Evaluation of Clinical Laboratory Methods for Plasma Cell-Free DNA Analysis in Suspected Septicaemia

Urosevic N Inglis Tjj

doi:10.4172/2168-9784.1000123

Abstract

Background: The amount and quality of plasma cell free DNA (cfDNA) changes in extreme physiological and pathological conditions. These changes could provide a basis for a novel prognostic biomarker in conditions as diverse as pregnancy, cancer, organ transplantation and septicaemia. Evaluation of current methods for cfDNA analysis is needed to identify the best practical approach to medical diagnostics. Methods: Initially, cf DNA was extracted from plasma of nine patients with febrile illness using both QIAamp Circulating Nucleic Acid and QI Aamp Mini Blood DNA kits. CfDNA concentration was determined by β-globin qPCR using PerfeCta and AmpliTaq mixes. Subsequently, Qubit fluorimetric and gel-on-a-chip assays were used to analyse plasma cfDNA from 64 additional patients with suspected septicaemia and positive blood cultures. Results: Determination of CfDNA concentration by β-globin gene qPCR using AmpliTaq mix was superior to the PerfeCta qPCR. Moreover, unlike PerfeCta AmpliTaq qPCR determined similar genome equivalent copy numbers in cfDNA isolated by either DNA extraction method. QIAamp Mini Blood kit and AmpliTaq qPCR were subsequently used in a larger prospective study for cfDNA isolation and quantification, respectively. However, qPCR was less suited for the detection of high plasma cfDNA levels when compared to direct DNA measurement by Qubit (mean 22.23 ng/ mL vs. 61.38 ng/mL, respectively) despite good correlation between the two methods. The DNA microfluidic chip method was then used to determine cfDNA fragments sizes and their relative concentrations revealing a presence of nucleosome-sized DNA fragments that were in strongly positive correlation with total cfDNA. Furthermore, apoptotic DNA was identified as a major DNA component in plasma with high cfDNA content. Conclusions: β-globin qPCR is more suited to detection of low plasma cfDNA concentrations, while the Qubit assay is a better choice for rapid identification of high plasma concentrations. Apoptosis is a major source of cfDNA in plasma with high cfDNA concentrations based on DNA chip analysis.

Highlights

Increased plasma levels of circulatory cell-free DNA occur in various medical conditions including sepsis [1]
DNA concentration was determined by β-globin gene qPCR using two different DNA polymerase mixes, PerfeCta and AmpliTaq (Figure 1)
When total plasma was used directly in qPCR, only the PerfeCta mix produced a positive signal while the qPCR reaction with AmpliTaq mix was consistently negative (Figure 1)

Summary

Introduction

Increased plasma levels of circulatory cell-free DNA occur in various medical conditions including sepsis [1]. Changes in blood coagulation and in function of endothelial cells can result in fibrin deposition, endothelial impairment leading to multiple organ failure and fatality [8]. Besides their decisive role in the pathophysiology of septicaemia, both immune cell loss and organ failure may variously contribute to circulating cfDNA potentially providing an independent prognostic tool for predicting the severity and outcome of sepsis. The amount and quality of plasma cell free DNA (cfDNA) changes in extreme physiological and pathological conditions These changes could provide a basis for a novel prognostic biomarker in conditions as diverse as pregnancy, cancer, organ transplantation and septicaemia. Evaluation of current methods for cfDNA analysis is needed to identify the best practical approach to medical diagnostics

Methods

Results

Conclusion