Nucleic acids amplification technique (NAT) screening for parvovirus B19: the first Italian routine experience.

Abstract

Dear Sir B19 can be transmitted through blood transfusions and plasma-derived products, but screening of blood donations for the presence of B19-DNA is not routine despite the fact that this virus is highly resistant (Prowse et al., 1997). In pooled plasma and bloodproducts, B19DNA levels lower than 10 IU mL may not be infectious, whereas those with viral load greater than 10 IU mL might be capable of transmitting infection (Daly et al., 2002). In 2004, the European Pharmacopoeia requested that plasma pools for production of anti-D immunoglobulin should not contain more than 10 IU mL of B19-DNA. Hence, highly viraemic blood donations have to be identified before the pooling process, and after the pooling process, the level of B19-DNAmust be determined (Hokymar et al., 2004; Koppelman et al., 2004). Moreover, European regulations (EMEA, 2004) for blood components require a risk assessment for B19 and an evaluation of potential infectivity of plasma source. In our Transfusion Service for biological qualification of blood units (BU), a triplex nucleic acids amplification technique (NAT) screening mini-pool (MP)-based strategy was adopted using Roche Ampliscreen methods (Roche Molecular System, Pleasaton, CA, USA). The aim of this study was to report the results of a 2-year study after introduction of a NAT screening for B19-DNA in a hospital-based Italian Transfusion Service. NAT screening was performed for hepatitis B virus (HBV), hepatitis C virus (HCV) and HIV using the Ampliscreen method (Roche Molecular System, Branchburg, NJ, USA). MP of 24 blood donations (BU) were prepared with Tecan Genesis pipettors (Tecan Trading AG, Mannedorf, Switzerland). Nucleic acids were isolated from plasma samples with the Multiprep Specimen Processing procedure for preparation of MP specimens and amplified and detected with the COBAS Ampliscreen HBV, HCV and HIV assay. As previously reported for B19-DNA extraction, we adopted the Multiprep Specimen Processing procedure (Gessoni et al., 2006). After the extraction process, an aliquot of purified nucleic acids was frozen at 280 C and stored until the tests for B19-DNAwere performed, before the shipment of plasma source to industrial fractionation process. All platelet and red cell concentrates confirmed to the Italian regulation and were transfusedwithout regard toB19-DNAscreening result. For quantitative detection of human parvovirus B19-DNA, we used a fully automated analyser for real-time PCR (Light Cycler) and a commercial method: Light Cycler Parvovirus B19 quantification kit; both analyser and kits were supplied by Roche. This method provides linear data between 10 and 10 IU mL (Schorling et al., 2004). By using the reported procedure for extraction and quantification ofB19-DNA,we achieved a satisfactory sensitivity: the detection limit 95% was 60 IU mL (Gessoni et al., 2006). In our Transfusion Service between April 2005 and March 2007, we tested for B19-DNA 51 274 BU in 2136 (MPs), each MP consisted of 24 BU. These MPs were tested in 93 analytical sessions (AS). Each AS consisted of an average of 23 MP (from 20 to 29), a positive control, a negative control and a multiparametric run control (RC) containing 10 IU mL for HBV-DNA, 20 IU mL for HCV-RNA, 50 IU mL forHIV-RNAand150 IU mL forB19-DNA). We considered an AS invalid if one of the kit s control or RC was incorrectly classified, and we considered an MP invalid if both the target and the internal control (IC) gave a negative result. In 2 years, we tested 51 274 BU in 2136 MPs of 24 BU. These MPs were tested in 93 AS, in each of these including testing of a positive control, a negative control, an RC and a mean of 23 MPs. We did not observe any invalidASbecause of incorrectly classified controls. Among 2136MPs, 41 (1 9%) gave an invalid result because of negativity of both target and IC. We observed 22 initially reactive MPs, of these 19 were repeatedly reactive. SeventeenMPs showed a low viral load for B19-DNA (from 8 2 10 to 2 9 10), and these BUs were shipped to the fractionation Correspondence: Gianluca Gessoni, Transfusional Service A-ULS 14, CommunityHospital, viaMadonnaMarina 500, 30015Chioggia (VE), Italy. Tel.: 0039 041 5534 400; fax: 0039 041 5534 401; e-mail: ggessoni@asl14chioggia.veneto.it or gianluca.gessoni@tin.it