from exposure to these antigens by transfusion, pregnancy or transplant, and may be associated with transfusion-related acute lung injury (TRALI) and immune neutropenias. Risk factors for acquisition of leukocyte antibodies are yet to be determined and present great importance for the prevention of severe and fatal transfusion reactions.Aims: To compare the prevalence of leukocyte antibodies in women with or without red blood cell (RBC) alloimmunization.Methods: This transversal study included women whose only stimulus to alloimmunization was pregnancy, with no transfusion history. We analyzed 147 blood samples from women with RBC alloimmunization, confirmed by the investigation and identification of RBC antibodies by the DG Gel-Card technique (Grifols-Spain). The control group consisted of 563 blood donors women with pregnancy history but without RBC alloimmunization. The identification of leukocyte antibodies was performed by: 1) granulocyte agglutination test (GAT), 2) white cell immunofluorescence test (Flow-WIFT), both tests using a panel of neutrophils obtained from three donors with previous HNA genotyping, and 3) bead-based assay - LABScreen Multi (LSM) (One Lambda), capable of detecting antibodies against HNA-1a, -1b, -1c, -2, -3a, -3b, -4a, -5a, -5b and HLA class I and II antigens. The identified HNA antibodies were confirmed by genotyping the corresponding antigen.Results: In the cohort of women with RBC alloimmunization we found 179 RBC antibodies: anti-D (30.7%), anti-Lea (21.3%), anti-C (15.1%), anti-E (8.9%), anti-K (5.6%), anti-M (5.6%) and other antibodies (12.8%). We identified 74/147 (50.3%) samples with anti-HLA antibodies and 11/147 (7.5%) samples with anti-HNA antibodies with the following specificities: 5/11 anti-HNA-1a, 2/11 anti-FCγRIIIb and 4/11 anti-HNA-3b. In the control group, we found 238/563 (42.3%) samples with anti-HLA and 24/563 (4.3%) samples with anti-HNA: 8/24 anti-HNA-1a, 01/24 anti-HNA-1c, 03/24 anti-HNA-2, 02/24 anti-HNA-3a, 5/24 anti-HNA-3b, 03/24 anti-HNA-5a and 02/24 anti-HNA-5b. Regarding the gestational history, 82/147 (55.8%) women in the group with RBC alloimmunization and 493/563 (87.6%) in control group had two or more pregnancies. The statistical analysis was performed in total samples, regardless the number of pregnancies, however no significant difference was observed in the prevalence rate of anti-HNA and anti-HLA between the two groups (p=0.10 and p=0.07, respectively). When the analysis was stratified including only multiparous women (two or more pregnancies) the prevalence of HNA alloimmunization was statistically significant in the group of women with RBC alloimmunization: 9/82 (11%) versus 23/493 (4.7%) blood donors, p=0.02, OR=2.52 (95% CI=1.15-3.79). Similarly a higher prevalence of HLA antibodies in the group of multiparous women with RBC alloimmunization was also observed: 51/82 (62.2%) versus 224/493 (45.4%), p=0.004, OR=1.97 (95% CI=1.18-2.71).Conclusions: The data show that RBC alloimmunization was significantly associated with the development of antibodies against leukocytes (anti-HNA and anti-HLA). A higher frequency of HNA alloimmunization (11%) observed in the group of multiparous women with RBC alloimmunization compared to the control group (4.7%), suggest that they are better immune responders and that they react strongly to allogeneic exposure. DisclosuresNo relevant conflicts of interest to declare.

Marginal Zone B Cells Facilitate RBC Alloimmunization Independent of Direct CD4+ T Cell Activation

Background: Blood transfusions decrease complications in patients with bone marrow failure syndromes, hemoglobinopathies, or neoplasia. However, patients who receive this often life-saving treatment can develop alloantibodies against non-ABO red blood cell (RBC) antigens. RBC alloimmunization can have severe clinical consequences, including increased morbidity and mortality secondary to difficulty obtaining compatible RBCs for future transfusion and an increased risk for hemolytic transfusion reactions.Aside from Rh(D) immune globulin, there are no prophylactic treatments available to prevent RBC alloimmunization. Improved understanding of the immune mechanism underlying RBC alloimmunization is central in developing additional prophylactic treatments. Recent studies suggest that marginal zone (MZ) B cells are critical for immune responses to RBC alloantigens. As a result, we sought to determine mechanisms whereby MZ B cells mediate RBC alloimmunization.Methods: RBCs expressing the HOD antigen [hen egg lysozyme, ovalbumin (OVA), and human Duffy] were labeled with DiI and transfused into C57BL/6 recipients, followed by confocal examination for HOD RBC localization by staining sections for CD1d, MadCAM and B220. HOD RBCs were also transfused into C57BL/6 recipients treated with anti-CD4 T cell (clone: GK 1.5), anti-MZ B cell (anti-CD11a and anti-CD49d) or isotype control followed by examination of anti-HOD IgM and IgG antibodies on days 5 and 14 post-transfusion by flow cross-match. To determine the impact of B cells in general and MZ B cells in particular on CD4+ T cell activation following HOD RBC transfusion, CFSE labeled splenocytes from OTII transgenic mice with CD4+ T cells specific for HOD RBCs were adoptively transferred into B-cell deficient μMT mice, MZ B cell depleted C57BL/6 recipients, C57BL/6 controls, and mice genetically deficient in MZ B cells followed by transfusion of HOD RBCs. On days 1 and 3 post-transfusion, OTII proliferation, activation and cytokine profile were assessed using flow cytometric analysis. To examine the role of MZ B cells in antigenic trafficking, C57BL/6 recipients were administered FTY720, which displaces MZ B cells from the marginal sinus, or PBS, followed by HOD RBC transfusion and analysis for anti-HOD antibody formation.Results: HOD RBCs initially localized within the marginal sinus following transfusion, where they displayed significant co-localization with MZ B cells. While depletion of MZ B cells inhibited anti-HOD IgM and IgG antibody formation, CD4+ T cell depletion only prevented anti-HOD IgG antibody development. Despite the requirement of MZ B cells in anti-HOD antibody formation, CFSE-labeled OTII CD4+ T cells showed proliferation as well as evidence of key markers of T cell activation following transfusion of HOD RBCs into MZ B cell depleted, genetically deleted or B cell deficient μMT recipients, strongly suggesting that the requirement of MZ B cells for IgG antibody formation does not reflect MZ B cell-mediated CD4+ T cell activation. Given the localization of HOD RBCs with MZ B cells and the ability of CD4+ T cells to proliferate in the absence of MZ B cells, we next determined whether the requirement of MZ B cells for anti-HOD IgG antibody formation may actually reflect MZ B cell-mediated trafficking of HOD RBCs to the B cell follicle. Consistent with this possibility, MZ B cell displacement from the marginal sinus to the splenic follicle, which prevents MZ B cell-mediated trafficking of antigen, prevented formation of anti-HOD IgM and IgG following HOD RBC transfusion.Conclusion: The ability of CD4+ T cells to undergo proliferation and activation following HOD RBC transfusion in the absence of MZ B cells suggests that the requirement of MZ B cells for antibody formation does not reflect MZ B cell-mediated CD4+ T cell activation. In contrast, the localization of HOD RBCs with MZ B cells, coupled with the ability of MZ B cell displacement to prevent HOD RBC-induced alloimmunization, strongly suggests that MZ B cells regulate RBC alloimmunization by initially trapping and the trafficking RBC alloantigens to the B cell follicle. In doing so, MZ B cells appear to be a key initiator and overall regulator of RBC alloimmunization and therefore a potential target for prophylactic measures designed to inhibit this process. DisclosuresNo relevant conflicts of interest to declare.

Epidemiologic Characterization of Transfusion Recipients with Red Blood Cell Alloantibodies from Four US Regions: Evidence from the REDS-III Recipient Database

Introduction: Red blood cell (RBC) alloimmunization is one of the most common complications of RBC transfusion. Despite the fact that alloimmunization impacts many transfusion recipients, the lack of standardized alloimmunization registries in the US has prevented large studies from being completed. Through the development of a uniform electronic research database, we investigated alloimmunization rates and diagnoses in inpatients and outpatients across the US.Methods: The REDS-III recipient database, established in 2013, houses demographic, diagnostic, transfusion, laboratory, and other information on inpatients and outpatients at 12 hospitals in 4 geographic regions. For this study, an antibody “responder” (R) was defined as an individual who had a putative transfusion (or pregnancy)-associated red cell antibody identified by screen at any point during the study period, independent of documented RBC exposure or pregnancy during the study period. An antibody “non-responder” (NR) was defined as an individual who was transfused with at least one unit of RBCs during the study period, and had a negative antibody screen at least 15 days post-transfusion. Anti-D antibodies were excluded from studied females, given an inability to distinguish passively administered anti-D in the database from authentic alloantibodies. Demographics, blood type, ICD9/10 codes, and antibody screen results were compared for R and NRs. Odds ratios were calculated for the likelihood of being a R or NR, based on the presence or absence of select chronic disease states.Results: 612,417 patients were recorded in the REDS recipient database from 2013-2016. 318,918 unique patients in the database had an RBC antibody screen during the study period. Of these, 6597 had at least one transfusion-associated RBC alloantibody identified by routine antibody screen (overall alloimmunization rate among all screened patients independent of RBC exposure = 2.07%). 63.8% of the R were female (alloimmunization rate 2.97%) and 36.2% were male (alloimmunization rate 1.35%). 30,468 unique patients (49.8% female and 50.2% male) were NR; the mean RBC exposure during the study period was similar between R and NR (mean, s.d., and median of 8.8, 18.2, and 4 units versus 9.3, 14.3, and 5 units). Approximately 60-70% of the antibodies in R were pre-existing, and the remainder were generated during the study period. Few children under 10 years of age were R compared to older age groups; an equation of R/R+NR = 26/26 + 467 = 5.3%. There were no differences in R vs NR by A/B/O blood type, though Rh negative patients (R/R+NR of 1175/1175 + 3901 = 23.1%) were significantly more likely to be R than Rh positive patients (R/R + NR of 5365/5365 + 26544 = 16.8%). .A total of 8892 clinically significant antibodies were detected, including 4749 (53.4%) in the Rh family (2588 anti-E, 780 anti-C, 556 anti-c, 105 anti-e, 525 anti-D), 1937 (21.8%) in the K family, 912 (10.3%) in the Kidd family, and 669 (7.5%) in the Duffy family, among others. Patients with sickle cell disease (n=554, OR 2.6, p<0.0001), sickle cell trait (n=117, OR 1.8, p=0.02), 'coronary atherosclerosis or heart disease (n=12,403, OR 1.2, p<0.0001), lupus (n=969, OR 1.3, p=0.002), rheumatoid arthritis (n=1213, OR 1.2, p=0.01), and myelodysplastic syndrome (n=1162, OR 1.3, p=0.0008) were more likely to be R than NR. In contrast, patients with leukemia (n=2801, OR 0.5, p<0.0001), solid tumors (n=17,513, OR 0.6, p<0.0001), and solid organ transplant (n=1491, OR 0.7, p=0.0001) were less likely to be R than NR.Conclusions: Data collected in this multi-center recipient database provide the largest RBC alloimmunized patient cohort ever studied in the US. The 2.07% overall rate of alloimmunization is similar to that reported in other retrospective studies, with females having higher alloimmunization rates than males. Rh negative individuals were significantly more likely to be alloimmunized than Rh positive patients, despite the exclusion of anti-D antibodies in females. Other unexpected findings include the predisposition to alloimmunization observed in patients with sickle cell trait and coronary artery disease. Ongoing studies are investigating the timing of RBC exposure to new antibody formation in this patient cohort, as well as potential physiologic explanations for the chronic disease associated findings. DisclosuresNo relevant conflicts of interest to declare.

Antibody-Mediated Immunosuppression Can Result from RBC Antigen Loss Independent of Fcγ Receptors in Mice

Background: Prophylaxis against red blood cell (RBC) alloimmunization can be achieved by Rh immune globulin (RhIg), a polyclonal anti-RhD antibody preparation used to prevent de novo anti-RhD alloantibody formation, a process commonly termed antibody-mediated immunosuppression (AMIS). As the availability of RhIg donors can be limited and the only available immunoprophylaxis is against RhD, there is a critical need to develop monoclonal antibody alternatives to RhD and immunoprophylaxis strategies in general to other RBC alloantigens. As different monoclonal antibodies to the RhD antigen can have differential immunological outcomes,understanding the mechanism regarding how monoclonal antibodies induce AMIS can facilitate the development and accurate assessment of monoclonal antibodies against RhD and other RBC alloantigens for appropriate therapeutic activity. Recent studies suggest that AMIS may occur through alterations in the target antigen, otherwise known as antigen modulation. However, several studies suggest that AMIS may occur independent of antigen modulation. In particular, AMIS to RBCs that transgenically express the HOD (hen egg lysozyme (HEL), ovalbumin and Duffy) antigen have been shown to occur independent of the activating Fcγ receptors thought to be required for modulation of the HEL antigen. Therefore, we sought to determine whether anti-HEL monoclonal antibodies can induce antigen modulation and whether AMIS to HOD RBCs occurs via monoclonal antibody-induced alterations to the target antigen.Methods: Using RBCs transgenically express the HOD antigen, we transferred HOD RBCs into C57BL/6 wild-type (WT) or FcγR-chain knockout recipients (FcγR KO) in the presence or absence of anti-HEL monoclonal antibodies individually or in combination. Serum was collected at days 3, 5, 7, 14, 21 and 28 post-transfusion. Antibody development in WT or FcγR KO recipients was examined by flow crossmatch, where serum was incubated with HOD RBCs. Following incubation, RBCs were washed and incubated with fluorescently-tagged secondary antibody and analyzed using flow cytometry. To determine the impact of antibody engagement, RBCs were sampled at 10 minutes and 2 hours, along with days 1, 2, 3 and 5 post-transfusion. The level of detectable HEL antigen, complement deposition, HOD RBC survival and bound antibody to the RBC surface was measured by flow cytometry.Results: A combination of anti-HEL antibodies suppressed anti-HOD IgM formation more effectively than monoclonal anti-HEL antibodies alone, which correlated with the rate of decrease in bound antibody and detectable HEL on HOD RBCs over time. Unexpectedly, combination or individual anti-HEL monoclonal antibodies not only induced AMIS, but also likewise induced antigen modulation in FcγR KO recipients. Loss of detectable antigen did not appear to reflect anti-HEL induced complement fixation and subsequent complement-mediated masking of the HEL antigen, as no complement could be detected on HOD RBCs when evaluated in parallel. Furthermore, regardless of anti-HEL antibody exposure, in combination or individually, anti-HEL antibodies failed to induce significant RBC clearance in WT or FcγR KO recipients. Pre-exposure of HOD RBCs to anti-HEL antibodies reduced antigen levels and likewise suppressed anti-HOD antibody formation following HOD RBC exposure.Conclusion: In summary, these results suggest that antibody-mediated antigen modulation may reflect a mechanism of AMIS that can occur independent of activating FcγRs and may provide a surrogate to identify antibodies capable of inducing AMIS against different RBC alloantigens. DisclosuresNo relevant conflicts of interest to declare.

Antigen matching for transfusion support in Brazilian female patients with sickle cell disease to reduce RBC alloimmunization.

Red blood cell (RBC) alloimmunization is a complication of patients with sickle cell disease (SCD) and it has a greater impact on pregnancy, leading to a risk of hemolytic disease of the newborn and reducing blood availability for pregnant women. This study proposed to evaluate antigen matching transfusion protocols, aiming to reduce RBC alloimmunization in Brazilian female patients with SCD. Samples from female patients with SCD (153) and self-declared Afro-Brazilian donors (307) were genotyped for RBC antigens and RH variants were investigated. The transfusion needs of patients during 1-year period and the number of compatible donors were assessed using three antigen-matching transfusion protocols: prophylactic CEK antigen-matched RBCs, prophylactic extended antigen-matched RBCs, and extended-matched red blood cells (RBCs) only for alloimmunized patients. In addition, RH molecular matching has been proposed for patients carrying variant RHCE. Provision of CEK antigen-matched donors would have been possible in 92.4% of transfusion events while provision of prophylactic extended antigen-matched RBCs would cover 88.7% of the transfusion events. Extended antigen matching for alloimmunized patients would be efficient in 99% of the cases. The presence of partial D in 10 patients increased the need of D-negative donors. Compatible donors could be enough for four of the five patients with altered RHCE genotypes in both alleles. In Brazilians, screening African descent donors allows the implementation of prophylactic CEK and extended antigen-matching transfusion protocols to female patients with SCD to reduce RBC alloimmunization; however, the supply of compatible blood can be impaired for patients with Rh variants.

Frequency Occurrence and Percentage Distribution of Rh C, Rh c, Rh E and Rh e Blood Group Amongst Pregnant Women Attending Antenatal Clinic in Port Harcourt, Nigeria

Background: The Rhesus (Rh) blood group is one of the most complex blood groups in humans comprising mainly of Rh D, C, c, E and e. However, only Rh D is routinely screened for in Nigeria despite the fact that other Rh antigens are clinically significant and can cause haemolytic disease of the newborn and delayed haemolytic transfusion reactions.&#x0D; Aim: The aim was to determine the frequency distribution of Rh C, c, E and e blood groups among pregnant women attending antenatal clinic in Port Harcourt, Nigeria.&#x0D; Study Design: The study consisted of 147 apparently healthy pregnant women within the age range of 18-45 years, attending a selected Primary Healthcare Centre (Obio Cottage Hospital) in Port Harcourt. The study was carried out from January 20, 2020 to March 27, 2020. The presence of Rh C, c, E and e blood groups were investigated using Anti-C, c, E and e monoclonal antibody in the same order.&#x0D; Results: Rh C, c, E and e were observed in 38.09%, 100%, 17.68% and 100% in the same order.&#x0D; Conclusion: The study indicated dominance of Rh c and Rh e over Rh C and Rh E among the studied pregnant women. It is necessary to take into cognizance the fact that the presence of Rh C, c, E and e antigens may be the cause of some delayed transfusion reactions and haemolytic disease of the foetus and newborn. Therefore, routine antigen typing for Rh antigens may help in decreasing red blood cell allo-immunization and delayed haemolytic transfusion reaction during pregnancy.

Screening for new red blood cell alloantibodies after transfusion in patients with sickle cell disease.

Patients with sickle cell disease (SCD) are frequent recipients of red blood cell (RBC) transfusions and are at risk for RBC alloimmunization. RBC alloimmunization is diagnosed by identifying RBC alloantibodies as part of pre-transfusion testing, but this testing fails to detect alloantibodies that have evanesced. It may be beneficial to screen for new RBC alloantibody development after transfusion before possible antibody evanescence. Our institution started a new initiative for episodically transfused patients with SCD to obtain at least one antibody screen 2-6 months after transfusion as part of their clinical care. A database was created to prospectively track all transfused patients for 1 year and their post-transfusion antibody screen results. Patients received prophylactically CEK-matched RBC units. During the study year, 138 patients with SCD received a total of 242 RBC transfusions. Patients with a history of an RBC alloantibody (n=13, 9.4%) had previously received more RBC units than non alloimmunized patients (median 11 vs. 2 RBC units, p=.0002). A total of 337 post-transfusion antibody screens were obtained in 127 patients (92.0%) with 110 patients (79.7%) having at least one antibody screen 2-6months post-transfusion. With this prospective testing, two new RBC alloantibodies (anti-C and -M) were identified in two patients. It is feasible to test for new RBC alloantibody development in most episodically transfused patients with SCD as part of their routine care. The yield of this screening appears low with CEK matching, but it could still provide important information for individual patients.

Maternal red blood cell alloimmunisation Working Party, literature review. RH blood group system: Rare specificities.

This report is part of a series reporting the GRADE review performed by the 2018-2020 French Working Party on maternal red blood cell alloimmunisation. This report focusses on the clinical significance in obstetrics, as published in the scientific literature, of the rare RH antibodies, variants and antigens (i.e. excluding conventional RH1 trough RH8 antigens, RH12, RH22 and RH27, which are discussed in other reports of this series). Extremely severe or severe haemolytic disease of the fetus and the newborn (HDFN), leading to death or requiring transfusions, have been reported for: anti-RH1 (-D) associated with DVI, DBT and DIVb phenotypes, RHD*12.04 (DOL4), RHD*03.03 (DIIIc), RHD*D-CE(2-5)-D, RHD*01EL.31 (RHD*148+1T), anti-RH9 (-CX), anti-RH11 (-EW), anti-RH17 (-Hr0), anti-RH18 (-Hr), anti-RH19 (-hrS), anti-RH23 (-DW), anti-RH29 ("total" Rh), anti-RH30 (-Goa), anti-RH32, anti-RH34 (-HrB), anti-RH36 (-Bea), anti-RH40 (-Tar), anti-RH46 (-Sec), anti-RH48 (-JAL), anti-RH54 (DAK), and antibodies to high prevalence antigens such as those associated with RHCE*02.08.02 (RHCE*CW-RHD(6-10)), RHCE*03N.01 (RHCE*cEMI). HDFN of moderate, mild or undetailed severity have been reported for: anti-RH1 associated with DHar, DIIIa and DIVa phenotypes, RHD*01EL.08 (RHD*486+1A),RHD*01EL.44 (RHD*D-CE(4-9)-D),RHD*25 (DNB), anti-RH20 (-VS), anti-RH31 (-hrB), anti-RH37 (-Evans), ani-RH42, anti-RH49 (-STEM), anti-RH51 (-MAR), anti-RH55 (-LOCR), anti-RH58 (-CELO). Positive direct antiglobulin test in the newborn but no clinically significant HDFN has been reported for anti-RH1 (-D) associated with RHD*10.05 (DAU5), RHD*12.02 (DOL2). Because so many specificities are associated with severe HDFN in the RH system, all RH antibodies should be considered as potentially able to cause HDFN, even if none has been reported yet.

Effect of intravenous immunoglobulins to postpone the gestational age of first intrauterine transfusion in very severe red blood cell alloimmunization: A case-control study

BackgroundEarly intrauterine transfusion (IUT) is associated with a higher risk of fetal loss. Our objective was to evaluate the efficiciency of intravenous immunoglobulins (IVIG) to postpone the gestational age at first IUT beyond 20 weeks of gestation (WG) compared to the previous pregnancy in case of very severe red blood cell (RBC) alloimmunization. Study design and methodsVery severe RBC alloimmunization was defined by a high titer of antibodies and a previous pregnancy complicated by a first IUT before 24 WG and/or perinatal death directly related to alloimmunization.We performed a single-center case-control study. Cases and controls were patients respectively treated with weekly IVIG infusions started before 13 WG, and without. ResultsTwenty cases and 21 controls were included. Gestational age (GA) at first IUT was postponed after 20 WG in 18/20 (90 %) of patients treated with IVIG and in 15/21 (71 %) in the control group (p = 0.24). Compared to the previous pregnancy, the GA at first IUT was postponed by a median of 22 [+11; +49] days in the IVIG group and occurred in average 2 days earlier [-17 ; +12] in the non-treated group (p = 0.02). There was no difference between number of IUT and need for exchange-transfusion. IVIG treatment was associated with a significant decrease of antibodies' quantitation. ConclusionIn our series, IVIG tends to differ first IUT beyond 20 WG and have a significant effect in postponing the gestational age of the first IUT in patients with very severe RBC alloimmunization.

Red blood cell alloimmunization in pregnancy: A 10-year single-center study

Background/Aim. Pregnancy-induced red blood cell (RBC) alloimmunization is important not only because of the possible negative effects on subsequent pregnancy outcomes in case the fetus carries the antigen but also because of the optimal trans-fusion management in cases of obstetric hemorrhage. Timely detection of RBC antibodies is part of testing, prevention, and treatment strategy, aimed at achieving better outcomes for alloimmunized mothers with an affected fetus. The aim of the study was to determine the frequency and specificity of alloantibodies among pregnant women from the South Backa District, Serbia, with special attention to the incidence of anti-D alloantibodies. Methods. A retrospective study was conducted in the Blood Transfusion Institute of Vojvodina and covered the period from January 1, 2010, to December 31, 2019. Screening and antibody identification were performed by an indirect antiglobulin test in gel-microcards (ID-Card Liss/Coombs) with two test RBC (ID-DiaCell I-II screening cells, Bio-Rad, Cressier, Switzerland) on an automated system (IH-500, Bio-Rad). Results. Among 25,694 tested pregnant women, 1.38% were actively immunized, while 1.12% of women acquired antibodies in the current pregnancy. Among 3,622 (14.09%) RhD-negative women, 1.77% produced anti-D antibodies during the ongoing pregnancy. Distribution of anti-body specificity was: anti-D 23.34%, anti-M 11.85%, anti-E 9.41%, anti-K 9.41%, anti-C 5.92%, anti-Fyb 5.92%, anti-c 3.13%, anti-S 3.13%, anti-Lea 3.13%, anti-Leb 3.13%, anti-Cw 1.75%, anti-Jka 1.40%, anti-P 1.05%, anti-Lub 0.70%, anti-Fya 0.35%, autoantibody of undetermined specificity 0.70%, and irregular antibodies of undetermined specificity 15.68%. Conclusion. Immunoglobulin prophylaxis has led to a significant reduction in the frequency of D-alloimmunization among pregnant women in the South Backa District over the last ten years. However, the incidence of anti-D antibodies is still significantly higher than in published data for developed countries. We also identified the other, less commonly present, clinically significant antibodies. There is a need to introduce uniform recommendations for immunohematological testing in pregnancy on the territory of the Republic of Serbia in accordance with modern requirements.

195 - Red Blood Cell (RBC) Allo-Immunization Risk Factors and Impact on Outcomes for Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) Patients

195 - Red Blood Cell (RBC) Allo-Immunization Risk Factors and Impact on Outcomes for Allogeneic Hematopoietic Stem Cell Transplantation (HSCT) Patients

Prevalence of Red Blood Cell Alloimmunization Among Beta-Thalassemia and Sickle Cell Anemia Patients: a Study from Saudi Arabia.

The periodic transfusion of red blood cells (RBCs) is an essential supportive therapy for beta-thalassemia and sickle cell anemia (SCA) patients. Nevertheless, this therapy can cause some adverse effects, such as RBC alloimmunization. This study aimed to determine the prevalence of RBC's alloimmunization and frequency of Rhesus (Rh) and Kell (K) antigens among SCA and beta-thalassemia patients in Al-Madinah region of Saudi Arabia. A cross-sectional study was conducted on 137 multiple transfusion patients with SCA and beta-thalassemia, who attended two of the largest hospitals located in Al-Madinah, Saudi Arabia. Demographic and medical data were collected from medical files of patients. Blood samples were collected from patients and Rh and Kell typing were done. Also, antibody screening and identification tests were performed. There were 66 (48.18%) pediatric patients with mean age of 7.8 years, while adult patients were 71 (51.82%) with mean age of 24.5 years. RBC alloantibodies were found in 9 patients (6.57%), and the most frequent alloantibody was anti-K. Antigen e (99.05%) was the most common antigen, while the least common was the antigen K (3.81%). The association between age (pediatric and adult) and alloimmunization rates was insignificant. Although the RBC alloimmunization rate among SCD and beta-thalassemia patients in this study is low compared to other studies in Saudi Arabia and countries all over the world, it still warrants more attention. Phenotyping of donors/recipients' RBCs for K and Rh subgroups may reduce the risk of alloimmunization and increase the efficiency of blood transfusion.

Association of multispecific red blood cell alloimmunization with HLA-Class II variants is related to Rh phenotypes.

It remains unclear, why only some patients form alloantibodies against foreign RBC antigens. Transfusion of red blood cell (RBC) products and pregnancy are the most relevant causes of immunization against RBC alloantigens. Here we investigated the relationship between RBC alloantibodies, Rh phenotype, and HLA phenotype among patients with multiple RBC alloantibodiesMETHODS: In a group of 124 multi-responders ‒ including both pregnant women and transplant recipients ‒ we analysed the distribution of HLA-Class II variants in subgroups of multi-responders to RBC alloantigens according to their Rh status. As expected, the RhD-negative phenotype was overrepresented in our alloimmunized group (49.2 %) compared to in the general population. Importantly, HLA-DRB1*15 carriers were significantly overrepresented among D-negative multi-responders compared to D-positive multi-responders (Pc = 0.045). Furthermore, the linked HLA-DRB1*13, HLA-DQB1*06, and HLA-DQA1*01 variants were more frequent in individuals with the DCCee phenotype than in other RhD-positive phenotypes. Our present findings showed that RBC multispecific alloimmunization was associated with particular HLA-Class II variants based on Rh status (Tab. 3, Ref. 22).

Association between renal failure and red blood cell alloimmunization among newly transfused patients.

BackgroundRenal failure and renal replacement therapy (RRT) affect the immune system and could therefore modulate red blood cell (RBC) alloimmunization after transfusion.Study Design and MethodsWe performed a nationwide multicenter case‐control study within a source population of newly transfused patients between 2005 and 2015. Using conditional multivariate logistic regression, we compared first‐time transfusion‐induced RBC alloantibody formers (N = 505) with two nonalloimmunized recipients with similar transfusion burden (N = 1010).ResultsRenal failure was observed in 17% of the control and 13% of the case patients. A total of 41% of the control patients and 34% of case patients underwent acute RRT. Renal failure without RRT was associated with lower alloimmunization risks after blood transfusion (moderate renal failure: adjusted relative rate [RR], 0.82 [95% confidence interval (CI), 0.67‐1.01]); severe renal failure, adjusted RR, 0.76 [95% CI, 0.55‐1.05]). With severe renal failure patients mainly receiving RRT, the lowest alloimmunization risk was found in particularly these patients [adjusted RR 0.48 (95% CI 0.39‐0.58)]. This was similar for patients receiving RRT for acute or chronic renal failure (adjusted RR, 0.59 [95% CI, 0.46‐0.75]); and adjusted RR, 0.62 [95% CI 0.45‐0.88], respectively).ConclusionThese findings are indicative of a weakened humoral response in acute as well as chronic renal failure, which appeared to be most pronounced when treated with RRT. Future research should focus on how renal failure and RRT mechanistically modulate RBC alloimmunization.

Red blood cell alloimmunization and sickle cell disease: a narrative review on antibody induction.

The high prevalence of red blood cell (RBC) alloantibodies in people with sickle cell disease (SCD) cannot be debated. Why people with SCD are so likely to form RBC alloantibodies, however, remains poorly understood. Over the past decade, a better understanding of non-ABO blood group antigen variants has emerged; RH genetic diversity and the role this diversity plays in RBC alloimmunization is discussed elsewhere. Outside of antigen variants, the immune systems of people with SCD are known to be different than those of people without SCD. Some of these differences are due to effects of free heme, whereas others are impacted by hyposplenism. Descriptive studies of differences in white blood cell (WBC) subsets, platelet counts and function, and complement activation between people with SCD and race-matched controls exist. Studies comparing the immune systems of alloimmunized people with SCD to non-alloimmunized people with SCD to race-matched controls without SCD have uncovered differences in T-cell subsets, monocytes, Fcγ receptor polymorphisms, and responses to free heme. Studies in murine models have documented the role that recipient inflammation plays in RBC alloantibody formation, with human studies reporting a similar association. Murine studies have also reported the importance of type 1 interferon (IFNα/β), known to play a pivotal role in autoimmunity, in RBC alloantibody formation. The goal of this manuscript is to review existing data on factors influencing RBC alloantibody induction in people with SCD with a focus on inflammation and other immune system considerations, from the bench to the bedside.

Erythrocyte Alloimmunization in Children with Sickle Cell Disease

Alloimmunization is an immune response against red blood cell (RBC) antigens due to sensitization during RBC transfusion. RBC alloimmunization is more common in sickle cell disease (SCD) with prevalence between 18 and 47%.

Genetic Basis of Erythrocyte Alloimmunization Among Children with Sickle Cell Anemia in the Dominican Republic

Introduction: In low-resource settings, red blood cell (RBC) transfusions for children with sickle cell anemia (SCA) may be cross-matched for only ABO and RhD antigens, so RBC alloimmunization can develop due to differences in Rh and other antigens between donors and recipients. In the Rh system, these differences can include absence of the antigen or the presence of partial or weak antigens, the latter more common with African ancestry. RBC alloimmunization in SCA has not been described in the Dominican Republic, and the genetic diversity of the RHD and RHCE loci have not been reported in this Hispanic-Afro-Caribbean population. Methods: A pediatric stroke prevention trial in the Dominican Republic (SACRED, NCT02769845) provided serum and dried blood spots from children with SCA, collected at enrollment and stored at -80C. Sera were screened for alloantibodies using a modified test-tube agglutination identification method with commercially available Panocell-20 (Immucor) RBC suspensions of known antigen phenotypes. Reaction patterns identified antigen specificity using standard blood bank methods. Patterns reactive with all cells tested were reported as pan-reactive and reactions without patterns were deemed low frequency antibodies. For all samples with an alloantibody, a matched SACRED control (age, sex, and transfusion history) was identified. Genomic DNA was analyzed by PCR and direct sequencing of each RHD and RHCE exon for single nucleotide polymorphisms (SNPs) that confer weak, partial, or variant phenotypes. Copy number variant PCR techniques with gene-specific primers were used to detect exonic substitutions in the RHD and RHCE gene loci that lead to negative or partial phenotypes. Results: A total of 272 SACRED participants were screened for RBC alloantibodies; 179 (66%) had at least one transfusion prior to enrollment, of whom 37 (21%) had a positive alloantibody screening result. Children with RBC alloantibodies (17 males, 20 females, average age 9.0 ± 2.4 years) had variable transfusion exposure with 23 (62%) having &amp;gt;10 prior transfusions, while 7 (19%) had ≤2 previous transfusions. Among the 37 positive samples, 18 sera had reactivity against Rh antigens including anti-E (12), anti-D (6, including 3 females), and anti-C specificity (5); 8 samples were consistent with a low frequency antibody and 3 were pan-reactive. Other alloantibodies had specificity to K (3, 1 female), Fya (4), Fyb (1), Lea (1), Leb (1), S (1), s (1), Jka (1), and U (1). More than one alloantibody was identified in 7 samples, of which 5 had two Rh antibodies. Genomic DNA analysis revealed numerous RHD variants, including several with one or more exonic SNPs that predict weak or partial RhD phenotypes, as well as predicted D-negative or partial phenotypes with hybrid RHD-CE-D genes or an insert pseudogene (RHDψ). Comparing the alloimmunized patients and controls, there was an equivalent frequency of RHD SNP variants (15/37 = 41%) in both cohorts, but more children with hybrid RHD-CE-D genes affecting multiple RHD exons (10 versus 4, p=0.077). All samples had the homozygous ccee genotype in the RHCE locus. There was a high frequency of RHCE SNP variants in both alloimmunized (31/37 = 84%) and control cohorts (32/37 = 86%), and only one control sample with possible RHD insertion in RHCE exon 5. All six children with anti-D alloantibodies had hybrid RHD-CE-D genes, typically typing as D-negative in tube testing. Among 17 children with anti-C or anti-E alloantibodies, 16 had at least one RHCE SNP, most frequently RHCE*ce.01, but no SNPs were associated with the development of Rh alloantibodies. Conclusions: RBC alloimmunization in children with SCA is common and often unrecognized in low-resource settings. In the Dominican Republic, 21% of children with SCA who entered SACRED with previous transfusions had RBC alloimmunization, including 10% with antibodies against Rh antigens. DNA analysis confirms extensive genetic complexity in both RHD and RHCE genes, with numerous SNPs that alter Rh antigen expression. Large RHD-CE-D insertions may also confer increased risks of anti-D alloimmunization. Lack of typing beyond serologic tube testing of ABO/RhD, and the presence of weak/partial RhD phenotypes in blood donors, may further increase the risk. Extending phenotyping for RhCE, providing RhD-negative blood when available, and performing enhanced screening for RhD-negative donors, will help reduce RBC alloimmunization. Disclosures No relevant conflicts of interest to declare.

Maternal Red Blood Cell Alloimmunization Managed with Intrauterine Blood Transfusion: Predictors of Poor Outcome

Background: The rhesus (Rh) and Kell blood group systems are the most common of over 50 different antigens capable of causing maternal red blood cell (RBC) alloimmunization and severe fetal hemolytic disease. Anti-K and anti-D are responsible for a significant proportion of fetal anemia requiring intrauterine transfusion (IUT). Whilst IUT of packed RBCs improves neonatal survival and morbidity, clinical prognostic indicators are lacking. Our primary objective was to identify predictors of adverse outcome. Methods: We conducted a retrospective single-center study at Mount Sinai Hospital (MSH), Toronto, Canada. All pregnant patients alloimmunized with anti-K and anti-D as a single antibody, between 1991 and 2018 were included. Data were obtained from patient medical records, ultrasound reports and information from the transfusion medicine laboratory. Data included maternal demographics, antibody titers, pregnancy history, number of IUTs, hemoglobin (HB) concentration at the beginning and end of all IUTs. Neonatal outcomes included survival, mode of delivery, gestational age at delivery, birth weight, HB at birth and need for neonatal transfusion, phototherapy or intravenous immunoglobulins (IVIG). Our primary outcome was the composite outcome of stillbirth or neonatal death (SB/NND). We also constructed a secondary outcome consisting of top-up neonatal transfusion, exchange transfusion, phototherapy, or use of IVIG. Medians and interquartile ranges (IQR) or mean±SD were used as summary statistics and compared by Mann-Whitney or t-test; p&amp;lt;0.05 was statistically significant. Outcome predictors were identified using multivariable logistic or linear regression analysis; accounting for patient level clustering by marginal Generalized Estimating Equation did not significantly affect variables. Data were analyzed using SPSS. Results: 116 women with 128 pregnancies and 425 IUTs with anti-K or anti-D as a single antibody were identified. Median maternal age was 31 years (27.0-35.0) for anti-K and 32 years (23.6-40.6) for anti-D. The gestational age at 1st IUT differed significantly between anti-K and anti-D (24.3 vs 28.7 weeks respectively, p=0.004). Women with anti-K antibodies required more IUTs than women with anti-D (3.84 vs 3.12 IUTs, p=0.036) and HB at 1st IUT was significantly lower in the anti-K group (5.10 vs 7.05 g/dL, p=0.001) (Table 1). Following initiation of IUT, the time from 1st IUT to delivery was 69.6 days in the anti-K group and 54.6 in the anti-D group (p=0.06). The daily decrease of HB between 1st and 2nd IUT (as a marker of disease severity), development of fetal hydrops and severe preterm birth did not differ significantly between the two groups. Mean gestation age at delivery was 35.0 weeks in the anti-K and 36.0 weeks in the anti-D group (p=0.28), with 87.1% and 93.9% survival (p=0.37), respectively. The proportion of neonates requiring phototherapy, IVIG and exchange/top-up transfusion was comparable across the two antibody groups (Table 1). Regression analysis showed that delivery occurred sooner if HB dropped more rapidly between the first two IUTs (p=0.01). Each additional transfusion gained on average 22.5 days in utero (Table 2). In multivariable analysis, gestational age at 1st IUT was the only predictor of a SB/NND outcome (adjusted OR 0.79 [95%CI 0.67-0.93]; p=0.006). With 1st IUT at 23 weeks, the risk of SB/NND was 8%, but only 2.5% at 28 weeks and &amp;lt;0.01% at 36 weeks (Table 3). Gestational age at delivery (adjusted OR 1.11 [95%CI 1.00 to 1.23]; p=0.046) and HB at 1st IUT (adjusted OR 0.84 [95%CI 0.72 to 0.99]; p=0.038) were the only predictors of a composite adverse blood product requirement/intervention outcome (Table 4). The odds of blood product requirement/intervention postnatally at 1st IUT (median HB at 1st IUT of 6.6g/dL) at 28 weeks were 43% and increased by a further 38% for delivery at 34 weeks. Conclusion: The earlier in gestation that IUTs are implemented, the higher the odds of a SB/NND; however the later the gestation at delivery, the greater the odds of the neonate requiring blood products post-partum. The greater the HB drop between the 1st and 2nd IUT, the shorter the 'time between the first IUT and delivery', which increases the odds of a SB/NND outcome. Disclosures Garbowski: Vifor Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Imara: Consultancy. Shehata:Ferring: Honoraria.

Type 1 Interferon Gene Signature Promotes RBC Alloimmunization in a Lupus Mouse Model.

Red blood cell (RBC) transfusion exposes recipients to hundreds of unmatched minor RBC antigens. This exposure can lead to production of alloantibodies that promote clinically significant hemolytic events. Multiple studies have reported an increased frequency of RBC alloimmunization in patients with autoimmunity. However, cellular and molecular mechanisms that underlie autoimmunity-induced alloimmunization have not been reported. Patients with systemic lupus erythematosus (SLE) have a high frequency of alloimmunization and express a type 1 interferon (IFNα/β) gene signature. Thus, we utilized the pristane-induced lupus mouse model to test the hypothesis that inflammation in lupus promotes RBC alloimmunization, and to examine the potential role of IFNα/β. Intraperitoneal injection of pristane, a hydrocarbon oil, led to autoantibody production, glomerulonephritis, and pulmonary hemorrhage in wild type (WT) mice. Pristane treatment significantly induced serum IFNα and expression of multiple interferon-stimulated genes (ISGs) in peripheral blood and peritoneal fluid cells, including inflammatory macrophages. Following transfusion with allogeneic RBCs expressing the KEL glycoprotein, pristane-treated WT mice produced significantly elevated levels of anti-KEL IgM and anti-KEL IgG, compared to untreated mice. Pristane induced comparable levels of inflammatory cells and cytokines in mice lacking the IFNα/β receptor (IFNAR1–/–) or the IFNα/β-inducing transcriptions factors (IRF3/7–/–), compared to WT mice. However, pristane-treated IFNAR1–/– and IRF3/7–/– mice failed to produce ISGs and produced significantly lower levels of transfusion-induced anti-KEL IgG, compared to WT mice. Thus, pristane induction of a lupus-like phenotype promoted alloimmunization to the KEL RBC antigen in an IFNα/β-dependent manner. To our knowledge, this is the first examination of molecular mechanisms contributing to RBC alloimmunization in a model of autoimmunity. These results warrant further investigation of the role of IFNα/β in alloimmunization to other RBC antigens and the contribution of the IFNα/β gene signature to the elevated frequency of alloimmunization in patients with SLE.