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).

Introduction. The identification of weak variants of the A antigen, as well as their differentiation, is necessary for the proper selection of erythrocyte-containing media for blood transfusions. To this end, selective anti-A1 reagents that react only with the A1 antigen are used in combination with anti-A reagents reacting equally with the A1 and A2 antigens. Given that the expression of the A antigen varies within the subgroups and there is no established standard for reagents and procedures, the interpretation of the obtained results presents difficulties.Aim. To develop a strategy for identifying the variants of the A antigen using available reagents in an agglutination reaction.Methods. We compared the effectiveness of four anti-A1 and two anti-H reagents using 23 blood samples (groups A2 and A2B) and control samples (groups A1 and A1 B). Two types of anti-A1 reagents were employed: Dolychos biflorus lectin and monoclonal antibodies. All of the reagents were designed for direct agglutination reactions. Belonging of the erythrocytes to the A2 subgroup was confirmed using genetic analysis.Results. It is shown that anti-A1 reagents did not interact with A2B red blood cells and often reacted with A2 red blood cells. The strength of the reaction with A2 red blood cells varied greatly and was weaker than with A1 red blood cells; however, it hindered the subgroup identification. Simultaneous tests conducted using an anti-H reagent allowed the authors to draw an unambiguous conclusion about blood belonging to a subgroup: a strong reaction indicated the A2 subgroup, whereas a negative or weak reaction indicated the A1 subgroup. A discrepancy was noted between the results obtained for two donors using serological and molecular methods: the A3 subgroup was identified serologically, whereas genotyping revealed the AB0*A1 allele. In both cases, direct sequencing showed a combination of mutant alleles giving the A3 phenotype. When using commercial kits to perform genotyping analysis through a polymerase chain reaction, it should be taken into consideration that primers are matched to the most common variants and cannot detect all mutations of the AB0 gene.Conclusion. Reliable identification of the A2 subgroup through serological methods is possible when using lectin or monoclonal anti-A1 antibodies in combination with a monoclonal anti-H reagent.Conflict of interest: the authors declare no conflict of interest.Financial disclosure: the study had no sponsorship.

Introduction. In patients after multiple blood transfusions, the serological determination of ABO and Rhesus blood groups becomes unreliable due to posttransfusion chimerism, i.e., circulation in the blood of two erythrocytes populations – own and donors. To solve this problem helps the genotyping of blood groups. The literature review includes 42 literature sources, including 9 Russian and 33 foreign articles. Materials and methods. The authors typed blood samples of 24 patients with hematological disorders after numerous erythrocyte-containing transfusions. Antigens A and B, Rh D, Rh C/c, Rh E/e were determined by the serological method using monoclonal antibodies anti-A, antiB, anti-D, anti-C, anti-Cw , anti-c, E and anti-e (Moabs) (Hematologist, Russia). The presence of chimerism was established by hemagglutination typing in gel columns ID-Cards «DiaClon ABO / D + Reverse Grouping» and «DiaClon Rh-subgroups + K». The DNA extract was examined by PCR-SSP using commercial primers ABO-TYPE and RH-TYPE (BAG, Germany). Results. In two patients with 50% chimerism with anti-A and anti-B monoclones, the ABO blood group was genotypically identified. Using molecular method presence of Rhesus system antigens was established in 24 patients with 20-95% chimerism for 1-5 antigens. Serological determination of blood groups at 4 months after the cessation of blood transfusions has confirmed all genotyping results. Conclusion. Genotyping is advisable to use to determine the blood groups in patients after numerous erythrocyte-containing transfusions, which allows increasing immunological safety and preventing alloimmunization to clinically significant erythrocytes antigens.

AIM:to estimate the spread of weak D antigen types of the Rhesus system in the citizens of the Russian Federation and a possibility of serologically identifying these types.SUBJECTS AND METHODS:The red blood cells and DNA of people with weakened expression of D antigen were investigated using erythrocyte agglutination reaction in salt medium (2 methods); agglutination reaction in the gel columns containing IgM + IgG anti-D antibodies, indirect antiglobulin test with IgG anti-D antibodies (2 methods); polymerase chain reaction to establish the type of weak D.RESULTS:A rhesus phenotype was determined in 5100 people in 2014-2015. The weakened agglutinable properties of red blood cells were detected in 102 (2%) examinees. 63 examinees underwent genotyping to identify the variants of the weak D antigen, which identified 6 weak D types. There were the most common weak D types 3 (n=31 (49.2%)) and weak D type 1 (n=18 (28.6%)), including weak D type 1.1 in one (1.6%) case. The other 4 weak D antigen types were as follows: weak D type 2 (14.3% (n=9)), weak D type 15 (4.8% (n=3)), weak D type 4.2 (DAR) (1.6% (n=1)) and weak D type 6 (1.6% (n=1)). The antiglobulin test in the gel column containing antiglobulin serum was the most sensitive serological assay to identify the weak D antigen. Only a molecular test could establish weak D type 15 in 2 samples of red blood cells with Ccdee and ccdEe phenotypes.CONCLUSION:The weak D antigen could be serologically identified in 96.8% of cases. When testing for weak D, particular attention should be given to people with the D-negative phenotype who had the C or E antigens. Our investigations conducted for the first time in Russia will be able to improve the immunological safety of red blood cell-containing medium transfusions for patients.

Serological methods of Rhesus antigens identification in humans cannot identify D-antigen variants. In this article the serological characteristics of Rhesus antigen D weak type 4.2. (Category DAR) are described.

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.

A rare variant of antigen D (type 15 Rh-D weak system) was detected in a pregnant woman. Serological methods for detection of Rh factor do not always identify the actual Rh appurtenance of the individual. Molecular methods of Rh appurtenance identification give an unambiguous result.

A rare variant of antigen D (type 15 Rh-D-weak system) was detected in a pregnant woman. Serological methods for detection of Rh factor do not always identify the actual Rh appurtenance of the individual. Molecular methods of Rh appurtenance identification give an unambiguous result.

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.