Rapado Inmaculada, Albizua Enriqueta, Ayala Rosa, Hernández Jose Angel, Garcia-Alonso Luis, Grande Silvia, Gallardo Miguel, Gilsanz Florinda, Martinez-Lopez Joaquin
Hematology Service, Hospital Universitario 12 de Octubre, Avenida Córdoba s/n, 28041 Madrid, Spain.
Ann Hematol. 2008 Sep;87(9):741-9. doi: 10.1007/s00277-008-0512-x. Epub 2008 Jun 25.
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.
最近已发表了几种检测JAK2 V617F突变的灵敏方法，其中大多数基于实时聚合酶链反应（PCR）。然而，只有部分方法进行了诊断有效性研究。本研究比较了三种基于实时PCR检测JAK2 V617F突变的方法：两种基于杂交探针（HP）和肽核酸探针（PNA），第三种采用等位基因特异性寡核苷酸引物进行JAK2 V617F定量分析。纳入了149名健康受试者、61例原发性血小板增多症（ET）、32例真性红细胞增多症（PV）、38例继发性血小板增多症和35例继发性红细胞增多症。PV中JAK2 617 HP PCR的有效性测试研究：敏感性（Se）为88%，特异性（Sp）为100%。在ET中，Se为57%，Sp为100%。对于PV中的JAK2 617 PNA PCR，Se为94%，Sp为97.8%。在ET中，Se为70%，Sp为95.7%。在JAK2 V671F等位基因特异性寡核苷酸（ASO）定量PCR（qPCR）中，通过接受操作特征（ROC）曲线确定截断点为1%。在PV中，Se为93.8%，Sp为98.5%。在ET中，Se为80%，Sp为95.9%。2%的健康受试者JAK2 617 PNA PCR呈阳性，2%的健康受试者JAK2 617 ASO qPCR呈阳性。通过克隆和测序在健康受试者中检测到JAK2 V617F突变。JAK2 617 HP在红细胞增多症和血小板增多症的鉴别诊断中是一种合适的检测方法。当JAK2 V617F等位基因负荷较低时，应进行JAK2 617 ASO qPCR。同时测定JAK2 V617F和PRV-1过表达并不能提高JAK2 V617F检测在骨髓增殖性疾病（MPD）中的诊断价值。
