[The evaluation and comparative characteristic of detection of clone of paroxysmal nocturnal hemoglobinuria on reticulocytes using the technique of flow cytometry].

The paroxysmal nocturnal hemoglobinuria is a rare clonal disease characterized by somatic mutation of gene PIG-A at the level of stem hematopoietic cell. This process results in disorder of synthesis of glycosil phosphatidyl innozitol (GPI) anchor fixing numerous molecules on membrane of blood cells which protect blood cells from impact of complement. The international society of clinical cytometry (2010) proposed the guidelines of detection of clone of paroxysmal nocturnal hemoglobinuria among erythrocytes, granulocytes and monocytes. The original technique is proposed to evaluate the clone of paroxysmal nocturnal hemoglobinuria in reticulocyte population of blood using method of flow cytofluorometry. The sampling of 160 samples of blood of patients with clinical symptoms of paroxysmal nocturnal hemoglobinuria and anemia was analyzed. Two modes of gatedrawing were applied--using monoclonal antibodies to CD71 (receptor to transferrin) and reagent BD ReticCount. The high correlation was established between size of reticulocytic clone of paroxysmal nocturnal hemoglobinuria evaluated by CD71 and size of granulocytic and monocytic clone of paroxysmal nocturnal hemoglobinuria. The developed panel (CD71/CD235a/CD59) can be applied for screening and monitoring of paroxysmal nocturnal hemoglobinuria.

[The characteristics of evaluation of expression of ZAP-70 in tumor cells under b-cell chronic lymphatic leukemia using the flow cytofluorometry technique].

The b-cell chronic lymphatic leukemia is the most common among all lymphatic proliferative diseases and is characterized by significant variability of its clinical course. The mutation status of genes of variable region of heavy chains of immunoglobulins (IgVH) is the most reliable prognostic factor forecasting time until beginning of treatment in case of b-cell chronic lymphatic leukemia. However its detection nowadays is inaccessible for routine diagnostics. Among surrogate markers of mutation status the indicator of expression of ZAP-70 by tumor cells estimated using flow cytofluorometry. However, in publications there are different guidelines concerning the technique of mentioned marker. To establish the optimal approach to evaluation of expression of ZAP-70 the peripheral blood samples of 5I patients with b-cell chronic lymphatic leukemia and 10 healthy persons were analyzed. The comparison with the results of detection of mutation status of IgVH-genes revealed the advantage of applying the technique of calculation of MFI ratio during interpretation of data of expression of ZAP-70 obtained with flow cytofluorometry. In this framework, the indicator of expression of ZAP-70 can be applied in assessing the course of disease and time until the beginning of treatment of b-cell chronic lymphatic leukemia.

[The reference values of indicators of total blood analysis of adult working population].

The application in laboratory diagnostics of the hematologic analyzers of different class functioning on the basis of various methods of calculation and identification of blood cells condition the need of establishing the reference intervals for the blood picture parameters. In the documents GOSTK 53002-2008, S28-A3 (CLSI) of 2008 the regulations are proposed to establish the reference intervals with detailed description of data statistical analysis. The processing of blood pictures of 1453 healthy patients the reference intervals were established for the indicators of clinical blood analysis. The results saved in the laboratory data base and obtained using analyzer Sysmex XT-2000i (Sysmex Corporation, Japan) were analyzed.

[The analysis of relationship between numbers of hemopoietic hematoblasts and laboratory indicators of state of hepatobiliary system in children with congenital and hereditary diseases of liver].

The content of CD34/CD45dim-positive cells in peripheral blood of children with congenital and hereditary diseases of hepatobiliary system is studied. The analysis of relationship between numbers of studied cells and level of C-reactive protein, sCD40L, sCD30 and laboratory parameters specific to liver functions is applied The number of CD34-positive hemopoietic hematoblasts in children with hepatocirrhosis correlated with the level of C-reactive protein, albumin, hemoglobin concentration and quantity of blood erythrocytes. No relationship was established with the levels of sDC40L and sDC30. The number ofstudied cells in children with liver diseases was higher than in healthy adult donors.

[Reference values of clinical donor blood analysis].

Basic hemogram parameters were analyzed in 835 blood donors (421 men and 414 women) aged 18 to 67 years) on an ADVIA 120 hematology analyzer. Reference ranges were calculated for each parameter in relation to their distribution (as X mean +/- 2alpha for normally distributed data and 2.5-97.5 per thousand for others). The findings suggest that the normal range should be extended for platelets, red blood cells, hemoglobin, and packed cell volume as compared with the earlier unified normal values, which must be taken into account when deciding whether a subject may be allowed to donate blood.

[The detection of minimal residual disease in patients with chronic B-cell lymphatic leukemia using patient-specified polymerase chain reaction].

The new effective protocols of treatment of chronic B-cell lymphatic leukemia, including purine analogs and monoclonal antibodies, provide robust remissions under this disease. Accordingly, the requirements to remission quality assessment are changed too. In particular the assessment of minimal residual disease is obligatory. To assess minimal residual disease in terms of quantity in case of chronic B-cell lymphatic leukemia the technique of polymerase chain reaction was applied in real time with patient-specific primers from the area of V-D-J combinations of genes of heavy chain of immunoglobulin. The study included samples from 60 patients suffering of chronic B-cell lymphatic leukemia. In 15 of them (25%), it was impossible to apply neither the sequence analysis of genes of heavy chain of immunoglobulin nor the fitting of patient-specific primer. The results of quantitative determination of minimal residual disease were obtained in 45 patients (55 tests). The minimal residual disease was detected in 30 of 55 samples (54.5%) and was not detected in 25 of 55 samples (45.5%). At the same time, the quantitative determination of minimal residual disease was implemented in regard to the initial level of neoplastic cells. The method sensitivity qualified by serial dilutions, consisted 10(-5) or 1 neoplastic cell to 100 000 normal cells. The comparative analysis was applied to the results of determination of minimal residual disease using two methods -polymerase chain reaction in real time using patient-specified primers and four-color flow cytofluometry. The determination of minimal residual disease with both methods was implemented in 37 patients (45 tests). The results of both methods matched in 93.3% (42 tests out of 45) with maximal disparity of one degree. Then Spearman factor consisted 0.87 (p < 0.0001). In 3 out of 45 tests (6.7%) neoplastic cells were detected with only one method. In the first case, it was the method of four-color flow cytofluometry and in other two cases it was polymerase chain reaction in real time. Therefore, the detection of minimal residual disease under chronic B-cell lymphatic leukemia using the method of polymerase chain reaction in real time is rather sensitive and specific and correlates with the results received with the method of four-color flow cytofluometry. The results are the same in the case of using anti-CD20 monoclonal antibodies under treatment.

[Principles and potentialities of the standardization of morphocytochemical diagnosis of acute leukemias].

Under the present conditions, the competitive capacity of a health care facility is provided by the high level and timeliness of diagnosis of disease. The diagnosis of the types of acute leukemia (AL) may be accomplished immunologically, by using a 33-marker panel and without consideration of the morphocytochemical parameters of blast cells. But such an approach complicates and prolongs the examination of patients with AL. Moreover, morphocytochemical data more exactly define the stages of blast cell differentiation than does the immunological phenotype. Their preparation methods are simple and cost-effective. Only M0, M6, and M7 forms of leukemia require compulsory blast cell phenotyping, particularly in the differential diagnosis of acute myeloid leukemia and acute lymphoblastic leukemia. Search for new markers of leukemia cells, including lesions at the chromosomal or molecular levels, is under way. Some of them are only of theoretical value while other markers have been already used by hematologists to diagnose leukemia. Standardization in this essence is the self-assessment of a facility and reference comparison, which are based on the principles of its orientation to the patient, the adjusted system for controlling the quality of health care that is up to the world standards rather than the compliance with the state-regulated standard. The present paper discusses the ways of establishing the uniform rates and requirements for the morphocytochemical diagnosis of acute leukemias.

[Significance of parameters of automated reticulocyte analysis in diagnosis and evaluation of treatment outcome in B12-deficient anemia]. / Znachenie pokazatelei avtomatizirovannogo analiza retikulotsitov dlia diagnostiki i otsenki éffektivnosti lecheniia B12-defitsitnoi anemii.

The results of examinations of reticulocytes in blood samples of 12 patients with the confirmed diagnosis of B12-deficit anemia and of 20 virtually healthy subjects (control group) by means of the GEN-S hematological analyzer (Beckman-Coulter) are described in the paper. Typical changes of the reticulocyte parameters are defined and their dynamics (with treatment by Vitamin B12) is studied. It was established that the additional parameters of the reticulocyte analysis, characterizing the quantity of immature reticulocytes and the fraction of immature reticulocytes, increase by 3 days before the total quantity of erythrocytes increases, which means that the above parameters can be used earlier as criteria in evaluating the treatment efficiency.

[Anemic syndrome in elderly patients with diabetic angiopathy: incidence rate, laboratory diagnosis]. / Anemicheskii sindrom u pozhilykh liudei s diabeticheskimi angiopatiiami: analiz vstrechaemosti, laboratornaia diagnostika.

Anemic syndrome rate in diabetic angiopathy reaching 1/3 in elderly patients, frequency increases after 70 years. The anemic syndrome rate in patients older 60 years suffering from the diabetes mellitus of the 1 type, complicated by "diabetic foot" syndrome and in cases with duration of the disease more than 10 years is elevated. In diabetic angiopathy anemia can be normo- or hypochromic or followed by aniso- or poykilocytosis. Among the causes of anemia in patients with diabetic angiopathies the important place is occupied by mediated by oxidative stress, endotoxicosis and protein glycation prolong hyperglycemia effect on the morphofunctional properties of the red blood cells.

[Anemia syndrome in patients with chronic renal insufficiency on peritoneal dialysis treated with recombinant erythropoietin]. / Anemicheskii sindrom u bol'nykh khronicheskoi pochechnoi nedostatochnost'iu, nakhodiashchikhsia na peritoneal'nom dialize, pri lechenii rekombinantnym éritropoétinom.

The paper presents the results of the studies of erythrocytic and iron metabolism parameters in the course of treatment with recombinant erythropoietin (RE) and iron preparations in 60 patients with chronic renal failure (CRF) on peritoneal dialysis (PD) aged 20 to 75 years. Pretreatment iron deficiency in CRF patients on PD persisted to the end of the third observation period. Hyperferritinemia registered in CRF patients on PD with frequent peritonitis may be considered as a result of active ferritin synthesis by the cells of the system of mononuclear phagocytes in response to inflammation. The only parameter indicating the presence of iron deficiency seems to be the percentage of hypochrome erythrocytes indirectly evidencing for insufficient supply and iron utilization in bone marrow. Pathogenetic mechanisms of anemia development in CRF patients on PD may be both low EP and high levels of proinflammatory cytokines as well as oversecretion of ferritin by activated by monocytes/macrophages ferrin, promoting apoptosis of late erythropoiesis precursors, development of ineffective erythropoiesis and having a suppressive effect on erythropoiesis cell precursors.

[Cytokine system in patients with chronic hepatitis C during treatment with interferon-alfa]. / Sistema tsitokinov u bol'nykh khronicheskim gepatitom C pri lechenii interferonom-al'fa.

AIM: To study changes in serum levels of interleukine-1 beta (IL-1b), IL-6, TNF-alpha (TNFa), HM-HMF and TFR-1 beta (TFR-1b), expression of surface antigens CD14 and CD95 on blood monocytes from patients with chronic hepatitis C (CHC) treated with interferon-alpha (INFa). MATERIAL AND METHODS: Examinations covered 25 CHC patients and 25 healthy controls. Concentrations of proinflammatory cytokines and growth factors in blood serum were measured with ELISA (kits by "R&D systems", USA). CD14 and CD95 antigen expression on monocytes of venous blood were studied using flow cytoflowmeter (Partes, USA) before and after a 12-week course of INFa. RESULTS: Before INFa treatment CHC patients had significantly elevated serum concentrations of TNFa, HM-KSF and TFR-1b. Coexpression of antigens CD14+ and CD95+ was found on 61% of blood monocytes. Three-month INFa treatment lowered levels of TNFa, GM-KSF and CD95+ expression on monocytes as well as TFR-1b concentration in the serum which correlated with a positive trend in the standard clinicolaboratory and virusological indices in the examinees. CONCLUSION: Changes in serum indices of proinflammatory cytokines and growth factors, in expression of CD95 on blood monocytes from CHC patients treated with INFa show an important role of cytokines system activation and mechanisms of programmed cell death in pathogenesis of chronic HCV infection.

[Cytokine production in patients with chronic viral hepatitis C during treatment with interferon-alpha]. / Produktsiia tsitokinov u bol'nykh khronicheskim virusnym gepatitom C na fone terapii interferonom-alpha.

Serum content of proinflammatory cytokines (IL-1 beta, IL-6, TNF-alpha) and growth factors (GM-CSF, TGF-1 beta) and expression of CD14 and CD95 antigens on peripheral blood monocytes before and after 12-day therapy with alpha-interferon were studied in 25 patients with chronic viral hepatitis C (VHC). The concentrations of TNF alpha, GM-CSF, and TGF-1 beta were significantly increased (p < 0.05) and coexpression of CD14+ and CD95+ antigens on monocytes was increased by 61% in VHC patients in comparison with the control. After 3 months of therapy with alpha-interferon, the content of TNF alpha, GM-CSF, and TGF-1 beta essentially decreased and that of IL-6 increased; this was paralleled by improvement of clinical and laboratory parameters and decrease of coexpression of CD14+ and CD95+ antigens on blood monocytes. Modulation of the functions of immunocompetent cells and changed production of cytokines are apparently one of the mechanisms of inhibitory effect of alpha-interferon on HCV infection. Study of proinflammatory cytokines and growth factors in the serum and expression of CD14 and CD 95 antigens on monocytes can serve as additional tests for evaluating the efficiency of interferon therapy in patients with VHC.