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.

Purpose: to study influence of platelet antigens system (HPA — Human Platelet Antigens) differences between donor and recipient on neutropenia and trombocytopenia duration after hematopoietic stem cells transplantation (HSCT).Materials and methods: 40 patients at the age from 19 till 55 years (a median age ~ 28.5) after allogeneic HSCT from HLA-identical sibling are included in the study. For the majority of patients (94%) a source of haematopoetic stem cells (HSC) were a bone marrow. 28 patients received myeloablative conditioning regimens and 12 patients — a lowered intensity conditioning. Achievement of 0.5×109/l or more considered as beginning of neutrophil count recovery. As the beginning of platelet count recovery considered achievement value of 50×109/l after platelet transfusions stopping. Identification of eight allele ≪a≫ and ≪b≫ genes HPA-1,-2,-3 and-5 locuses performed by polymerase chain reaction (PCR) with use allele-specific primers. Serological HlAtyping performed by microlymphocytotoxic test with use of specific serum panels.Results: the basic criterion of patient groups distribution (HPA-identical — 1st group; HPA-compatible — 2nd group; HPA-incompatible — 3rd group) was identity or qualitative characteristic of their differences with donor for HPA-genes. Earlier neutrophils and platelet counts recovery in patients transplanted from HPA-identical/compatible donor in comparison with patients with HPA-incompatible transplant, it is shown. In first two groups neutropenia duration was 13.45 and 14.3 days, respectively; in 3 groups — 19.0 days. This pattern of recovery was shown irrespective of conditioning regimens and leukemia type. Platelet recovery occurred earlier in patients of 1st (16.22 days) and 2nd groups (18.2 days) in comparison with patients of 3rd groups (24.2 days).Conclusion: HPA-incompatibility can influence neutrophils and platelet recovery duration after allogeneic HSCT.

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.

The article provides a literature review about RHD and RHCE polymorphisms which encode different RhD and RhC antigen variants. The data about genes RHD and RHCE polymorphisms, RhD weak types, RhD partial types and RhC variants in Russians is presented for the first time. The molecular and serological characteristics of rare RhD and RhC antigens are summarized. The role of serological and molecular methods in Rhesus system antigens identifying is shown.

Introduction. Neonatal thrombocytopenia presents a serious clinical problem, due to the possible development of dangerous bleeding in the fetus and the newborn. Aim. To elucidate pathogenesis, methods of laboratory diagnostics as well as markers and predictors of neonatal immune thrombocytopenia (NAIT). General findings. NAIT develops due to a mismatch between the mother and the fetus in terms of platelet alloantigens (HPA, Human Platelet Alloantigens). The mother produces alloantibodies against alloantigen, absent on her platelets, but expressed on the platelets of the fetus and the father. Antibodies enter the bloodstream of the fetus, thus causing destruction of the platelets of the fetus/newborn. Neonatal transimmune thrombocytopenia (NTIT) is diagnosed in some newborns (20-40%) from mothers with immune thrombocytopenic purpura (ITP). In this case, the platelets of the fetus/newborn are affected by ITP maternal autoantibodies. The following methods are used to diagnose neonatal immune thrombocytopenia: measurement of platelet-associated immunoglobulins G (TA-IgG); determination of antiplatelet circulating (serum) antibodies (cAB); identification of cAB antigens. In the case of NAIT, the mother has neither thrombocytopenia nor an increase in TA-IgG; however, cABs are detected that react with the father's platelets carrying conflicting HPA alloantigen. In newborns, thrombocytopenia and increased TA-IgG are observed. Alloimmune conflict is confirmed by genotyping HPA of maternal and child alloantigens, and/or by determining the specificity of cABs using alloantigens of HPA-typed donors. In the Russian population, the most common causes of NAIT are conflicts with respect to HPA-1a, HPA-1b (33 % and 33%, respectively) and HPA-15a/b (25%) conflicts. In the case of NTIT, ITP mothers demonstrate reduced platelet count and increased TA-IgG, and thrombocytopenic newborns shows increased TA-IgG. The predictor of NTIT is the presence of antiplatelet cAB in pregnant women with ITP.

Adherence to proper indications for red blood cells (RBC) transfusion is essential because of its potential adverse effects and costs of therapy. Aim of these recommendations is to summarize typed of RBC concentrates and indications for RBC transfusions among different categories of the patients. Methods. The methodological approaches are based on the recommendations of the Russian expert council (leading specialists of the Russian Federation) and literature search for randomized clinical trials evaluating RBC storage duration, hemoglobin thresholds and clinical indications for RBC transfusion without language restrictions. Results. The draft clinical guidelines were reviewed on February 1, 2018 at First Russian Transfusiology Congress of the (Vladivostok). The main types of RBC concentrates, storage duration, transport conditions and indications for RBC transfusions are presented. The indications for RBC transfusions are analyzed for various clinical conditions (in obstetrics, neonatology, hematology, cardiology, neurosurgery, nephrology, in patients with sepsis and septic shock, patients with acute blood loss, in patients after hematopoietic stem cell and organ transplantation). Conclusion. The recommendations are intended for doctors of various specialties, health administrators, medical students.

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.

The aim. Mechanisms underlying the development of neonatal alloimmune thrombocytopenia (NAIT) in in Russia have been studied. Materials and methods. Genetic polymorphisms of human platelet alloantigens (HPA) -1, -2, -3, -4, -5, and -15 were evaluated in 27 families having the newborns with NAIT. NAIT was diagnosed according to the following criteria: (1) newborn with thrombocytopenia; (2) mother with no thrombocytopenia and no increase of platelet associated IgG, (3) presence of antibodies reacting with paternal platelets in maternal plasma / serum. HPA genotyping revealed incompatibilities in 23 out of 27 tested families. In these 23 families HPA-1 conflicts were detected in 16 ones (70%). In 8 cases mothers were homozygous carriers of rare HPA-1 b allele and in another 8 cases - of HPA-1 a allele which cased incompatibilities with fetal HPA-1 a and HPA-1 b respectively. In 5 out of 23 families (22%) there were incompatibilities with fetal HPA-15 (HPA-15a, n=2 and HPA-15b, n=3), in 1 family - with HPA-5b (4%), and in 1 family - with HPA-3b (4%) alloantigens. In conclusion the main causes of NAIT in Russia were HPA-1a and -1b conflicts and HPA-15 conflicts were the second frequent ones.

Daratumumab, the first therapeutic monoclonal antibody that binds to CD38, is used for treatment of patients with multiple myeloma. The CD38 transmembrane protein is highly expressed on the surface of malignant myeloma cells, but also, to a lesser extent, is expressed by many other types of cells, including red blood cells (RBCs). When daratumumab binds to CD38 expressed on RBCs, this may result in positive indirect antiglobulin tests (IAT) performed to identify immunological compatibility before RBC transfusions. Anti-CD38-specific agglutination may also variably affect autocontrol, direct antiglobulin test (DAT), and eluate tests. The aim of the study was to develop a protocol for preventing CD38-specific agglutination in antiglobulin tests by treatment of RBCs with dithiothreitol (DTT). Materials and methods. Between July 2016 and April 2017, 9 multiple myeloma patients undergoing daratumumab treatment were tested using routine methods in the Immunohematology Laboratory of the National Hematological Research Center in Moscow. The tests included ABO and Rh typing, extended phenotype matching, DAT, and IAT in LISS/Coombs gel cards. Tests were performed before and after treatment of RBCs with DTT. Results. No daratumumab interference was observed in ABO, Rh, Kell, MNS or Fy typing with IgM antibodies. DAT and auto control were also negative in all patients. On the other hand, all patients showed daratumumabmediated positive IAT, which was resolved by treatment of RBCs with DTT. Conclusions. Anti-CD38 antibodies interfere with serological tests. This interference can be resolved by treating RBCs with DTT. Blood banks and immunohematological laboratories should be informed that a patient is receiving treatment with anti-CD38.

Adherence to proper indications for red blood cells (RBC) transfusion is essential because of its potential adverse effects and costs of therapy. Aim of these recommendations is to summarize typed of RBC concentrates and indications for RBC transfusions among different categories of the patients. Methods. The methodological approaches are based on the recommendations of the Russian expert council (leading specialists of the Russian Federation) and literature search for randomized clinical trials evaluating RBC storage duration, hemoglobin thresholds and clinical indications for RBC transfusion without language restrictions. Results. The draft clinical guidelines were reviewed on February 1, 2018 at First Russian Transfusiology Congress of the (Vladivostok). The main types of RBC concentrates, storage duration, transport conditions and indications for RBC transfusions are presented. The indications for RBC transfusions are analyzed for various clinical conditions (in obstetrics, neonatology, hematology, cardiology, neurosurgery, nephrology, in patients with sepsis and septic shock, patients with acute blood loss, in patients after hematopoietic stem cell and organ transplantation). Conclusion. The recommendations are intended for doctors of various specialties, health administrators, medical students.

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.