Storage conditions of blood samples and primer selection affect the yield of cDNA polymerase chain reaction products of hepatitis C virus.

Author(s): Cuypers HT, Bresters D, Winkel IN, Reesink HW, Weiner AJ, Houghton M, van der Poel CL, Lelie PN

Publication: J Clin Microbiol, 1992, Vol. 30, Page 3220-4

PubMed ID: 1333489 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the use of fresh-frozen plasma with serum for the detection of HCV by RT-PCR and to examine the effectiveness of different primers.

   Summary of Findings:

   In fresh-frozen plasma specimens, the 5'-TR PCR primers were 10-100 times more effective at amplifying HCV RNA than the NS3/NS4 PCR primers. HCV detection occurred in 67% of plasma specimens using the NS3/NS4 PCR primers, but only in 50% of serum specimens. The use of the 5'TR PCR primers increased detection of HCV to 92% in plasma and to 75% in serum. 5'TR PCR products from two of the three serum specimens which were negative by ethidium bromide staining but positive in plasma specimens were detected in serum by southern transfer. In conclusion, RT-PCR detection of HCV is more sensitive in fresh-frozen plasma than in serum.

   Biospecimens

   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Hepatitis

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   RT-PCR Specific	Targeted nucleic acid	NS3/NS4 5' terminal region
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Serum
   Biospecimen Preservation	Type of fixation/preservation	Frozen Snap frozen
   Storage	Storage temperature	-20 degrees C -70 degrees C

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2. Study Purpose

   The purpose of this study was to determine the effects of storage of EDTA-whole blood at room temperature or serum at room temperature or 4 degrees C on the detection of HCV by RT-PCR with two different primer sets. After the experimental storage of whole blood or serum, plasma was prepared from whole blood and both serum and plasma were stored at -70 degrees C.

   Summary of Findings:

   Serum specimens were more stable than EDTA whole blood when stored at either 4 degrees C or room temperature. For one plasma specimen, the NS3/NS4 RT-PCR product was lost after storage of EDTA-whole blood for only 1 day at room temperature. In contrast, the NS3/NS4 product was detected in all 3 serum specimens after 14 days of storage at 4 degrees C or 3 days of storage at room temperature. The 5'TR PCR product was detected in plasma when whole blood specimens were stored at room temperature for at least 6 days and in serum stored at room temperature for at least 8 days. No loss of the 5'TR PCR product was observed in serum stored at 4 degrees C for 14 days.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Hepatitis

   Platform:

   Analyte	Technology Platform
   RNA	RT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	4 degrees C
   Storage	Storage duration	0 days 1 day 2 days 3 days 4 days 6 days 8 days 14 days
   RT-PCR Specific	Targeted nucleic acid	NS3/NS4 5' terminal region
   Biospecimen Aliquots and Components	Blood and blood products	Serum Whole blood Plasma
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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