Evaluation of the JAK2V617F Mutational Burden in Patients with Philadelphia Chromosome Negative Myeloproliferative Neoplasms: A Single-center Experience.

The identification of the JAK2V617F mutation in several distinct myeloproliferative neoplasms (MPNs) raised the question how one single mutation incites expression of at least three different clinical phenotypes, i.e., polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). In order to further evaluate already published data on the correlation between mutant JAK2V617F allele burden and specific hematological and clinical parameters, we tested the level of the JAK2 mutation in 134 JAK2+ patients with different MPNs. The patients were diagnosed according to the 2008 WHO criteria and followed for a median of 48 months. The JAK2 V617F quantification was done with a real time polymerase chain reaction (real time-PCR) method. The median allele burden was lowest in ET (25.8%), followed by 34.6% in PV and 51.8% in PMF patients (p<0.01). There was statistically significant association between the mutational load of 10.0-50.0% and blood count parameters in the PV patients (p<0.05). In PMF patients the mutational load was in correlation with older age and leukocyte count that were higher in patients with the mutational load of 10.0-50.0% and >50.0% compared to those with a mutational load of <10.0%. There were no statistically significant associations between the allele burden and blood counts in the ET cohort. Our study confirmed an association between the JAK2V617F allele burden and the distinct MPN phenotypes, indicating unfavorable prognosis in patients with a higher JAK2 allele burden. Our results suggest that JAK2 quantification should be incorporated in the diagnostic work-up of MPN patients as a useful tool for optimal treatment decision.

Haematology in the Republic of Macedonia: present situation and brief history.

The development of clinical haematology in Macedonia has taken place over the past nine decades. The greatest expansion of its development took place in the second half of the 20th century. The oficial start of clinical haematology dates from 1956, when the Department of Haematology was founded within the framework of the Internal Medicine Clinic in Skopje. In the beginning, haematology represented a form of virtual sub-specialty, but its expansion was so progressive and rapid that it reached the highest peaks of Yugoslav haematology in those times. The period from 1968 to 1979 was a period of integral development of haematology and blood-transfusion science in Macedonia. Nowadays, the autonomous Public Health Institution, the University Hematology Clinic, is a unique healthcare, educational and scientific establishment in the Republic of Macedonia in its field of work. The diagnostics algorithm comprises cyto-morphologic and cyto-chemical analysis, through immunologic characterization with the assistance of a flow cytometer, to sophisticated molecular analysis for detecting genetic abnormalities. The therapeutic approach is based upon modern poly-haemotherapeutic protocols, application of monoclonal antibodies, immuno-modulatory agents, molecular target therapy and the use of alogeneic and autologous transplantation of fresh bone-marrow and frozen haemopoietic stem-cells. The current motto of the Haematology Clinic is: always help those who seek help, provide precise and early diagnostics, and apply all up-to-date therapeutic strategies, scientific research, continual education and day-to-day implementation of the latest achievements in the field of haematology in daily practice.

Minimal screening analysis based algorithm for diagnosis and clinical stratification of patients with acute myeloid leukaemia (AML): single centre experience.

In this paper we present our results from a study designed in order to establish and standardize a diagnostic algorithm for acute myeloid leukaemia (AML) in the Republic of Macedonia. A total of 146 consecutive adult patients (>15 years) were enrolled in the study. First, we determined the correct lineage assignment of the blast cells and evaluated the incidence of the favourable PML/RARα, AML1/ETO, CBFß/MYH11 genetic markers among the AML cases. Additionally, the obtained results were correlated with patients' age, comorbidities, and performance status, and each single AML patient was stratified to effective treatment strategy. Our results showed that morphology and cytochemistry established a lineage in 132 (89.1%) of the patients, but not in 16 cases that presented as acute leukaemia, of which 7 were assigned as myeloid, and in two a non-haematopoietic malignancy was indicated with immunophenotyping. Mulitparameter flow cytometry immunophenotyping also changed the assigned lineage based on morphology and cytochemistry in 5 (3.3%) of the patients from lymphoid to myeloid and improved diagnosis in 21 (14.1%) cases. By using a reverse transcriptase-polymerase chain reaction (RT-PCR) essay 28 (23.1%) patients were classified in the prognostically favourable AML genetic group; 8 patients expressed the fusion transcript PML/RARα AML1/ETO and 15 CBFß/MYH11. Moreover, analyses of the age, performance status and comorbidities further strtified an additional 12.5% of the patients to a different risk-adapted therapy. The applied minimal screening-analysis-based diagnostic algorithm enabled improved and more precise diagnosis and clinical stratification in 37.2 % of AML patients from our study group.

A case report of aggressive adult neuroblastoma mimicking acute leukemia with fulminant course and fatal outcome.

A case of aggressive adult neuroblastoma mimicking acute leukemia with fulminant course and fatal outcome is described. Pancytopenia and circulating blasts cells at presentation suggested the diagnosis of acute leukemia in the previously healthy 38 years old Caucasian male patient, but flow-cytometry analysis of the bone marrow disclosed the correct diagnosis of neuroblastoma. The immunophenotype was CD45-/CD56+/CD9+ in around 50% of the mononuclear cells, indicating neuroectodermal origin of the malignant cells. Subsequently, the diagnosis was confirmed by immunohistochemical staining of a bone marrow biopsy. A review of the reported cases of neuroblastoma with leukemic features showed that several of them were misdiagnosed as having leukemia and that the diagnosis of neuroblastoma was made at autopsy examination, indicating that misdiagnosis may happen more often than is appreciated. It is in our opinion that the diagnosis of neuroblastoma should be considered in all cases of acute leukemia and pancytopenia, regardless of the age group of the patients.

Prognostic value of immunoglobulin variable heavy chain gene mutation status: long term follow-up in a series of chronic lymphocytic leukemia patients.

B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease, with many patients surviving for decades with minimal or no treatment, whereas others succumb rapidly to their disease despite therapy. Classical staging systems and laboratory features help predict survival in CLL, but they do not distinguish patients who will progress from those whose disease will remain indolent. In recent years, new molecular prognostic factors have emerged that have significantly improved prediction of the risk for disease progression. The mutational status of the immunoglobulin variable heavy chain genes (VH) is one of the major molecular prognostic factors. In this study we evaluated the association between the immunoglobulin VH gene mutation status and the clinical characteristics and outcome in 65 CLL patients that had been followed for a considerably long period at our institution. At diagnosis, patients with unmutated VH genes had higher median lymphocyte counts (P=0.001), higher total tumor mass score (P=0.001) and more often presented at an advance clinical stage (P=0.005) compared to patients utilizing mutated VH genes. Moreover, the median survival of patients with unmutated VH genes was considerably shorter (VH unmutated, 56 months, VH mutated, 125 months; P<0.001). These data confirmed the prognostic value of immunoglobulin VH genes mutational status in CLL, which divides the disease in two prognostic subsets in terms of overall survival and clinical characteristics of the disease. Analysis of the mutational status of the immunoglobulin VH genes may allow for an individualized approach to CLL treatment in the near future.

Imbalance of tissue-type plasminogen activator (t-PA) and its specific inhibitor (PAI-1) in patients with rheumatoid arthritis associated with disease activity.

The relationship between plasma fibrinogen, D-dimer (DD), t-PA and PAI-1 and their correlation with disease activity (DA) were studied in 45 patients with rheumatoid arthritis (RA) (group B) to further understand the implication of fibrinolysis in the pathophysiology of RA. The control group constituted 24 healthy subjects (group A). A Stoke index (SI) of DA was assigned to each patient. Patients were divided into two groups: C, minimal-mild DA (SI 1-7); D, moderate-severe DA (SI 8-17). Fibrinogen was elevated in RA correlating positively with SI and CRP. Hypercoagulability counteracted by reactive fibrinolysis was inferred from a 10-fold increase of DD in group B as compared to group A. The relatively lower levels of DD in group D compared to group C and their negative correlation with SI (r(s) = -0.49, p = 0.0006) indicate the tendency of fibrinolysis to decrease with the increase of DA. Significant elevation of t-PA and PAI-1 were found in group B compared to group A. While t-PA progressively decreased with the increase of DA (r(s) = -0.45, p = 0.0019), a positive relation of PAI-1 to DA was observed (r = 0.42, p = 0.0042). A 2-fold increase of PAI-1/t-PA molar ratio in group D compared to groups A and C as well as its positive correlation with SI (r(s) = 0.63, p = 0.0001) indicate the displacement of balance between t-PA and PAI-1 in favour of the inhibitor with the increase of DA in RA. The involvement of inflammatory mediators in PAI-1/t-PA imbalance was proposed from the relation of fibrinolytic abnormalities with the activity of systemic inflammatory process.

Calmodulin-related changes in microsomal membrane fluidity during liver regeneration.

Based on our previous findings on the modifying effect of calmodulin (CaM) on the physiochemical properties of biomembrane, we have investigated the possible relationship between intracellular CaM content and endoplasmic reticulum (ER) membrane fluidity and function during liver regeneration. The degree of ER membrane fluidity was estimated by fluorescence polarization analysis with the 1,6-diphenyl-1,3,5-hexatriene probe. Microsomal guanylate cyclase (GC) was used as a functional parameter. The kinetics of the increase in the ER membrane fluidity during liver regeneration was strictly parallel to the CaM surge and was matched by an increase in GC activity. The stimulative effect of splenectomy on liver regeneration and its inhibition by Walker-256 tumor, inferred from the corresponding alterations of CaM levels, were mirrored by the modulation in GC activity. The fluidizing effect of CaM on ER membrane was concluded from the drop in thermotropic transition temperature from 28.3 +/- 1.6 degrees C in control membranes to 17.8 +/- 1.1 degrees C membranes from regenerating livers and to 19.8 +/- 1.2 degrees C in control membranes treated with CaM. Arrhenius plots of GC activity exhibited a transition temperature of 25.5 +/- 1.25 degrees C in controls, which shifted to 20.5 +/- 0.9 degrees C in ER membranes from regenerating livers and to 21.7 +/- 1.1 degrees C in control membranes treated with CaM. The Hill coefficient for the allosteric activation of the GC by Mn.GTP decreased from 1.49 +/- 0.16 in controls to 0.93 +/- 0.085 in membranes from regenerating cells and to 0.86 +/- 0.073 in CaM-treated membranes. Both effects of CaM were consistent with a fluidity increase in the enzyme's lipid microenvironment. The results of the present study suggest that an early key event in liver regeneration may be the CaM-induced modulation of ER membrane fluidity and function.

Electropharmacology of the bradycardic agents alinidine and zatebradine (UL-FS 49) in a conscious canine ventricular arrhythmia model of permanent coronary artery occlusion.

Myocardial infarction was produced in 27 anesthetized dogs by ligating the left anterior descending (LAD) coronary artery proximal to the septal branch. Nineteen of these animals survived the operation and were studied by programmed stimulation in a random sequence between the third and seventh days after the infarct. Complete electrophysiologic testing was implemented in each animal prior to and after single doses of either alinidine (1 mg/kg IV) or zatebradine (0.5 mg/kg IV). Alinidine prevented reinduction of sustained ventricular tachycardia (SVT) in only 2 of 9 dogs and zatebradine in 1 of 8 dogs. The SVT cycle length was not significantly changed in all cases in which it was still inducible despite drug administration (p > 0.05). Alinidine lengthened the effective refractory period (ERP) in the AV node (p < 0.01), whereas zatebradine did not induce a statistically significant prolongation. Conversely, zatebradine increased the left ventricular ERP, while alinidine left it almost unchanged. The rate-corrected QT interval (QTc) did not significantly differ from control values after the administration of either agents. Also, the duration and the ERP of infarctzone potentials, defined as late potentials, remained unaltered. The results indicate that the bradycardic agents alinidine and zatebradine do not exert antiarrhythmic efficacy against SVT induced during subacute myocardial infarction in conscious dogs. None of these drugs substantially changed ventricular electrophysiology or showed a drug-specific proarrhythmic effect.

Thrombin generation and neutralization test (TGNT) - a simple, practical, and sensitive assay for plasma heparin quantitation.

A simple, quantitative method for detection of small amounts of heparin in human plasma is described. This method is based on the activation of coagulation factor X to its enzymatic form (Xa), by a mixture of RVV platelet substitute (Esnouf and Williams 1962, Esnouf and Jobin 1967) in a plasma rendered fibrinogen free without affecting other clotting factores (Bell et al. 1968, Bell 1973), by Arvin (Ancrod). Activation of factor X, results in thrombin generation. Thrombin inactivation by its natural inhibitors depends on the time elapsed from the start-point of activation. The inactivation process is enhanced by heparin (Biggs et al. 1970, Blombäck et al. 1963) and this enhancement depends on the quantity of heparin in the plasma.