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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