Application of dye-ligand chromatography for purification of coagulation factor VIII

The problem of processing of donor plasma for creating an effective medications is important. Nowadays a big number of methods for fractionation of plasma separation and purification components, especially coagulation factors and fibrinolysis were described. The methods of biospecifical chromatography are especially attractive. The chromatographic purification of factor VIII is a trouble due to potential risks of activation and instability in the presence of plasma proteases. The anion exchange, gel-filtration and affinity chromatography are widely used for purification of coagulation factor VIII. The synthetic active triazine dyes have a number of advantages as ligands. We explored the possibility of using dye-ligand affinity chromatography on silica carriers for the isolation and purification of clotting factor VIII. The results of studies of sorption this factor with different chromatographic sorbents and the method of its purification from cryoprecipitate by binding of additional proteins on Diasorb aminopropyl with immobilized triazine active dyes are described. As a result of this work, we have selected a group of sorbents are the best for purification of factor VIII of blood coagulation: Diasorb-Active scarlet damask 4GT, Diasorb-Procion gelb M4R, Diasorb-Procion blue HB, Diasorb-Procion blue MXR and Diasorb-Active bright blue K. The purification of factor VIII occured due to the phenomenon of negative affinity chromatography. The specific activity of factor increased approximately tenfold

Informative Value of Combination of Molecular Genetic and Cytogenetic Diagnostic Methods in Myelofibrosis

Introduction. Myelofibrosis is a rare disease affecting myeloid progenitor stem cells. According to the modern criteria of myelofibrosis diagnostics, the confirmation of the clonality of pathological myeloproliferative process is needed. However, the unique pathognomonic marker for this disease is not revealed. Point missense mutation of JAK2 gene in exon 12 – the JAK2V617F mutation, and karyotype abnormalities are the most frequently found in myelofibrosis patients. In case of additional anomalies the disease prognosis worsens dramatically, in particular, because of higher risk of the transformation to acute leukemia. The purpose of the study was to identify informative combination of molecular genetic and cytogenetic clonal markers for diagnosis and prognosis of myelofibrosis. Materials and research methods. 37 patients were recruited in the study (33 with primary myelofibrosis and 4 with secondary post-polycythemia vera myelofibrosis). Cytogenetic studies were performed for 34 patients. All the patients underwent molecular genetic studies of blood to detect the JAK2V167F mutation. The mutational status of JAK2 gene was reexamined in 10 patients from 1 to 2 years after the initial examination. Results of the investigation and their discussion. The presence of the mutation JAK2V167F was confirmed in 59.5 % of the patients. Chromosomal abnormalities were revealed in 38.2 % of patients. The spectrum of abnormalities included deletions and translocations of chromosome 1, deletions of 5q and 20q, trisomies of chromosomes 3, 8, 9 and 12, monosomies of chromosomes 3, 5, 7, 9, 11, 13, 15 and 17, appearance of іsochromosome 17q, and polyploidy. 4 patients had a complex karyotype. The coincidence of investigated point mutation and karyotype abnormalities was revealed in the total of 8 (21.6 %) patients. The presence of the mutant allele V617F was associated with higher frequency of cytogenetical abnormalities. The complete molecular response to the therapy with hydroxyurea and interferon-α was not achieved in any of the 10 patients who underwent the molecular genetic reexamination. The overall mortality of the patients with JAK2V167F mutation was 27.3 % in 24 months. In the absence of the mutation the mortality rate was slightly lower and constituted 20. 0%. Сytogenetic abnormalities were the only detected clonal markers that allowed to confirm the chronic myeloproliferative process in 13.5 % of patients. Conclusions. Simultaneous cytogenetic analysis and the identification of JAK2V167F mutation increased the rate of clonal confirmation of the pathological process in myelofibrosis patients to 73.0 %, in contrast to 38.2–59.5 % using only one method of investigation. The presence of such cytogenetic anomalies as polyploidy and trisomy of chromosome 9 could result in increase of the copy number of JAK2 gene mutant alleles in pathological cells. The patients having both no detectable level of “wild type” allele of JAK2 gene and considerable quantity of lymphocytes in the blood, could possibly be of higher risk of further transformation of myelofibrosis to acute lymphoid, and not only myeloid leukemia