Some plasma fractionators, mostly those who use Source Plasma, have begun to use nucleic acid testing (NAT) methods to test minipools for parvovirus B19 DNA to exclude donations containing high titers of the virus. FDA considers such NAT screening as an in-process control test and hence we have formulated a working standard of B19 DNA targeted to contain 10e6 geq/mL. The standard was accepted as one of the four candidate samples in a WHO/NIBSC collaborative study. As a result of the study, an International Standard (IS) for B19 DNA, 5 x 10e5 IU/vial, has been established, and our working standard has been assigned a value of 10e6 IU/mL. We continued to investigate the viral safety of plasma derivatives with respect to parvovirus B19 by our in-house NAT assay method. B19 DNA was detected in some lots of Immune Globulin Intravenous (IGIV), Immune Globulin (IG), and albumin products. The prevalence and contaminating levels in those products were much lower than those found in coagulation products. Many lots of the three manufacturers' anti-D immunoglobulin products, to treat Rh-negative pregnant women to prevent hemolytic disease of the newborn, were tested but no lot was found positive. Positive NAT results for B19 may not equate with infectivity. A cell culture system using an erythroid cell line has been set up to quantify virion infectivity. Preliminary results indicate that a window-period plasma donation can infect the cells when its concentrations were at or above 10e5 IU of B19 DNA/mL. Further studies are needed so that the system can be more sensitive and can be used to characterize B19 neutralizing antibodies and to evaluate efficient viral inactivation/removal procedures for B19. In collaboration with OTRR scientists, we continued to investigate whether a commercial NAT kit utilizing a hybridization probe to detect the RT-PCR amplified products for HCV RNA might have detected false positives, i.e., detected non-specific, cross-reactivities in vector or human genomic sequences used in a gene therapy product. Further study is needed to resolve the issues. We participated in a WHO/NIBSC collaborative study, to calibrate HCV genotypes 2-6 against the HCV IS (genotype 1). We were one of the 17 laboratories participated. We found that there was general good agreement between laboratories and assays for IS and genotypes 2, 3 and 6, but poorer agreement for genotypes 4 and 5. Further collaborative study is needed in order to determine whether our in-house HCV NAT assay method as well as other methods can detect all genotypes with equivalent efficiency. Studies are on-going to establish and validate both the real-time quanti-tative and semi-automated qualitative methods for both HCV and B19 NAT. Like B19, Hepatitis A virus (HAV) is a non-enveloped virus and difficult to be inactivated. Some fractionators have also begun to use NAT methods to limit the viral load for HAV RNA in the manufacturing pools. Similar to B19 NAT, FDA also considers such HAV NAT screening as a in-process control test and a working standard is needed. We developed a sensitive, in-house nested NAT method. In addition, we formulated a HAV RNA working standard, ca. 10e4 copies/mL. The consensus level of this working standard, however, is being determined by a WHO/NIBSC collaborative study that is still in progress. In collaboration with the CDC, we investigated whether a reported (MedWatch) case of B19 was causally related to two implicated lots of human plasma derived factor VIII concentrate (AHF) from the same manufacturer. We found that the AHF lot derived from the B19 NAT screened minipools had no detectable B19 DNA whereas the other lot from the B19 NAT unscreened plasma was positive for B19 DNA. Both a pre- and a post-infusion serum specimens are available. Further studies, especially sequencing data, are needed to establish whether the patient was infected with B19 due to the AHF infusion.
