Nucleic acid test (NAT) methods are being used to screen blood and plasma donors for HCV RNA because these tests can detect viral RNA even when serological tests are negative. Some plasma fractionators have begun to use NATs to test plasma mini-pools to lower the viral load of HCV. However, such testing is considered by the FDA to be donor screens. Appropriate standards are needed to evaluate the analytical sensitivity because of diverse NAT methods and varied pool sizes. In collaboration with Dr. Tabor~s group, an HCV dilutional panel for licensing NATs to detect HCV RNA has been formulated from a plasma unit obtained from a window-period collection. The unit was estimated to contain 5 x 107 HCV RNA by us and 5 other testing laboratories using different NAT methods, and was determined by sequencing to be of genotype 1b. The panel was then diluted with a defibrinated human plasma pool to construct 10 members containing HCV RNA concentrations ranging from 0 to 105 copies/mL,. For panel member #1, 4,000 vials (0.75 mL/vial) were filled while for the rest, 2,000 vials per member were similarly filled. The panel members were then sent to 8 other testing laboratories for testing. Not all results were received. Nevertheless, based upon the preliminary data from us and others, the measured level of HCV RNA was close to the targeted level for each panel member. The panel can potentially be used as a sensitivity panel for licensing NATs. We were one of the 20 some laboratories participating in a collaborative study sponsored by NIBSC. The study was to calibrate 5 internationally available HCV working reagents against the newly established WHO~s HCV International Standard. Our HCV panel member #1 (150 vials of which was provided to NIBSC) was included as one of the 5 working reagents. The study has just been completed and soon the level of HCV RNA for panel member #1 can be expressed as International Units (IU). Our study of human parvovirus B19 in factor VIII concentrates (AHF) revealed that B19 DNA was prevalent in human plasma derived AHF. B19 DNA was not detected in either recombinant AHF or porcine AHF. The effects of various viral inactivation and purification methods were studied. We found that B19 DNA was less prevalent in one of 5 manufacturers~ AHF products and the level of B19 DNA was also lower. The manufacturer uses a wet-heat treatment in combination with an immunoaffinity purification. Since B19 DNA was prevalent and the levels were high in another manufacturer~ lots subjected to the wet-heat treatment alone, the immunoaffinity purification may be an efficient step in removing B19 which we need to explore further. Interestingly, B19 DNA was still found in AHF lots made by another manufacturer utilizing a S/D treatment in combination with a different type of immunoaffinity chromatography. We also began to investigate whether TT virus, a newly found non-enveloped virus, is present in plasma derivatives currently licensed in the US. In collaboration with Dr. Harvey Alter~s group in NIH, we found that TTV DNA was only prevalent in AHF (Human). However, the prevalence rate varied among 5 manufacturers' AHF lots. TTV was not found in products purified by either type of immunoaffinity chromatography. Thus, we found that such a chromatography step can not only serve to purify Factor VIII but also to remove TTV DNA. The prevalence of TTV DNA was less in products virally inactivated by wet heat than those by S/D treatment. Therefore, even though both B19 and TTV are non-enveloped viruses, they are varied in physicochemical characteristics, i.e., the former is not susceptible to wet heating. We continued to perform lot release testing for HCV RNA testing in 2 IGIM products not yet subjected to validated viral clearance procedures in their manufacture.
