| Objective:The mutation rate of JAK2V617 F gene is high in patients with myeloproliferative neoplasms(MPN).The main method is to detect mutant load by real-time fluorescence quantitative PCR(RT-qPCR),but it will not achieve absolute quantification.According to the new diagnostic classification of WHO in 2016,pre-PMF was separated from PMF and became a new disease.However,its diagnosis is complex,mainly based on the megakaryocyte morphology in bone marrow biopsy,and it is not easy to differentiate from ET.The purpose of this study is to detect the mutation load of JAK2V617 F in ET,PV and PMF quantitatively by using droplet digital PCR(ddPCR),and provide strong laboratory evidence for the diagnosis of the three.Methods:1.Using ddPCR technology based on MGB probe,we detected the mutation load of JAK2V617 F in 149 MPN patients at DNA level,and compared the difference between ET,PV and PMF groups.2.According to the diagnostic criteria of MPN in 2016 and combined with the results of bone marrow biopsy,all MPN patients were rediagnosed.The patients with ET,PV,and prePMF,which were in accordance with the diagnostic criteria,were selected to compare the mutation load of their JAK2V617 F.3.According to the 2016 WHO diagnostic criteria for MPN and the difference of JAK2V617 F mutation load,the indeterminate MPN was rediagnosed.Results:1.We collected the only JAK2V617 F positive MPN patients 149 cases from 2013.11 to 2017.10 in the second hospital of Shanxi Medical University,.according to the diagnostic criteria of WHO in 2008,there were 86 patients in ET,27 patients in PV,19 patients in PMF,17 patients in the uncertain type of MPN group.2.According to the diagnostic criteria of MPN in 2008,we carry out quantitative JAK2V617 F mutation load test in 132 cases with clear diagnosis by ddPCR,including 86 cases of ET,27 cases of PV and 19 cases of PMF.Among the three groups,the mean value of JAK2V617 F mutation was 31.68 ± 19.70 in group ET,83.44±6.77 in group PV and 59.47±26.10 in group PMF.There was a difference between group ET and group PV,group ET and group PMF,which was statistically significant,p<0.001.There was no statistical difference between group PV and group PMF,p=0.68.The JAK2V617 F mutation load Cut-off value of group PV and group ET was 54.7 by ROC curve statistics.3.In strict accordance with the diagnostic criteria of WHO in 2016,we diagnosed 44 cases from all the collected cases,including 26 cases of ET group,PV group of 11 cases,7 cases of group pre-PMF.JAK2V617 F mutation load average respectively: group ET 26.33±9.49,PV group 81.87±5.95,pre-PMF group 68.33±14.06.There was a statistical difference between group ET and group PV,group ET and group pre-PMF.There was no statistical difference between group p<0.001,PV and pre-PMF,p=0.1.4.The JAK2V617 F mutation load of 17 patients was analyzed,and 5 cases of < 54.7%,12 cases > 54.7% were obtained.According to bone marrow morphology and follow-up data,17 patients were rediagnosed.5 patients with a load of less than 54.7% were diagnosed as ET,12 cases with medium load > 54.7% were diagnosed as PV,and 2 were diagnosed as pre-PMF.Conclusions:1.A new method of JAK2V617 F mutation load detection based on ddPCR technology has been established.2.The absolute quantitative detection of JAK2V617 F mutation load by dd PCR technique can provide a powerful laboratory basis for the differential diagnosis of PV,ET,PMF and atypical MPN,and the JAK2V617 F mutation load can be used as an auxiliary diagnostic indicator.3.For the differential diagnosis of ET and pre-PMF patients,the 50%JAK2V617F mutation load can be used as a diagnostic reference.When the JAK2V617 F mutation load is more than 50%,the diagnosis is pre-PMF;when the JAK2V617 F mutation load is less than 50%,the diagnosis is ET. |
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