[The development and comparative approbation of methods of increasing sensitivity of detection of mutation V617F in gene JAK2 by pyro-sequencing].

The possibilities of early detection of chronic myelo-proliferative tumors (MPT) are determined by sensitivity of techniques implemented for finding somatic mutation V617F in gene JAK2. The mutation V617F can also be found in individuals without unfolded picture of hematological diseases. The detection of mutation even in low concentrations is associated with increasing of risk of cerebral stroke and thrombosis of arterial and venous vessels. The study was carried out to develop techniques based on COLD polymerase chain reaction and allele-specific polymerase chain reaction targeted to increasing of sensitivity of finding mutation V617F detected using pyro-sequencing. The analytical sensitivity of techniques was evaluated by control samples with different ratio of alleles. For allele-specific polymerase chain reaction analytical sensitivity amounted to 0.25% of mutant allele at concentration of analyzed control sample 10 copies of DNA per mkl. For COLD polymerase chain reaction sensitivity amounted to 0.5% at concentration 10 copies of DNA per mkl. The comparative approbation of techniques was implemented using clinical material obtained from 106 patients with suspicion on MPT. The analysis of clinical samples using COLD polymerase chain reaction revealed 13 (14%) and using technique of allele-specific polymerase chain reaction - 15 (16%) positive samples. In all 15 cases of detection of mutation clinical confirmations of diagnosis of MPT was received. The proposed techniques permit increasing efficiency of amplification of mutant DNA in analyzed samples and hence to increase sensitivity of subsequent analysis of products of amplification using technique of pyro-sequencing. Therefore, the mentioned techniques can be recommended to be applied for confirmation of diagnosis of MPT and early identification of individuals with increased risk of development of venous and arterial thromboses.

[ON THE ISSUE OF SELECTION OF COAGULANTS UNDER ADP-INDUCED IMPEDANCE TEST OF AGGREGATION OF THROMBOCYTES IN PATIENTS WITH CHRONIC MYELO-PROLIFERATIVE TUMORS].

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 AG & Co) 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.

[On issue of detection of limits of normal reaction of thrombocytes in testing of impedance agregometry].

The study was carried out to evaluate analytical characteristics of impedance agregometry technique and aspirin test. The sampling included 89 healthy volunteers i.e. 55 females and 34 males. The analysis of aggregation of thrombocytes was implemented using aggregometer Chrono Log 700,. The ADP in ultimate concentration of 5 mkM was used as inductor. The coefficients were calculated concerning analytical, intra- and inter-individual variation (CVA, CV1, and CVG accordingly), index of individuality and critical level of difference (RCV). The study established that ADP-aggreagtion of thrombocytes was significantly higher in females as compared with males. The necessity of stratification of population by gender for calculation of reference limits was established using method of criterion Z calculation (Z=3.6; Zcrit=1.3). The intra-individual and analytical variation of impedance aggregometry made up to 5.8% and 8.1% correspondingly. Overall, parameters of aggregation in whole blood are characterized by high degree of individuality (II<0.06) that limits application of reference intervals in agregometry for differentiating norm and pathology. The statistically reliable alteration of functional activity of thrombocytes can be considered only at change of amplitude of aggregation no less than up to 26.9% as comparing with previous test in particular individual.