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. 2001 Apr;39(4):1211-6.
doi: 10.1128/JCM.39.4.1211-1216.2001.

"V体育ios版" Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen

S J Stevens  1 , I Pronk, J M Middeldorp
Affiliations

Toward standardization of Epstein-Barr virus DNA load monitoring: unfractionated whole blood as preferred clinical specimen

S J Stevens (V体育平台登录) et al. J Clin Microbiol. 2001 Apr.

Abstract

Epstein-Barr virus (EBV) DNA load monitoring in peripheral blood has been shown to be a useful tool for the diagnosis of aberrant EBV infections. In the present study we compared the relative diagnostic values of EBV DNA load monitoring in unfractionated whole blood and simultaneously obtained serum or plasma samples from Burkitt's lymphoma (BL) patients, transplant recipients, human immunodeficiency virus (HIV)-infected individuals, and infectious mononucleosis (IM) patients by a quantitative competitive PCR (Q-PCR). The EBV DNA load in BL patients was mainly situated in the cellular blood compartment (up to 4. 5 x 10(6) copies/ml). EBV DNA loads in unfractionated whole blood and parallel serum samples showed no correlation. In transplant recipients, IM patients, and HIV-infected patients, the EBV burden in the circulation was almost exclusively restricted to the cellular blood compartment, because serum or plasma samples from these patients yielded negative results by Q-PCR, despite high viral loads in corresponding whole-blood samples. A 10-fold more sensitive but qualitative BamHI-W-repeat PCR occasionally revealed the presence of EBV at <2,000 copies of EBV DNA per ml of serum. Spiking of 100 copies of EBV DNA in samples with negative Q-PCR results excluded the presence of inhibitory factors in serum or plasma that influenced the Q-PCR result. Serum samples from all populations were often positive for beta-globin DNA, indicating cell damage in vivo or during serum preparation. We conclude that serum is an undesirable clinical specimen for EBV DNA load monitoring because it omits the presence of cell-associated virus and uncontrolled cell lysis may give irreproducible results or overestimation of the DNA load. Unfractionated whole blood is strongly preferred since it combines all blood compartments that may harbor EBV and it best reflects the absolute viral burden in the patient's circulation. VSports手机版.

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Figures

FIG. 1
FIG. 1
Experimental approach for EBV DNA load monitoring in whole-blood, serum, and plasma samples. After DNA isolation by silica-based DNA extraction, samples were prescreened by qualitative EBNA-1 PCR. The EBV DNA load in positive samples was subsequently quantified by Q-PCR. Samples negative by the qualitative EBNA-1 PCR were additionally amplified by the BamHI-W-repeat PCR in order to detect putative small (trace) amounts of EBV. DNA quality was assessed by β-globin PCR.
FIG. 2
FIG. 2
Comparison of EBV DNA load in unfractionated whole blood and simultaneously obtained serum of Burkitt's lymphoma patients and African controls.
See this image and copyright information in PMC

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