Realtime ebv assay

The amplification target is a highly conserved region of the BLLF1 gene which encodes the gp350/220 envelope glycoprotein. The Abbott RealTime EBV assay (Abbott Molecular Inc., Des Plaines, IL, USA) uses three reagent kits, the amplification reagent kit, the calibrator kit for the standard curve and the control kit for external control. An internal control is also supplied to check the overall internal process, including DNA extraction and possible PCR inhibition. Extraction of DNA was done on the m2000sp system. DNA extraction was performed from 300 μL of WB and eluted in 250 μL. Extraction was followed by automated addition of 25 μL of master mix and 35 μL of purified DNA into the PCR plate. In each run, one negative control and two positive controls (Low and High) were included. Two calibrators (A and B) were used to determine the standard curve. The results are expressed in IU/mL. Manufacturer lower limit of quantification (LLQ) is reported as 150 IU/mL and limit of detection (LOD) as 115 IU/mL.

The cobas EBV test was performed on either a cobas 6800 System or a cobas 8800 System (13 ). As these systems are fully automated molecular analyzers, all steps, including primary sample handling, DNA extraction, and PCR amplification, were performed in the same system without further manual intervention. The comparator assay, namely, the CE-IVD-approved (but not FDA-cleared) Abbott RealTime EBV assay, was performed on an m2000 RealTime System (m2000sp instrument for DNA extraction and 2000rt for PCR amplification), according to the manufacturer’s instructions (14 ).

Leftover EDTA K2 tube (BD Vacutainer®) samples sent to the Virology unit of Saint Louis Hospital for EBV monitoring were used in this study. A total of 282 whole blood (WB) specimens received in the laboratory for EBV load quantification were collected from 196 patients including 95 hematopoietic stem cell transplant recipients, 22 kidney transplant recipients, 29 patients with immunological or heamatological diseases, 27 patients from general medicine, 10 HIV infected patients, 9 patients from intensive care unit and 4 patients hospitalized in infectious disease department. The clinical samples were selected prospectively and retrospectively from aliquots frozen at − 80 °C. All clinical consecutive whole blood samples received for EBV quantification within 7 days (124) were tested with the two assays. In order to obtain sufficient positive samples to enable a correlation analysis of EBV loads between the two assays, additional positive whole blood samples were selected retrospectively within 8 months. The samples were tested in separate runs both with the Abbott RealTime EBV assay (RT assay) and EBV PCR Kit V1 assay (V1 assay). Furthermore, 75 additional frozen samples at − 80 °C of 11 HSCT recipients were tested in order to analyze EBV DNA kinetics after rituximab injection in the two quantitative real time PCR assays.