Simultaneous quantitative assessment of circulating cell-free mitochondrial and nuclear DNA by multiplex real-time PCR

Quantification of circulating nucleic acids in plasma and serum could be used as a non-invasive diagnostic tool for monitoring a wide variety of diseases and conditions. We describe here a rapid, simple and accurate multiplex real-time PCR method for direct synchronized analysis of circulating cell-free (ccf) mitochondrial (mtDNA) and nuclear (nDNA) DNA in plasma and serum samples. The method is based on one-step multiplex real-time PCR using a FAM-labeled MGB probe and primers to amplify the mtDNA sequence of the ATP 8 gene, and a VIC-labeled MGB probe and primers to amplify the nDNA sequence of the glycerinaldehyde-3-phosphate-dehydrogenase (GAPDH) gene, in plasma and serum samples simultaneously. The efficiencies of the multiplex assays were measured in serial dilutions. Based on the simulation of the PCR reaction kinetics, the relative quantities of ccf mtDNA were calculated using a very simple equation. Using our optimised real-time PCR conditions, close to 100% efficiency was obtained from the two assays. The two assays performed in the dilution series showed very good and reproducible correlation to each other. This optimised multiplex real-time PCR protocol can be widely used for synchronized quantification of mtDNA and nDNA in different samples, with a very high rate of efficiency

Accuracy of frozen section analysis versus specimen radiography during breast-conserving surgery for nonpalpable lesions

BACKGROUND: Whereas specimen radiography (SR) is an established strategy for intraoperative resection margin analysis during breast-conserving surgery for nonpalpable lesions, the use of frozen section analysis (FSA) is still a matter of debate. METHODS: A retrospective review was conducted of 115 consecutive operations in which the two objectives sought were the excision of nonpalpable malignant lesions and breast conservation. Breast surgery was performed in the Gynecology and the Surgery Departments at the Basel University Hospital Breast Center. Whereas one department preferably uses SR for intraoperative margin assessments of lesions involving ductal carcinoma in situ (DCIS) or atypical ductal hyperplasia, the other uses FSA to increase the rate of complete removal of these lesions with a single procedure. The respective accuracy and therapeutic impact of these two techniques are compared here. RESULTS: Intraoperative resection margin assessments were performed with FSA in 80 and SR in 35 of a total of 115 operations performed on 111 patients with pTis, pT1, or pT2 nonpalpable breast cancer. FSA diagnostic accuracy, sensitivity, and specificity were 83.8%, 80.0%, and 87.5%, respectively, compared to 60%, 60%, and 60%, respectively, for SR. FSA tended to have a stronger therapeutic impact than SR in terms of the number of patients in whom initially positive margins were rendered margin-negative thanks to intraoperative analysis and immediate reexcision or mastectomy (27.5% vs. 14.3%; p = 0.124). More importantly, significantly fewer secondary reexcisions were performed in the FSA series than in the SR series (12.5% vs. 37.1%; p = 0.002). Finally, the intraoperative detection of invasive cancer with FSA led to a significantly lower number of secondary procedures for axillary lymph node staging (5% vs. 25.7%; p = 0.001). CONCLUSIONS: The present results suggest that FSA may be more accurate than SR for analyzing intraoperative resection margins during breast-conserving surgery for nonpalpable lesions