Features of interpretation of the results of studies of ABО and Rhesus antigens and antibodies in patients with hematological diseases

Aim. To assess the aspects of interpretation of pre-transfusion tests in patients with hematological diseases. Material and Methods. We performed an analysis of the results of serological studies of ABО, Rh blood groups in blood samples of 857 patients with oncohematological diseases. ABO blood group determination and typing of D, C, c, E, e, К antigens were carried out using a gel agglutination test. Results. The decrease in strength of the agglutination of standard red blood cells by the patient’s anti-A and/or anti-B antibodies was observed in 112 patients (13.07% of the total number of patients). Abnormal agglutination strength in ABO and Rh antigens testing was observed in 17 patients (1.98% of the total number of patients), among them were 7 patients with acute myeloid leukemia (AML), 6 - with сhronic myeloid leukemia (CML), 2 – myelodysplastic syndrome (MDS), 2 – polycythemia vera (PV). Double populations of red blood cells were mainly detected in patients with MDS (45.61 %), aplastic anemia (AA) (27.27 %), primary myelofibrosis (PMF) (22.73 %), acute leukemia (AL) (22.2 %). In most cases double populations were associated with previous transfusions of blood products, meanwhile, three patients from this group (two patients with CML and one patient with PV) had never received blood transfusions before. Conclusion. Differences in anti-A and anti-B antibodies content were much more common in patients with lymphoproliferative disorders (LPDs) than in patients with myeloproliferative neoplasms (MPNs) (85.71% and 8.04%, respectively), while decrease of expression of red blood cell antigens was more typical for MPNs and did not occur in patients with LPDs

CHEMOKINE RECEPTORS AT DISTINCT DIFFERENTIATION STAGES OF T-HELPERS FROM PERIPHERAL BLOOD

Expression of chemokine receptors (CCR4, CCR6, CXCR3 and CXCR5) on T-helper (Th) cells at various levels of differentiation in a group of healthy volunteers (n = 52) was assessed on the basis of CD45RA and CD62L expression, using the eight-color flow cytometry. It was found that the “naive” T helper cells (N) with CD45RA+CD62L+ phenotype express CXCR3 (4.94±0.39%), and CXCR5 (3.63±0.25%). About 50% of central memory T helpers (CD45RA–CD62L+, CM) were CXCR3 positive, and 43.72±1.27% of CM cells expressed CCR6, whereas CXCR5 and CCR4 levels were about 30%. Furthermore, CXCR3 was expressed by 76.76±0.75% of the CD3+CD4+CD45RA–CD62L– (EM) population, and similar values were obtained for CCR6, while the relative abundance of CXCR5+ cells decreased to 13.68±0.50%, and CCR4 levels did not change and accounted for 33.26±1.13% positive cells. Likewise, co-expression of the chemokine receptors was studied for the abovementioned subpopulations of T helper cells. Among the CXCR5– Th, Th1 cells were identified as CXCR3+CCR6–CCR4– (this subset also contained Th9), and CXCR3+CCR6+CCR4– subsets, referred to as Th1/Th17. Th2 were detected on the basis of CCR4 expression in absence of all other chemokine receptors. In addition to the mentioned Th1/Th17 populations, Th 17 cells were found in the subsets of Th17 CXCR3–CCR6+CCR4– and CXCR3–CR6+CCR4+. The latter also contained a Th22 population. Follicular Th cell populations (CXCR5+) consisted of, at least, six different subsets: CXCR3–CCR6–CCR4– (Tfh/Tfh2), CXCR3–CCR6–CCR4+ (Tfh2), CXCR3-CCR6+CCR4–(Tfh17), CXCR3–CCR6+CCR4+ (Tfh17), CXCR3+CCR6–CCR4– (Tfh1) and CXCR3+CCR6+CCR4–(Tfh1/Tfh17). The cells with Th1/Th9 and Th1/Th17 phenotypes dominated among CM (about 13%), whereas their relative abundance within EM increased to 22.37±1.69% and 31.69±1.52%, respectively. The amounts of Th2 were 8.15±0.46% within CM, and only 1.72±0.15% for EM population. For the cells with Th17/Th22 phenotype, these values are 8.07±0.30% and 12.03±0.57%, respectively. The main Tfh subsets were represented among the CM T-helpers: the relative content of Tfh/Tfh2 was 5.79 0.26%, Tfh2, 1.34±0.07%; Tfh17 with CXCR3-CCR6+CCR4– and CXCR3-CCR6+CCR4+ phenotypes made up to 6.22±0.28% and 3.28±0.16%, as well as Tfh1 (7.68±0.31%), and Tfh1/Tfh17 (4.02±0.17%), respectively. Relative content of the mentioned Tfh subsets was decreased > 2-fold within effector memory Th subpopulation. The data obtained may be applied for diagnostics of different immunopathological conditions and could be used as a comparison group in further studies

Platelet antigens and antibodies. Literature review

Platelet antigens structure, role of platelet antibodies in the pathogenesis of various clinical conditions, characteristic of modern antibodies detection methods are presented in this article

Allosensibilisation to erythrocyte antigens (literature review)

In this article literature review of the causes of allosensibilisation to erythrocyte antigens are presented. It is shown that the ability to produce antierythrocyte antibodies is affected by many factors, principal of whom it is difficult to identify. For the allosensibilisation development requires genetically determined differences in erythrocyte antigens phenotypes of donor and recipient, mother and fetus, which can lead to immune response and antibodies production. The biochemical nature of erythrocyte antigens, antigen dose (the amount of transfused doses, the number of antigens determinants on donor and fetus erythrocytes, the number of pregnancies) are important. Individual patient characteristics: age, gender, diseases, the use of immunosuppressive therapy and the presence of inflammatory processes, are also relevant. Note that antibody to one erythrocyte antigens have clinical value, and to the other – have no. The actual data about frequency of clinically significant antibodies contribute to the development of post-transfusion hemolytic complications prophylaxis as well as the improvement of laboratory diagnosis of hemolytic disease of the newborn in the presence of maternal antierythrocyte antibodies

Platelet antigens and antibodies. Literature review

Platelet antigens structure, role of platelet antibodies in the pathogenesis of various clinical conditions, characteristic of modern antibodies detection methods are presented in this article.</p

THE USE OF DIRECT ANTIGLOBULIN TEST TO DETECT AUTOANTIBODIES IN PATIENTS WITH ANEMIA OF VARIOUS ORIGINS

Introduction. Autoantibodies directed against red blood cells (RBCs) are the main cause of hemolysis in patients with autoimmune hemolytic anemia (AIHA) and they can also complicate the course of certain diseases. Various methods are used to detect autoantibodies, but most of all, a polyand monospecific direct antiglobulin test (DAT) is applied. The method is based on the detection of immunoglobulins of G, M, A classes and С3 complement components bound with RBCs surfaces. DAT is used to differentiate between immuno-dependent and immuno-independent anemia. Objective of this study was the analysis of DAT results in patients with various diseases accompanied by anemia.Materials And Methods. Blood samples of patients aged between 5 to 90 years with anemia who underwent examination and/or treatment at the Russian Research Institute of Hematology and Transfusiology of Russian Federal Medical-biological Agency were used as a study material. The results of laboratory testing for the period from 2013 to 2016 were examined and data analysis was performed. The determination of classes of antibodies directed against RBCs was carried out in a direct Coombs reaction using a gel system DiaMed-ID (DiaMed Micro Typing System, Switzerland).Results. The DAT results were positive in patients with AIHA (53 %), paroxysmal nocturnal hemoglobinuria (PNH, 100 %), cryoglobulinemia (62.5 %), chronic lymphoproliferative diseases (CLPD, 27.5 %), multiple myeloma (MM, 23.6 %), anemia of unknown origin (21.4 %), and other autoimmune diseases (19 %). In blood samples of DAT-positive patients, monospecific IgG or C3 complement components, as well as various combinations of Ig with a complement component: IgG + C3, IgG + IgM + C3, IgM + C3, and IgG + IgA, were detected. In our study, positive DAT result was not always associated with laboratory evidence of hemolysis. Among all DAT-positive patients, signs of hemolysis were observed in 53.2 % of cases. Only in patients with AIHA and PNH hemolysis was observed in 100 % of cases.Conclusions. A positive DAT result is one of the criteria for diagnosing of immune hemolytic anemia, however, it does not always indicate an autoimmune pathogenesis of anemia, the presence of specific autoantibodies, and thus is not always associated with hemolysis. Only a comprehensive assessment of a clinical picture and DAT results makes it possible to diagnose the disease

MULTICOLOR FLOW CYTOMETRIC ANALYSIS OF CYTOTOXIC T CELL SUBSETS

Multiparameteric flow analysis has offered an ability of simultaneous analysis of multiple molecules at the single-cell level. Peripheral blood cells from 110 healthy subjects aged 18-65 years (59 males and 51 females) were stained with antibodies to CD3, CD4, CD8, CD27, CD28, CD45, CD45RA and CD62L, and analyzed using different gating strategies. The first one was based on initial analysis of CD45RA and CD62L expression, and CD3+CD8+ cells were divided into naïve population (CD45RA+CD62L+) comprising approx. 30% of the CD3+CD8+ subset; central memory cells (CD45RA–CD62L+, ~11%), effector memory cells (EM; CD45RA–CD62L–, ~35%) and «terminally differentiated» effector memory cells (TEMRA, CD45RA+CD62L–, ~24% of total CD8+ subset). As based on expression of CD27 and CD28 in EM and TEMRA, some further populations were distinguished, i.e., CD27+CD28+ (termed as EM1, about 19% from CD3+CD8+); CD27+CD28– (EM2, ~5%), CD27–CD28– (EM3, ~9%) and CD27–CD28+ (EM4, ~2%). Appropriate subsets were identified within TEMRA population, as follows: CD27+CD28+ (pE1, ~3%), CD27+CD28– (pE2, ~5%) and CD27–CD28– (E, ~15%). The second approach was based on initialexpression of CD27 and CD28, followed by analysis of CD45RA and CD62L expression on CD27+CD28+subset. Total cytotoxic T cell population was divided into naïve – CD27+CD28+CD45RA+CD62L+ (~30% from CD3+CD8+ subset), central memory (CD27+CD28+CD45RA–CD62L+, ca.~12% of total), transitional memory cells (CD27+CD28+CD45RA–CD62L–, approx.~12%), as well as effector memory cells and effectorcells (CD27+CD28я, ~11% и CD27–CD28–, ~24%, respectively). Expression of CD45RA and CD62L was not analyzed for the latter two populations. Frequencies of all cell populations, identified by means of two different gating strategies, were expressed as percentages of the total CD3+CD8+ and absolute cell counts. Using the gating strategy based on initial analysis of CD45RA and CD62L, some correlations between naïve CD3+CD8+frequencies and donor age were revealed (r = -0.646, р &lt; 0.001, and r = -0.562, р &lt; 0.001, respectively). Relative and absolute counts of ЕМ3 (r = 0.474, р &lt; 0.001 and r = 0.435, р &lt; 0.001, respectively) and Е subsets (r = 0.393, р &lt; 0.001 and r = 0.375, р &lt; 0.001, respectively) CD3+CD8+ subsets showed linear increase with age. Usage of another gating strategy based on CD27 and CD28 expression revealed age-dependent changes in relative and absolute frequencies of naïve CD3+CD8+ (r = -0.638, р &lt; 0.001 and r = -0.530, р &lt; 0.001, respectively). Meanwhile, CD27–CD28– subset accumulated linearly with age (r = 0.495, р &lt; 0.001 and r = 0.442, р &lt; 0.001, respectively). The results suggest that differences in subset distribution are responsible for age-related changes in CD8+ cells

Frequency of red cell, leukocytic and platelet alloantibodies in patients with hematological diseases

History of multiple transfusions in patients with hematological diseases increases the likelihood of immunization to donor blood cells antigensand immunological complications development. Incidence of alloantibodies development in this patients was assessed in this work. Alloantibodies detection was performed in patients with aplastic anemia, acute leukemia, chronic lymphocytic leukemia, and autoimmune thrombocytopenia. 9696 patients were included in this study. Frequency of alloantibodies to red cell antigens was 3.8 %, with 0.9 % of the antibody belong to the immunoglobulin G, and 2.9 % of the cases – to immunoglobulin M. Most of the IgG antibodies had following specificity:monospecific anti-D antibody (21 cases), anti-DC and anti-DE antibodies (4 cases), anti-C (8 cases), anti-E (15), anti-c (13), and anti-K (11). Anti-e (1), anti-Fya (2), anti-Lea (4), anti-S (2), anti-s (2), anti-Jka (2) antibodies were less common. Granulocytes antibodies were found in 66.7 % of 384 patients, with results dependent on the detection method used. The presence of antiplatelet alloantibodies studied in 285 serum samples, of which antibodies were detected in 99 patients (34.7 %). Specificity of platelet antibodies was determined in three serum samples only: anti-2b, anti-1a, anti-1b. In other patients, probably present antibodies to several antigens simultaneously, and to identify them was not possible.</p

Frequency of red cell, leukocytic and platelet alloantibodies in patients with hematological diseases

History of multiple transfusions in patients with hematological diseases increases the likelihood of immunization to donor blood cells antigensand immunological complications development. Incidence of alloantibodies development in this patients was assessed in this work. Alloantibodies detection was performed in patients with aplastic anemia, acute leukemia, chronic lymphocytic leukemia, and autoimmune thrombocytopenia. 9696 patients were included in this study. Frequency of alloantibodies to red cell antigens was 3.8 %, with 0.9 % of the antibody belong to the immunoglobulin G, and 2.9 % of the cases – to immunoglobulin M. Most of the IgG antibodies had following specificity:monospecific anti-D antibody (21 cases), anti-DC and anti-DE antibodies (4 cases), anti-C (8 cases), anti-E (15), anti-c (13), and anti-K (11). Anti-e (1), anti-Fya (2), anti-Lea (4), anti-S (2), anti-s (2), anti-Jka (2) antibodies were less common. Granulocytes antibodies were found in 66.7 % of 384 patients, with results dependent on the detection method used. The presence of antiplatelet alloantibodies studied in 285 serum samples, of which antibodies were detected in 99 patients (34.7 %). Specificity of platelet antibodies was determined in three serum samples only: anti-2b, anti-1a, anti-1b. In other patients, probably present antibodies to several antigens simultaneously, and to identify them was not possible