No abstract

Hereditary hemolytic anemia, a dominantly transmitted disorder, has affected 12 family members spanning three generations. The concentration of adenosine triphosphate in the red cells was about half that of comparably reticulocyte-rich blood. Since adenosine deaminase and adenosine kinase compete for a common substrate, the greatly increased activity of the former may interfere with nucleotide salvage via the latter.

Umbilical cord blood transplantation (UCBT) has been used increasingly in both pediatric and adult patients. The total nucleated cell (NC) dose infused is the most critical factor in determining speed of engraftment and survival. Using standard collection techniques, the mean NC content of UCB units is about 10 × 10 8, and only 25% of these units reach the target cell dose of 2 × 10 7 /kg in UCBT patients weighing 50-70 kg. We have designed a modified placental/umbilical two-step collection method in which a standard blood fraction obtained by umbilical venipuncture is combined with a second fraction harvested after placental perfusion with 50 ml heparinized 0.9% saline. This second fraction contributed 32% volume and 15% NCs to the whole UCB unit (123.7 ± 50.1 ml and 1.26 ± 0.52 × 10 9 NC). The proportion of progenitor cells in both fractions was not significantly different, indicating that the hematopoietic potential of these larger units is 20% (range, 2%-100%) higher than UCB units collected by standard methods. In addition, the bacterial contamination rate associated with this novel collection method (2.78%) compares favorably. Since 1998 we have further enriched our units by processing only UCB units over 0.8 × 10 9 NCs, resulting in a 36% cell increment (1.46 ± 0.52 × 10 9 NCs). Thus, 84% and 54% of the Madrid UCB Bank inventory would fulfill the target cell dose of 2 × 10 7 /kg in patients weighing 50 and 65 kg, respectively. This significant UCB banking improvement gives larger pediatric and adult patients a greater chance of finding adequate grafts in order to achieve better clinical outcomes after UCBT. Stem Cells 2005;23:324-334

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.