Platelets may affect detection and quantitation of HIV RNA in plasma samples with low viral loads.

Abstract

To the Editor: There is evidence that HIV binds to and may even replicate in megakaryocytes and platelets.1 Although the pathophysiologic significance of this association is not known, viral replication or binding to platelets could play a role in viral dissemination, clearance, or persistence as well as in HIV-associated thrombocytopenia. Lee and colleagues2,3 have shown that HIV particles are physically associated with platelets ex vivo and after in vitro incubation of HIV-positive plasma or cultured virus with normal donor platelets and have suggested that residual platelets in plasma could interfere with the results of viral load assays. Piatak et al4 did not confirm this finding; however, these studies were performed using different in-house assays and on samples with high viral loads. We have recently observed unexpectedly high rates of persistent viremia in samples from HIV-infected patients on highly active antiretroviral therapy when the plasma was prepared with plasma preparation tubes (PPTs; Becton Dickinson, Franklin Lakes, NJ) and believe this is related to the high platelet content in plasma prepared using this tube. Commercial viral load tests have generally been performed on plasma collected in EDTA tubes.5 Because initial studies suggested that the time between collection and separation of plasma from cells could affect the results, the kit manufacturers recommended that plasma should be separated from cells within 6 hours of collection.5 PPTs are EDTA-coated tubes containing a barrier that is able to maintain separation of red cells and leukocytes (below the gel separator) from plasma (above the gel separator) after centrifugation. The advantage of using this tube is that the assay is performed on the primary tube, thus avoiding tube transfer and possible clerical errors, and the plasma is stable for a longer period of time with refrigeration and frozen storage.6 The PPT has therefore been increasingly used for viral load testing and blood donor nucleic acid amplification testing (NAT) for HIV and hepatitis C virus (HCV) screening. Recently Squires and colleagues7 presented at the Bristol-Myers Squibb (BMS) Symposium at the International AIDS Society Conference in Paris in 2003 that in the BMS Atazanavir Study 034, the proportion of subjects with nondetectable viral load samples was significantly higher when the assay was performed using plasma collected from EDTA tubes rather than from PPTs. We tested the hypothesis that for samples with low viral load, the proportion of virus particles associated with platelets versus free cell particles is high and that the preanalytical conditions used to obtain the plasma may interfere with the test results depending on the amount of platelets present in the plasma. Twenty samples of blood submitted to our laboratory for routine HIV viral load testing were used in this study. The samples had been collected in PPTs and centrifuged at 1100g for 10 minutes within 1 hour of blood collection as suggested by the manufacturer. The plasma present in the upper part of the tube was mixed by inverting the tube, removed, and divided into 2 aliquots. One aliquot was centrifuged again in an Eppendorf tube at 1100g for 10 minutes. The platelet count was significantly higher in the plasma aliquots that had not been submitted to a second centrifugation step than in the plasma subjected to the second spin (mean of 330,000 cells/μL, 95% confidence interval [CI]: 155,000–258,000 cells/μL vs. mean of 25,600 cells/μL, 95% CI: 5000–51,000 cells/μL). This shows that the plasma collected in PPTs is rich in platelets that can be removed by a second centrifugation in an Eppendorf tube. To test the effect of the second spin on HIV viral load results, we performed viral load testing (Roche Amplicor HIV-1 Monitor assay) in 20 plasma samples collected in PPTs before and after a second centrifugation step (Fig. 1). Of the 15 samples with a viral load below 3500 copies/mL, the viral load loads became nondetectable (below 400 copies/mL) in 13 after the second centrifugation step. Values did not decrease substantially in samples with a higher viral load.FIGURE 1.: HIV-1 viral load before (protocol A) and after (protocol B) a second centrifugation step.Our data suggest that preanalytical conditions affecting platelet counts may interfere with the results of the viral load assay. The discrepancy in the results obtained in the BMS Atazanavir Study 034 could probably be related to differences in the preanalytical conditions used to obtain the plasma. We believe that this finding is also important for donor screening NAT testing; in the early window phase, when the viral load is low, conditions that increase platelets in the plasma may increase the sensitivity of the assay. Ester Cerdeira Sabino, MD, PhD Nelson Gaburo Jr, MSc José Paulo Leite, PhD Mauricio Viecili, MSc Luiz Gastão Rosenfeld, MD, PhD Diagnósticos da America, São Paulo, Brazil