RBC Antigens Research Articles

P1628: ALLOIMMUNIZATION DURING ASSISTED REPRODUCTIVE TECNOLOGY: CASE REPORT OF A PREGNANCY WITH IRREGULAR ANTIBODIES PROBABILY RELATED TO EGG DONOR ANTIGEN.

Background: Alloimmunization is a well known complication during pregnancy. It consists in an immune response of the mother against non-self antigens expressed by the fetus, which derive from the male partner, and its manifestation is an hemolytic disease of the fetus and newborn (HDFN). The most common antigen involved in alloimmunization came from the AB0 group and affect 15% to 25% of all pregnancies. However, the response caused by AB0 incompatibly is modest most likely due to the expression of AB0 blood type antigen in several organs. Other red cell antigens have been implicated in the HDFN; the Rh(D) antigen is the most immunogenic and the main cause of HDFN because the antigen is well expressed on the surface of the erythrocytes in the fetus. Previously, HDFN was known to cause fetal death in 1% of all pregnancies; with the advent of immunoprophylactic therapies, HDFN is currently well managed with fewer complications. In Italy, RBC iantibody screenings are typically performed at first and third trimester of pregnancy. Affected women are followed up to ensure the timely identification of those antibodies who require therapeutic interventions to prevent perinatal morbidity and mortality. The italian guidelines aim to direct these women to third-level centers where management protocols are shared by Immuno-hematology and Obstetrics Units in order to monitor the pregnancy with laboratory tests and clinical interventions. Aims: Identification of risk factor for alloimmunization during pregnancy Methods: Blood group, undirect and direct antiglobulin test during prregnancy mounthly based. After delivery blood group and DAT on Newborn Results: We report a case of alloimmunization during pregnancy of a woman that underwent a medically assisted procreation with egg donation. The subject, in apparently good health, had no chronic pathologies and was not taking medications. Her blood group was B Neg ccdee kk and the partner was 0 Pos CcDee Kk During the last triemster of the pregnancy, the woman developed alloimmunization towards many antigens of different blood systems,Rh (anti-D, antiC, anti-E), and the Kell (anti-Kell) systems. at low titers At week 37 of the pregnancy, she was admitted to the hospital for obstetrical problems and delivery was induced without clinical complications, neither for her nor for the fetus. Newborn blood group was 0 Pos CcDee Kk and Direct antiglobulin test was positive (++++). Anti-RHD and Kell antibodies were identified after elution. Additional studies are underway to characterize the egg donor RBC Phenotype to verify the presence of E antigen Summary/Conclusion: Alloimmunization during pregnancy is a condition caused by a response of the mother to paternal antigenes exhibited by the fetus and can be monitored with standard laboratory and clinical approaches However, the possibility to carry out pregnancies with egg donor that has a different RBC antigen profile of the mother open a new immuno-hematological scenarios previously not pointed. This could lead to potentially increased risk for the mother and the newborn.

P1387: MONITORING OF DONOR CHIMERISM ON RED BLOOD CELLS ANTIGENS AFTER HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment for diseases of the blood system. One of the indicators reflecting the functioning of donor hematopoietic tissue is posttransplant chimerism. Aims: To evaluate the effect of ABO belonging of the donor and recipient on the timing of the formation of red blood cells (RBS) donor chimerism after HSCT. Methods: The study included 57 patients who underwent HSCT in 2013-2021. Patient age ranged from 3 to 60 years (median - 34). Diagnosis of the patients: acute leukemia - 42 persons, Hodgkin’s lymphoma - 6, aplastic anemia - 4 and other blood diseases - 5. Before HSCT, the study of RBC antigens of the ABO, Rh, Kell systems in donors and recipients was performed by gel agglutination using reagents and equipment BioRad (USA). Antigens identified in the recipient’s phenotype and absent in the donor’s phenotype were considered informative, or vice versa. For statistical data processing, the nonparametric Kruskal-Wallis test was used. Results: Thirty-two (56.1%) of 57 pairs differed in ABO system antigens. Of these, 16 pairs (28.1%) had a major ABO incompatibility, 16 pairs (28.1%) had a minor incompatibility. The time of appearance of donor’s RBS in patients with ABO incompatibility had a wider range (from 14 to 201 days) and occurred significantly later (Me - 28 days) than in recipients ABO identical with donors (Me - 20 days), and from minor ABO incompatibility (Me - 19 days) (Table 1). The periods of occurrence of 50% and 100% donor chimerism did not differ significantly in all groups. In the group of recipients with major ABO incompatibility a case of pure red cell aplasia was revealed. The first appearance of RBC carrying donor antigens was recorded on day 201. 100% donor chimerism was formed on day 278. Antibodies of the ABO system (anti-A) IgG were present in the recipient’s blood for 189 days, IgM - for 230 days. Table 1. - The emergence of chimerism depending on ABO compatibility of donor-recipient pairs ABO identical Minor ABO incompatibility Major ABO incompatibility р (n=25) (n=16) (n=16) First appearance of donor chimerism 20 (12-54) 19 (11-41) 28 (14-201) р<0.05 Ме (Min-Max days) 50% donor chimerism 41 (19-142) 41 (26-74) 54 (34-227) p>0.05 Ме (Min-Max days) 100% donor chimerism 138 (40-316) 110 (70-150) 141 (90-278) p>0.05 Ме (Min-Max days) Summary/Conclusion: In recipients with a major ABO incompatibility the first appearance of donor’s RBS after HSCT occurs significantly later than in couples ABO identical and couples with a minor ABO incompatibility.

Impact of magrolimab treatment in combination with azacitidine on red blood cells in patients with higher-risk myelodysplastic syndrome (HR-MDS).

7054 Background: Magrolimab is a monoclonal antibody that blocks CD47, a “don’t eat me” signal expressed on cancer cells to escape immune surveillance and macrophage-mediated clearance. Prior preclinical studies have shown that CD47 is critical to RBC homeostasis, with CD47 deficiency decreasing RBC half-life. Fc-mediated opsonization also depletes RBCs, raising concerns for potential on-target anemia from anti-CD47 agents via multiple mechanisms. Notwithstanding, several clinical trials have demonstrated that magrolimab can be safely administered as a monotherapy with initial lower “priming” dose yielding transient anemia with compensatory reticulocytosis, with anemia not observed at subsequent higher maintenance doses. However, the mechanism underlying this observed protection has not been fully defined. Here we describe manageable anemia in patients (pts) with HR-MDS treated with magrolimab in combination with azacitidine (AZA) (NCT03248479) and further investigate these underlying mechanisms in preclinical models. Methods: In a multicenter prospective study, CBCs, peripheral blood, and bone marrow (BM) were collected at prespecified timepoints from HR-MDS pts (n = 57) treated with magrolimab in combination with AZA. CBCs were measured, and blood and BM samples were analyzed by flow cytometry for expression of CD47 on RBCs and WBCs. Magrolimab was initially dosed with a priming dose (1mg/kg) followed by an initial weekly maintenance dosing (30mg/kg) before transitioning to every 2 weeks maintenance dosing. AZA 75mg/m2 was administered on days 1-7 of the 28-day cycle. Preclinical modeling studies were conducted with intact and Fc-deficient anti-mouse CD47 (MIAP410) and anti-human CD47 (magrolimab) antibodies in murine models, including C57BL/6J B-hSIRPA/hCD47 mice. Results: Combination treatment of magrolimab with AZA resulted in a tolerable anemia that correlated with rapid, near complete loss of CD47 from RBCs, but not WBCs. The initial 1mg/kg priming dose was sufficient for this CD47 loss, which persisted under subsequent 30mg/kg maintenance doses. Both findings are consistent with prior clinical observations in solid tumor pts with magrolimab monotherapy and lymphoma pts in combination with rituximab. Our preclinical studies with mouse models revealed that the CD47 removal is mechanistically independent of previously described RBC antigen modulation mechanisms and cellular compartments. Instead, this CD47 loss requires anti-CD47 crosslinking between RBCs and non-RBCs. Conclusions: Overall, these results support that on-target magrolimab mediated anemia is mitigated by a near complete loss of RBC CD47. HR-MDS patients treated with magrolimab in combination with AZA exhibit a tolerable anemia through priming and maintenance doses. Clinical trial information: NCT03248479.

Serological Detection of Helicobacter pylori Infection in Pregnant Women Related to ABO Blood Group.

Helicobacter pylori was known as a pathogen related to peptic ulcers and gastric carcinoma. Some researches confirmed that the infected pregnant women with H. pylori have poor pregnancy outcomes so that its effects extended to other systems other than gastrointestinal tracts. This study aimed to evaluate H. pylori infection in pregnant women who had morning sickness (nausea and vomiting) related to the ABO blood group. In total, 202 pregnant women within the age range of 15-45 years with severe nausea and vomiting attended the outpatient and specialized clinic. The seroprevalence of H. pylori was 62% in pregnant women, especially at the age group of 20-24 years with 32.5% of the cases who had epigastric pain, nausea, vomiting, flatulence, and burning of the stomach, the majority of which related to O+ (33.3%), followed by A+ and B+ (25.39%) blood groups. Most infected pregnant women with H. pylori were during the first (41.26%) and second trimesters (34.12%), especially in multigravida (68.25%) cases. This study found that hyperemesis (severe nausea and vomiting), dyspepsia, and other gastrointestinal symptoms during pregnancy were related to the infection with H. pylori; therefore, it is a risk factor for complications in pregnancy and its poor outcomes, especially in developing countries, such as Iraq. These results can be minimized by improving the socioeconomic and sanitation conditions. H. pylori infection in pregnancy is considered a health problem and should be treated before and during pregnancy. Further investigations are required in this regard and researchers are recommended to conduct studies on the RBC antigens to recognize the pathophysiology related to H. pylori infection.

Studies on Lectin Mediated Agglutination Reaction on Red Blood Cell Surface Antigens Using Hot and Cold Water Plant Extracts

Lectin has various physiological roles in cell agglutination, based on their carbohydrate-binding properties, plant lectins are widely used for the detection, segregation, and characterization of glycoconjugates. Rhesus (Rh) factor is a protein that is inherited and found on the surface of red blood cells. If the surface protein is present, the RBC is Rh positive; otherwise, it is Rh-negative in nature. In this paper, we use agglutination reactions to investigate the effect of different cold and hot water extracted plants on RBC antigens as an alternative to commercial monoclonal antibodies. Extensive research on the sequence homology and 3-D structure of various plant lectins suggests that they have been conserved throughout evolution and may play important physiological roles that are still unknown.

Sickle cell disease patients in two London trusts: Genotyping including RH variants.

All SCD patients need extended RBC antigen typing (by serology or genotyping) for provision of extended RH, K matched blood and to guide RBC selection in those with complex transfusion requirements. Genotyping can also identify RH variants which can cause sensitisation even when extended RH phenotypically matched blood is provided and alloantibodies associated with RH variants can cause HTRs. To review the use of RBC genotyping in SCD patients at two London trusts (ICHNT, LNWH) with a focus on RH variants. Retrospective review with data collected from clinical notes, local and national pathology reporting systems. A 311/482 (64%) ICHNT patients and 181/346 (52%) LNWH patients had extended genotyping. Of genotyped patients, 68 (22%) ICHNT and 31 (17%) LNWH patients had RH variants. Eight ICHNT patients had RH variants and corresponding antibodies associated with RH variants; 4/8 received multiple transfusions with antigen positive RBCs but had no evidence of haemolysis. One LNWH patient had a RH variant with corresponding alloantibody but could not be investigated further for possible HTR. Most patients (59%) had genotyping and a significant number had RH variants (99, 20%). A small proportion (9, 9%) had antibodies associated with RH variants, but with no evidence of clinically significant HTRs despite transfusions in four of them with antigen positive RBCs. All SCD patients should have RBC genotyping including RH variants (preferentially over extended phenotyping) to guide better selection of RBC units. However, where antigen negative blood cannot be provided, the risk of alloimmunisation is not inevitable and subsequent HTRs from antibodies associated with RH variants might not always occur.

Impact of Alloimmunization in Patients with Sickle Cell Disease

INTRODUCTIONBlood transfusion is an essential therapeutic and prophylactic component in the management of sickle cell disease (SCD) and associated complications. Prolonged transfusion therapy can lead to the development of antibodies to the donor's RBC antigens (alloimmunization), causing complications such as delayed hemolytic transfusion reactions, hyperhemolysis, worsening vaso-occlusive episodes, and end-organ damage. There have been only a few case series highlighting the impact of RBC alloimmunization on SCD morbidity and mortality, proposing a pathway involving RBC alloimmunization and decreased survival associated with hemolytic reactions or difficulty obtaining compatible blood when needed. However, apart from the consequences of iron overload, there is no long-term data for alloimmunization highlighting the clinical consequences, multiorgan damage, or associated morbidity in sickle cell patients.AIMThe primary aim is to investigate the incidence of alloimmunization in SCD patients in an academic health system. The secondary aim is to elucidate the differences in demographics, frequency of vaso-occlusive crisis, end-organ damage, and inflammatory markers between alloimmunized and non-alloimmunized SCD patients.METHODSWe conducted a retrospective multicentric descriptive study, including all sickle cell patients treated in an academic health system from January 1st, 2009, to December 31st, 2020, in Maryland, Virginia, and Washington, DC. An exemption from the Institutional Review Board for obtaining individual subjects' consent was procured. Patients included in the study were older than 18 years and diagnosed with sickle cell disease. Patients who did not have sickle cell disease were excluded from the study. Statistical analysis was reported using means for descriptive data, t-test for continuous variables, and chi-square for categorical variables.RESULTSA total of 94 patients with sickle cell disease were included in the study. Of these, 24 (25.5%) patients were found to have alloimmunization, whereas 70 (74.4%) patients did not. Of the alloimmunized patients, the average age, BMI and BSA were 30.15 years (p=0.037), 23.15 kg/m2 (p=0.040), and 1.65 m2 (p=0.003) compared to 37.07 years, 26.17 kg/m2 and, 1.84 m2 respectively among the non-alloimmunized group. 83% of the alloimmunized patients had sickle cell anemia (Hb SS), and 17% had a sickle thalassemia phenotype (p=0.005). A lower baseline hemoglobin (Hb) value of 8.01 g/dL was seen among alloimmunized patients compared to a higher Hb value of 9.63 g/dL (p=0.001) among the non-alloimmunized. Alloimmunized patients had an average of 5.55 alloantibodies. The average number of vaso-occlusive crises per year and related hospitalizations was statistically significantly higher in the alloimmunized group with 4.82 and 3.78, respectively, compared to 2.34 (p=0.035) 1.01 (p=0.0005) in the non-alloimmunized group. Similarly, the incidence of other sickle cell-related complications were higher among the alloimmunized patients, such as priapism (29% vs. 9%; p=0.0139), pulmonary hypertension (38% vs. 9%; p=0.0038) with no statistical difference in the iron overload (25% vs. 11%; p=0.150) or ferritin levels (. 83% of alloimmunized patients had a history of narcotic use vs. 34% among the non-alloimmunized (p=0.0001). Higher use of disease-modifying therapies including hydroxyurea (71% vs. 31%; p=0.0009) and voxelotor (13%vs0; p=0.0029), were also seen among alloimmunized patients. While no statistically significant difference was seen in the mean number of lifetime transfusions, there was a difference in the mean number of lifetime exchanges (3.67 vs. 0.0; p=0.0208).CONCLUSIONThe prevalence of alloimmunization in sickle cell patients in our study population (25.5%) was higher than in the literature (7- 59%) and the general population (2%). An increase in alloimmunization was associated with an increased number of exchanges but not with simple transfusions. Independent from the iron overload, alloimmunization was associated with increasing end-organ damage and sickle cell complications such as priapism, pulmonary hypertension. Strategies to decrease alloimmunization are needed to prevent these complications. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Bleeding Patient with Anti-PP1Pk – Revisiting Autologous Frozen Red Blood Cells

Abstract Introduction/Objective A 34-year-old Amish woman G11P8A2L8, with a known PP1Pk antibody, presented to her OB with vaginal hemorrhage secondary to an incomplete spontaneous abortion. Despite medical treatment, her hemoglobin decreased from 12 g/dL to 8.4 g/dL. She was air transferred to a tertiary care hospital for further management and antigen negative RBC transfusion. Upon arrival, her hemoglobin had decreased to 7.1 g/dL and her BP decreased to 92/64 mm Hg. Additional blood draws were discontinued to save blood wastage. Her blood pressure continued to decrease over the next several hours to a low of 78/36. The patient was briefly stabilized, and a successful dilation and curettage was performed. Methods/Case Report A national search was conducted for PP1Pk antigen negative blood. Her RBC phenotype: C+, c-, E-, e+, K-k-, Fy(a + b-), Jk(a+ b+), MN+, p, Le(a-b-). There was no history of blood product transfusion and the prior miscarriages where thought to be the source of alloimmunization. There was no known family history of the p (P- P1- Pk-) phenotype. Results (if a Case Study enter NA) One unit of PP1Pk antigen negative PRBCs was located approximately 48 hours after the patient’s admission, however, the consulting hematology team suggested withholding transfusion due the possibility of alloimmunization from other blood groups complicating future transfusions. She was treated with IV iron and discharged four days later with a hemoglobin level of 6 g/dL. She was asymptomatic upon discharge. Conclusion Anti-PP1Pk is a rare antibody (5.8 per 1 million people). In the Amish population, the incidence is approximately 1 in 5000 individuals. Our patient is planning for future pregnancies. Upon our suggestion, the patient was encouraged to consider frozen autologous blood donation after recovery and before the next pregnancy as well as close monitoring in high-risk maternal fetal medicine settings. In a patients and donors with anti PP1PK, we recommend advance planning including frozen autologous blood donation and family members RBC antigen studies. They should be encouraged to become regular blood donors for themselves and others.

Transfusion requirements and alloimmunization to red blood cell antigens in orthotopic liver transplantation.

Orthotopic liver transplantation (OLT) has been associated with high blood transfusion requirements. We evaluated the transfusion needs and frequency of alloimmunization to RBC antigens among OLT recipients pre- and post-transplantation. We reviewed the medical records of patients who underwent a first OLT between January 2007 and June 2017. Transfusions given only during the perioperative period, defined by 1week before OLT until 2 weeks following OLT, were included in this study. Records were reviewed in June 2019 for updated antibody testing results. A total of 970 patients underwent OLT during the study period. The median age of patients was 57 years; 608(62.7%) were male. During the perioperative period, transfused patients received an average of 10.7 (±10.7) RBC units, 15.6 (±16.2) thawed plasma units and 4.1 (±4.3) platelet units. At the time of OLT, a total of 101 clinically significant RBC alloantibodies were documented in 58(5.98%) patients. Fifty-three of these antibodies were directed against Rh blood group antigens. Twenty-two (37.9%) patients had more than one alloantibody. Patients with alloimmunization before OLT (N=58) received perioperatively comparable number of RBCs to non-alloimmunized patients (10.5 ± 10.6 vs. 9.6 ± 10.7; p=0.52). There was no significant difference in perioperative or intraoperative RBC transfusion between patients with one alloantibody and those with multiple alloantibodies. Only 16 patients (16/737; 2.17%) developed new alloantibodies at a median of 61 days after OLT. The overall alloimmunization rate was 9.8% (72/737), and female patients were more likely to be alloimmunized. Blood transfusion requirements in OLT remain high. However, the rate of RBC alloimmunization was not higher than the general patient population.

Red blood cell frequencies and genetic polymorphisms of various blood grouping systems in the emirati population

AbstractBackground and objectivesData on the frequencies of RBC antigens and their variants in the Arab world are limited. Thus, determining the antigenic profile of the United Arab Emirates (UAE) population by serologic and molecular methods is significant to enhance operational efficiency in donor centres and transfusion services.Study design and methodsThe study is a retrospective retrieval of serologic and genotype data. The serological ABO type data of 30 954 UAE national donors, the serological RHD and RHCE data of 10 036 UAE national donors, and the genotype data of 354 patients and donors of admixed nationalities for determining different blood group systems were retrieved and analysed.ResultsRh D negative (dccee) phenotype in the UAE national donor population is lower compared to the reported Rh D negative phenotype in the European population (6·8% vs. 18%) and there were a high number of variants observed in the patient and donor populations, especially in the RhCE and Duffy blood group systems (12·1% and 59·3%, respectively).ConclusionHigh number of variants and rare blood group profiles were observed and the gene pool of the Emirati nationals, as well as their antigenic frequencies, is an intermediate between the European and African populations rather than the Asian gene pool.

Impact of Extended Molecular RBC Antigen Matching between Blood Donors and Patients with Hematological Diseases

Background: Programs to prevent alloimmunization against red RBC antigens aims to provide antigen-compatible blood transfusions for patients with hematological diseases. Recently, there is an emerging role of molecular methods in donor-receptor compatibility for major blood group polymorphisms and for Rh variants to improve transfusion safety.Aims: We aimed to determine the genotype and allele frequencies of major blood-group systems and RHD and RHCE variants in blood donors and patients with SCD, Myelodysplastic Syndromes (MDS), Acute Myeloid Leukemia (AML) or Autoimmune Hemolytic Anemia (AIHA) to verify if the genotyping results are in concordance with serologic typing results and to perform molecular compatibility between donors and patients.Methods: Samples from 198 blood donors were phenotyped for five clinically relevant blood-group system antigens (RH, KEL, JK, FY and MNS). Samples from 158 patients with hematological diseases (101 with SCD, 14 with MDS, 26 with AIHA and 17 with AML) and from 198 blood donors were evaluated molecularly for 18 blood-group systems alleles (RH, CO, CROM, DI, DO, FY, GE, IN, JK, KEL, KN, LE, LU, LW, MNS, OK, SC and YT) using the Blood-MLPA assay (Multiplex Ligation-dependent Probe Amplification). RHD and/or RHCE exon-specific sequencing was performed to discriminate RHD and RHCE subtypes not distinguished by MLPA. Computerized molecular matching was performed between all patients and donors for ABO, RH, MNS, KEL, FY, JK and DI blood-group systems (Level 1). Afterwards, we extended the matching degree [ABO, RH (further variants), MNS, KEL, FY, JK and DI (Level 2)] for patients who inherit clinically relevant RHD and/or RHCE genotypes predicting partial Rh antigens.Results: We found statistically significant differences in the frequencies of the blood group alleles MNS, FY, JK, DO, CO, GE and RH (RHD*DAU0, RHCE*ceVS.01, RHCE*Ce and RHCE*CE) between donors and patients with hematological diseases. The allelic profile of AML patients was the most similar to the allelic profile of donors, while that of SCD patients was the most different. Eight donors and nine patients had clinically relevant RHD genotypes, while 14 donors and 22 patients had clinically relevant RHCE genotypes. There was 99.2% concordance between the results of the serologic typing and the results of the predicted phenotypes by the blood-MLPA. Considering the Level 1 matching It would be possible to find at least one compatible blood donor for 91.1% patients with hematological diseases. From recently transfused patients (i.e., those transfused up to one year prior to participation in the study), 70.3% would have enough blood units for an annual transfusion cycle. The MDS patient group were the least attended with matched blood (55.6%) and the one with the lowest number of compatible donors (85.7%). Taking into consideration the Level 2 matching the average number of available donors per patient reduced from 19.7 to 3.5 when the degree of matching was extended (i.e., from Level 1 to Level 2). Besides, the number of compatible donors was not sufficient to fulfil the transfusion needs of patients with Rh variants.Conclusion: We reliably determine the genotype and allele frequencies of clinically relevant blood-group systems in donors and in patients using molecular methods. It would be possible to meet the transfusion needs of most patients with hematological diseases through molecular compatibility, but the current donor population would not fill the transfusion requirements of patients with Rh variants. Molecular RBC matching increases the safety of patients with SCD, MDS, AML and AIHA by providing more accurate compatibility with genotyped donors and by reducing the risk of transfusion reactions. DisclosuresNo relevant conflicts of interest to declare.

Blood group genotyping in alloimmunized multi-transfused thalassemia patients from Iran.

ObjectivesSerological methods may not be reliable for RBC antigen typing, especially in multi‐transfused patients. The blood group systems provoking the most severe transfusion reactions are mainly Rh, Kell, Kidd, and Duffy. We intended to determine the genotype of these blood group system antigens among Iranian alloimmunized thalassemia patients using molecular methods and compare the results with serological phenotyping.MethodsTwo hundred patients participated in this study. Blood group phenotype and genotype were determined using the serological method and PCR‐SSP, respectively. The genotypes of patients with incompatibility between phenotype and genotype were re‐evaluated by RFLP‐PCR and confirmed by DNA sequencing.ResultsDiscrepancies between phenotype and genotype results were found in 132 alleles and 83 (41.5%) patients; however, there was complete accordance between the three genotyping methods. Most discrepancies were detected in Rh and Duffy systems with 47 and 45 cases, respectively, and the main discrepancy was in the FY*B/FY*B allele when serologically showed Fy(a+b+). All 39 undetermined phenotypes, due to mixed‐field reactions, were resolved by molecular genotyping.ConclusionMolecular genotyping is more reliable compared with the serological method, especially in multi‐transfused patients. Therefore, the addition of blood group genotyping to serological assays can lead to an antigen‐matched transfusion in these patients.

Marginal zone B cells mediate a CD4 T-cell–dependent extrafollicular antibody response following RBC transfusion in mice

AbstractRed blood cell (RBC) transfusions can result in alloimmunization toward RBC alloantigens that can increase the probability of complications following subsequent transfusion. An improved understanding of the immune mechanisms that underlie RBC alloimmunization is critical if future strategies capable of preventing or even reducing this process are to be realized. Using the HOD (hen egg lysozyme [HEL] and ovalbumin [OVA] fused with the human RBC antigen Duffy) model system, we aimed to identify initiating immune factors that may govern early anti-HOD alloantibody formation. Our findings demonstrate that HOD RBCs continuously localize to the marginal sinus following transfusion, where they colocalize with marginal zone (MZ) B cells. Depletion of MZ B cells inhibited immunoglobulin M (IgM) and IgG anti-HOD antibody formation, whereas CD4 T-cell depletion only prevented IgG anti-HOD antibody development. HOD-specific CD4 T cells displayed similar proliferation and activation following transfusion of HOD RBCs into wild-type or MZ B-cell–deficient recipients, suggesting that IgG formation is not dependent on MZ B-cell–mediated CD4 T-cell activation. Moreover, depletion of follicular B cells failed to substantially impact the anti-HOD antibody response, and no increase in antigen-specific germinal center B cells was detected following HOD RBC transfusion, suggesting that antibody formation is not dependent on the splenic follicle. Despite this, anti-HOD antibodies persisted for several months following HOD RBC transfusion. Overall, these data suggest that MZ B cells can initiate and then contribute to RBC alloantibody formation, highlighting a unique immune pathway that can be engaged following RBC transfusion.

Pure red blood cell aplasia: patient management pitfalls in major ABO-incompatible haematopoietic cell transplantation.

In this issue of British Journal of Haematology, Longval and colleagues report on the successes and pitfalls of prediction, diagnosis and management of pure red blood cell aplasia (PRCA) after major ABO-incompatible allogeneic stem cell transplantation.1 Their data confirm prior reports indicating a paucity of cases of PRCA after umbilical cord blood transplantation.2 Among the remaining 587 patients receiving major ABO-incompatible donor grafts, 66 cases of PRCA were identified. In this series, high pre-transplant isohaemagglutinin titre was the only pre-transplant variable associated with subsequent development of PRCA. Despite the various methodologies for performing isohaemagglutinin titre assays,4, 5 this observation highlights the need for pre-transplant quantitation of isohaemagglutinin titre in instances of major ABO mismatch in order to identify patients at highest risk. Furthermore, at some centres pre-conditioning therapeutic plasma exchange is considered for patients with high titres.6 In some patients, residual recipient plasma cells may secrete sufficient quantities of antibody to prevent nascent erythroblast precursors from achieving timely maturation,3 forming a rationale for consideration of plasma cell-directed therapy in patients with persistently high isohaemagglutinin titres. Interestingly, several low titre isohaemagglutinin recipient–donor pairs resulting in post-transplant PRCA were identified, pointing to the need to identify additional predictors of PRCA. This work also exposes a gap in knowledge within the transplant field, namely when can we be sure that red blood cell engraftment has transpired? To date, no universal standard for red blood cell engraftment exists, and across institutions several working definitions exist including red cell transfusion independence, ABO blood type conversion, and absence of reticulocytopenia. Advantages and disadvantages of various case definitions are outlined in Table I. Standard diagnostic assessments for profound anaemia, reticulocytopenia, and absence of red blood cell precursors in the bone marrow are certainly essential when patients display prolonged red cell transfusion dependence,7 but in the absence of alternative causes, the presumptive diagnosis in this setting is PRCA due to ABO incompatibility, and persistence of some level of the corresponding isohaemagglutinin is typical. In this series, over 10% of patients receiving transplants from donors with a major ABO incompatibility developed PRCA, but this immunologic insult did not significantly affect overall survival, which is in line with results of previous studies.8, 9 As such, any therapeutic intervention must be considered in the context of the inherent risk of the intervention itself. Introduction of rituximab,10 donor lymphocyte infusions,11 tapering of immunosuppressive medications, or use of a proteasome inhibitor12 all carry risk–benefit calculations, and to date have demonstrated mixed results in treating post-transplant PRCA. Anecdotal reports of the successful use of the anti-CD38 monoclonal antibody daratumumab for management of post-transplant PRCA13, 14 deserve mention, particularly given that the physiology of prolonged PRCA after transplant is presumably a reflection of residual host plasma cell clones producing the isohaemagglutinin. In the current study, no particular intervention was associated with more rapid resolution of PRCA, although no patients in this series received daratumumab and only a handful received a proteasome inhibitor. Management of patients undergoing major ABO-incompatible allogeneic stem cell transplantation requires close collaboration with the blood bank to identify patients at greatest risk prior to transplantation, and to optimize transfusion strategies during the post-transplant period. This may include pre-transplant minor red blood cell antigen typing to reduce the likelihood of alloimmunization,15 and consideration of pre-transplant plasma exchange for patients with very high isohaemagglutinin titres. It is fair to say that there is no ‘gold standard’ for treatment of active post-transplant PRCA, and this study does not inform us regarding how best to manage these patients. Fortunately, the situation typically resolves over time, but further studies to establish effective treatment strategies are sorely needed given the significant morbidity and symptom burden associated with this not so rare complication. As a final thought, we might speculate two distinct (but not mutually exclusive) mechanisms for what we call ‘PRCA’ in this setting: residual host isohaemagglutinin levels that simply take time to dissipate because of high pre-transplant titres versus persistence of host isohaemagglutinin-producing plasma cells. We have the molecular and blood banking tools to systematically study these post-transplant events, which could better elucidate management approaches most appropriate for individual patients. Variability in RBC products Complicated by suspected transfusion reactions Provider/institutional variability for transfusion thresholds Time to independence is not a universal definition (e.g,, 30 days without transfusion versus 60 days) Inter-observer variability in scoring of agglutination Interference between residual transfused isohaemagglutinins from platelets/plasma No established grading system for mixed field observations Interference from intravenous immune-globulin treatment Results often not reported in patient’s medical record Difficulty in interpretation for non-transfusion medicine personnel Monoclonal antibodies for minor RBC antigens are licensed Low cost and quick turn-around time Only viable if minor antigen discrepancy exists between donor/recipient. If patient has positive Direct Coombs’ test, results could be invalid based on reagent used

Antibodies to Low-Copy Number RBC Alloantigen Convert a Tolerogenic Stimulus to an Immunogenic Stimulus in Mice.

Red blood cells expressing alloantigens are well known to be capable of inducing robust humoral alloantibody responses both in transfusion and pregnancy. However, the majority of transfusion recipients and pregnant women never make alloantibodies, even after repeat exposure to foreign RBCs. More recently, RBCs have been used as a cellular therapeutic—very much like transfusion, engineered RBCs are highly immunogenic in some cases but not others. In animal models of both transfusion and RBC based therapeutics, RBCs that do not induce an immune response also cause tolerance. Despite a robust phenomenology, the mechanisms of what regulates immunity vs. tolerance to RBCs remains unclear. However, it has been reported that copy number of alloantigens on the RBCs is a critical factor, with a very low copy number causing non-responsiveness (in both humans and mice) and also leading to tolerance in mice. Recently, we reported that an IgG2c specific for an RBC antigen can substantially enhance the humoral immune response upon transfusion of RBCs expressing that antigen. Herein, we report that an IgG2c converts RBCs with low antigen copy number from a tolerogenic to an immunogenic stimulus. These findings report the first known stimulus that induces humoral alloimmunization to a low copy number RBC alloantigen and identify a previously undescribed molecular switch that has the ability to affect responder vs. non-responder phenotypes of transfusion recipients.

Autoimmune Hemolytic Anemia in the Pediatric Setting.

Autoimmune hemolytic anemia (AIHA) is a rare disease in children, presenting with variable severity. Most commonly, warm-reactive IgG antibodies bind erythrocytes at 37 °C and induce opsonization and phagocytosis mainly by the splenic macrophages, causing warm AIHA (w-AIHA). Post-infectious cold-reactive antibodies can also lead to hemolysis following the patient’s exposure to cold temperatures, causing cold agglutinin syndrome (CAS) due to IgM autoantibodies, or paroxysmal cold hemoglobinuria (PCH) due to atypical IgG autoantibodies which bind their target RBC antigen and fix complement at 4 °C. Cold-reactive antibodies mainly induce intravascular hemolysis after complement activation. Direct antiglobulin test (DAT) is the gold standard for AIHA diagnosis; however, DAT negative results are seen in up to 11% of warm AIHA, highlighting the need to pursue further evaluation in cases with a phenotype compatible with immune-mediated hemolytic anemia despite negative DAT. Prompt supportive care, initiation of treatment with steroids for w-AIHA, and transfusion if necessary for symptomatic or fast-evolving anemia is crucial for a positive outcome. w-AIHA in children is often secondary to underlying immune dysregulation syndromes and thus, screening for such disorders is recommended at presentation, before initiating treatment with immunosuppressants, to determine prognosis and optimize long-term management potentially with novel targeted medications.

PREVALENCE OF IRREGULAR RED CELLANTIBODIES IN HEALTHY BLOOD DONORS ATTENDING A TERTIARY CARE HOSPITAL IN SOUTH INDIA

Introduction: Red cell antibodies that are found normally in human serum are considered naturally occurring and those are anti A and anti B. All other antibodies directed against RBC antigens are considered “unexpected or irregular". Aim: This study is aimed to evaluate the prevalence of the anti-red blood cell antibodies among healthy blood donors. Material and Methods: Antibody screening and identification was done using commercially available 3 cell and 11 cell reagent cells (0.8% Surgiscreen, Ortho Clinical Diagnostics Limited, USA and Low ionic Strength Saline Ortho Bliss with AHG Cassettes) in antihuman globulin phase. Results: A total of 36,684 donors were screened for the presence of irregular erythrocyte antibodies. Among these donors, twenty donors showed presence of alloantibodies in their serum (0.054%). Most frequent alloantibodies identified were from Lewis blood group system. The results showed statistically a higher prevalence of RBC alloantibodies in males than in females. Conclusion: Screening for presence of alloantibodies in donor blood is important to provide compatible blood products and to avoid transfusion reactions.

Association Between RBC Antigen Allo-Antibodies and Immune-Related Adverse Events During Immune Checkpoint Inhibitor Treatment for Advanced Cancers.

IntroductionImmune checkpoint inhibitors (ICI) have become a primary treatment modality for patients with a variety of malignancies. Given their increasing use, it is essential to be familiar with their immune-related adverse events (irAEs). Here we report a severe case of autoimmune hemolytic anemia (AIHA) associated with cold agglutinin precipitated by pembrolizumab, and a retrospective study of patients treated with ICI utilizing an institutional database where we analyzed the patterns of anti-RBC testing and their ability to predict irAE.MethodsPatients treated with at least one dose of ICI (PD-1, PD-L1, CTLA-4 inhibitors) for advanced cancer between November 2012 and September 2017 at our institution were included. Electronic Medical Records were reviewed to abstract data. Medians and 95% CIs were estimated using Kaplan–Meier method and differences compared using the Log Rank test. Fisher’s exact test and Chi square test were used to analyze clinical associations.ResultsWe identified 1065 patients who received at least one dose of ICI: 180/1065 (17%) underwent direct antiglobulin test (DAT) or allo-antibody (alloAb) testing at any time; 127/1065 (12%) had either DAT or alloAb testing pre-ICI; 129 had either DAT or alloAb testing after ICI initiation; and 76 had either DAT or alloAb testing at both time points. There was a significant association between positive alloAb pre-ICI and the development of irAE while on ICI (p = 0.04).ConclusionGiven the increasing use of ICI, oncologists should be aware of potential irAEs with ICI. We found an association between the presence of an alloAb pre-ICI and the development of irAE, indicating that this previous non-self antigen response may predict immune adverse events. A larger prospective study is needed for systematic evaluation of the association between alloAb testing and irAE, and whether routine testing may inform clinical decision-making for patients.

Pregnancy-Related RBC Alloimmunization in Sickle Cell Trait Patients; A Single-Center Experience

Abstract Background Sickle Cell Disease (SCD) is an autosomal recessive disorder, which results from a point mutation in the β-globin gene. The production of mutant Hb S (V6E) leads to hemolytic anemia and numerous clinical complications. Patients homozygous for HbS gene (SS) typically rely on life-long transfusion therapy. Alloimmunization to foreign RBC antigens in multiply transfused patients is significantly more frequent in SS patients (observed in 7–47%) compared to patients with HbA (AA), and can pose a significant hurdle in finding compatible RBCs. However, the mechanism for enhanced alloimmunization in SS patients has not yet been elucidated. Patients heterozygous for HbS (SA) have a no distinct clinical phenotype and do not typically require transfusions. We investigated the rate of RBC antigen alloimmunization and specificity in SA patients. Aim The aim of this study was to determine and compare the rate and specificities of RBC antigen pregnancy-related alloimmunization in SA and AA patients. Methods In our retrospective study, we identified females who delivered a newborn between January 2014 to October 2019, for whom prenatal hemoglobin electrophoresis testing, and prenatal and perinatal antibody screening and identification was performed. R (V.3.6.2) statistical computing program was used for analysis. Results A total of 41735 subjects were identified: 40058 (AA) and 1677 (SA). African Americans were more prevalent in the SA compared to the AA group (55.8% vs 29.4%, P&amp;lt;0.01)and were more likely to have received ≥ 1 transfusion during pregnancy (4.5% vs 3.3%, P&amp;lt;0.01). A total number of 267 alloantibodies were detected in 246 patients (0.6% of the studied population) during pregnancy, 228 patients (0.6%) in the AA group and 18 patients (1.1%) in the SA group.229/246 (93%) of patients with antibodies did not receive transfusion during pregnancy, and thus antibodies were considered to represent pregnancy-related alloimmunization. In non-transfused subjects the SA group were more likely to have developed alloantibodies in pregnancy compared to the AA group [OR= 1.94, P=0.015]. The median age of patients with antibodies in the SA was older [33.5 vs 27 (p=0.008)] as compared to those in the AA group [29 vs 27 (p=0.002)]. The most common antibody identified was anti-E (SA 42% vs AA 28%), anti-Lea (SA 21% vs AA 13%) and anti-C (SA 5% vs AA 12%). Subjects with pre-existing alloantibodies had a 5.4x increased risk of forming a new allo-antibody. However only 0.1% of subjects had a history of prior allo-antibodies, with no significant difference identified between AA and SA. Conclusions SA patients were found to have an increased rate of pregnancy-related alloimmunization (OR 1.75, P=0.02) after adjusting for the effects of increasing age, RH type, ethnicity, number of previously existing antibodies and number of transfusions in pregnancy.

Prevalence of Ca Blood Type and Alloantibodies in a Population of Horses from Italy.

Simple SummaryIndications for whole blood transfusion in equine critical care include severe anemia from surgical blood loss or acute hemorrhage, hemolysis and neonatal isoerythrolysis. In horses, as in other animals, transfusions are associated with a number of inherent risks such as transfusion reactions. Pretransfusion screening and blood typing are indicated to minimize the risk of incompatible red cell transfusions. Equine blood types include seven systems, namely A, C, D, K, P, Q, and U. The major RBC antigens that warrant identification before packed RBC or whole blood transfusions in horses are Ca and Aa. The frequencies of blood groups can vary from one population to another and from one breed to another. In some situations where testing compatibility is not possible, such as in rural practice, the knowledge of the breed blood type frequencies may help selecting the best donor candidate. The aims of this study were to: estimate the prevalence of Ca blood type in horses from northern Italy; estimate the association between Ca blood type sex and breed of horse; estimate the prevalence of anti-Ca alloantibodies in Ca− horses. The prevalence of the Ca+ blood type was 79.1%. No significant association was found between blood type Ca and sex. The total number of Ca− samples with detectable anti-Ca alloantibodies was 7/23 (30.4%). A knowledge of the blood groups and alloantibodies present is essential for the safe transfusion of blood products in horses. Pre-transfusion screening and blood typing minimizes the risk of incompatible RBC transfusions and prevents immunization of the recipient against incompatible RBC antigens. The frequencies of blood groups can vary among different breeds. Knowledge of a breed’s blood group prevalence can be very useful for identifying the best blood donors during transfusion in clinical practice. The aims of this study were to estimate the prevalence of the Ca blood type in horses from Italy using a monoclonal immunocromatographic method and to estimate the prevalence of anti-Ca alloantibodies in Ca− horses using agglutination on gel technique. Ca blood type was determined on 110 whole blood samples. The prevalence of the Ca+ blood type was 79.1%. This study also provides data about the prevalence of Ca+ blood group in Italian Saddle Horses (77,3%) and Dutch Warmblood (58,3%). No significant association was found between Ca blood type and sex with 79.5% and 78.8% of females and males testing Ca+, respectively. The total number of Ca− samples with detectable anti-Ca alloantibodies was 7/23 (30.4%).