JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis , which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera , secondary erythrocytoses , essential thrombocythemia , or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera , and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra-and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia , clinical sensitivities were 93.5% and 67.9% , clinical specificities were 98.8% and 97.0% , positive predictive values were 93.5% and 79.2% , and negative predictive values were 98.8% and 94.6 , respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis , and the three mutations in exon 12 were detected by both methods. Hence , HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition , HRM analysis has the advantage of detecting unknown mutations. High resolution melting (HRM) analysis represents a new generation of mutation scanning technology. HRM molecular methods based on real-time PCR have recently been developed and allow screening of known and unknown mutations, including 1-bp substitutions. Furthermore, melting curve analysis with a high-resolution melting instrument is a simple, high performance, time saving, and low labor-intensive technique that is a sensitive and specific tool for detecting DNA variations.

Several sensitive methods for the detection of JAK2 V617F mutation have been published recently, most of them based on Real Time polymerase chain reaction (PCR). However, only some of them have performed studies of diagnostic validity. This study compares three methods based on Real Time PCR to detect JAK2 V617F mutation: two based on hybridization probes (HP) and peptide nucleic acid probe (PNA) and a third employing allele specific oligonucleotide primers for JAK2 V617F quantification. One hundred forty-nine healthy subjects, 61 essential thrombocythemia (ET), 32 polycythemia vera (PV), 38 secondary thrombocytoses, and 35 secondary erythrocytoses were included. Validity test study for JAK2 617 HP PCR in PV Sensitivity (Se) was 88% and in Specificity (Sp), 100%. In ET, Se was 57% and Sp, 100%. For JAK2 617 PNA PCR in PV, Se was 94% and Sp, 97.8%. In ET, Se was 70% and Sp, 95.7%. In JAK2 V671F allelo-specific-oligonucleotide (ASO) quantitative PCR (qPCR), cutoff point of 1% was established by receiving operating characteristic (ROC) curves. In PV, Se was 93.8% and Sp, 98.5%. In ET, Se was 80% and Sp, 95.9%. Two percent of the healthy subjects were positive by JAK2 617 PNA PCR and 2% by JAK2 617 ASO qPCR. JAK2 V617F mutation was detected in healthy subjects by cloning and sequencing. JAK2 617 HP is an adequate test in differential diagnosis for both erythrocytosis and thrombocytosis. When JAK2 V617F allele burden is low, JAK2 617 ASO qPCR should be performed. Simultaneous determination of JAK2 V617F and PRV-1 overexpression does not improve the diagnostic value of JAK2 V617F tests in MPD.

BackgroundPortal vein thrombosis is a frequent complication in end-stage cirrhosis with a considerable peri-operative risk for liver transplant candidates. We aimed to characterize the pre-transplant portal vein thrombosis in a cohort of liver transplant recipients, and to identify independent risk factors for this complication.Methods380 consecutive primary orthotopic liver transplants were performed in the Digestive Surgery Department of “12 de Octubre” Hospital (Madrid, Spain), between January 2001 and December 2006. The main risk factors considered were smoking, obesity, metabolic disorders, previous immobility, surgery or trauma, nephrotic syndrome, associated tumor, inflammatory disease, neoplasm myeloprolipherative. Furthermore we have reported genetic thrombophilia results for 271 recipients.ResultsSixty-two (16.3%) patients developed pre-transplant portal vein thrombosis and its presence had no impact in the overall survival of liver recipients. Obesity was the only independent risk factor for pre-transplant portal vein thrombosis.ConclusionWe recommend close control of cardiovascular factors in patients with liver cirrhosis in order to avoid associated thrombosis.

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We aimed to quantify peripheral donor chimerism (DC) and to analyze its association with graft and recipient outcome. Forty-two liver transplant recipients and their respective donors were studied, providing a total of 148 posttransplantation serum samples. DC was assessed with real-time quantitative polymerase chain reaction (qPCR) to detect polymorphic markers. DC did not decrease with time post-transplantation and was higher in child recipients versus adults and in recipients of deceased donor liver transplants versus recipients of live donor liver transplants. Higher levels of DC were detected in Rh-positive blood group donors, in O blood group recipients versus A blood group recipients, and in recipients with hepatitis C virus versus recipients with alcoholic cirrhosis. High DC was associated with patients with organ damage due to recurrent disease and rejection. Stable, high levels of DC, in the absence of other major clinical events, may thus be a marker of transplantation tolerance, and this knowledge may help to tailor immunosuppressive treatment. In conclusion, qPCR is a useful technique for DC follow-up in liver transplantation, although the evolution of DC levels should be analyzed in accordance with the clinical outcome of the patient. Liver Transpl 15: 581-591, 2009.