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Simultaneous quantitative measurement of mRNA of the WT1, BAALC, EVI1, PRAME and HMGA2 genes in whole blood samples reflects the specific pathological proliferative activity in acute leukemia and their ratio is promising as a diagnostic marker. The transcriptome profile of acute leukemia cells is usually assessed using NGS or microarray techniques after a preliminary procedure for isolation of mononuclear cells. However, the results of using the multiplex PCR reaction for the simultaneous determination of all above mRNAs in whole blood samples have not been published so far. Determination of mRNA of WT1, BAALC, EVI1, PRAME and HMGA2 genes in venous blood level samples by multiplex RT-PCR. The study included 127 blood samples from patients who diagnosis of acute leukemia was subsequently confirmed. In the comparison group, 87 samples of patients without oncohematological diagnosis were selected, including 31 samples (K1) with a normal blood formula and 56 samples (K2) with a violation of the cellular composition - anemia, leukocytosis and thrombocytopenia. RNA isolation and reverse transcription were performed using the Ribozol-D and Reverta-L kits (TsNIIE, Russia). Determination of the mRNA expression level of the WT1, BAALC, EVI1, PRAME and HMGA2 genes by multiplex real-time PCR using a homemade multiplex PCR kit. The mRNA level was characterized by high interindividual variation and did not correlate with the rate of circulating leukocytes or blood blasts. Expression of WT1 mRNA was observed in whole blood only in one patient from the control group and in 112 (88%) patients with leukemia and was combined with a decrease in the level of HMGA2 mRNA expression and BAALC mRNA values. In contrast to the control groups, patients with leukemia had higher levels of BAALC mRNA in AML and ALL, increased PRAME mRNA in AML and APL, but lower levels of HMGA2 in APL.

Overactive JAK pathway signaling is a hallmark of immune diseases and critically affects on inflammation and coagulation. A number of mutations in the JAK2 gene act as driving forces of myeloproliferative neoplasms (MPN), the pathogenesis of certain variants of acute leukemia, a number of solid malignancies and cardiovascular diseases. Assays for quantifying JAK2 mRNA in circulating blood cells can be used as a marker associated with the activity of this enzyme. Development of an original method for detecting JAK2 mRNA in venous blood and assessment of the possible diagnostic value in chronic oncohematological diseases. The development of an RT-PCR method for determining the expression of the JAK2 gene mRNA in venous blood samples was carried out in accordance with the MIQE requirements. Primers and TaqMan probes were designed using the Primer3 program, taking into account the possibility of excluding subsequent DNase treatment. The stability of the investigated mRNA was assessed in vacutainers with different anticoagulants and depending on the storage time of the samples. The study of the expression of JAK2 mRNA in blood leukocytes of 41 patients with B-CLL, 16 patients with CML, 12 patients with multiple myeloma and 39 donors using the developed "real-time" PCR method. The study revealed a decrease in the level of JAK2 mRNA in venous blood samples in patients with primary CLL, but not with CML or with multiple myeloma. The level of the marker in the majority of patients with CLL after the start of therapy returned to the range typical for healthy people. It has been shown that the values of the relative expression of JAK2 mRNA are most stable in the range of 2 - 7 hours after taking blood in a vacutainer with EDTA. An original RT-PCR method was developed for the quantitative determination of JAK2 mRNA in venous blood samples, which meets the requirements of the MIQE system. Determination of JAK2 mRNA can be useful for clarifying the pathogenesis features of certain diseases involving impaired Janus kinase activity and can become a promising marker for prognosis and assessment of the effectiveness of therapy.

The JAK2 V617F somatic mutation is one of the most frequent markers of CHIP (clonal hematopoiesis of indeterminate potential). CHIP is characterized by the presence of a myeloid cells clone in peripheral blood in the absence of the sufficient reasons to diagnose the hematologic disease. The CHIP is proposed as a potential independent risk factor for vascular pathology. The aim of this study is to identify carriers of JAK2 V617F mutation among patients admitted for planned hospitalization at the Federal Center of Cardiovascular Surgery of Krasnoyarsk.The study included 930 venous blood samples. JAK2 V617F mutation was detected by using the allele - specific real time polymerase chain reaction.JAK2 V617F mutation was detected in 15 (1.6%) patients, but only two of them had blood cell count that could cause a hematological disease to be suspected.The inclusion of the JAK2 V617F mutation detection in the complex of laboratory tests of the cardiovascular patients can facilitate the timely identification of patients with increased thrombotic risk, as well as the timely diagnosis of myeloproliferative diseases.Соматическая мутация V617F в гене JAK2 является одним из наиболее частых маркеров клонального гемопоэза «неопределенного потенциала», так называемого синдрома CHIP (clonal hematopoiesis of indeterminate potential). Синдром CHIP характеризуется наличием в периферической крови клона миелоидных клеток при отсутствии достаточных оснований для диагноза онкогематологического заболевания. Наличие CHIP предложено рассматривать как потенциальный независимый фактор риска сосудистой патологии. Цель работы - выявление мутации V617F в гене JAK2 у пациентов, поступивших на плановую госпитализацию в ФГБУ «Федеральный центр сердечно - сосудистой хирургии» Красноярска. Материалы и методы. В исследование включено 930 образцов венозной крови пациентов. Выявление мутации V617F в гене JAK2 выполняли методом аллель - специфической полимеразной цепной реакции в режиме реального времени. Результаты. Среди обследованного контингента выявлены 15 (1,6%) пациентов с мутацией V617F в гене JAK2, при этом только у двух из них показатели гемограммы могли указать на возможность гематологического заболевания. Заключение. Включение теста выявления мутации V617F гена JAK2 в меню лабораторных исследований пациентов кардиологического профиля может способствовать своевременному выявлению больных с повышенным тромбогенным риском, а также своевременной диагностике миелопролиферативных заболеваний.

The evaluation was implemented concerning impact of anticoagulants used during venous blood sampling, on aggregation of thrombocytes and acetylsalicylic acid effect in vitro in 111 patients with suspicion to chronic myelo-proliferative tumors and 16 healthy volunteers. The vacutainers (Becton Dickinson) with 3.2% citrate, with heparin (Becton Dickinson) and S-Monovette system (Sarstedt AGCo) with recombinant herudin were applied. The analysis of aggregation was implemented using the technique of impedance in whole blood before and after preliminary incubation of blood samples with acetylsalicylic acid effect in 0.1 mM concentration. The induction was implemented by ATP in final concentration of 5 mkM. It is demonstrated that ATP-induced amplitude of aggregation under application of heparin and hirudin is significantly higher in comparison with its level in samples of citrate blood. At that, acetylsalicylic acid effect paradoxically increases amplitude of aggregation in samples with heparin but not in samples with citrate or hirudin. The application of hirudin permits evaluating impact of acetylsalicylic acid effect both at aggregation and disaggregation component of thrombocyte functions under erythrocytosis and thrombocytosis and can be recommended as a preferable approach in testing individual sensitivity of patients to acetylsalicylic acid effect.

The detection of somatic mutations in the 9 exon of the calreticulin gene (CALR) is regulated by the clinical recommendations as a diagnostic criterion for chronic Ph-negative myeloproliferative neoplasms (MPN). Some methods of nucleic acids testing are used to identify CALR gene mutations with different requirements for special skills of personnel and expensive equipment. The purpose of this work is to compare the results of the detection of CALR gene mutations in venous blood samples by allele-specific RT-PCR with subsequent electrophoresis, fragment analysis and Sanger- or pyro- sequencing. We used 1284 blood samples of patients with suspected MPN and 20 blood donor samples. Mutations in the CALR gene of the I and II type were identified using PCR-RT with the original primers and TaqMan probes. Also, all samples were tested for mutations in the CALR gene by electrophoretic detection of PCR results in an agarose gel. The use of allele-specific RT-PCR followed by electrophoretic detection made it possible to determine clinically significant mutations in the CALR gene in 81 venous blood samples of JAK2- and MPL-negative patients, including 42 cases of type I mutation, 33 cases of type II mutation and 8 rare CALR mutations. Mutations in the 9 exon of the CALR gene were not detected in any of the 20 blood donor samples or in 121 blood samples of patients with polycythemia vera. In randomly selected 20 negative samples, CALR gene mutations were also not detected using Sanger sequencing. All positive samples were confirmed by fragment analysis, as well as with Sanger- sequencing and pyro- sequencing. The described combined approach to detect mutations of the CALR gene in peripheral blood samples can be used in clinical diagnostic laboratories that have a standard set of equipment for electrophoresis of nucleic acids and a PCR-RT. We also propose a confirmatory test based on the pyrosequencing of DNA using the system of genetic analysis "PyroMark Q24".