[The diagnostic screening of acute leukemia using new technologies of automated blood analysis.]

The leukemia is a neoplastic clonal disease of hematopoietic system with primary affection of bone marrow. The modern technologies of automated blood analysis permit to implement a quick primary screening of pathological samples of blood suspicious for presence of blast cells. The application of various analysis techniques (optical, cytochemical, immune phenotypical) with the purpose of detecting blast cells demonstrates their different distribution at the graphics.

[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 prognostic value of evaluation of minimal residual disease using technique of flow cytofluorometry during application of therapy of chronic lymphatic leukemia].

The achievement of molecular remission is associated with increasing of survival of patients with chronic lymphatic leukemia. The important direction of research is seeking of parameters applicable to forecast of response to therapy. The purpose of the study was evaluating prognostic significance of indicator of minimal residual disease detected by technique of flow cytofluorometry of peripheral blood of patients with chronic lymphatic leukemia during therapy application. The sampling included 112 patients with chronic lymphatic leukemia aged from 43 to 82 years. All patients were given treatment consisted of 6 courses of immune chemotherapy combined with fludarabine with cyclophosphan and rituximab. The samples of peripheral blood were analyzed after 3 courses during therapy and after 6 courses after completion of treatment. The cells were analyzed using 5 and 6 color flow cytometry for the purpose of detection of immune phenotype associated with chronic lymphatic leukemia. The evaluation of minimal residual disease was implemented according international standardized protocol (Rawstron A.C. et al. 2007; 21 (5): 956-64). The minimal residual disease negative status was reached in 87 (78%) patients during evaluation of response after 6th course of treatment. The implementation of indicators of residual disease after 3 courses with fludarabine, cyclophosphan and rituximab permitted to sort out two groups of patients with chronic lymphatic leukemia i.e 67 patients with low (< 0.12%) level of minimal residual disease and 45 patients with high (> 0.12%) level of tumor cells. The rate of molecular remission after completion of treatment. in the given groups consisted 100% and 44% correspondingly. The study demonstrates possibilities of early immune phenotype evaluation of minimal residual disease to forecast differences in response to treatment in patients with chronic lymphatic leukemia that makes it possible to avoid undesirable toxicity of therapy or to choose method of consolidation.

[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 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.

[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.

[A comparative evaluation of the differential count of peripheral blood leukocytes using the Cell-Dyn 3500 and NE-7000 hematology analyzers]. / Sravnitel'naia otsenka differentsirovannogo podscheta leikotsitov perifericheskoi krovi gematologicheskimi analizatorami Cell-Dyn 3500 i NE-7000.

Two blood analyzers, Cell-Dyn 3500 (Abbott) and NE-7000 (Sysmex) are compared as regards their use in clinics of a general profile. Both devices can differentiate the leukocytic formula by 5 parameters (neutrophils, lymphocytes, monocytes, eosinophils, and basophils) and possess a system of alarm signals indicating changes in the leukocytic formula. A total of 165 peripheral blood samples stabilized with K3-EDTA were examined. Visual assessment of stained preparations, 200 cells per smear, was the reference method. Both devices yielded similar results of leukocyte counting, were in good correlation with the visual method in assessment of neutrophils, lymphocytes, and eosinophils. Both devices proved to be inaccurate in counting monocytes, which was due to a great variety of morphologic forms of these cells. In addition, the number of false-positive and false-negative results was assessed. No abnormalities were detected in the 20 samples which were assessed as "negative" by the analyzers. Study of the peripheral blood by automated blood analyzers Cell-Dyn 3500 and NE-7000 helps detect a pathological leukocytic formula in 100% cases, although the type of shifts is not always precisely assessed by these devices. Therefore, morphological control is needed in case of alarm signals.

[The prognostic criteria in the primary myelodysplastic syndrome]. / Prognosticheskie kriterii pri pervichnom mielodisplasticheskom sindrome.

Basing on computer processing of 126 primary clinical and laboratory parameters obtained from 92 patients with myelodysplasia and using multifactorial regression analysis, the authors have developed prognostic models of life span and probability of transformation into acute leukemia. The model of life span enabled recognition of 3 prognostic groups of myelodysplasia patients: of high (median 10 months), moderate (median 22 months) and low (median 35 months) risk. This makes it possible to prognosticate the disease and assume optimal therapeutic policy.