HLA and red blood cell antigen genotyping in SARS-CoV-2 convalescent plasma donors.

Aim: More data is required regarding the association between HLA allele and red blood cell (RBC) antigen expression in regard to SARS-CoV-2 infection and COVID-19 susceptibility. Methods: ABO, RhD, 37 other RBC antigens and HLA-A, B, C, DRB1, DQB1 and DPB1 were determined using high throughput platforms in 90 Caucasian convalescent plasma donors. Results: The AB group was significantly increased (1.5×, p = 0.018) and some HLA alleles were found to be significantly overrepresented (HLA-B*44:02, C*05:01, DPB1*04:01, DRB1*04:01 and DRB1*07:01) or underrepresented (A*01:01, B51:01 and DPB1*04:02) in convalescent individuals compared with the local bone marrow registry population. Conclusion: Our study of infection-susceptible but non-hospitalized Caucasian COVID-19 patients contributes to the global understanding of host genetic factors associated with SARS-CoV-2 infection and severity.

Minor red blood cell antigen phenotyping of athletes sampled in international competitions.

Blood transfusion is performed by cheating athletes to rapidly increase oxygen delivery to exercise muscles and enhance their performance. This method is banned by the World Anti-doping Agency (WADA). Heterologous or allogenic blood transfusion happens when blood from a different person is transfused. The method used to detect this type of doping is based on flow cytometry, by identifying variations in blood group minor antigens present on the red blood cells' surface. Transfusion practices have regained interest since the introduction of human recombinant erythropoietin detection method. It has been reported that the number of occurrences of two athletes sharing an identical phenotype in the same sport was five times higher than the theoretical populational probability. The present work describes the prevalence of 10 erythrocytes surface antigens in a population of 261 athletes from all five continents. The matching phenotype per sport is also described.

Determination of the Rh/Kell phenotypes in donor as well as patients might be significant to provide phenotype-matched blood to cancer patients: A retrospective analysis from a tertiary care oncology center in North India.

BACKGROUND: Multiple reports are available from different parts of the globe indicating the incidences of alloimmunization and blood transfusion-related reactions, which emphasizes the need for phenotyping and providing antigen-matched safe blood. AIMS AND OBJECTIVES: This study aims to determine the frequency of Rh and Kell antigens and phenotype for both donors and patients to propose the importance of providing Rh Kell phenotype cross-matched packed red blood cell (RBC) units to minimize the alloimmunization and transfusion reactions. MATERIALS AND METHODS: Ten thousand blood donors and four thousand patients were investigated between October 2017 and July 2019. Each donor unit was tested for blood grouping, antibody screening, and Rh Kell antigen Phenotyping, and the blood unit was issued after the patient's blood grouping, antibody screening by 3 cell panels, and Rh Kell antigen phenotyping followed by cross-matching with an Rh Kell-matched phenotype RBC unit. RESULTS: Nine thousand four hundred and fifty-two donors were D positive (94.5%) while 548 tested D negative (5.5%). Overall Rh and K antigens frequencies in donors were: "e" (98%) >"D" (94.5%) >"C" (86.6%) > "c" (57.5%) >"E" (18.8%) >K (0.98%). Among patients, 3762 tested D positive (94.05%), and 238 tested D negative (5.95%). Overall Rh and K antigens frequencies in patients were: "e" (98.5%) >"D" (94.05%) >"C" (90.2%) >"c" (51%) >"E" (18.2%) >K (1.8%). CONCLUSION: Our study has given us more clarity on the prevalence of major Rh and K antigens in our donor as well as patient populations, highlighting the similarities as well as differences. This variance holds a great significance, since such donor units when transfused into patients may lead to alloimmunization and adverse transfusion reactions. Hence, the determination of Rh and Kell phenotypes and providing phenotype-matched blood will help prevent such events.

Application of blood group genotyping in complex cases of immunohematology.

BACKGROUND: Red blood cell (RBC) group systems are depicted by antigens on the surface of RBCs, which when transfused to a recipient that lacks them, can result in alloimmunization. Thus, transfusion of matched RBC components to the recipient is recommended, especially for the more immunogenic blood group antigens, such as Rh (E, e, C, and c), Kell, Kidd, Duffy, and MNS. AIMS: The aim of this study was to perform the blood group genotyping from blood samples of 12 polytransfused patients whose phenotyping was inconclusive or incomplete. METHODS: The amplicons were amplified by polymerase chain reaction-sequence-specific primers for the following alleles: RHCE (RHCE * C, RHCE * c, RHCE * E, and RHCE * e), KEL (KEL * 01 and KEL * 02), FY (FY * 01 and FY * 02), and KID (JK * 01 and JK * 02), in addition to the GATA1-mutated gene (FY * 02N.01). RESULTS: Discrepancies were found in the Rh (E) and Kidd systems, in addition to cases of Fyb antigen silencing attributed to the GATA mutation, which was present in all individuals with Fy (a-b-) phenotype. The technique also solved the inconclusive phenotyping caused by mixed-field agglutination. CONCLUSION: The results show the contribution of blood group genotyping in complex immunohematology cases, optimizing the delivery of RBC components suitable for transfusion safety, and expanding the number of compatible donors for patients with the Fy (a-b) phenotype related to the FY (02N.01) allele.

Procuring rare (しい)* Japanese red blood cell units for a bleeding patient with anti-K11 requiring a life-saving procedure.

Alloimmunization to K11 is an extremely rare event. However, given the potential clinical significance of K11 alloantibodies, allocating antigen-negative red blood cell (RBC) units is a clinical necessity. In brief, we report a 39-year-old woman with multiple comorbidities including a right lower-extremity, below-the-knee amputation, who developed aggressive osteomyelitis associated with continuous bloody oozing, leading to anemia. To address these issues, the patient required extremity amputation. Surgery required addressing the concomitant critical anemia (hemoglobin <5 g/dL). However, with anti-K11 (in addition to anti-Jka) identified, no compatible units were immediately on hand and transfusing crossmatch-incompatible, antigen-positive units was deemed too high a risk. After a national search by the American Rare Donor Program (ARDP) was unsuccessful, the ARDP identified 2 irradiated, group O, K0 (Kellnull), Jk(a-) RBC units in Japan that were predicted to be crossmatch-compatible with the patient's plasma. The units were successfully procured and infused, without evidence of adverse reactions, and the patient was able to safely undergo amputation to save her life. This case report reviews the complexities of anti-K11 detection and confirmation, as well as the processes by which K11- RBC units may be procured, which could help others in the global transfusion community should they be faced with similar challenging cases.

RBCeq: A robust and scalable algorithm for accurate genetic blood typing.

BACKGROUND: While blood transfusion is an essential cornerstone of hematological care, patients requiring repetitive transfusion remain at persistent risk of alloimmunization due to the diversity of human blood group polymorphisms. Despite the promise, user friendly methods to accurately identify blood types from next-generation sequencing data are currently lacking. To address this unmet need, we have developed RBCeq, a novel genetic blood typing algorithm to accurately identify 36 blood group systems. METHODS: RBCeq can predict complex blood groups such as RH, and ABO that require identification of small indels and copy number variants. RBCeq also reports clinically significant, rare, and novel variants with potential clinical relevance that may lead to the identification of novel blood group alleles. FINDINGS: The RBCeq algorithm demonstrated 99·07% concordance when validated on 402 samples which included 29 antigens with serology and 9 antigens with SNP-array validation in 14 blood group systems and 59 antigens validation on manual predicted phenotype from variant call files. We have also developed a user-friendly web server that generates detailed blood typing reports with advanced visualization (https://www.rbceq.org/). INTERPRETATION: RBCeq will assist blood banks and immunohematology laboratories by overcoming existing methodological limitations like scalability, reproducibility, and accuracy when genotyping and phenotyping in multi-ethnic populations. This Amazon Web Services (AWS) cloud based platform has the potential to reduce pre-transfusion testing time and to increase sample processing throughput, ultimately improving quality of patient care. FUNDING: This work was supported in part by Advance Queensland Research Fellowship, MRFF Genomics Health Futures Mission (76,757), and the Australian Red Cross LifeBlood. The Australian governments fund the Australian Red Cross Lifeblood for the provision of blood, blood products and services to the Australian community.

Major and minor subgroup profile of blood in patients receiving multiple transfusions and donors.

OBJECTIVE: It was aimed to profile the blood subgroups of our region and to reveal the prevalence of auto/alloimmune sensitization in patients who had to undergo multiple erythrocyte transfusions and to establish the sensitization profile by screening major and minor subgroups. MATERIALS AND METHODS: In this study, the distribution of ABO and Rh system major subgroups was studied in 100 donor blood. As the patient group, 50 patients who received three or more red blood cell transfusions were included. In addition to this group, Kell, Lewis, Duffy blood group systems were studied. RESULTS: According to the ABO system, 35% of the donors were in O, 33% in A, 17% in AB, and 15% in B. According to the Rh system, 75% is Dvi positive. Rh system is 99% e positive and 33% E positive in major subgroups and Kell1 positivity is 8%. In the patient group, 22% D(-) was determined compared to Rh blood group. Among the major subgroups of Rh, C was 68%, E was 14%, c was 76%, and e positivity was found to be 100%. The Kell1 negativity rate is 96%. The highest negativity was found in 86% Lea antigen in Lewis system, in 36% S antigen in MNS system, 34% Fyb antigen in Duffy system, and 24% Jka antigen in Kidd system. When inappropriate blood is given for any antigen, a double population is formed. The double negativities we detected in our study occurred as follows according to blood group systems: E 18%, C 12%, c 8%, Cw 2%, Kell 1 2%, M 8%, N 4%, S 18%, s 6%, Fya 8%, Jka 6%, Jkb 22%. Indirect Agglutination Test (IAT) was negative in all patients. CONCLUSION: IAT negativity in all patient groups suggests that we do not develop alloimmunization, but the high rates of double population suggest a high risk of alloimmunization.

Silent red blood cell autoantibodies: Are they naturally occurring or an effect of tolerance loss for a subsequent autoimmune process?

Unexpected anti-red blood cell (RBC) alloantibodies are routinely investigated in immunohematology and blood banking since their existence in pregnant women may induce haemolytic disease of the foetus and newborn, and their presence in donors may induce haemolytic transfusion reactions or hyperacute rejection in solid organ transplantation. Unexpected anti-RBC alloantibodies may target antigens of the most blood types excluding the expected antibodies targeting the ABO antigens. Their incidence in humans was originally linked to alloimmunization events such as blood transfusions, transplants, or pregnancies. But later, many findings revealed their existence in pathogenic processes such as malignancies, infections, and autoimmune diseases; and usually (but not always) associated to autoimmune haemolytic anaemia (AIHA). Nevertheless, unexpected anti-RBC autoantibodies are also occasionally found in healthy individuals in the absence of AIHA and with no history of alloimmunization or the associated pathologic processes. Hence, they are generally known as non-clinically significant, are excluded for typification and called "silent red blood cell autoantibodies (SRBCAA)". This review highlights evidence related to genetic predisposition, molecular mimicry, immune dysregulation, and immune tolerance loss surrounding the existence of anti-RBC antibodies, describing the presence of SRBCAA as possible early witnesses of the development of autoimmune diseases.

Red cell alloimmunization in transfusion-dependent and transfusion-independent beta thalassemia: A review from the Eastern Mediterranean Region (EMRO).

BACKGROUND: ß-Thalassemia is considered one of the common hemoglobin disorders in the Arabian Peninsula. Red blood cell (RBC) transfusion is a crucial component of the management of transfusion-dependent ß-Thalassemia patients. Patients with Thalassemia Intermedia (TI), also known as non-transfusion dependent ß-thalassemia, have a wide clinical presentation and variable transfusion dependence. Rates of RBC alloimmunization and its risk factors in transfusion-dependent ß-thalassemia patients varied between different reports. Risk of alloimmunization is higher in TI patients. MATERIAL AND METHODS: A literature review on existing reports on alloimmunization rates and risk factors in transfusion dependent and non-transfusion dependent ß-thalassemia in the Eastern Mediterranean region was performed. RESULTS: A total of 17 publications were found. Reported rates of alloimmunization among transfusion-dependent ß-Thalassemia patients ranged between 2.87 and 30 % and between 6.8 and 19.5 % among TI patients. Most centers utilize ABO and RhD matched RBCs. The most common antibodies described are anti-K and anti-E. The risk factors described included age at onset of transfusion, gender, history of splenectomy, duration of transfusion and number of units transfused. Rate of autoantibody formation ranged between 0.1 and 45 %. CONCLUSION: Our review showed variable alloimmunization rates and risk factors in thalassemia patients and scant data on TI patients. The commonest antibodies are anti-K and anti-E. Further studies are required in addressing the rate of alloimmunization, cross-match requirements and role of genotyping in this group of patients. Transfusion support of patients with thalassemia necessitates the availability of blood bank facilities and specialized expertise.

ABO, Rhesus, and Kell Antigens, Alleles, and Haplotypes in West Bengal, India.

BACKGROUND: Few studies have documented the blood group antigens in the population of eastern India. Frequencies of some common alleles and haplotypes were unknown. We describe phenotype, allele, and haplotype frequencies in the state of West Bengal, India. METHODS: We tested 1,528 blood donors at the Medical College Hospital, Kolkata. The common antigens of the ABO, Rhesus, and Kell blood group systems were determined by standard serologic methods in tubes. Allele and haplotype frequencies were calculated with an iterative method that yielded maximum-likelihood estimates under the assumption of a Hardy-Weinberg equilibrium. RESULTS: The prevalence of ABO antigens were B (34%), O (32%), A (25%), and AB (9%) with ABO allele frequencies for O = 0.567, A = 0.189, and B = 0.244. The D antigen (RH1) was observed in 96.6% of the blood donors with RH haplotype frequencies, such as for CDe = 0.688809, cde = 0.16983 and CdE = 0.000654. The K antigen (K1) was observed in 12 donors (0.79%) with KEL allele frequencies for K = 0.004 and k = 0.996. Conclusions: For the Bengali population living in the south of West Bengal, we established the frequencies of the major clinically relevant antigens in the ABO, Rhesus, and Kell blood group systems and derived estimates for the underlying ABO and KEL alleles and RH haplotypes. Such blood donor screening will improve the availability of compatible red cell units for transfusion. Our approach using widely available routine methods can readily be applied in other regions, where the sufficient supply of blood typed for the Rh and K antigens is lacking.

The relationship between immunogenic red blood cell antigens and Human Immunodeficiency Virus infection.

INTRODUCTION: Evidence suggests that red cell antigens may act as receptors for viruses and bacteria and therefore could be associated with HIV infection. Previous studies have been controversial and therefore the aim of this exploratory study was to analyse the expression of immunogenic red cell antigens in HIV-seropositive individuals and to compare the results to negative donors from South Africa. METHODS: The expression of ABO, Rh, Kell and Duffy antigens from 119 HIV-seropositive patients was compared to 317 HIV-seronegative blood donors. Nucleic acid amplification testing and PCR were used to determine the HIV status and the ID-Gel Card Technology was used to determine the blood group antigen profile. RESULTS: There was no significant difference in the expression of A, B, AB, Duffy or Kel antigens between the two groups but significantly lower numbers of HIV+ individuals were O Rh Negative (p = ,0.0001). Analysis of those with a Duffy null phenotype revealed a significantly higher incidence of blood type A RH1-Positive, Dce/R0r and B RH1-Positive, DcEe/R2r within the HIV-seropositive group (p = < 0.05). None of the HIV-seropositive individuals were O RH1-Negative, dce/rr. CONCLUSION: In conclusion these initial findings have demonstrated a decreased incidence of blood type O Rh1-negative in HIV + individuals which suggests that red blood cell antigens may play an important role in susceptibility to HIV infection. The relationship between red cell antigens and HIV infection however remains complex and therefore larger studies are required to confirm these results.

Prophylactic RhCE and Kell antigen matching: impact on alloimmunization in transfusion-dependent patients with myelodysplastic syndromes.

BACKGROUND AND OBJECTIVES: Thirty to 80 per cent of patients with myelodysplastic syndromes (MDS) become transfusion-dependent and are at risk for red blood cell (RBC) alloimmunization. This study compared alloimmunization rates in transfusion-dependent patients with MDS at an institution with a policy of prophylactic antigen matching for RhCE and K (PAM) with those transfused at institutions without such a policy (non-PAM). MATERIALS AND METHODS: Transfusion records were retrospectively reviewed to determine total number of RBC transfusions received, whether RBC phenotyping was performed, the type and date of first alloantibody development and receipt of prophylactic antigen matching for RhCE and K. RESULTS: In 176 transfusion-dependent patients with MDS, the overall rate of new alloimmunization was 17%; the majority of patients (87%) developed at least one alloantibody to Rh or Kell antigens. The alloimmunization rate at the institution with a PAM policy was 11% compared with 23% at non-PAM institutions (P = 0·06). The rate of Rh/K alloimmunization was 7 vs. 22%, respectively (P = 0·008). No patient who received PAM developed a Rh/K alloantibody. CONCLUSION: The rate of alloimmunization was 11% at an institution with a PAM policy which was non-significantly lower than 23% at institutions without a PAM policy. However, rates of Rh/K alloimmunization were significantly lower. Such a policy should be considered in transfusion-dependent patients with MDS, although further studies on cost-effectiveness and careful consideration of resource availability in the local context are required.

Association of HLA-DRB1 and HLA-DQB1 with red-blood-cell alloimmunization in the Czech population.

BACKGROUND AND OBJECTIVES: Alloimmune antibodies against red-blood-cell (RBC) antigens induced in susceptible individuals (responders) by transfusion, pregnancy or transplantation may have serious clinical consequences. The aim of this study was to investigate association of alloimmunization against selected RBC antigens with HLA-Class II. MATERIALS AND METHODS: A total of 230 responders (106 monoresponders and 124 multiresponders) were enrolled into the study. HLA-DRB1 and HLA-DQB1 variants were determined by PCR-SSO and their frequencies compared between the patients (patient subgroups) and 375 ethnically and regionally matched controls. RESULTS: Development of multiple RBC antibodies was associated with HLA-DRB1*15 and HLA-DQB1*06 allelic groups in the patients, with the relationship being particularly apparent in those with anti-C+D antibodies. Furthermore, DRB1*13 and DQB1*06 were more frequent in multiresponders with anti-E+c antibodies and DRB1*03 and DQB1*02 in those with anti-E+Cw. CONCLUSION: For the first time, we confirmed the association of HLA-DRB1*15 with RBC antibody multiresponder status and found HLA-Class II associations for three frequent RBC antibody combinations. Our data support the concept that HLA restriction plays an important role in the response to RBC alloantigens.

Impact of a confirmatory RhD test on the correct serologic typing of blood donors

BACKGROUND: The RHD gene is highly polymorphic, which results in a large number of RhD variant phenotypes. Discrepancies in RhD typing are still a problem in blood banks and increase the risk of alloimmunization. In this study, the RhD typing strategy at a blood bank in Brazil was evaluated.METHODS: One-hundred and fifty-two samples typed as RhD negative and C or E positive by routine tests (automated system and indirect antiglobulin test using the tube technique) were reevaluated for RhD status by three methods. The method with the best performance was implemented and evaluated for a period of one year (n = 4897 samples). Samples that were D positive exclusively in the confirmatory test were submitted to molecular analysis.RESULTS: The gel test for indirect antiglobulin testing with anti-D immunoglobulin G (clone ESD1) presented the best results. Seventy samples (1.43%) previously typed as RhD negative showed reactivity in the gel test for indirect antiglobulin testing and were reclassified as D positive. D variants that may cause alloimmunization, such as weak D type 2 and partial DVI, were detected.CONCLUSION: The confirmatory RhD test using the gel test for indirect antiglobulin testing represents a breakthrough in transfusion safety in this blood center. Our results emphasize the importance of assessing the blood group typing strategy in blood banks.

Impact of a confirmatory RhD test on the correct serologic typing of blood donors.

BACKGROUND: The RHD gene is highly polymorphic, which results in a large number of RhD variant phenotypes. Discrepancies in RhD typing are still a problem in blood banks and increase the risk of alloimmunization. In this study, the RhD typing strategy at a blood bank in Brazil was evaluated. METHODS: One-hundred and fifty-two samples typed as RhD negative and C or E positive by routine tests (automated system and indirect antiglobulin test using the tube technique) were reevaluated for RhD status by three methods. The method with the best performance was implemented and evaluated for a period of one year (n=4897 samples). Samples that were D positive exclusively in the confirmatory test were submitted to molecular analysis. RESULTS: The gel test for indirect antiglobulin testing with anti-D immunoglobulin G (clone ESD1) presented the best results. Seventy samples (1.43%) previously typed as RhD negative showed reactivity in the gel test for indirect antiglobulin testing and were reclassified as D positive. D variants that may cause alloimmunization, such as weak D type 2 and partial D(VI), were detected. CONCLUSION: The confirmatory RhD test using the gel test for indirect antiglobulin testing represents a breakthrough in transfusion safety in this blood center. Our results emphasize the importance of assessing the blood group typing strategy in blood banks.

REGGI and the American Rare Donor Program.

The American Rare Donor Program (ARDP) was formed in 1998 to provide rare blood units for patients in need. Members of the program identify rare donors and submit donor information for entrance into a database, REGGI. Information on patients in need of rare blood is also submitted and entered into REGGI. REGGI serves to match phenotypes of registered donors with patients having the respective antibodies. A search process for available units ensues, and blood is provided to the patient. This report provides information on REGGI and its use in the ARDP.

Responder individuality in red blood cell alloimmunization.

Many different factors influence the propensity of transfusion recipients and pregnant women to form red blood cell alloantibodies (RBCA). RBCA may cause hemolytic transfusion reactions, hemolytic disease of the fetus and newborn and may be a complication in transplantation medicine. Antigenic differences between responder and foreign erythrocytes may lead to such an immune answer, in part with suspected specific HLA class II associations. Biochemical and conformational characteristics of red blood cell (RBC) antigens, their dose (number of transfusions and pregnancies, absolute number of antigens per RBC) and the mode of exposure impact on RBCA rates. In addition, individual circumstances determine the risk to form RBCA. Responder individuality in terms of age, sex, severity of underlying disease, disease- or therapy-induced immunosuppression and inflammation are discussed with respect to influencing RBC alloimmunization. For particular high-risk patients, extended phenotype matching of transfusion and recipient efficiently decreases RBCA induction and associated clinical risks.