Background. when transplantation of hematopoietic stem cells (HSC) is performing, it is necessary to take into account the incompatibility of the donor and recipient in terms of erythrocyte antigens in order to assess the possibility of immunological complications during HSC transfusion and/or graft engraftment (acute hemolysis, delayed hemolysis, etc.). The results of serological research methods do not always allow identifying the true group affiliation due to posttransfusion chimerism in patients and/or the presence of antigen allelic polymorphism.Aim. To establish the frequency of ABO-incompatible allo-HSC transplantations in the National Research Center for Hematology, to determine by molecular methods the group affiliation of patients with a weakened expression of antigens and/or after multiple blood transfusions before HSC transplantation, to clarify the blood type of HSC donors with a weakened expression of antigens.Materials and methods. The blood of 270 HSC donor-recipient couples was examined. The blood group of the ABO, Rhesus, MNS, Kell systems was determined in a plane agglutination test using the corresponding IgM class Tsoliclones and in gel cards. Genotyping was performed by polymerase chain reaction with primers to identify the genes of the ABO, Rhesus, Kell, and MNS systems.Results. In 2018-2020 270 HSC transplantations were performed at the National Research Center for Hematology. In 141 (52.22 %) couples, incompatibility of the donor and recipient according to the ABO system was revealed: major - 23.33 %, minor - 20 %; bidirectional - 8.89 %. problems in assessing of serological results were observed in 97 (36.3 %) patients: in 78 patients with post-transfusion chimerism and 19 patients with weakened antigen expression; in 15 (5.56 %) HSC donors: in 4 due to the lack of information about the blood group of cryopreserved cells, in 10 due to weakened antigen expression, in 1 to search for informative markers for monitoring HSC engraftment. The results of the study demonstrated that the percentage of agglutinated erythrocytes in post-transfusion chimerism cannot be a reliable criterion for establishing the true phenotype of a patient. In donors and patients with weakened expression of antigens, the presence of ABO*O1, -A1, -A2, -B1, RHD weak type 1, RHD weak type 2, RHD weak type 3, RHCE*Cw genes was confirmed. for the first time in Russia gene RHCE*01.38 was found.Conclusion. The prevalence of ABO-incompatible HSC transplants was noted. problems with serological determination of the blood group in a third of patients before HSC transplantation arose due to the presence of post-transfusion chimerism and weakened expression of antigens. Determining of the genotypes of HSC donors is necessary when the expression of antigens is weakened and cryopreserved cells are received. The percentage of agglutinated erythrocytes in post-transfusion chimerism cannot be a reliable criterion for establishing the true phenotype of a patient. Detection of mixed chimerism in the determination of group factors by serological methods is an indication for genotyping, especially in the context of the predominance of incompatible HSC transplantations.

Background. One of the polymorphic antigens in the ABO system is antigen A, which includes many allelic variants with different expression. Immunological methods for determining the blood group of the ABO system have limitations in their use, including due to the presence of weekly expressed antigens in humans. For the correct determination of blood group according to the ABO system, genetic typing is becoming increasingly important. 89 alleles of the ABO*A gene are known. Knowledge of ABO*A gene polymorphisms and their prevalence contributes to the prevention of errors in determining the blood group of donors and recipients.Objective: to describe variants of ABO*A gene alleles in Russians and serological characteristics of the antigens encoded by them.Materials and methods. The blood of 14,000 people was examined. The blood group was determined using anti-A, anti- Aweak, anti-B, lectin (anti-A1) and gel cards. A molecular study of ABO*A gene polymorphisms was conducted in 151 people. Polymerase chain reaction with sequence-specific primers and direct Sanger sequencing were used.Results. 7 different ABO*A alleles were detected, including the ABO*A1.01 and ABO*A1.02 alleles. In 118 individuals with a weak A antigen, the ABO*A2.01 allele was the most frequent (87.29 %). Rare alleles ABO*A2.06 (5.93 %), ABO*AW.06 (4.23 %), ABO*A2.09 (0.85 %) and ABO*Ax (1.70 %) were found. Serological characteristics of A antigens variants depending on genotypes are described, variants A1, A2, A3 and very weak A were detected. Extraagglutinins α1 were absent in all persons with weakened A antigen.Conclusion. Small or mixed agglutination with Coliclones or red blood cell stratification in the gel suggest the presence of antigen A with weakened expression. Modern molecular methods make it possible to identify rare gene alleles and genotypes. Erythrocyte genomics helps to resolve the ambiguity of the serological results allows understanding the true mechanisms of particular phenotype formation and makes a contribution to ensuring the immunological safety of blood components transfusions.

Background. 62 ABO*O alleles of the ABO system are known. Some ABO*O alleles may be accompanied by the presence of residual A-glycosyltransferase activity in people of group O, which may lead to errors in determining the blood group. This confirms the important clinical significance of the ABO*O allele polymorphism. Knowledge of ABO*O gene polymorphisms and their prevalence contributes to the prevention of errors in determining the blood group of the ABO system.Objective: to study allele variants of the ABO*O gene in Russians.Materials and methods. The blood samples of 14,000 people were examined. The blood group was determined using anti-A, anti-Aweak, anti-B, lectin (anti-A1) and gel cards, as well as by cross-sectional method using standard red blood cells of O, A, and B groups. In one patient, the method of adsorption-elution with cold elution was used to identify a weak variant of antigen A, and the method of thermal elution was used to eliminate antigen- blocking plasma factors. Molecular determination of ABO*O alleles was performed in 130 individuals by polymerase chain reaction with sequence- specific primers and Sanger direct sequencing.Results. 13 allelic variants of the ABO*O gene were identified (10 with a typical deletion of c.261delG / N and 3 nondeletional alleles with polymorphism c.802G>A). Deletion alleles of ABO*O.01 were found in 92.85 % of the examined patients, nondeletion alleles of АВО*О.02 group – in 7.15 % of cases. The ABO*O.01.01 allele was detected with a frequency of 67.14 %, other deletion alleles – much less frequently: ABO*O.01.02 and ABO*O.01.11 – 5.71 %, ABO*O.01.26 – 5.00 %, ABO*O.01.12 – 4.30 %, ABO*O.01.13 and ABO*O.01.44 – 1.43 %, ABO*O.01.05, ABO*O.01.46, ABO*O.01.68 – 0.71 % each. Non-deletional alleles were found with the following frequencies: ABO*O.02.01 – 4.3 %, ABO*O.02.03 allele – 2.14 %, ABO*O.02.02 – 0.71 %. All individuals with the O group with the nondeletional allele had the Oαβ group, except for one patient (with the ABO*O.01.02 O.02.02 genotype), who had the Oβ group.Conclusion. For the first time, the immunogenetic characteristics of Russians are given according to ABO*O genes. Erythrocyte genomics helps to resolve the ambiguity of serological methods results and allows understanding mechanisms of different phenotypes formation. For the correct definition of natural isohemagglutinins and weak antigens variants should be used at least two different serological methods.

Background. Rhesus phenotype has been determined in 404 persons which have problems with blood groups identification. Genetic typing of antigen RhD variants was performed in 73 individuals. Objective of the work was to give molecular and serological characteristics of the antigen RhD weak types.Materials and methods. Method of rhesus phenotype determination in direct agglutination test on plane by using of anti-D, anti-C, anti-c, anti-Cw, anti-E and anti-e monoclonal antibodies; gel method of rhesus phenotype determination; methods of genetic typing of RhD; methods of antigen RhD determination in the classic indirect antiglobulin test and in the gel indirect antiglobulin test; method of antigen RhD determination in the saline agglutination test.Results. Serological methods identified 73 red blood samples with the weakened expression of RhD antigen. Molecular methods showed the reasons of weakness of antigen expression. Three RHD*D weak types which are common in Russians (RHD*D weak type 1–3) were identified and for the first time 3 types were found – RHD*D weak type 67, RHD(G255R) and RHD(JVS5-38del4). Serological characteristic of RhD weak types was given. It was shown that combined using of monoclonal antibodies in direct agglutination test and in gel is the most effective serological method of the antigen variants detection. Red blood cells with weak RhD antigens can be recognized by weakness or absence of agglutination with monoclonal antibodies on plane if agglutination in gel was 3+4+.Conclusion. Concrete weak RhD variants can be determined only by genetic typing. Serologically weak antigen variants can be detected by using of at least two series of monoclonal antibodies or by using of two different methods (it is preferable).

The molecular basis of the D-negative phenotype formation in humans is presented in this article. Causes of true and false D-negative phenotype appearance are described. The basis of true D-negative phenotype are changes in the genome, that lead to complete lack of RhD antigen expression on the red blood cells surface, or defective expression of RhD antigen, not detectable by serological methods. The reason for the false D-negative phenotype is the insufficient sensitivity of routine serological methods. Cases of true and false D-negative phenotype identified during the examination of the Russia residents are described. We were able to identify one case of true (RHDψ) and five cases of false D-negative phenotype (RHD weak type 2 – two cases, RHD weak type 15 – one case and RHD weak type 20 – two cases) by molecular method.

Incorporation of H-3-thymidine in the lymphocytes of short-term cultures was evaluated by MicroBeta Plus and 1409 DSA beta-scintillation counters ("Wallac", Finland). Comparable values of lymphocyte proliferative activity were obtained by both counters in specimens from 16 individuals in response to phytohemagglutinin (PHA) stimulation and in 8 donor-recipient pairs in mixed lymphocyte culture (MLC). MicroBeta Plus counter detected a trend to reduction of proliferative responses to stimulation by PHA and by allogeneic cells in MLC (p < 0.05); an increase of lymphocyte proliferation was detected in cultures without stimulation (p = 0.05). The advantages of the desktop MicroBeta Plus counter are multidetector system, convenient 96-pipe cell harvester and solid scintillator and rapid data processing. This recommends the counter for evaluation of proliferative activity of cultured lymphocytes.

The informative value of mixed lymphocyte culture (MLC) test in complex with gene typing for selection of an HLA-identical sibling donor for bone marrow transplantation (BMT) was studied and the HLA genotypical significance of proliferative response in MLC test was evaluated. Compatibility of 443 recipients (hematological patients) with their 474 sibling donors before BMT was studied in 1981-2010 by the MLC test, evaluated by H-3 thymidine incorporation. Two cases of false HLA identity were detected by the MLC test in 200 recipients who had HLA-DRB1, DQB1 identical donors. The relationship between the proliferative response in MLC test and donor-recipient genetic non-identity by HLA class II was detected. It is useful to include the MLC test in the complex of HLA identity evaluation when selecting a donor for BMT. The proliferative response in MLC test detects the donor-recipient HLA non-identity by class II HLA nontyped genes and locus alleles.