Expansion of CD8+ cells in autoimmune hemolytic anemia.

Autoimmune hemolytic anemia (AIHA) is a rare blood disease associated with the production of auto-antibodies and autoimmune hemolysis. A critical role of B-cells in the development of AIHA has been demonstrated before. Here, we present the analysis of the clonal T-cell populations in patients with AIHA. Thirty-three patients with AIHA were included in this study. Thirteen patients with other anemias, 14 patients with other autoimmune conditions (SLE - 6, RA - 8) and 20 healthy donors were included in the study as a control group. The clonality of T-cell was evaluated by the assessment of the T-cell receptor gamma and beta chain gene rearrangements (TCRG and TCRB). The incidence of T-cell monoclonality detected in patients with AIHA was significantly higher compared to the control group. The persistence of T-cell clones did not correlate with the level of hemoglobin and other signs of remission or relapse and did not disappear after the therapy and clinical improvement (observation period was between 1 and 10 years). There was no correlation between the T-cell clonality and the gender, age, splenectomy, duration or severity of the disease. Fractionation of T-lymphocytes (CD4+, CD8+, CD4+25+) revealed that the monoclonal T-cells belonged to the CD8+ sub-population. We assume that besides a possible causative role of the T-cell clones in AIHA to autoimmune process, these clones do not directly participate in the development and maintenance of hemolysis. Most of the AIHA patients (48.5%) demonstrated a T-cell monoclonality, which requires monitoring and should be distinguished from T-cell tumors.

Eculizumab effect on the hemostatic state in patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a hypercoagulable state associated with acute hemolysis. Eculizumab is used to reduce the intensity of intravascular hemolysis in PNH patients. The hemostatic status of three patients with PNH was assessed during eculizumab treatment by D-dimer assay and the global assays: thromboelastography (TEG), thrombin generation test (TGТ), and thrombodynamics (TD). In the state of hemolytic crisis before the therapy D-dimer concentration was increased in two patients accompanied by hypercoagulation changes in TEG parameter angle (α). TD parameter the clot growth velocity (V) revealed hypercoagulability while TGT parameter ETP was within the normal range in all patients. The lactate dehydrogenase (LDH) activity decreased during the 8months of eculizumab therapy. The physical health was improved, the frequency of hemolytic crisis decreased. Patients periodically exhibited hypercoagulable state: the mean values α=38±11° (with normal range 20-40°), ETP=1311±442nM·min (with normal range 800-1560nM·min), V=31±4µm/min (with normal range 20-29µm/min). During the eculizumab therapy two patients had the repeated clinical manifestation of acute hemolytic crisis, the parameters of the global tests were increased compared to the previous measurement. The global hemostasis tests TEG, TGT and TD revealed hypercoagulability in patients with PNH during eculizumab therapy.

[The method of analysis of distribution of erythrocytes by density: practical guidelines].

The article describes the phthalate method of analysis of distribution of erythrocytes by density and demonstrates its possibility. The distribution of erythrocytes by density is implemented using centrifugation of blood in micro-hematocrit capillaries in presence of compounds of dimethyl- and dibuthylphthalates of known density. The acquisition of such clinically reliable parameters of distribution of erythrocytes by density as mean density of erythrocytes, width of distribution of erythrocytes by density, light and heavy fraction of erythrocytes and maximum of curve of distribution of erythrocytes by density is described. The causes of deviation of distribution of erythrocytes by density from standard values under various pathological conditions are considered. The syndrome of dehydration of erythrocytes is described in details. The simple and accessible method of acquisition of distribution of erythrocytes by density is described. It is demonstrated that analysis of distribution of erythrocytes by density makes it possible to determine character of changes occurring with erythrocytes. The monitoring of parameters of distribution of erythrocytes by density allows evaluating dynamics of pathological process and effectiveness of therapy.

Filtration method for studies of the kinetics of hypo-osmotic pore closure in erythrocyte.

Filterability of erythrocytes through small (3 µ) pores decreases with decreasing osmolarity of suspension medium because of hypo-osmotic swelling of cells. After appearance of lytic pores, erythrocyte filterability increases for some time, while after recovery of membrane integrity it decreases again. We suggest filtration method for studies of the kinetics of hypo-osmotic lytic pores closure. The dynamics of changes in erythrocyte filterability was studied in 2 patients with paroxysmal nocturnal hemoglobinuria and 6 donors (Ht 0.01%, Na phosphate buffer 5 mM, pH 7.4, 35 mOsm, 24°C). The method can be used for studies of erythrocyte membrane characteristics in various diseases and for evaluation of the membranotropic effects of drugs, infusion media, hemolysins, ethanol, etc.

[Density-specific distribution of erythrocytes in different types of anemia].

AIM: To study density-specific distribution of erythrocytes (DSDE) in different types of anemia. MATERIAL AND METHODS: DSDE was determined in anemic patients by fractionation of the whole blood in hematocritic capillaries in the presence of mixtures of dimethyl- and dibutylphthalates with known density. RESULTS: Parameters are proposed which characterize DSDE changes typical for each type of anemia: mean erythrocyte density (MED)--mean density of total erythrocytic population; DSDE width (W)--a characteristic of erythrocytic population heterogeneity; light fraction of erythrocytes (LEF)--% of the cells with density less than 1.086 g/ml (hypochromic cells and reticulocytes); dense fraction of erythrocytes (DEF)--% of cells with density over 1.112 g/ml (hyperchromic cells forming as a result of erythrocyte dehydration). DSDE parameters for different types of anemia differed: reduced MED was typical for iron deficiency anemia (IDA) and paroxysmal nocturnal hemoglobinuria (PNH), increased DEF was seen in microspherocytic anemia (MSA), autoimmune hemolytic anemia (AHA), deficiency of glucose-6-phosphate dehydrogenase, increased LEF was observed in reticulocytosis in all anemia types except MSA, DSDE W was larger in MSA, AHA, PNA. CONCLUSION: DSDE is determined by proportion of erythropoiesis and sequestration of erythrocytes as well as pathological impacts leading to impairment of membrane permeability for cations and erythrocytic metabolism. Informative value of DSDE parameters makes them effective for diagnostic screening of anemias and control over course of different diseases.

Detection of stages of autoimmune hemolytic anemia by evaluating erythrocyte deformability and density.

Study of erythrocyte density and deformability in patients with hemolytic anemia, including long-term monitoring of 5 patients, helped us to characterize the pathological processes leading to changes in the erythrocyte population at different terms of the disease and to detect its main stages (agglutination, pathological dehydration, combination of pathological dehydration and microvesiculation, hemolytic crisis, and remission).