Background. In addition to anti-HLA-I and anti-HPA-antibodies and specific cytotoxic T-lymphocytes, another cause of immune refractoriness to donor's platelet transfusions could be a platelet-associated different classes immunoglobulins PAIg (G, M, A) and C3 / C4‑components of complement system (PAC3, PAC4). These markers can be detected by flow cytofluorometry of double-stained platelets. The fixation density of immunoglobulins and components of complement systems were measured by the mean fluorescence intensity (MFI).Objective: to study additional factors that aggravate the course of refractoriness to donor's platelet transfusions in patients with aplastic anemia (AA) and hemoblastosis.Materials and methods. 77 patients (AA – 47, myelodysplastic syndrome (MDS) – 10, acute myeloid leukemia (AML) – 20) admitted to National Research Centre for Hematology during 11.09.2016–04.28.2018 were enrolled in the study. M / f ratio was 33 / 44, median age was 36 yrs. (19–71 yrs.). Plasmapheresis and cross-matching for PRP selection were used for patients with refractoriness to donor's platelet transfusion. PAIg (G, M, A) and PAC3 / C4 detection and density (MFI) were evaluated in all patients by flow cytofluorometry of doublestained platelets (CD41a-PE; IgA, M, G-FITC; C3 / C4‑FITC) and MFI measurement. Patients with AA were investigated on different stages of therapy and if refractoriness to donor's platelet transfusion is developed. Blood donors (n = 28) MFI measurement results were established as negative control.Results. It was found that MFI PAIgG/M/А and PAC3/С4 was higher in all groups of the patients (АА, MDS, AML), as compared with donors. MFI of PAIgM and PAIgA in patients were significant higher than MFI of PAIgG and PAC3 / C4. Combination of PAIgM / A, PAIgM / C3 / C4 and PAIgA / C3 / C4 were more frequent. Multiple transfusions of PRP were associated with PAIgA and PAC3 detection. Development of refractoriness to donor's platelet transfusions was accompanied by alloantibodies (HLA-I, HPA) and PAIgM, PAC4 detection. In patients of AA group during development of refractoriness to donor's platelet transfusions and multiple infection complications the high density of PAIgM and PAIgA were identified. Relapse of AA was accompanied MFI of PAC3 density increment.Conclusion. In addition to application of a certain transfusion therapy algorithm it is also necessary to detect PAIg (G, M, A) and PAC3 / C4 for prediction of severe refractoriness to donor's platelet transfusions.

The radical-producing activity of human platelets has been studied using the enhanced chemiluminescence method. It is shown that chemiluminescence of isolated platelets is observed only in the presence of lucigenin, a selective probe for superoxide anion; the luminescence is amplified many times upon the addition of NADH and NADPH, the substrates of oxidative chains. The chemiluminescence is not affected by diphenyliodonium, an inhibitor of NADPH oxidase, but it is inhibited in a dose-dependent manner by the oxidative phosphorylation uncouplers dinitrophenol and rotenone. Thus, a superoxide anion radical is the main free radical generated by platelets, and mitochondria are one of the superoxide anion radical sources in platelets.

Refractoriness to transfusions of platelet concentrates (PC) adversely affects the conduct of complex therapy in hematological patients. Individual selection of platelets is recommended for such patients. In cases of high degree of alloimmunization with the formation of polyspecific antibodies, when individual selection is difficult, procedures plasmapheresis (PPs) is included in the treatment program.Aims: to evaluate the effectiveness of PC transfusions by individual selection in patients refractory to transfusions and the use of PPs as a second line therapy in combination with individual platelet selection.Materials and methods: from September 2015 to December 2017, 91 patients with refractory to PC transfusions from 1263 patients who received PC transfusion were observed in the center’s clinics. The median age was 43 (18–71) years. M/F – 38/53. Patients: 20 – aplastic anemia (AA), 17 – myelodysplastic syndrome (MDS), 45 – acute myeloid leukemia (AML), 9 – acute lymphoblastic leukemia (ALL). All patients underwent PC transfusion by individual selection (HLA/HPA) Immucor’s Capture-P solid phase technology. In 28 (30 %) of 91 patients, due to the inability to select, there was a need for PP as a second line therapy. Patients: AA – 4 (20 %); MDS – 8 (47 %); AML – 12 (26 %); ALL– 4 (44 %). The median age was 48 (23–71) years. M/F – 8/20. From 2 to 15 procedures were performed (on average – 6) for each patient. All patients received PC transfusions by individual selection by cross-matching immediately after the PP procedure. The efficacy of PC transfusions was assessed by Absolute Platelet Increment (API) and Corrected Count Increment (CCI), relief of hemorrhagic syndrome.Results: in 26 of 28 refractory to PC transfusions patients, in the absence of compatible donor platelets, carrying out PPs in combination with subsequent individual platelet selection promoted relief of hemorrhagic syndrome, increase in API from 3.3 × 109/L at 29.5 × 109/L and CCI from 1.3 to 10.7. Against the background of PPs, combined with individual selection, the degree of alloimmunization (the percentage of incompatible pairs) decreased on average: AA (n = 4) – from 91.7 to 50.2 %; MDS (n = 8) – from 89.6 to 31.6 %; AML (n = 12) – 86.0 to 40.5 % and ALL (n = 4) – from 91.7 to 37.7 %. In 2 patients with a high degree of alloimmunization and after carrying out PPs, it was not possible to select compatible platelets, PC transfusions were ineffective (API = 5 × 109/L, CCI = 1), and hemorrhagic syndrome was not completely managed, but its severity was reduced.Conclusions. With the development of refractoriness to PC transfusions and the ineffectiveness of individual platelet selection, PPs should be used as the second line of therapy, which, combined with individual selection, increases the likelihood of compatible donor-recipient pairs and increases the clinical efficacy of PC transfusions. When PPs is ineffective in combination with individual selection, it is necessary to exclude the syndrome of increased consumption and other mechanisms of refractoriness.

The radical-producing activity of human platelets was studied using the method of activated chemiluminescence. Lucigenin, a selective probe for superoxide anion, enhanced the chemiluminescence of isolated platelets. The addition of NADH and NADPH as substrates of the oxidative systems significantly amplified the production of superoxide. The NADH- and NADPH-stimulated chemiluminescence was not affected by diphenyliodonium, an inhibitor of NADPH-oxidase, but it was lowered by 2,4-dinitrophenol. Thus, superoxide anion radical is a key free radical generated by the platelets, and one of the sources of superoxide anion radical is the mitochondria of platelets.

Modern therapy of hematological diseases requires the intensive use of maintenance of high-quality and safe platelet concentrate (PC) transfusions. Transfusions of PCs are the main method of arresting hemorrhagic complications in thrombocytopenia. The highest need to PC transfusions is noted in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS), acute leukemia and after transplantation of hematopoietic stem cells (THSC). Multiple PC transfusions may be a risk factor for the development of alloimmunization by leukocyte (HLA) and specific platelet (HPA) antigens. It is advisable to carry out such PC transfusion in alloimmunized patients on the basis of the individual selection with the use of immunological compatibility tests of donor-recipient pairs. In cases of multiple alloimmunization, the selection of a "donor-recipient" pair is difficult or impossible. Circulating alloantibodies and autoantibodies can be eliminated by plasmapheresis.

The aim of this work is to develop a method for ethanol determination in some alcoholic beverages, by using an alcohol dehydrogenase/peroxidase-based amperometric enzyme electrode. The enzymes alcohol dehydrogenase and horseradish peroxidase, as well as the coenzyme nicotinamide adenine dinucleotide (NAD+) were immobilized on a nylon (Biodyne A) membrane. The enzyme-membrane was consecutively attached to the polyethylene membrane of a Clark oxygen electrode, which functioned as a transducer. Ethanol is oxidized by NAD+ in the presence of alcohol dehydrogenase; the NADH produced is aerobically oxidized by horseradish peroxidase. The rate of molecular oxygen consumption, which is directly proportional to the alcohol concentration in the sample, is amperometrically monitored with the oxygen electrode-based biosensor.The analytical characteristics of the biosensor (linear range, sensitivity, selectivity, response time, stability) were investigated. The value of the current intensity was monitored as a function of time, for different ethanol concentrations. The obtained calibration graph was linear within the range 10–80 mM. The values of the ethanol content obtained for the analysed beverages ranged between 4.56% (v/v) for Belheimer beer and 41.83% for Stalinskaya vodka.