Vol. 41, No. 9S, p. 77-78S September 2001 Supplement

The Journal of the American Association of Blood Banks

TRANSFUSION

Development And Testing Of An Intrinsic Fluorescence Assay For Identifying HCV-Infected Plasma

Theodore E. Maione, Yongwu Yang, Wang Long Zhou, Shaoqing Peng, Jeffrey Baler, Victor Sapirstein and Joseph Canter;
Seroptix, Inc., Woburn, MA


Background: Blood from diseased subjects often possesses aberrant arrays of biological molecules which produce modified spectral signals. As an alternative to current systems, which detect antibody responses or viral components, an assay has been developed that directly identifies HCV-infected samples based on specific changes in the profile of plasma fluorophores (intrinsic fluorescence) associated with viral infection.

Methods: Citrated plasma was obtained from normal volunteers and untreated patients recently diagnosed with HCV. Plasma was extracted with 50%(v/v) isopropyl alcohol and centrifuged to remove insoluble material. The fluorescence spectra of extracted supernatants were obtained with laser excitation at 355nm. Data were accumulated and averaged for ten seconds per sample and analyzed by Principal Component Analysis (PCA).

Results: The evaluation of panels of plasma obtained from normal and known-infected donors provided a spectral database that was subjected to advanced computational analysis. PCA identified several specific spectral features that when combined, fully distinguished between the normal and infected groups. Using the standard preparative method and these defined spectral parameters, an additional panel of 13 recently drawn HCV-positive plasma samples obtained from screened blood donations were evaluated, and all scored positive by objective computational methods. An additional panel of 10 HIV-pos/HCV-neg plasma samples obtained from treatment naïve patients were evaluated and tested negative by this spectral analysis. The spectra of these test panels and additional recently drawn samples were combined with the original database to form a revised database for higher order statistical evaluation. PCA evaluation of the revised database confirmed the discriminatory information carried by PCA Factors 2 and 4 to be of clear statistical significance, with other vectors contributing information of lower significance. From this analysis, an eight factor linear Discriminator Function was generated and a simulation was conducted which projected the assay to offer both sensitivity and specificity greater than 98%. Sample analysis and database compilation are continuing which will be used to further optimize the discriminator function and establish projections of assay performance more precisely.

Conclusions: Based on the level of spectral discrimination, simplicity of sample preparation, and ease of direct fluorescence spectral analysis, the intrinsic fluorescence assay described here may provide a useful, cost effective screening or supplemental analysis method for detecting HCV-infected plasma in donated units.

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