scholarly journals Test Verification and Validation for Molecular Diagnostic Assays

2012 ◽  
Vol 136 (1) ◽  
pp. 11-13 ◽  
Author(s):  
Kevin C Halling ◽  
Iris Schrijver ◽  
Diane L Persons
Keyword(s):  
Clinical Laboratory ◽  
Molecular Diagnostic ◽  
Molecular Assay ◽  
Validation And Verification ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Clinical Laboratories ◽  
Hereditary Disorders ◽  
Test Verification

With our ever-increasing understanding of the molecular basis of disease, clinical laboratories are implementing a variety of molecular diagnostic tests to aid in the diagnosis of hereditary disorders, detection and monitoring of cancer, determination of prognosis and guidance for cancer therapy, and detection and monitoring of infectious diseases. Before introducing any new test into the clinical laboratory, the performance characteristics of the assay must be “verified,” if it is a US Food and Drug Administration (FDA)–approved or FDA-cleared test, or “validated,” if it is a laboratory-developed test. Although guidelines exist for how validation and verification studies may be addressed for molecular assays, the specific details of the approach used by individual laboratories is rarely published. Many laboratories, especially those introducing new types of molecular assays, would welcome additional guidance, especially in the form of specific examples, on the process of preparing a new molecular assay for clinical use.

scholarly journals Challenges and Controversies to Testing for COVID-19

2020 ◽  
Vol 58 (11) ◽  
Author(s):  
Matthew J. Binnicker
Keyword(s):  
Clinical Laboratory ◽  
Threshold Value ◽  
Current Debate ◽  
Research Staff ◽  
Viral Pathogen ◽  
Patient Reports ◽  
Molecular Assays ◽  
Clinical Laboratories ◽  
Laboratory Operation

ABSTRACT The coronavirus disease (COVID-19) pandemic has placed the clinical laboratory and testing for SARS-CoV-2 front and center in the worldwide discussion of how to end the outbreak. Clinical laboratories have responded by developing, validating, and implementing a variety of molecular and serologic assays to test for SARS-CoV-2 infection. This has played an essential role in identifying cases, informing isolation decisions, and helping to curb the spread of disease. However, as the demand for COVID-19 testing has increased, laboratory professionals have faced a growing list of challenges, uncertainties, and, in some situations, controversy, as they have attempted to balance the need for increasing test capacity with maintaining a high-quality laboratory operation. The emergence of this new viral pathogen has raised unique diagnostic questions for which there have not always been straightforward answers. In this commentary, the author addresses several areas of current debate, including (i) the role of molecular assays in defining the duration of isolation/quarantine, (ii) whether the PCR cycle threshold value should be included on patient reports, (iii) if specimen pooling and testing by research staff represent acceptable solutions to expand screening, and (iv) whether testing a large percentage of the population is feasible and represents a viable strategy to end the pandemic.

scholarly journals Validation of Laboratory-Developed Molecular Assays for Infectious Diseases

2010 ◽  
Vol 23 (3) ◽  
pp. 550-576 ◽  
Author(s):  
Eileen M. Burd
Keyword(s):  
Infectious Diseases ◽  
Clinical Laboratory ◽  
Reference Interval ◽  
Attractive Alternative ◽  
Analytical Sensitivity ◽  
Additional Specimen ◽  
Molecular Assays ◽  
Clinical Laboratories ◽  
Regulatory Standards ◽  
Sensitivity Specificity

SUMMARY Molecular technology has changed the way that clinical laboratories diagnose and manage many infectious diseases. Excellent sensitivity, specificity, and speed have made molecular assays an attractive alternative to culture or enzyme immunoassay methods. Many molecular assays are commercially available and FDA approved. Others, especially those that test for less common analytes, are often laboratory developed. Laboratories also often modify FDA-approved assays to include different extraction systems or additional specimen types. The Clinical Laboratory Improvement Amendments (CLIA) federal regulatory standards require clinical laboratories to establish and document their own performance specifications for laboratory-developed tests to ensure accurate and precise results prior to implementation of the test. The performance characteristics that must be established include accuracy, precision, reportable range, reference interval, analytical sensitivity, and analytical specificity. Clinical laboratories are challenged to understand the requirements and determine the types of experiments and analyses necessary to meet the requirements. A variety of protocols and guidelines are available in various texts and documents. Many of the guidelines are general and more appropriate for assays in chemistry sections of the laboratory but are applied in principle to molecular assays. This review presents information that laboratories may consider in their efforts to meet regulatory requirements.

scholarly journals Improved RT-PCR SARS-Cov2 results interpretation by indirect determination of cut-off cycle threshold value

2020 ◽  
Author(s):  
Khelil Mohamed Mokhtar
Keyword(s):  
Real Time ◽  
Clinical Laboratory ◽  
Threshold Value ◽  
Mixed Data ◽  
Rt Pcr ◽  
Data Set ◽  
Iso 15189 ◽  
Indirect Determination ◽  
Clinical Laboratories

ABSTRACTClinical laboratories of the developing world are overwhelmed with RT-PCR SARS-Cov2 testing demands. It is of paramount importance that each clinical laboratory use an appropriate cut-off value in the interpretation of SARS-Cov2 real-time RT–PCR results, which is specific to their laboratory performances as ISO 15189 recommendations stipulate. We applied an indirect statistical method to a large mixed data set of Ct values (ORF1ab and N) to estimate cut-off Ct value (∼32 cycles).we conclude that the use of indirect statistical approaches to estimate cut-off value in the interpretation of SARS-Cov2 real-time RT–PCR results may improve differential diagnosis of COVID-19 cases with low risk of infectivity, and may help to better estimates of the burden of COVID-19 disease.

scholarly journals Comparison of Serological and Molecular Assays for Bartonella Species in Dogs with Hemangiosarcoma

Pathogens ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 794
Author(s):  
Erin Lashnits ◽  
Pradeep Neupane ◽  
Julie M. Bradley ◽  
Toni Richardson ◽  
Ricardo G. Maggi ◽  
...  
Keyword(s):  
Diagnostic Tests ◽  
Latent Class ◽  
Fluorescent Antibody ◽  
Molecular Diagnostic ◽  
Infection Prevalence ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Tissue Biopsy ◽  
Frozen Tissue ◽  
Fresh Frozen

Currently, a gold standard diagnostic test for Bartonella infection in dogs is lacking. This represents a critical limitation for the development and evaluation of new diagnostic tests, as well as for the diagnosis of, and research on, bartonellosis in dogs. This retrospective observational study aims to compare the results of commonly performed and newly-reported Bartonella spp. diagnostic tests in banked clinical specimens from 90 dogs with hemangiosarcoma (HSA) using composite reference standard (CRS) and random effects latent class analysis (RE-LCA) techniques. Samples from each dog were tested using six serological or molecular diagnostic assays, including indirect fluorescent antibody (IFA) and Western blot (WB) for the detection of antibodies in serum, and qPCR and droplet digital PCR (ddPCR) in blood and fresh frozen tissue biopsy samples (mainly splenic HSA tumors and histopathologically normal spleen or skin/adipose tissue). Bartonella infection prevalence was estimated to be 78% based on the CRS (parallel testing with all six assays), and 64% based on the RE-LCA model. The assay with the highest diagnostic accuracy was qPCR performed on fresh frozen tissue biopsy samples (sensitivity: 94% by RE-LCA and 80% by CRS; specificity: 100%). When comparing newly-reported to traditional Bartonella diagnostic assays, ddPCR was more sensitive for the detection of Bartonella DNA than qPCR when testing blood samples (36% vs. 0%, p < 0.0001). Dogs that were positive on serological assays alone with negative molecular assays were highly unlikely (<3%) to be classified as infected by the RE-LCA model. These data indicate that Bartonella spp. DNA can be PCR amplified from fresh frozen tissues from a majority of dogs with HSA using both qPCR and ddPCR, supporting the use of these methods for future controlled studies comparing the prevalence of Bartonella spp. DNA in the tissue of dogs with HSA to that of unaffected controls.

Reporting of Vancomycin-Resistant Enterococci in Connecticut: Implementation and Validation of a State-Based Surveillance System

1999 ◽  
Vol 20 (10) ◽  
pp. 671-675 ◽  
Author(s):  
Zygmunt F. Dembek ◽  
Scott E. Kellerman ◽  
Lisa Ganley ◽  
Constance M. Capacchione ◽  
Fred C. Tenover ◽  
...  
Keyword(s):  
Outcome Measures ◽  
Proficiency Testing ◽  
Clinical Laboratory ◽  
Surveillance Systems ◽  
Passive Surveillance ◽  
Emerging Pathogens ◽  
Clinical Laboratories ◽  
Vancomycin Resistant Enterococci ◽  
Vancomycin Resistant

AbstractObjective:To assess state-based surveillance for isolation from a sterile site of vancomycin-resistant enterococci (VRE) in Connecticut.Design:Clinical laboratory reporting (passive surveillance) of VRE isolates to the Connecticut Department of Public Health (CDPH) was followed by state-initiated validation, laboratory proficiency testing, and review of hospital demographic characteristics.Settings:All 45 clinical laboratories and all 37 (36 for 1995 and 1996) acute-care hospitals in Connecticut were included in the study.Main Outcome Measures:The outcome measures included determination of the statewide incidence of VRE and the accuracy of passive reporting, determination of clinical laboratory proficiency in detecting VRE, and analysis of hospital characteristics that might be associated with an increased incidence of VRE.Results:During 1994 through 1996, 29 (78%) of 37 hospital-affiliated clinical laboratories and 1 (11%) of 9 commercial or other laboratories in Connecticut reported to the CDPH the isolation of VRE from sterile sites; 158 isolates were reported for these 3 years. Based on verification, we discovered that these laboratories actually detected 58 VRE isolates in 1994, 104 in 1995, and 104 in 1996 (total, 266). The age-standardized incidence rate of VRE was 14.1 cases per million population in 1994 and 26.8 cases per million population for both 1995 and 1996. Laboratory proficiency testing revealed that high-level vancomycin resistance was identified accurately and that low- and moderate-level resistance was not detected. The incidence of VRE isolates was three times greater in hospitals with over 300 beds compared with categories of hospitals with fewer beds. Increases in the number of VRE isolates were at least twice as likely in hospitals located in areas with a higher population density, or with a residency program or trauma center in the hospital.Conclusions:Passive reporting of VRE isolates from sterile sites markedly underestimated the actual number of isolates, as determined in a statewide reporting system. Statewide passive surveillance systems for routine or emerging pathogens must be validated and laboratory proficiency ensured if results are to be accurate and substantial underreporting is to be corrected.

DETERMINATION OF WHITE BLOOD CELLS USING FOLDSCOPE WITH SMARTPHONE

2019 ◽  
pp. 10-13
Author(s):  
Ranu Kumar ◽  
Prasad Kapildeo
Keyword(s):  
Human Blood ◽  
Blood Cells ◽  
Blood Smear ◽  
Clinical Laboratory ◽  
White Blood Cells ◽  
Healthcare Services ◽  
Field Level ◽  
Morphology Analysis

We are traditionally used Microscope in clinical laboratory for determination of white blood cells of human blood smear. Now, in this study we were used Foldscope with Smartphone in the place of Microscope and examine many samples of human blood smear which was collected from local diagnostic centers. We were very easily quantity & morphology analysis of all types of WBC cells such as Neutrophils, Lymphocytes, Monocytes, Eosionophils, Basophils in blood smear with the help of Foldscope & image taken by Smartphone. The main objective of this study is to use Foldscope for quantity & morphology analysis of human WBCs at field level especially poor resource area where healthcare services or centers is not available & where carry of microscope is not possible.

scholarly journals Comparison of nine different commercially available molecular assays for detection of SARS-CoV-2 RNA

2021 ◽  
Author(s):  
Ute Eberle ◽  
◽  
Clara Wimmer ◽  
Ingrid Huber ◽  
Antonie Neubauer-Juric ◽  
...  
Keyword(s):  
Real Time ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Molecular Assays ◽  
Pcr Assays ◽  
Laboratory Experiences

AbstractTo face the COVID-19 pandemic, the need for fast and reliable diagnostic assays for the detection of SARS-CoV-2 is immense. We describe our laboratory experiences evaluating nine commercially available real-time RT-PCR assays. We found that assays differed considerably in performance and validation before routine use is mandatory.

scholarly journals Diagnostic Challenges on the Laboratory Detection of Lupus Anticoagulant

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 844
Author(s):  
Armando Tripodi
Keyword(s):  
Lupus Anticoagulant ◽  
Clinical Laboratory ◽  
External Quality Assessment ◽  
State Of The Art ◽  
Diagnostic Procedures ◽  
External Quality ◽  
Glycoprotein I ◽  
Current State ◽  
Clinical Laboratories

Lupus anticoagulant (LA) is one of the three laboratory parameters (the others being antibodies to either cardiolipin or β2-glycoprotein I) which defines the rare but potentially devastating condition known as antiphospholipid syndrome (APS). Testing for LA is a challenging task for the clinical laboratory because specific tests for its detection are not available. However, proper LA detection is paramount for patients’ management, as its persistent positivity in the presence of (previous or current) thrombotic events, candidate for long term anticoagulation. Guidelines for LA detection have been established and updated over the last two decades. Implementation of these guidelines across laboratories and participation to external quality assessment schemes are required to help standardize the diagnostic procedures and help clinicians for appropriate management of APS. This article aims to review the current state of the art and the challenges that clinical laboratories incur in the detection of LA.

scholarly journals The pathway through LC-MS method development: in-house or ready-to-use kit-based methods?

2020 ◽  
Vol 58 (6) ◽  
pp. 1002-1009 ◽  
Author(s):  
Caroline Le Goff ◽  
Jordi Farre-Segura ◽  
Violeta Stojkovic ◽  
Patrice Dufour ◽  
Stéphanie Peeters ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Liquid Chromatography ◽  
Tandem Mass Spectrometry ◽  
Method Development ◽  
Clinical Laboratory ◽  
Cross Reactivity ◽  
Tandem Mass ◽  
Chromatography Tandem Mass Spectrometry ◽  
Liquid Chromatography Tandem Mass

AbstractHistorically, the determination of low concentration analytes was initially made possible by the development of rapid and easy-to-perform immunoassays (IAs). Unfortunately, typical problems inherent to IA technologies rapidly appeared (e.g. elevated cost, cross-reactivity, lot-to-lot variability, etc.). In turn, liquid chromatography tandem mass spectrometry (LC-MS/MS) methods are sensitive and specific enough for such analyses. Therefore, they would seem to be the most promising candidates to replace IAs. There are two main choices when implementing a new LC-MS/MS method in a clinical laboratory: (1) Developing an in-house method or (2) purchasing ready-to-use kits. In this paper, we discuss some of the respective advantages, disadvantages and mandatory requirements of each choice. Additionally, we also share our experiences when developing an in-house method for cortisol determination and the implementation of an “ready-to-use” (RTU) kit for steroids analysis.

scholarly journals Analysis and phylogenetic comparisons of full-length VP2 genes of the 24 bluetongue virus serotypes

2007 ◽  
Vol 88 (2) ◽  
pp. 621-630 ◽  
Author(s):  
S. Maan ◽  
N. S. Maan ◽  
A. R. Samuel ◽  
S. Rao ◽  
H. Attoui ◽  
...  
Keyword(s):  
Bluetongue Virus ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Full Length ◽  
Molecular Diagnostic ◽  
Mammalian Host ◽  
Reading Frame ◽  
Diagnostic Assays ◽  
Primary Determinant ◽  
Serotype Identification

The outer capsid protein VP2 of Bluetongue virus (BTV) is a target for the protective immune response generated by the mammalian host. VP2 contains the majority of epitopes that are recognized by neutralizing antibodies and is therefore also the primary determinant of BTV serotype. Full-length cDNA copies of genome segment 2 (Seg-2, which encodes VP2) from the reference strains of each of the 24 BTV serotypes were synthesized, cloned and sequenced. This represents the first complete set of full-length BTV VP2 genes (from the 24 serotypes) that has been analysed. Each Seg-2 has a single open reading frame, with short inverted repeats adjacent to conserved terminal hexanucleotide sequences. These data demonstrated overall inter-serotype variations in Seg-2 of 29 % (BTV-8 and BTV-18) to 59 % (BTV-16 and BTV-22), while the deduced amino acid sequence of VP2 varied from 22.4 % (BTV-4 and BTV-20) to 73 % (BTV-6 and BTV-22). Ten distinct Seg-2 lineages (nucleotypes) were detected, with greatest sequence similarities between those serotypes that had previously been reported as serologically ‘related’. Fewer similarities were observed between different serotypes in regions of VP2 that have been reported as antigenically important, suggesting that they may play a role in the neutralizing antibody response. The data presented form an initial basis for BTV serotype identification by sequence analyses and comparison of Seg-2, and for development of molecular diagnostic assays for individual BTV serotypes (by RT-PCR).

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