Inhibition of human immunodeficiency virus gene amplification by heparin

Abstract

Gene amplification of virus-specific sequences is widely used as a method to detect or confirm human immunodeficiency virus (HIV) infection. In this study we used an enzyme-linked affinity assay to quantify polymerase chain reaction products from whole blood, plasma, and separated mononuclear cells collected in the presence of four common anticoagulants: acid citrate dextrose, sodium EDTA, potassium oxalate, and sodium heparin. Attenuation of the product signal was observed after amplification of nucleic acid extraction from whole blood, washed mononuclear cells, and plasma from specimens collected in sodium heparin. These inhibitory effects on gene amplification could be reversed with heparinase. The addition of as little as 0.05 U of heparin completely inhibited amplification of an HLA-DQa sequence from placental DNA. We conclude that heparin can cause attenuation or inhibition of gene amplification. Acid citrate dextrose and EDTA, which lack inhibitory activity, are the most appropriate anticoagulants for clinical blood samples when polymerase chain reaction amplification is anticipated.

The T helper cell response in Lyme arthritis: differential recognition of Borrelia burgdorferi outer surface protein A in patients with treatment-resistant or treatment-responsive Lyme arthritis.

“…During this study, it became possible to test for B~ burgdorferi DNA in SF by PCR (18). Before this time, joint fluid was obtained in the presence of heparin, a known inhibitor of PCR amplification (22), and therefore these samples could not be used for PCR. In the other five patients, an aliquot of SF was obtained without heparin for PCR testing.…”

A three-center European external quality control study of PCR for detection of cytomegalovirus DNA in blood

“…It would be expected that the former source, representing a dilution of a CMV DNA-positive preparation extracted from two million cells with the CMV DNA dispersed throughout, would yield a higher frequency of positives than the latter, a dilution of the number of cells from which the DNA was initially extracted, since any CMV DNApositive cell probably contains more than one DNA copy. We know from studies using the antigenemia test that expression of the CMV pp65 protein is restricted to a very small proportion of blood neutrophils (9), with often as few as 1 of 200,000 leukocytes being pp65 positive. If this is also true for the number of leukocytes containing CMV DNA, then the number of cells from which the DNA was initially extracted will prove to be a critical factor in determining the sensitivity of the PCR test, as suggested by our findings here.…”

“…The disparate sensitivities observed might also be due to factors other than the different amounts of leukocyte DNA added to the PCR, such as the anticoagulant used, the choice of primers, and the specificity controls employed. For example, the collection of blood into heparin has been shown to profoundly inhibit the PCR detection of HIV and hepatitis C virus and the PCR amplification of cellular genes (9,16,18), while additional hybridization steps might amplify the signal. Transport of the samples did not appear to have adversely affected the results, since there was no bias towards detecting positives in the center shipping the specimen.…”

Molecular methods used in clinical laboratory: prospects and pitfalls

“…Inhibitory substances in the specimen may also pose challenges. Substances such as heparinized blood, triglycerides, haemoglobin, some therapeutic drugs or extraction reagents, may cause false‐negative results; no PCR product is detected (Holodniy et al , 1991; Elbeik et al , 2004) even though the infectious agent is actually present. In some cases, this can be corrected by diluting the sample.…”