Effects of Storage and Type of Blood Collection Tubes on Hepatitis C Virus Level in Whole Blood Samples

Kessler, Harald H.; Stelzl, Evelyn; Raggam, Reinhard B.; Haas, Josef; Kirchmeir, Franz; Hegenbarth, Karin; Daghofer, Elisabeth; Santner, Brigitte I.; Marth, Egon; Stauber, Rudolf

doi:10.1128/jcm.39.5.1788-1790.2001

Cited by 23 publications

(13 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Conditions for handling, storage, and extraction have been found to be crucial to the performance of PCR assays of patient material (6,9,20). Our study demonstrated that B. burgdorferi DNA can be detected in urine by PCR assay when certain conditions are fulfilled, such as avoidance of the first morning urine, centrifugation at 36,000 ϫ g, extraction by DNAzol, and avoidance of freezing for Ͼ3 months.…”

Section: Resultsmentioning

confidence: 99%

Importance of Sample Preparation for Molecular Diagnosis of Lyme Borreliosis from Urine

et al. 2002

View full text Add to dashboard Cite

Urine PCR has been used for the diagnosis of Borrelia burgdorferi infection in recent years but has been abandoned because of its low sensitivity and the irreproducibility of the results. Our study aimed to analyze technical details related to sample preparation and detection methods. Crucial for a successful urine PCR were (i) avoidance of the first morning urine sample; (ii) centrifugation at 36,000 ؋ g; and (iii) the extraction method, with only DNAzol of the seven different extraction methods used yielding positive results with patient urine specimens. Furthermore, storage of frozen urine samples at ؊80°C reduced the sensitivity of a positive urine PCR result obtained with samples from 72 untreated erythema migrans (EM) patients from 85% in the first 3 months to <30% after more than 3 months. Bands were detected at 276 bp on ethidium bromide-stained agarose gels after amplification by a nested PCR. The specificity of bands for 32 of 33 samples was proven by hybridization with a GEN-ETI-K-DEIA kit and for a 10 further positive amplicons by sequencing. By using all of these steps to optimize the urine PCR technique, B. burgdorferi infection could be diagnosed by using urine samples from EM patients with a sensitivity (85%) substantially better than that of serological methods (50%). This improved method could be of future importance as an additional laboratory technique for the diagnosis of unclear, unrecognized borrelia infections and diseases possibly related to Lyme borreliosis.Laboratory methods for the diagnosis of Lyme borreliosis (LB) are still a matter of debate. Serological enzyme-linked immunosorbent assay (ELISA) and immunoblotting (IB) methods are now the tests of choice for confirming the diagnosis of a Borrelia burgdorferi infection (21). In early LB, however, serologic testing is not suitable because of its low sensitivity of ϳ40%, especially since erythema migrans (EM) is a clinical diagnosis (11,19). Recent studies compared several diagnostic modalities for early LB. The most sensitive method was quantitative PCR assay of skin biopsies (80.9%), followed by two-stage serologic testing of convalescent-phase samples (66.0%), nested PCR assay of skin biopsies (63.8%), and culture (51.1%) (11). In several other studies, PCR assays for B. burgdorferi had been performed worldwide with various organs, cultures, culture supernatants, and body fluids in clinical studies, with animal models, and with ticks by using different targets for amplification derived from chromosome-and plasmidrelated molecules (17). Whereas amplification of borrelia DNA from synovial fluid and cerebrospinal fluid has become an important diagnostic tool (7,8,10,14,18), the urine PCR assay is now done only sporadically (14). In previous work, we found that ϳ90% of EM patients showed a positive urine PCR assay result before treatment (15, 16). These results obtained by using the same technique, however, could not be confirmed either by us or by other researchers (personal communications; 2, 8). Recent recommendations thus rule out ...

show abstract

Section: Resultsmentioning

confidence: 99%

Importance of Sample Preparation for Molecular Diagnosis of Lyme Borreliosis from Urine

et al. 2002

View full text Add to dashboard Cite

show abstract

“…One limitation of this study is that we did not analyze RNA stability in EDTA blood samples. However, a previous study found HCV RNA stable in EDTA blood for up to 96 h (9), similar to what we observed for DNA targets.…”

mentioning

confidence: 36%

“…Although there are published guidelines for the collection, transport, and storage of specimens for molecular tests, described by CLSI (4), they are based mostly on opinions and limited experimental data. Published data on the stability of pathogen-specific amplification targets are also limited to individual pathogens and/or specimen types (3,4,(6)(7)(8)(9)(10)12). Therefore, in this study, we undertook a broader study to compare the stability of extracted and unextracted clinical samples over time and at different temperatures.…”

mentioning

confidence: 99%

Short-Term Stability of Pathogen-Specific Nucleic Acid Targets in Clinical Samples

et al. 2012

View full text Add to dashboard Cite

bThe stability of pathogen-specific DNA or RNA amplification targets in clinical samples following short-term storage at room temperature, 4°C, and ؊80°C was assessed by real-time PCR. In purified nucleic acid extracts, both DNA and RNA targets were stable for up to 30 days, irrespective of storage temperature. In unextracted samples, temperature-dependent loss of targets (P < 0.05) was observed in serum and cerebrospinal fluid specimens, while no changes were observed for EDTA blood samples. N ucleic acid amplification tests (NAATs) to detect infectious pathogens are now widely used in clinical microbiology laboratories. Although these assays provide high sensitivity, high specificity, and rapid turnaround time, special laboratory practice and procedures are required to avoid false-positive and false-negative results (1, 2). Optimal application of NAATs depends on a number of preanalytical factors, such as specimen collection, transport, and storage conditions (3, 4). These factors impact the stability of pathogen-associated nucleic acids (NA), which must be protected from physical, chemical, or enzymatic degradation during specimen transport and handling. The most important threats to the stability of NA targets in clinical samples are nucleases, such as DNases and RNases, which may be released from lysed human cells and microorganisms (5). In real-life clinical situations, questions arise over whether clinical samples or NA extracts, left at room temperature (RT) for a few days or stored in the refrigerator for a week, are still valid for molecular testing. Although there are published guidelines for the collection, transport, and storage of specimens for molecular tests, described by CLSI (4), they are based mostly on opinions and limited experimental data. Published data on the stability of pathogen-specific amplification targets are also limited to individual pathogens and/or specimen types (3,4,(6)(7)(8)(9)(10)12). Therefore, in this study, we undertook a broader study to compare the stability of extracted and unextracted clinical samples over time and at different temperatures.To determine the short-term storage stability of purified DNA and RNA, NA was extracted from known positive specimens containing the following: Neisseria meningitidis in EDTA blood, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in serum, and influenza A virus in nasopharyngeal washes (NPW). All samples were extracted using the QIAsymphony virus/bacteria kit in an automated DNA extraction platform QIAsymphony SP (Qiagen) except for EDTA blood, which was extracted manually using the QIAamp DNA blood minikit (Qiagen). Purified eluates were stored in aliquots under sterile conditions at room temperature (RT) (22 to 25°C), 4°C, and Ϫ80°C for 0 to 30 days.For unextracted samples, the target pathogen and the specimen types were chosen so that they (i) represent the most common specimen type for the respective pathogen and (ii) had not previously been studied. EDTA blood, serum, cerebrospinal fluid (CSF), and NPW samples were spiked w...

show abstract

“…The clinical utility of RNA amplification based assays by RT-PCR depends on the specificity, sensitivity, reproducibility, and stability of viral RNA in blood [4]. The stability of viral RNA in blood is dependent on sample storage and shipping conditions and the type of blood collection device [5]. Poor specimen handling, sample processing, storage conditions or use of an incompatible blood collection tube may result in viral RNA degradation which may lead to false-negatives or an underestimation of viral RNA concentration.…”

Section: Introductionmentioning

confidence: 99%

Viral Load Stability of an RNA virus In Stabilized Blood Samples

Das¹

2014

J Bioanal Biomed

View full text Add to dashboard Cite

Background: The stability of viral load in a patient blood sample during storage and shipping is an important pre-analytical variable that affects the accuracy of viral pathogen quantitation. Therefore, there is a need for developing a blood collection tube with stabilizing reagents which can not only inactivate the viral agent but also stabilize the viral nucleic acids for accurate viral load determination in blood samples post blood draw. This study was undertaken to investigate the ability of Cyto-Chex ® BCT (BCT) blood collection device to stabilize the nucleic acids of the Mason-Pfizer monkey virus, an RNA virus, in collected blood specimens during storage and shipping for viral load determination at a later time point.Findings: Blood was drawn from each donor into K 3 EDTA and BCT tubes, spiked with Mason-Pfizer monkey virus particles and stored at room temperature. Viral RNA was extracted using RNeasy ® FFPE Kit. Our results demonstrate that the viral RNA was stable in blood drawn into BCT after storage at 22°C for 7 days. Virus stored in blood drawn into K 3 EDTA tubes showed a decrease in viral load over time and a statistically significant decrease was observed at day 7. Shipping blood samples which took 3 days did not have adverse effect on viral load measurements for blood samples collected in BCT. Conclusion:Our results demonstrate that BCT can stabilize viral load in blood during shipping and storage at 22°C for 7 days.

show abstract

Effects of Storage and Type of Blood Collection Tubes on Hepatitis C Virus Level in Whole Blood Samples

Cited by 23 publications

References 21 publications

Importance of Sample Preparation for Molecular Diagnosis of Lyme Borreliosis from Urine

Importance of Sample Preparation for Molecular Diagnosis of Lyme Borreliosis from Urine

Short-Term Stability of Pathogen-Specific Nucleic Acid Targets in Clinical Samples

Viral Load Stability of an RNA virus In Stabilized Blood Samples

Contact Info

Product

Resources

About