Regulation of Molecular Diagnostics.

Molecular diagnostic tests enable rapid analysis of genomic and proteomic markers. These tests are subject to diverging premarket access and postmarket surveillance requirements and mechanisms in the United States and the European Union. Each of these jurisdictions has its own challenges in keeping the regulations up to date with technological developments. A specific area of attention is that of laboratory-developed tests in the United States and health institution in-house-produced tests in the European Union, for which the United States and the European Union have markedly different regulatory approaches. Both jurisdictions have specific but differing requirements for the use of test samples and test-related data under their rules regarding the protection of (personal) health data, which can cause complexity when moving samples or sample-related data from one jurisdiction to the other.

Performance of the cobas EBV and cobas BKV assays: multi-site comparison of standardized quantitation.

Guidelines recommend monitoring of Epstein-Barr virus (EBV) and BK virus (BKV) in solid organ and hematopoietic stem cell transplant patients. The majority of quantitative DNA testing for EBV and BKV employs unstandardized individual laboratory-developed testing solutions (LDTs), with implications for accuracy, reproducibility, and comparability between laboratories. The performance of the cobas EBV and cobas BKV assays was assessed across five laboratories, using the World Health Organization International Standards (WHO IS) for EBV and BKV, and the National Institute of Standards and Technology Quantitative Standard for BKV, and results were compared with the LDTs in use at the time. Methods were also compared using locally sourced clinical specimens. Variation was high when laboratories reported EBV or BKV DNA values using LDTs, where quantitative values were observed to differ by up to 1.5 log10 unit/mL between sites. Conversely, results from the cobas EBV and cobas BKV assays were accurate and reproducible across sites and on different testing days. Adjustment of LDTs using the international standards led to closer alignment between the assays; however, day-to-day reproducibility of LDTs remained high. In addition, BKV continued to show bias, indicating challenges with the commutability of the BKV International Standard. The cobas EBV and cobas BKV assays are automated, aligned to the WHO IS, and have the potential to reduce the variability in viral load testing introduced by differences in LDTs. Standardization of reporting values may eventually allow different centers to compare data to allow clinical decision thresholds to be established supporting improvements in patient management.IMPORTANCEThe application of center-specific cut-offs for clinical decisions and the variability of LDTs often hinder interpretation; thus, the findings reported here support the need for standardization in the field of post-transplant monitoring of EBV and BKV to improve patient management. Alongside the choice of assay, it is also important to consider which standard to use when deciding upon a testing methodology. This is a call to action for standardization, as treatment for EBV and BKV is driven by viral load test results, and the more accurate and comparable the test results are across institutions, the more informed and better the treatment decisions can be.

Cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of Shigella flexneri.

Pathogen infections including Shigella flexneri have posed a significant threat to human health for numerous years. Although culturing and qPCR were the gold standards for pathogen detection, time-consuming and instrument-dependent restrict their application in rapid diagnosis and economically less-developed regions. Thus, it is urgently needed to develop rapid, simple, sensitive, accurate, and low-cost detection methods for pathogen detection. In this study, an immunomagnetic beads-recombinase polymerase amplification-CRISPR/Cas12a (IMB-RPA-CRISPR/Cas12a) method was built based on a cascaded signal amplification strategy for ultra-specific, ultra-sensitive, and visual detection of S. flexneri in the laboratory. Firstly, S. flexneri was specifically captured and enriched by IMB (Shigella antibody-coated magnetic beads), and the genomic DNA was released and used as the template in the RPA reaction. Then, the RPA products were mixed with the pre-loaded CRISPR/Cas12a for fluorescence visualization. The results were observed by naked eyes under LED blue light, with a sensitivity of 5 CFU/mL in a time of 70 min. With no specialized equipment or complicated technical requirements, the IMB-RPA-CRISPR/Cas12a diagnostic method can be used for visual, rapid, and simple detection of S. flexneri and can be easily adapted to monitoring other pathogens.

Evaluation of analytical performance of homocysteine LC-MS/MS assay and design of internal quality control strategy.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become a common technique in clinical laboratories in recent years. Because most methods are laboratory-developed tests (LDTs), their reproducibility and quality control (QC) have been controversial. In this study, Westgard Sigma Rules were used to evaluate the analytical performance and establish an individualised internal QC (IQC) strategy for these LDTs. METHODS: Taking the LC-MS/MS LDT method for homocysteine (Hcy) as an example, the 'desirable specifications' from the Biological Variation Database were used as quality goals. Based on the external quality assessment (EQA) samples, bias was calculated and the coefficient of variation (CV) was also calculated by IQC measurements for six consecutive months. The analytical performance was evaluated by calculated sigma metrics and an IQC strategy was designed using the Westgard Sigma Rules with run size. RESULTS: Over 116 days within 6 months, a total of 850 data points were collected for each of IQC 1 and IQC 2. The monthly coefficient of variation CV% was 2.57-4.01%, which was non-significant (p-value: 0.75). The absolute bias% for IQC1 and IQC2 was 1.23 and 1.87%, respectively. The allowable total error (TEa) was selected as 15.5%, Sigma metrics were 4.02 and 4.30, and the analytical performance was 'Good'. The 13s/22s/R4s/41s multi rules (n=4, r=1) with a run size of 200 samples were suggested for the Hcy IQC scheme. The quality goal index (QGI) values were over 1.2, indicating that trueness needed to be improved. CONCLUSIONS: The analytical performance of the Hcy LC-MS/MS LDT conformed to the Six Sigma rating level, achieving 'good' (four Sigma). Clinical practice indicated that calibration bias was the primary factor affecting trueness.

ISO 15189 is a sufficient instrument to guarantee high-quality manufacture of laboratory developed tests for in-house-use conform requirements of the European In-Vitro-Diagnostics Regulation.

The EU In-Vitro Diagnostic Device Regulation (IVDR) aims for transparent risk-and purpose-based validation of diagnostic devices, traceability of results to uniquely identified devices, and post-market surveillance. The IVDR regulates design, manufacture and putting into use of devices, but not medical services using these devices. In the absence of suitable commercial devices, the laboratory can resort to laboratory-developed tests (LDT) for in-house use. Documentary obligations (IVDR Art 5.5), the performance and safety specifications of ANNEX I, and development and manufacture under an ISO 15189-equivalent quality system apply. LDTs serve specific clinical needs, often for low volume niche applications, or correspond to the translational phase of new tests and treatments, often extremely relevant for patient care. As some commercial tests may disappear with the IVDR roll-out, many will require urgent LDT replacement. The workload will also depend on which modifications to commercial tests turns them into an LDT, and on how national legislators and competent authorities (CA) will handle new competences and responsibilities. We discuss appropriate interpretation of ISO 15189 to cover IVDR requirements. Selected cases illustrate LDT implementation covering medical needs with commensurate management of risk emanating from intended use and/or design of devices. Unintended collateral damage of the IVDR comprises loss of non-profitable niche applications, increases of costs and wasted resources, and migration of innovative research to more cost-efficient environments. Taking into account local specifics, the legislative framework should reduce the burden on and associated opportunity costs for the health care system, by making diligent use of existing frameworks.

Validation of a Molecular Diagnostic Test for Circulating Tumor DNA by Next-Gen Sequencing.

A modified version of the PGDx elioTM Plasma Resolve assay was validated as a laboratory-developed test (LDT) for clinical use in the Molecular Diagnostics Laboratory at Fox Chase Cancer Center. The test detects single nucleotide variants (SNVs) and small insertions and deletions (indels) in 33 target genes using fragmented genomic DNA extracted from plasma. The analytical performance of this assay was assessed with reference standard DNA and 29 samples from cancer patients and detected 66 SNVs and 23 indels. Using 50 ng of input DNA, the sensitivity was 95.5% to detect SNVs at 0.5% allele frequency, and the specificity was 92.3%. The sensitivity to detect indels at 1% allele frequency was 70.4%. A cutoff of 0.25% variant allele frequency (VAF) was set up for diagnostic reporting. An inter-laboratory study of concordance with an orthologous test resulted in a positive percent agreement (PPA) of 91.7%.

At-Home Specimen Self-Collection and Self-Testing for Sexually Transmitted Infection Screening Demand Accelerated by the COVID-19 Pandemic: a Review of Laboratory Implementation Issues.

The coronavirus disease 2019 (COVID-19) pandemic reduced the sexually transmitted infection (STI) testing volume due to social-distancing and stay-at-home orders, among other reasons. These events highlighted previously known benefits of at-home STI self-testing or specimen self-collection and accelerated testing demand via telemedicine. We review testing outside traditional clinical settings. We focus on three curable bacterial STIs among the top 10 U.S. nationally notifiable conditions with screening recommendations: syphilis, gonorrhea (Neisseria gonorrhoeae, also known as the gonococcus [GC]), and chlamydia (Chlamydia trachomatis). At least 19 million GC/C. trachomatis (GC/CT) screening or diagnostic tests are performed annually, presenting a considerable challenge during the pandemic. Unlike for HIV, STI at-home tests are currently not commercially available. However, innovative telemedicine providers currently offer services where specimen self-collection kits are mailed to patients at home who then ship them to laboratories for processing. We discuss technical and regulatory aspects of modifications for home-based specimen self-collection. The telemedicine provider typically manages and communicates results, provides linkage to care, and is responsible for billing and case reporting. We also describe rapid testing devices in development that may present an opportunity for future self-testing. In summary, COVID-19 has accelerated the evaluation and development of STI self-tests and specimen self-collection. The remaining obstacles are high price, regulatory approval, support for laboratories offering the service, and uncertainty regarding whether target populations with the greatest need are reached effectively. However, increased testing, convenience, and privacy are potential benefits that may enhance uptake and outlast the pandemic.

Analytical Validation of Telomere Analysis Technology® for the High-Throughput Analysis of Multiple Telomere-Associated Variables.

BACKGROUND: A large number of studies have suggested a correlation between the status of telomeres and disease risk. High-throughput quantitative fluorescence in situ hybridization (HT Q-FISH) is a highly accurate telomere measurement technique that can be applied to the study of large cell populations. Here we describe the analytical performance testing and validation of Telomere Analysis Technology (TAT®), a laboratory-developed HT Q-FISH-based methodology that includes HT imaging and software workflows that provide a highly detailed view of telomere populations. METHODS: TAT was developed for the analysis of telomeres in peripheral blood mononuclear cells (PBMCs). TAT was compared with Terminal Restriction Fragment (TRF) length analysis, and tested for accuracy, precision, limits of detection (LOD) and specificity, reportable range and reference range. RESULTS: Using 6 different lymphocyte cell lines, we found a high correlation between TAT and TRF for telomere length (R2 ≥ 0.99). The standard variation (assay error) of TAT was 454 base pairs, and the limit of detection of 800 base pairs. A standard curve was constructed to cover human median reportable range values and defined its lower limit at 4700 bp and upper limits at 14,400 bp. Using TAT, up to 223 telomere associated variables (TAVs) can be obtained from a single sample. A pilot, population study, of telomere analysis using TAT revealed high accuracy and reliability of the methodology. CONCLUSIONS: Analytical validation of TAT shows that is a robust and reliable technique for the characterization of a detailed telomere profile in large cell populations. The combination of high-throughput imaging and software workflows allows for the collection of a large number of telomere-associated variables from each sample, which can then be used in epidemiological and clinical studies.

Comparison of the Accula SARS-CoV-2 Test with a Laboratory-Developed Assay for Detection of SARS-CoV-2 RNA in Clinical Nasopharyngeal Specimens.

Several point-of-care (POC) molecular tests have received emergency use authorization (EUA) from the Food and Drug Administration (FDA) for the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The test performance characteristics of the Accula (Mesa Biotech) SARS-CoV-2 POC test need to be evaluated to inform its optimal use. The aim of this study was to assess the test performance of the Accula SARS-CoV-2 test. The performance of the Accula test was assessed by comparing results of 100 nasopharyngeal swab samples previously characterized by the Stanford Health Care EUA laboratory-developed test (SHC-LDT), targeting the envelope (E) gene. Assay concordance was assessed by overall percent agreement, positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa coefficient. Overall percent agreement between the assays was 84.0% (95% confidence interval [CI], 75.3 to 90.6%), PPA was 68.0% (95% CI, 53.3 to 80.5%), and the kappa coefficient was 0.68 (95% CI, 0.54 to 0.82). Sixteen specimens detected by the SHC-LDT were not detected by the Accula test and showed low viral load burden, with a median cycle threshold value of 37.7. NPA was 100% (95% CI, 94.2 to 100%). Compared to the SHC-LDT, the Accula SARS-CoV-2 test showed excellent negative agreement. However, positive agreement was low for samples with low viral load. The false-negative rate of the Accula POC test calls for a more thorough evaluation of POC test performance characteristics in clinical settings and for confirmatory testing in individuals with moderate to high pretest probability of SARS-CoV-2 who test negative on Accula.

Evaluation of a laboratory-developed test for simultaneous detection of norovirus and rotavirus by real-time RT-PCR on the Panther Fusion® system.

The Hologic Panther Fusion® Open Access™ functionality allows implementation of laboratory-developed tests (LDTs), with fully automated sample extraction, real-time PCR, and result interpretation. We report the development and validation of a multiplex LDT for norovirus G1, norovirus G2, and rotavirus from stool samples on this system. The LDT was optimized for primer and probe sequences, salt concentration, and PCR annealing temperature. Reproducibility of the PCR and extraction process was assessed. Performance of the multiplex LDT assay was evaluated with external quality assessment (EQA) samples and compared to a commercial multiplex assay (Allplex™ GI-Virus Assay, Seegene) in clinical samples. Salt concentrations and annealing/extension temperature were optimized to 4 mM MgCl2, 70 mM KCl, 20 mM Tris, and 60 °C, respectively. The user-prepared part of the LDT PCR mix (containing salts, probes, and primers) was stable for ≥ 11 days onboard the instrument. We observed reproducible results of PCR and the extraction process. The LDT had a sensitivity comparable to or greater than the commercial Allplex™ assay and showed excellent linearity. Forty-five EQA samples yielded the expected result with the LDT. There was 100% concordance between LDT and Allplex™ results in 160 clinical samples. Results from the suspension and direct swab stool sample preparation methods were highly concordant in the LDT. We report the successful development and validation of a multiplex PCR LDT for detection of norovirus G1, norovirus G2, and rotavirus from stool samples on the Panther Fusion® system.

The end of the laboratory developed test as we know it? Recommendations from a national multidisciplinary taskforce of laboratory specialists on the interpretation of the IVDR and its complications.

The in vitro diagnostic medical devices regulation (IVDR) will take effect in May 2022. This regulation has a large impact on both the manufacturers of in vitro diagnostic medical devices (IVD) and clinical laboratories. For clinical laboratories, the IVDR poses restrictions on the use of laboratory developed tests (LDTs). To provide a uniform interpretation of the IVDR for colleagues in clinical practice, the IVDR Task Force was created by the scientific societies of laboratory specialties in the Netherlands. A guidance document with explanations and interpretations of relevant passages of the IVDR was drafted to help laboratories prepare for the impact of this new legislation. Feedback from interested parties and stakeholders was collected and used to further improve the document. Here we would like to present our approach to our European colleagues and inform them about the impact of the IVDR and, importantly we would like to present potentially useful approaches to fulfill the requirements of the IVDR for LDTs.

Anaplastic lymphoma kinase 5A4 immunohistochemistry as a diagnostic assay in lung cancer: A Canadian reference testing center's results in population-based reflex testing.

BACKGROUND: The presence of anaplastic lymphoma kinase (ALK) rearrangement predicts response to ALK tyrosine kinase inhibitor (TKI) therapy. Fluorescence in situ hybridization (FISH) was the initial reference standard to detect ALK rearrangement, but immunohistochemistry (IHC) using D5F3 has gained acceptance as an alternative diagnostic method. ALK IHC assays using other ALK antibodies have also been used as screening methods, but data supporting their utility as diagnostic tests have not been widely reported. METHODS: Data from reflexive clinical ALK IHC test using the 5A4 clone concurrent with epidermal growth factor receptor (EGFR) mutation testing were analyzed. ALK IHC results were reported as negative (-), equivocal, or positive (+), with equivocal or positive staining validated by FISH break-apart probe testing. Treatment outcomes were reviewed for ALK IHC+ patients. RESULTS: Between 2012 and 2015, 146 (2.5%) cases were reported as ALK IHC+, 188 (3.2%) were reported as equivocal, and 5624 (94.4%) were reported as ALK IHC-. Of the ALK IHC+ cases, 131/143(91.6%) were ALK FISH+. Excluding 6 cases in which FISH was inconclusive or not performed, the positive predictive value was 95.6%, and the negative predictive value was 100%. Most specimens (n = 5352 [89.6%]) were also successfully tested for EGFR. Clinical responses to ALK TKIs were noted in 49 ALK IHC+ patients, with a median progression-free survival of 9.9 months. CONCLUSIONS: ALK 5A4 IHC can serve as a robust diagnostic test for ALK-rearranged lung cancer and is associated with treatment response and survival. Optimized tissue allocation resulted in high success rates of combined reflex EGFR and ALK testing.

Validation and Implementation of Clinical Laboratory Improvements Act-Compliant Whole-Genome Sequencing in the Public Health Microbiology Laboratory.

Public health microbiology laboratories (PHLs) are on the cusp of unprecedented improvements in pathogen identification, antibiotic resistance detection, and outbreak investigation by using whole-genome sequencing (WGS). However, considerable challenges remain due to the lack of common standards. Here, we describe the validation of WGS on the Illumina platform for routine use in PHLs according to Clinical Laboratory Improvements Act (CLIA) guidelines for laboratory-developed tests (LDTs). We developed a validation panel comprising 10 Enterobacteriaceae isolates, 5 Gram-positive cocci, 5 Gram-negative nonfermenting species, 9 Mycobacterium tuberculosis isolates, and 5 miscellaneous bacteria. The genome coverage range was 15.71× to 216.4× (average, 79.72×; median, 71.55×); the limit of detection (LOD) for single nucleotide polymorphisms (SNPs) was 60×. The accuracy, reproducibility, and repeatability of base calling were >99.9%. The accuracy of phylogenetic analysis was 100%. The specificity and sensitivity inferred from multilocus sequence typing (MLST) and genome-wide SNP-based phylogenetic assays were 100%. The following objectives were accomplished: (i) the establishment of the performance specifications for WGS applications in PHLs according to CLIA guidelines, (ii) the development of quality assurance and quality control measures, (iii) the development of a reporting format for end users with or without WGS expertise, (iv) the availability of a validation set of microorganisms, and (v) the creation of a modular template for the validation of WGS processes in PHLs. The validation panel, sequencing analytics, and raw sequences could facilitate multilaboratory comparisons of WGS data. Additionally, the WGS performance specifications and modular template are adaptable for the validation of other platforms and reagent kits.

[Comparison of Classifcation and Regulation of in Vitro Diagnostic Products for Clinical Pathology in China and Foreign Country].

The classification and the regulatory requirement among U.S., E.U., Japan and China were summarized and compared for the immunohistochemistry and in situ hybridization products. The results indicate that:the regulatory classifications of the related products are higher in Japan and China, than U.S.and E.U.; the classification and regulatory requirement are adjusted more flexibly in the centralized system. The difference was discussed and accordingly some suggestions and implications were given for the China's regulation.

Development and performance evaluation of a qPCR-based assay for the fully automated detection of group B Streptococcus (GBS) on the Panther Fusion Open Access system.

Streptococcus agalactiae [group B Streptococcus (GBS)] poses a major threat as the primary cause of early-onset neonatal invasive disease, particularly when mothers are colonized rectovaginally. Although culture remains the gold standard for antepartum GBS screening, quantitative polymerase chain reaction (qPCR) offers advantages in terms of sensitivity and turnaround time. The aim of this study was to validate the clinical utility of an automated qPCR laboratory-developed test (LDT) for antepartum GBS screening using the Panther Fusion Open Access system (Hologic, California, USA). The LDT targeted a conserved region of the GBS surface immunogenic protein gene, demonstrating no cross-reactivity and high coverage (99.82%-99.99%). The limit of detection (LoD) was 118 CFU/mL. Comparison with commercial qPCR assays (Panther Fusion GBS and VIASURE Streptococcus B Real-Time) revealed an overall agreement of 99.7%, with a robust Cohen's kappa coefficient of 0.992. Testing of 285 rectovaginal swabs from pregnant women and 15 external quality assessment samples demonstrated exceptional diagnostic performance of the LDT, achieving a diagnostic sensitivity and specificity of 100%, underscoring its accuracy. Prevalence and predictive values were also determined to reinforce test reliability. Our research highlights the limitations of culture-based screening and supports the suitability of our qPCR-based LDT for GBS detection in a clinical setting.IMPORTANCERectovaginal colonization by GBS is a major risk factor for early-onset invasive neonatal disease. The most effective approach to reducing the incidence of early-onset disease (EOD) has been described as universal screening, involving assessment of GBS colonization status in late pregnancy and intrapartum antibiotic prophylaxis. Despite its turnaround time and sensitivity limitations, culture remains the gold standard method for GBS screening. However, nucleic acid amplification-based tests, such as qPCR, have been utilized due to their speed and high sensitivity and specificity. This study validated the clinical usefulness of an automated qPCR-LDT for antepartum GBS screening through the Panther Fusion Open Access system (Hologic). Our study addresses the critical need for more robust, sensitive, and rapid strategies for GBS screening in pregnant women that could favorably impact the incidence of EOD.

Analytical evaluation of the automated interleukin-6 assay on the Roche cobas e602 analyzer.

BACKGROUND: Interleukin-6 (IL-6) is a proinflammatory cytokine that is associated with many inflammatory diseases. This validation study evaluates the automated Roche Elecsys IL-6 electrochemiluminescent immunoassay that has been granted emergency use authorization by the US Food and Drug Administration. METHODS: The Elecsys IL-6 assay was evaluated for precision, linearity, interference (by hemoglobin, bilirubin, triglycerides, and biotin) and clinical performance was compared to the V-PLEX Human IL-6 immunoassay (Meso Scale Discovery), performed by a reference laboratory. RESULTS: The Elecsys IL-6 assay is precise (intra-assay <3% coefficient of variation [CV], interassay <5% CV), exhibits an analytical measurable range of 1.5-4790 pg/mL, and is tolerant of significant interferences (H < 2522, I <62, L<2101, biotin <50 ng/mL). Comparison with the V-PLEX assay revealed a 2.95 slope bias in patient samples evaluated for IL-6 concentration (n = 43, range = 1.5-1891 pg/mL, y = 2.95x - 32.7, r2 = 0.84). Bland-Altman analysis revealed an absolute mean bias of 152 pg/mL (SD = 254 pg/mL), or a mean percentage difference of 73%. CONCLUSION: The Roche IL-6 assay showed good analytical performance. The large systematic bias compared with another reference method precludes using multiple methods to monitor IL-6 response. The random-access nature of an automated IL-6 assay on the Roche platform makes the test available on demand.

Automated molecular detection of West Nile Virus in mosquito pools using the Panther Fusion system.

West Nile Virus (WNV) is an arthropod-borne virus that is spread through mosquito vectors. WNV emerged in the US in 1999 and has since become endemic in the US, causing the most domestically acquired arboviral disease in the country. Mosquito surveillance for WNV is useful to monitor arboviral disease burden over time and across different locations. RT-qPCR is the preferred method for WNV surveillance, but these methods are labor-intensive. The Panther Fusion System has an Open Access feature that allows for laboratory-developed tests (LDTs) to run on a fully automated system for nucleic acid extraction, RT-qPCR, and result generation. This study demonstrates the successful optimization of a WNV multiplex LDT (assay targets: ENV and NS1 genes) for high-throughput environmental surveillance testing of mosquito pool homogenates on the Panther Fusion System. Analytical sensitivity of the assay was 186 and 744 copies/PCR reaction for the ENV and NS1 targets, respectively. To assess the performance of this assay, a total of 80 mosquito pools were tested, including 60 previously tested pools and 20 spiked negative mosquito pools. Among the 60 previously tested specimens, the Panther Fusion WNV LDT demonstrated 100% positive and negative agreement with the CDC West Nile RT-qPCR assay. The Panther Fusion WNV LDT also detected all 20 spiked specimens. The Panther Fusion WNV LDT assay was successfully developed and optimized for high throughput testing with similar performance to the previously used CDC West Nile RT-qPCR assay.

[Use of the ETV6/RUNX1 probe to verify the performance of the fluorescence in situ hybridization probe before clinical detection].

Objective: To explore the standardized performance of a FISH probe before clinical detection. Methods: The probe sensitivity and specificity of ETV6/RUNX1 were analyzed via interphase and metaphase FISH in 20 discarded healthy bone marrow samples. The threshold system of the probe was established using an inverse beta distribution, and an interpretation standard was established. Finally, a parallel-controlled polymerase chain reaction detection study was conducted on 286 bone marrow samples from patients at our hospital. The clinical sensitivity, specificity, and diagnostic coincidence rate of ETV6/RUNX1 FISH detection were analyzed, and the diagnostic consistency of the two methods was analyzed by the kappa test. Results: The probe sensitivity and specificity of the ETV6/RUNX1 probe were 98.47% and 100%, respectively. When 50, 100, and 200 cells were counted, the typical positive signal pattern cutoffs were 5.81%, 2.95%, and 1.49%, respectively, and the atypical positive signal pattern cutoffs were 13.98%, 9.75%, and 6.26%, respectively. The clinical sensitivity of FISH was 96.1%, clinical specificity was 99.6%, diagnostic coincidence rate was 99.00%, diagnostic consistency test kappa value was 0.964, and P value was <0.001. Conclusion: For FISH probes without a national medical device registration certificate, standardized performance verification and methodology performance verification can be performed using laboratory developed test verification standards to ensure a reliable and accurate reference basis for clinical diagnosis and treatment.

Development and validation of a fully automated laboratory-developed test for detection of HSV-1/2 and VZV in clinical samples run on the Panther Fusion® system.

The purpose of this study was to develop a laboratory developed test (LDT) for HSV1/2 and VZV to run on fully automated Hologic Panther Fusion® System. The Panther Fusion System is a fully automated walkaway system, providing end-to-end workflow from sample input to DNA/RNA extraction, amplification, automated analysis, and reporting to a laboratory information system (LIS). The LDT was developed and validated on 230 clinical and 20 reference samples (n = 250) and compared to a commercially available kit. Assessment of the analytical and diagnostic performances of the LDT revealed >98% accuracy, sensitivity, and specificity, which is consistent with or better than many of the commercial or laboratory-developed tests available. The advantage of this LDT is that it is designed to perform a single-run full female health screening in parallel with 4 commercially available Hologic kits for Chlamydia trachomatis/Neisseria gonorrhea (CT/NG), Trichomonas vaginalis (TV), Mycoplasma genitalium (MG), and bacterial vaginosis (BV).