Effects Of Preanalytical Factors Research Articles

Reference intervals and stability of haptocorrin and holotranscobalamin in Danish children and elderly

BackgroundHaptocorrin (HC) and holotranscobalamin (holoTC) carry vitamin B12 (B12) in the circulation and can be useful biomarkers for evaluating B12 status. The concentration of both proteins depends on age, but data on reference intervals for children and the elderly are sparse. Similarly, not much is known about the effect of preanalytical factors. MethodsHC plasma samples from healthy elderly > 65 years (n = 124) were analysed, and both HC and holoTC were analysed in paediatric serum samples ≤ 18 years (n = 400). Furthermore, we investigated assay precision and stability. ResultsHC and holoTC were effected by age. We established reference intervals for HC: 2–10 years, 369–1237 pmol/L; 11–18 years, 314–1128 pmol/L; 65–82 years, 242–680 pmol/L and for holoTC: 2–10 years, 46–206 pmol/L; 11-18 years, 30–178 pmol/L. Analytical coefficients of variations of 6.0–6.8% and 7.9–15.7% were found for HC and holoTC, respectively. HC were affected when stored at room temperature and by freeze/thaw. HoloTC was stable at room temperature and after delayed centrifugation. ConclusionWe present novel 95% age-related reference limits for HC and HoloTC in children, and for HC both in children and elderly. Moreover, we found HoloTC to be fairly stable when stored, whereas HC was more vulnerable to preanalytical factors.

Serum GFAP- reference interval and preanalytical properties in Danish adults.

Glial fibrillary acidic protein (GFAP) is a promising biomarker that could potentially contribute to diagnosis and prognosis in neurological diseases. The biomarker is approaching clinical use but the reference interval for serum GFAP remains to be established, and knowledge about the effect of preanalytical factors is also limited. Serum samples from 371 apparently healthy reference subjects, 21-90 years of age, were measured by a single-molecule array (Simoa) assay. Continuous reference intervals were modelled using non-parametric quantile regression and compared with traditional age-partitioned non-parametric reference intervals established according to the Clinical and Laboratory Standards Institute (CLSI) guideline C28-A3. The following preanalytical conditions were also examined: stability in whole blood at room temperature (RT), stability in serum at RT and-20°C, repeated freeze-thaw cycles, and haemolysis. The continuous reference interval showed good overall agreement with the traditional age-partitioned reference intervals of 25-136ng/L, 34-242ng/L, and 5-438ng/L for the age groups 20-39, 40-64, and 65-90 years, respectively. Both types of reference intervals showed increasing levels and variability of serum GFAP with age. In the preanalytical tests, the mean changes from baseline were 2.3% (95% CI:-2.4%, 6.9%) in whole blood after 9h at RT, 3.1% (95% CI:-4.5%, 10.7%) in serum after 7days at RT, 10.4% (95% CI:-6.0%, 26.8%) in serum after 133days at-20°C, and 10.4% (95% CI: 9.5%, 11.4%) after three freeze-thaw cycles. The study establishes age-dependent reference ranges for serum GFAP in adults and demonstrates overall good stability of the biomarker.

Influence of preanalytical factors on feline proteinuria.

To date, little information is available about the effect of preanalytical factors on the urinary protein-to-creatinine (UPC) ratio in cats. We aimed to evaluate the effect of a commercially available cat litter, creatinine measurements at three different dilutions of urine, and different storage conditions on the UPC ratio in cats. Feline urine specimens were prospectively collected. Twenty-two whole-urine specimens were placed uncovered and in contact with cat litter for 1hour; 25 urine supernatants were diluted 1:10, 1:20, and 1:100 for creatinine measurements. The correlation, difference, agreement, and concordance in classifying specimens according to International Renal Interest Society staging were determined. Storage effects on UPC ratios were assessed in specimens stored for 6hours at +20℃ (n=20), 1week at +4℃ (n=20), and 3months at -20℃ (n=25). Specimens were also subjected to four freeze-thaw cycles (n=20). Results were compared, and clinical significance was assessed by comparing each UPC ratio to the inter-assay range of the baseline value. Exposure to cat litter did not affect UPC ratios. A positive proportional bias was found in the 1:100 dilution compared with the 1:20 dilution; however, concordance was high for all comparisons. At +20, +4℃, and after four repeated freeze-thaw cycles, UPC ratios were stable. Compared with baseline values, UPC ratios decreased (P<.01) after 8 and 12weeks at -20℃. However, all UPC ratios were within the inter-assay variability of the baseline value. Exposure to cat litter did not affect UPC ratios, but further studies are necessary to evaluate other potential variables. The effects of the dilutions and storage conditions were clinically acceptable, although the 1:20 and 1:100 dilutions were not perfectly comparable.

Pre-analytical protocol for measuring Alzheimer's disease biomarkers in fresh CSF.

IntroductionWe aimed to establish a standardized, routine‐use pre‐analytical protocol for measuring Alzheimer's disease (AD) biomarkers in cerebrospinal fluid (CSF).MethodsThe effect of pre‐analytical factors (sample collection/handling/storage/transportation) on biomarker levels was assessed using freshly collected CSF. Tube type/sterilization was assessed using previously frozen samples. A low‐bind false‐bottom tube (FBT, Sarstedt) was used for all experiments, except tube types/sterilization experiments. Biomarkers were measured using Elecsys CSF assays.ResultsAmyloid beta (Aβ)1‐42 levels varied by tube type, using a low‐bind FBT reduced variation. Aβ1‐42 levels were higher with no mixing versus roller/inversion mixing. Aβ1‐42 levels were lower with horizontal versus upright transportation; this was resolved by maximal tube filling and storage at 2°C to 8°C. Aβ1‐40 levels were less strongly affected. Phospho‐tau and total‐tau levels were largely unaffected.DiscussionWe propose an easy‐to‐use, standardized, routine‐use pre‐analytical protocol, using low‐bind FBTs, for measuring AD CSF biomarkers in clinical practice.

Abstract P4-08-20: Pre-analytical effects of FFPE extraction methods on targeted and whole transcriptome sequencing assays for endocrine sensitivity in metastatic breast cancer

Abstract Background: The clinical management of patients with metastatic HR-positive breast cancer is often uncertain due to decreased sensitivity to anti-estrogen therapy over time. Recently, we developed a targeted RNAseq based 18-transcript SET ER/PR assay of endocrine sensitivity from biopsies of metastatic cancer. In this work we assess the effect of pre-analytical factors, specifically RNA extraction methods for FFPE tissue samples, on the reliability of the targeted RNAseq assay. Methods: FFPE blocks and matched fresh frozen (FF) sections from 12 tumors were collected at MD Anderson Cancer Center. RNA from FFPE slides was extracted in duplicate using three kits (Norgen, Qiagen, Roche), and RNAseq libraries from all samples were prepared using Kapa Total RNAseq kit. Targeted RNA libraries were prepared using droplet-based PCR (RainDance), and also by transcriptome-wide RNAseq for comparison. Reads were mapped to genomic sequence using STAR and expression was quantified using RSEM. Expression data were normalized based on expression of 10 reference genes. The effect of FFPE RNA extraction kit on the reliability of the SET index was assessed using linear mixed effects model (LME) analysis, and agreement with FF was assessed using the concordance correlation coefficient (CCC). Results: Analysis of the whole transcriptome RNAseq data confirmed minimal 3'-end transcript bias from FFPE samples, irrespective of transcript size or FFPE kit. All 18 genes included in the SET index had high overall concordance between FFPE and FF (median CCC percentile=98.8, range 57.2-99.9 for Norgen; similar for the other two kits) and relatively consistent bias across genes, as estimated by the random effects of the LME model. Furthermore, compared to random 18-gene indices, concordance in the SET index values between FF and FFPE was higher than 99.8% of the random samples, verifying the analytical reliability of the selected genes. For the targeted RNAseq assay, RNA from FFPE extracted with the Norgen kit showed the highest concordance compared to FF (CCC=0.956, 95%CI 0.871-0.985). In general, the analytical variation of SET from FFPE samples was greater than that from FF (1.71-2.71 fold greater), with the lowest variation associated with the Norgen kit. The SET index values from targeted RNAseq for both FF and FFPE samples were consistently lower compared to transcriptome-wide RNAseq but were highly correlated, with the Norgen kit having the highest correlation between targeted and transcriptome-wide RNAseq (rho=0.915). Conclusions: All three FFPE RNA extraction kits have excellent analytical performance compared to FF samples. The Norgen kit may be marginally better yielding higher concordance with FF and lower analytical variation between replicates. All genes in the SET ER/PR showed very good analytical performance in comparison to random indices and individual genes. Targeted gene RNA sequencing appears very promising as a platform for clinical deployment of quantitative assays, showing only a small (fixable) bias compared to RNAseq. Citation Format: Marczyk M, Fu C, Lau R, Du L, Trevarton AJ, Sinn BV, Gould RE, Symmans WF, Hatzis C. Pre-analytical effects of FFPE extraction methods on targeted and whole transcriptome sequencing assays for endocrine sensitivity in metastatic breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-08-20.

Molecular Profiling of Malignant Pleural Effusion in Metastatic Non-Small-Cell Lung Carcinoma. The Effect of Preanalytical Factors.

Non-small-cell lung cancer (NSCLC)-associated malignant pleural effusions (MPEs) are sometimes the only available specimens for molecular analysis. This study evaluates diagnostic yield of NSCLC-associated MPE, its adequacy for molecular profiling and the potential influence of MPE volume/cellularity on the analytic sensitivity of our assays. Molecular results of 50 NSCLC-associated MPE cases during a 5-year period were evaluated. Molecular profiling was performed on cell blocks and consisted of fluorescent in situ hybridization (FISH) for ALK gene rearrangements and the following sequencing platforms: Sanger sequencing (for EGFR) and high-throughput pyrosequencing (for KRAS and BRAF) during the first 4 years of the study period, and targeted next-generation sequencing performed thereafter. A total of 50 NSCLC-associated MPE cases were identified where molecular testing was requested. Of these, 17 cases were excluded: 14 cases (28%) due to inadequate tumor cellularity and 3 cases due to unavailability of the slides to review. A total of 27 out of 50 MPE cases (54%) underwent at least EGFR and KRAS sequencing and FISH for ALK rearrangement. Of the 27 cases with molecular testing results available, a genetic abnormality was detected in 16 cases (59%). The most common genetic aberrations identified involved EGFR ( 9 ) and KRAS ( 7 ). Six cases had ALK FISH only, of which one showed rearrangement. MPE volume was not associated with overall cellularity or tumor cellularity (P = 0.360). Molecular profiling of MPE is a viable alternative to testing solid tissue in NSCLC. This study shows successful detection of genetic aberrations in 59% of samples with minimal risk of false negative.

Effect of Preanalytical Factors on the Stability of Maternal Serum Biomarkers and Calculated Risk for Trisomy 21, Trisomy 18, and Open Neural Tube Defect.

Biochemical prenatal screening tests are used to determine the risk of fetal aneuploidy based on the concentration of several biomarkers. The concentration of these biomarkers could be affected by preanalytical factors (PAFs) such as sample type (whole blood vs serum), storage time, and storage temperature. The impact of these factors on posttest risk is unknown. Blood samples were collected from 25 pregnant patients. Each sample was divided into 24 aliquots, and each aliquot was subjected to 1 of 24 different treatments (2 sample types × 2 temperatures × 6 storage times). The impact of each PAF on calculated risk was estimated using mixed-effects regression and simulation analysis. PAFs were associated with statistically significant changes in concentration for some analytes. Simulation studies showed that PAFs accounted for 6% of the variation in posttest risk, and analytical imprecision accounted for 94% of the variation. We estimated that the background misclassification rate due to analytical imprecision is approximately 1.37% for trisomy 21 and 0.12% for trisomy 18. Preanalytical factors increased the probability of misclassification by 0.46% and 0.06% for trisomies 21 and 18, respectively. Relaxing sample specifications for biochemical prenatal serum screening tests to permit analysis of serum samples stored for up to 72 h at room temperature or 4 °C as well as serum obtained from whole blood stored similarly has a small impact in calculated posttest aneuploidy risk.

Abstract 1377: Impact of biospecimen pre-analytical factors on molecular analysis

Abstract The Biospecimen Pre-analytical Variables (BPV) Program of the Biorepositories and Biospecimen Research Branch (BBRB) at the National Cancer Institute (NCI) is designed to systematically investigate the effects of preanalytical factors on the molecular integrity of biospecimens. Specific parameters related to formalin fixation and paraffin embedding (FFPE) of cancer tissues were examined, including the effects of cold ischemic time (delay to fixation (DTF)) and time in fixative (TIF). Additional preanalytical studies focus on snap freezing method for tissue specimens (dry Ice vs. LN2 vapor), storage temperature (-80°C vs. LN2 vapor), and duration of storage for frozen specimens. Biospecimens for the BPV program were collected at four medical centers, within a tightly regulated infrastructure and strict adherence to SOPs, to enable consistent collection and handling across all sites. Biospecimens were collected from research participants undergoing surgical treatment for renal cell carcinoma; ovarian, fallopian tube, and peritoneal carcinoma; lung adenocarcinoma and squamous cell carcinoma; and colorectal adenocarcinoma. Biospecimens were subjected to specific experimental protocols to systematically vary the preanalytical factors of interest. Extensive annotation was performed with 300+ data elements that include steps in the collection, handling, and processing of the biospecimens, pathological evaluation of the tumor, and clinical information collected from donors. Biospecimen collections concluded in April 2015 with a total of 364 tumor tissue cases collected. The BPV program has conducted multiple, simultaneous molecular analyses to understand the impact of FFPE preanalytical factors on the expression and detection of various molecular analytes. Initial efforts were focused on evaluating the impact of DTF and TIF on the quality of DNA and RNA from FFPE samples using multiple methods and approaches such as NanoDrop 8000 UV-Vis spectrophotometer, Qubit 2.0 Fluorometer, Agilent Bioanalyzer and KAPA Human Genomic DNA Quantification. The QC data from both RIN and Kappa assays showed that FFPE samples have a significant drop in RNA and DNA quality compared with matched frozen samples. 72 hr TIF samples showed a significant drop in both RNA and DNA quality compared to shorter time points (6, 12, or 23 hr TIF), as measured by DV200 and Kappa assays. No significant differences were observed in RNA/DNA quality between the shorter TIF time points or between the DTF time points (1, 2, 3, 12 hr DTF). Further studies are now underway to evaluate additional preanalytical factors. The data from BPV studies will be widely shared with the research community through publication and deposition at a public data repository. The results from these studies will be used to develop evidence-based protocols and best practices for fit-for-purpose collection, processing, and storage of biospecimens. This project is funded by NCI Contract No. HHSN261200800001E. Citation Format: Rachana Agarwal, Ping Guan, Mary Barcus, Jasmin Bavarva, Robin Burges, Philip Branton, Latarsha Carithers, Corinne Camalier, Biswajit Das, Jason Lih, Hana Odeh, Nancy Roche, Dan Rohrer, Michael Sachs, Leslie Sobin, Jewell Scott, Anna Smith, Conrado Soria, Kimberly Valentino, Dana Valley, Mickey Williams, Helen Moore. Impact of biospecimen pre-analytical factors on molecular analysis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1377.

The Effect of Preanalytical Factors on Stability of the Proteome and Selected Metabolites in Cerebrospinal Fluid (CSF)

To standardize the use of cerebrospinal fluid (CSF) for biomarker research, a set of stability studies have been performed on porcine samples to investigate the influence of common sample handling procedures on proteins, peptides, metabolites and free amino acids. This study focuses at the effect on proteins and peptides, analyzed by applying label-free quantitation using microfluidics nanoscale liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (chipLC-MS) as well as matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FT-ICR-MS) and Orbitrap LC-MS/MS to trypsin-digested CSF samples. The factors assessed were a 30 or 120 min time delay at room temperature before storage at -80 degrees C after the collection of CSF in order to mimic potential delays in the clinic (delayed storage), storage at 4 degrees C after trypsin digestion to mimic the time that samples remain in the cooled autosampler of the analyzer, and repeated freeze-thaw cycles to mimic storage and handling procedures in the laboratory. The delayed storage factor was also analyzed by gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for changes of metabolites and free amino acids, respectively. Our results show that repeated freeze/thawing introduced changes in transthyretin peptide levels. The trypsin digested samples left at 4 degrees C in the autosampler showed a time-dependent decrease of peak areas for peptides from prostaglandin D-synthase and serotransferrin. Delayed storage of CSF led to changes in prostaglandin D-synthase derived peptides as well as to increased levels of certain amino acids and metabolites. The changes of metabolites, amino acids and proteins in the delayed storage study appear to be related to remaining white blood cells. Our recommendations are to centrifuge CSF samples immediately after collection to remove white blood cells, aliquot, and then snap-freeze the supernatant in liquid nitrogen for storage at -80 degrees C. Preferably samples should not be left in the autosampler for more than 24 h and freeze/thaw cycles should be avoided if at all possible.

Liquid chromatography–tandem mass spectrometry method for routine measurement of oxalic acid in human plasma

A solid phase extraction (SPE)-LC–MSMS method for the routine determination of oxalic acid (OX) in plasma, a diagnostic marker of primary hyperoxaluria (PH), was developed and validated. The normal range of OX was found to be 3–11 μmol/L ( n = 67), with no differences attributable to gender or age. The effect of pre-analytical factors on the in vitro production of OX was investigated, and plasma was found to be stable for 1–2 h at room temperature, less after ingestion of vitamin C; the process was not completely stopped by preservation at either −20 or −70 °C.

Preanalytical Influences on DPC IMMULITE 2000 Intact PTH Assays of Plasma and Serum from Dialysis Patients

Measurement of intact parathyroid hormone (PTH) is essential to the diagnosis and management of metabolic bone disease (1), hypercalcemia, hypocalcemia (2), and renal osteodystrophy (3). The effects of specimen type, collection temperature, and storage temperature on the in vitro stability of PTH differ by method and platform (4)(5)(6)(7)(8)(9)(10). Characterization of preanalytical effects unique to each method, platform, and patient population is important to prevent potential clinical misclassification. The Diagnostics Product Corporation (DPC) IMMULITE 2000 intact PTH assay is a solid-phase two-site chemiluminescent immunoassay with a monoclonal mouse capture antibody and a polyclonal goat signal antibody conjugated to alkaline phosphatase. The effects of preanalytical factors on this assay have been partially investigated. Underfilling of EDTA-plasma tubes decreased measured PTH in samples from both healthy persons and those with chronic kidney disease (CKD) (11). Storage of SST serum samples for 3 days at room temperature decreased measured PTH in SST serum samples, an effect not seen with EDTA plasma (6). Results from EDTA plasma paradoxically increased after storage at 4 °C (12). Despite evidence that PTH is more stable in EDTA-anticoagulated specimens (5)(6)(7)(9)(10), the manufacturer describes an instability associated with EDTA-plasma samples and therefore recommends cold (2–8 °C) collection, centrifugation, and storage until analysis (13). At our institution, PTH is most frequently measured for management of CKD and helps direct therapies, including calcium, vitamin D, bisphosphonates, and parathyroidectomy. Preanalytical influences on PTH assays may be atypical in CKD because samples contain increased concentrations of N-terminal truncated PTH fragments that cross-react with intact PTH assays (14). Accordingly, we have investigated preanalytical variables in a dialysis-dependent CKD population. The study was approved by the St. Paul’s Hospital ethics committee. Five predialysis samples were …

Effects of Preanalytical Factors on the Molecular Size of Cell-Free DNA in Blood

Recently, plasma DNA analysis has been increasingly explored for different clinical diagnostic purposes. Previous studies have shown that the concentration of plasma DNA is increased in patients with neoplastic diseases (1), pregnancy-related complications (2)(3), trauma(4), and certain autoimmune diseases (5). In addition to quantitative analysis, qualitative changes in plasma DNA have also been investigated in different physiologic and pathologic conditions. In this regard, our group has shown that fetal DNA is shorter than the maternal counterpart in the plasma of pregnant women (6), and this has allowed the enrichment of fetal DNA from maternal plasma by size fractionation (7). It has also been shown that plasma DNA fragments are longer in patients suffering from a variety of neoplastic diseases (8). In addition, circulating Epstein–Barr virus DNA molecules in patients with nasopharyngeal carcinoma have been found to consist mainly of short DNA fragments <180 bp in size (9). Because the samples collected from patients and healthy controls in these studies were usually collected at different time points and under different conditions, it is important to ensure that the observed variations in the sizes of plasma DNA are the result of biological changes rather than artifacts attributable to differences in sample handling. Therefore, in this study, we investigated the effects of several preanalytical factors, including clotting, delayed separation of blood cells from plasma, freezing–thawing, and storage, on the integrity of circulating cell-free DNA. We recruited 27 healthy volunteers for modules 1 to 4 of the study. Venous blood (30 mL) was collected from each volunteer into eight tubes containing EDTA and one plain tube. The samples serving as references were processed immediately after collection. Plasma was separated from the blood cells by centrifugation at 1600 g for 10 min, and the plasma was then microcentrifuged at 16 000 g …

Measurement of Prothrombin Time in EDTA Plasma with Combined Thromboplastin Reagent

Prothrombin time (PT) tests usually are performed on blood collected with citrate. Because blood samples for other common tests use EDTA as anticoagulant, the use of EDTA plasma for PT tests could be convenient, and we wished to explore this possibility. PT tests are needed at regular intervals for control of anticoagulant therapy with vitamin K antagonists (1), and they are the most commonly performed coagulation test in our laboratory. Two major reagent systems are used to measure PT: the Quick method and the Owren method (combined thromboplastin reagent). The latter is the main method in Nordic countries, Benelux, and Japan. The Owren method measures only coagulation factors II, VII, and X because fibrinogen and factor V are in the reagent. We studied only the Owren method, and our results may not apply to the Quick method, in which the sample constitutes a much larger portion of the final reaction volume. The effects of preanalytical factors on PT depend, in part, on the units in which PT results are expressed. In the Nordic countries, percentage has previously been the unit used. When percentages or seconds are used as the unit, the result depends on the reagent. Clinical laboratories are moving toward the use of a new International Normalized Ratio (INR) unit. The use of INR units, INR = (samples/normals)ISI, where “normal” is a plasma pool obtained from healthy persons, ISI is the International Sensitivity Index, is designed to eliminate dependence on sensitivity of the reagents so that the international result is the same and recommendations for care are uniform. INR units are not without problems: reagents do not behave in the same way, and in some cases, the citrate concentration in the sample affects results (2)(3)(4). In our study, we …

A Highly Sensitive Enzyme-amplified Lanthanide Luminescence Immunoassay for Interleukin 6

Interleukin 6 (IL-6) is a cytokine that is produced by a wide variety of cells and has pleiotropic biological functions, such as the induction of acute phase proteins in hepatocytes, the terminal differentiation and growth promotion of B cells, the differentiation and activation of T cells and macrophages, and the regulation of expression of other cytokines (1). IL-6 has been implicated in the pathology of several diseases, and its role in inflammation, viral infection, autoimmunity, and cancer has been reviewed recently (1)(2). Serum IL-6 increases markedly in many pathological conditions, and its accurate and precise determination in biological fluids is of great importance for the early diagnosis of many diseases (3)(4)(5)(6)(7). Measurement of IL-6 in biological fluids is mainly based on bioassays and immunoassays (8)(9)(10)(11)(12)(13). The presence of natural compounds known to bind IL-6 in biological fluids (14)(15)(16)(17)(18) and the effect of preanalytical factors (19), as well as the use of an internationally accepted IL-6 standard preparation (20)(21), is of great importance for the performance of IL-6 immunoassays. Here we describe a highly sensitive enzyme immunoassay for the determination of IL-6 in serum and plasma, based on the enzyme-amplified lanthanide luminescence (EALL) detection approach (22)(23)(24)(25). An ultra-low detection limit is obtained by the powerful detection system that is based on the combination of enzymatic amplification introduced by the enzyme alkaline phosphatase (ALP) and the formation of a highly fluorescent terbium complex (26)(27), which is monitored by time-resolved or conventional fluorometry. A monoclonal antibody (anti-IL-6, CLB IL-6/16) and a biotinylated goat polyclonal antibody against human IL-6 were kindly donated by Prof. L. …

Polymerase chain reaction assay for hepatitis C virus RNA using a single tube for reverse transcription and serial rounds of amplification with nested primer pairs.

A procedure is described for the detection of hepatitis C virus (HCV) RNA in blood by means of the polymerase chain reaction (PCR) in which the reverse transcription step and two rounds of amplification are carried out in a single tube. This results in fewer manipulations, reduced risk of contamination, and economy of time. The procedures are generally applicable to other assays based on the PCR. We describe the preparation (from 100 microL serum) of test samples that remain stable for at least 6 days under specified conditions and an assay that employs nested primer pairs homologous to conserved sequences in the 5' noncoding region. The method was tested on 107 sera from the United States and Japan. Correlation with first-generation anti-HCV was 77%. Two sets of nested primer pairs homologous to sequences in the 5' noncoding region and one set based on structural region sequences showed differences in their reactivities with serum HCV RNA. The recommended single tube procedure specified a primer for reverse transcription that was conserved in all reported HCV genomes but absent from pestivirus genomic sequences. The effects of preanalytical factors on the detection of HCV RNA were studied. Qualitatively, there was no change in the HCV RNA-positivity of sera that were exposed to room temperature for 24 hours. Quantitative studies showed a decrease in titer in some specimens. Three cycles of freeze-thawing had no detectable effects on the titers of HCV RNA.

Effects of Preanalytical Factors on the Intraindividual Variation of Analytes in the Blood of Healthy Subjects: Consideration of Preparation of the Subject and Time of Venipuncture

(1977). Effects of Preanalytical Factors on the Intraindividual Variation of Analytes in the Blood of Healthy Subjects: Consideration of Preparation of the Subject and Time of Venipuncture. CRC Critical Reviews in Clinical Laboratory Sciences: Vol. 8, No. 2, pp. 105-144.