Book contents
- Frontmatter
- Contents
- Preface
- Foreword
- List of abbreviations
- Part I Basic principles
- Part II Recent advances
- Part III Experimental approaches
- Part IV Protocols and techniques
- 18 Sample collection, preparation, and storage
- 19 Hepatitis C RNA in blood and tissue samples
- 20 Hepatitis C genotypes and quasispecies
- 21 Replication of hepatitis C in tissue culture
- 22 Enzyme activities of nonstructural protein 3
- 23 Characterization of nonstructural protein 5B
- Part V Some outstanding questions and emerging areas for investigation
- References
- Index
18 - Sample collection, preparation, and storage
Published online by Cambridge University Press: 27 August 2009
- Frontmatter
- Contents
- Preface
- Foreword
- List of abbreviations
- Part I Basic principles
- Part II Recent advances
- Part III Experimental approaches
- Part IV Protocols and techniques
- 18 Sample collection, preparation, and storage
- 19 Hepatitis C RNA in blood and tissue samples
- 20 Hepatitis C genotypes and quasispecies
- 21 Replication of hepatitis C in tissue culture
- 22 Enzyme activities of nonstructural protein 3
- 23 Characterization of nonstructural protein 5B
- Part V Some outstanding questions and emerging areas for investigation
- References
- Index
Summary
The low levels of HCV RNA in the blood requires sensitive template or signal amplification methods for detection. These procedures are highly sensitive and are prone to crossover contamination, especially when the procedures are carried out manually. To limit contamination, laboratories that are going to be doing amplification on a regular basis need to take a number of precautions (Table 18.1). One of the driving forces for automation of the amplification process is to eliminate virtually all contamination by reducing the number of steps that need to be performed manually. Even so, a major source of variability is sample collection, handling, and storage, especially prior to nucleic acid extraction and amplification. For HCV, it is very important to use a dedicated vacutainer tube that remains unopened until received by the molecular diagnostic laboratory. There does not seem to be a difference in HCV RNA levels between serum and plasma samples obtained from the same patient on the same day (Cuypers et al., 1992; Krajden et al., 1996). However, other independent observations have shown that fresh frozen plasma reproducibly yielded almost twofold higher levels of HCV RNA compared with a corresponding serum sample from the same patient (Busch et al., 1992). HCV RNA levels in clinical samples were also sensitive to storage at room temperature as well as to repeated freezing and thawing in some but not all studies (Busch et al., 1992; Wang et al., 1992; Fong et al., 1993; Quan et al., 1993; Halfon et al., 1996).
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- Chapter
- Information
- Hepatitis C VirusFrom Laboratory to Clinic, pp. 143 - 144Publisher: Cambridge University PressPrint publication year: 2002