Cytomegalovirus Quantitation by Real-Time PCR Is Unaffected by Delayed Separation of Plasma from Whole Blood

Abstract

A previous study suggested that delayed sample preparation can cause a false-positive plasma cytomegalovirus (CMV) PCR. We evaluated 78 samples submitted for CMV testing, and we report that a 24-h delay in plasma separation did not affect viral quantitation by real-time PCR. Laboratories should evaluate specimen handling requirements for their own PCR technology.Cytomegalovirus (CMV) is an important cause of morbidity and mortality in immunocompromised individuals (2). Accordingly, monitoring of patients for CMV viremia has become an important laboratory tool for transplantation services. We recently developed a real-time PCR assay for detection of CMV. Studies comparing our CMV PCR assay with the older CMV antigenemia assay (fluorescent antibody stain for expression of pp65 on mononuclear cells) have demonstrated that PCR is superior for early detection of CMV-related disease (1). After discharge from our institution, posttransplant patients are monitored for CMV viremia for up to 1 year. Because this often requires shipping of samples from distant sites, it is critical to validate specimen handling strategies to ensure correct results.For in-house patients, our PCR specimen handling protocols call for plasma to be separated immediately from EDTA-anticoagulated whole blood and stored frozen prior to testing. In contrast, the antigenemia test previously used for outpatients utilized EDTA-anticoagulated whole blood that was shipped and stored at room temperature for up to 48 h before processing. Although room temperature shipping of EDTA-anticoagulated whole blood is logistically simpler than immediate separation of plasma, it is unclear whether such handling allows accurate quantitation of CMV by real-time PCR. Shafer et al. reported that delayed separation of plasma increased the frequency of positive PCR results for CMV in latently infected patients (3). Those authors suggested that lysis of latently infected leukocytes released CMV into the plasma and thus increased the number of positive results. Shafer et al. used semiquantitative nested PCR followed by hybridization to radiolabeled probe, gel electrophoresis, and autoradiographic detection of amplified product. In contrast, our laboratory has used a rapid real-time TaqMan assay performed on the ABI 7700 (Boeckh et al., submitted). Schafer's assay amplified a region within the coding sequence of glycoprotein B (gB), while our amplicon spanned the junction between gB and UL123. Because of these differences, we felt it necessary to investigate whether delayed separation of plasma from EDTAanticoagulated blood would increase the frequency of detection of CMV DNA by our assay.To evaluate the potential release of CMV DNA from cells in whole blood, it was necessary to establish the stability of CMV DNA in plasma after separation from cells. Ten previously PCR-positive plasma samples were chosen to encompass our clinical measuring range. These samples, stored at Ϫ20°C, were thawed, and a portion was separated for extraction and CMV DNA quantity determination. The ...

Prediction of Cytomegalovirus (CMV) Plasma Load from Evaluation of CMV Whole-Blood Load in Samples from Renal Transplant Recipients

“…Since CMV replication starts in cells and is followed by the release of viral particles into plasma, CMV DNA in plasma can represent a valuable indicator for viral replication providing that specimens are prepared without excessive delay so as to avoid positive plasma results due to cell lysis (20,32). We aimed at predicting plasma CMV loads from WB CMV loads in order to identify active CMV infections.…”

Interlaboratory Comparison of Cytomegalovirus Viral Load Assays

“…QNAT to define a reference standard: In order to obtain a consensus result that would define a quantitative reference laboratory standard (QRS) for the constructed samples in the panel, two aliquots of a defined dilution of the stock virus preparation were shipped to seven reference laboratories and tested using a range of CMV PCR assays. Results were obtained using three commercial assay/reagent sets (Roche COBAS R Amplicor TM , Roche ASR for LightCycler R and artus-Biotech ABI SDS format CMV assay from Qiagen) and four laboratory-developed assays (10,(15)(16)(17). The consensus result [or QRS; geometric mean (GM) ± SD (copies/mL) of the reported results] was 5.0 ± 0.5 log 10.…”

“…for the LightCycler R and ABI sequence detection systems) (4). Many laboratorydeveloped assays have been described for detection and monitoring of CMV load in plasma of 'at risk' patients and are used in different clinical diagnostic laboratories (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Each of these assays was calibrated with proprietary standards, and assay parameters and performance were published.…”

Cytomegalovirus DNA stability in EDTA anti-coagulated whole blood and plasma samples

“…However Schafer et al concluded an elevation of CMV viral load in plasma was due to WBC lysis in whole blood stored 24 hours before separation 3 , while Boom et al concluded that plasma separated within 48 hours of collection from EDTA whole blood did not affect CMV DNA level when stored 4°C 4 . In a 2004 study Nesbitt et al further demonstrated that a 24 hour delay in separation did not affect plasma viral load levels 5 . None of these studies evaluated CMV DNA stability when storing whole blood or plasma for greater than 72 hours prior to testing.…”