Development of E-ice-COLD-PCR assay combined with HRM analysis for Nucleophosmin1 gene mutation detection in acute myelogenous leukemia.

Mutations of the nucleophosmin1 (NPM1) gene represent the most frequent molecular alteration in acute myelogenous leukemia (AML), especially in patients with AML who have a normal karyotype. These alterations have been shown to carry favorable prognostic significance in patients with AML. Several methods have been developed for detection of NPM1 gene mutations. However, their ability to detect low levels of mutations in a wild-type background is limited. In this study, the Enhance improved and complete enrichment Co-amplification at Lower Denaturation temperature Polymerase Chain Reaction (E-ice-COLD-PCR) assay combined with High Resolution Melting (HRM) analysis was developed and validated for highly specific and sensitive screening for NPM1 gene mutations. A total of 83 blood samples from patients with AML were collected, and their DNA was extracted. For mutational analysis, the E-ice-COLD-PCR assay for the detection of NPM1 gene mutations was developed. PCR products were analyzed by HRM analysis. All positive samples were confirmed by direct sequencing. This assay enabled detection specificity and sensitivity of NPM1 mutations in 9/83 patients with AML. Direct sequencing results were 100% concordant with this method. In addition, the limit of detection was 12.5% mutant in the final concentration of 5 ng genomic DNA. The E-ice-COLD-PCR assay with HRM analysis is a highly specific and sensitive screening method for enrichment of detecting NPM1 gene mutations. This method has both a short turn around time and easier interpretation compared to those of other methods.

Biomarkers of disseminated intravascular coagulation in pediatric intensive care unit in Thailand.

INTRODUCTION: Disseminated intravascular coagulation (DIC) is a systemic activation of hemostatic system caused by several causes. Biomarkers including antithrombin (AT), protein C (PC), and thrombomodulin (TM) were reported as the additional markers for DIC in adults. This study aimed to determine the association between biomarkers among patients with overt DIC (ODIC) and nonovert DIC (NDIC) in children in PICU. METHODS: We enrolled 103 subjects, aged 1 month-18 years, who were admitted to PICU at Chiang Mai University (CMU) Hospital >24 hours with underlying conditions predisposing to DIC were enrolled. Biomarkers were tested after 24 hours of admission. Subject who had NDIC on the 1st investigations would have other tests on days 3-5 of admission. RESULTS: The incidence of ODIC by the International Society on Thrombosis and Hemostasis (ISTH) DIC score was found 24%. The bleeding, thrombosis, and death were significantly higher in ODIC group (P < 0.05). Mean levels of AT and PC in ODIC group were significantly different from NDIC one (66.9% vs 79.9%, P < 0.001 and 46.1% vs 59.2%, P = 0.004, respectively) while mean level of TM was not different between two groups. Adding AT to DIC score was better than the original score for predict mortality [area under curve (AUC) = 0.662 vs AUC = 0.65] and bleeding (AUC = 0.751 vs AUC = 0.732). CONCLUSIONS: ODIC is prevalent among critically ill children. Adverse outcomes were more commonly found in children with ODIC. AT and PC levels after 24 hours of PICU admission seem to be the useful biomarkers for ODIC in PICU.