QUANTITATIVE DETECTION OF HEPATITIS-C VIRUS-RNA WITH A SOLID-PHASE SIGNAL AMPLIFICATION METHOD - DEFINITION OF OPTIMAL CONDITIONS FOR SPECIMEN COLLECTION AND CLINICAL-APPLICATION IN INTERFERON-TREATED PATIENTS

To determine the optimal conditions for preparation of serum specimens for quantitative hepatitis C virus RNA determination, patient samples were processed such that differences in time from clot formation to c entrifugation, centrifugation to separation of serum and collection of serum until freezing could be independently assessed. The effects of multiple cycles of freezing and thawing were also determined. There wa s progressive and significant loss of hepatitis C virus RNA activity w hen the time from the formation of the clot until centrifugation was l onger than 2 hr. This reduction reached 32% after 6 hr and 49% after 2 4 hr. if centrifugation was performed immediately after formation of t he clot, loss of hepatitis C virus RNA activity was reduced to less th an 10% even though the serum remained unseparated from the clot for up to 6 hr. Centrifugation of blood through a paraffin plug (serum separ ator tube) prevented loss of hepatitis C virus RNA activity for up to 24 hr. There was no loss of hepatitis C virus RNA activity with up to three freeze-thaw cycles. When patient specimens were prepared under t hese optimal conditions, the sensitivity of the quantitative branched DNA signal amplification assay in patients with hepatitis C virus infe ction was 83% and the specificity in patients with liver disease was 1 00%. Fluctuations in hepatitis C virus RNA levels were shown to correl ate with biochemical changes observed in patients treated with recombi nant interferon-alpha(2b). These data demonstrate that improper or inc onsistent methods of serum preparation may result in falsely low and u nreliable levels of hepatitis C virus RNA. Clinical applications of qu antitative hepatitis C virus RNA assays will only be possible when ser a are properly prepared.