R. Zadro

Discrepancies between APTT results determined with different evaluation modes on automated coagulation analyzers

Modern photo‐optical coagulometers collect optical data in the form of reaction curves and offer a possibility to determine clotting times at different points of clot formation by using different evaluation modes. The objectives of this study were to determine the possible impact of an evaluation mode on activated partial thromboplastin time (APTT) results and to investigate potential benefits from visual inspection of obtained reaction curves. APTT was determined by using actin FS as reagent on two coagulometers (Siemens Medical Solutions) in 174 plasma samples with three different evaluation modes: fixed absorbance (FA), drifting baseline (DB), and point of inflexion (POI) on Behring coagulation timer (BCT), and with DB mode on Behring coagulation system (BCS). Statistically significant difference of APTT results applying the Friedman’s test (P < 0.0001) followed by Dunn’s multiple comparison test (P < 0.05) was obtained in all tested samples between POI mode and all other evaluation modes, independently of analyzer used. The differences obtained indicated that laboratory professionals must be aware of possible different evaluation modes on different analyzers, and establish evaluation mode‐specific reference intervals. Moreover, correctness of a reported result could be confirmed only by visual analysis of the reaction curve.

19 Inherited prothrombotic risk factors in children with perinatal arterial stroke

Perinatal arterial stroke (PAS), defined as a cerebrovascular event which occurs between 28 weeks of gestation and 28 days of postnatal age with either pathological or radiological evidence of focal arterial infarction, has received increased attention as an important cause of cerebral palsy and other neurologic disabilities, including epilepsy and cognitive impairment. Although sensitive neuroimaging techniques have dramatically improved the detection of PAS in recent years, the cause of PAS is still poorly understood. The increasing evidence that inherited or acquired prothrombotic disorders may be implicated in the pathogenesis of PAS prompted us to investigate the prevalence of genetic polymorphisms that encode proteins associated with thrombosis (factor V G1619A [FVL], factor II G20210A [PT], homocysteine metabolism (methylenetetrahydrofolate reductase C677T [MTHFR]), and human platelet antigens (HPA) in children with PAS. Polymorphisms were investigated in 24 children (11 boys, 13 girls) with PAS confirmed by brain imaging and in 103 children (72 boys, 31 girls) from the same geographical region that represented the control group, with standard laboratory techniques. At least 1 prothrombotic abnormality was identified in 18/24 (75 %) children with PAS: 3 were heterozygous for FVL, 16 were carriers of the MTHFR mutation (13 heterozygotes, 3 homozygotes) and 1 was double heterozygous for FVL and MTHFR ; the presence of PT 20210A was not detected. Similar genotype and allele frequencies of HPA-1, HPA-2 and HPA-5 were observed in both study groups. Although higher frequencies of HPA-3a/a (50%), HPA-3a allele (68.7%) and FVL (12.5%) were observed in children with PAS compared to the control group (28.2%, 53.4% and 1.9% respectively), these differences failed to reach statistical significance. On the contrary, homozygosity for MTHFR (12.5% in patients and 1.9% in controls) was found to be the only statistically significant difference between the studied groups.

P035 JAK2 V617F mutation and the clinical features in myeloproliferative disorders

Myeloproliferative disorders share clinical and biological similarities that may be influenced by presence of recently discovered point mutation in JAK2 gene (JAK2 V617F). The aim of this study was to determine the correlation between JAK2 V617F mutation and the clinical features of patients with polycythemia vera (PV) and essential thrombocythemia (ET). The study included 36 patients with PV (PV group) and 38 patients with ET (ET group). The diagnosis was established according to the Polycythemia Vera Study Group criteria. The detection of JAK2 V617F was performed by allele specific PCR (Baxter et al., Lancet 2005) on DNA isolated from bone marrow cells or peripheral blood cells. JAK2 V617F positive and negative members of PV and ET groups were analyzed for peripheral blood counts and bone marrow aspiration. JAK2 V617F positive PV patients exhibited statistically significantly higher number of red blood cells, white blood cells and platelets when compared to PV patients negative for JAK2 V617F. No statistically significant difference was found in hemoglobin concentration and hematocrit value. In contrast, JAK2 V617F positive subjects of the ET group had statistically significantly increased number of red blood cells, hemoglobin concentration and hematocrit value, and no significant difference in platelet or white blood cell count. Bone marrow evaluation revealed that JAK2 V617F positive patients of the PV group had myeloid hyperplasia with the excess of eosinophils and platelet clumps, which is consistent with the findings of the peripheral blood analysis. On the contrary, bone marrow evaluation of the ET group did not reveal any significant differences between JAK2 V617F positive and negative patients. Overall our data suggest that JAK2 V617F mutation status could serve as a criterion to categorize both PV and ET patients into distinct subgroups. Further studies are needed to determine whether JAK2 V617F mutation status in myeloproliferative disorders should be used as a criterion for therapy approach.

P019 JAK2 V617F and FLT3 ITD mutation in myelodysplastic syndrome

Molecular basis of myelodysplastic syndrome (MDS) is characterized by a number of frequently occurring abnormalities. Cytogenetic abnormalities in MDS are among the most important routine parts of diagnosis and prognosis, but mutations like internal tandem duplication (ITD) in FLT3 gene and point mutation V617F in JAK2 gene may provide supplementary information. Since some forms of MDS show a significant overlap with chronic myeloproliferative disease (MDS classified as MDS/MPD-U) or acute myeloid leukemia (AML transformed from MDS), this prompted us to determine the prevalence of FLT3 ITD and JAK2 V617F mutations in patients with MDS. The study included a total number od 50 MDS patients classified according to the World Health Organization classification: 4 patients with refractory anemia (RA), 13 patients with myelodysplastic/myeloproliferative disease-unclassifiable (MDS/MPD-U), 4 patients with chronic myelomonocytic leukemia (CMMoL), 8 patients with RA with excess of blasts (RAEB) and 21 patients with secondary AML (AML transformed from MDS, but not therapy-related MDS). Analysis of bone marrow was done by conventional cytogenetic and fluorescence in situ hybridization (FISH) for del(5q), del(7q) and rearrangement of MLL gene. DNA and RNA were isolated from bone marrow cells or peripheral blood cells. The detection of JAK2 V617F was performed by allele specific PCR (Baxter et al, Lancet 2005) and FLT3 ITD by RT-PCR (Nakao et al, Leukemia 1996). Structural and numerical cytogenetic abnormalities were frequent in patients with secondary AML and RAEB as expected. There were no patients with isolated del(5q) or del(7q). Patients with CMMoL did not have any karyotypic abnormalities with exception of one patient with del(20q) and izo(17q). The presence of JAK2 V617F was detected in one of four patients with RA and one out of thirteen patients with MDS/MPD-U. There was no mutation detected in patients with RAEB, CMMoL or secondary AML. On the contrary, the only two positive FLT3 ITD patients were those with secondary AML. In conclusion, there is a need to analyze the mutational status of JAK2 and FLT3 gene in order to properly differentiate MDS/MPD-U and to predict possible transformation of MDS toward AML.

P015 Outcome in a small series of biphenotypic acute leukemia (BAL) patients

Background: Less than 5% of all cases of acute leukemia are classified as biphenotypic acute leukemia (BAL). Being a distinct entity recognized by the WHO classification, BAL is immunophenotypically defined by the European Group for the Immunological Classification of Leukemia (EGIL) scoring system, whereas according to FAB classification BAL may present as one of the ALL or AML subtypes. Since BAL is both a rare form of acute leukemia and shows diverse biological features, there is no consensus on the best treatment approach in these patients. Aim: Our aim was to analyze the laboratory characteristics and the outcome of patients diagnosed with BAL. Patients and methods: Using the EGIL system, we identified 21 cases (4%) of BAL from 535 newly diagnosed acute leukemia patients in the Zagreb Clinical Hospital Center in the period from end 1994-2006. Results: There were 16 male and 5 female patients with median age of 44 years (16- 74). Among them, there were 12 cases of B+myeloid leukemia (55%), 8 cases of T+myeloid (36%), 1 case of B+T lymphoid (5%) and 1 case of trilineage B+T+myeloid leukemia (5%). Morphologic assessment showed myeloid features in 9, lymphoid features in 6 and undifferentiated in 6 patients. Cytogenetic findings revealed normal as well as a wide range of aberrant karyotypes. The patients were treated according to the protocols for AML or ALL or with low-dose chemotherapy - 8, 10 and 3 patients, respectively. In the majority of patients overall survival was poor with a median of 7 months (1- 100) and with a probability of survival at two years of 35%. Conclusion: Despite the progress in the treatment of acute leukemia, the definition and the prognosis of BAL remains poor. Current treatment approach is heterogeneous and still based on cytomorphology. Treatment protocols designed specifically for this type of leukemia should be devised and studied in larger groups of patients.