Evaluation of Viasure SARS-CoV-2 RT-qPCR kit (CerTest Biotec) using CDC FDA EUA RT-qPCR kit as a gold standard

BackgroundSeveral RT-qPCR kits are available for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by FDA, but most of them lacking of proper evaluation studies due to covid19 emergency.ObjectiveWe evaluated Viasure RT-qPCR kit (CerTest Biotec, Spain) for SARS-CoV-2 diagnosis using CDC FDA EUA kit as gold standard.ResultsAlthough we found the lack of RNA quality control probe as the main limitation for Viasure kit, the sensitivity was up to 97.5% and specificity was 100%.ConclusionsViasure RT-qPCR kit is a reliable tool for SARS-CoV-2 diagnosis but improvement of an alternative RT-qPCR reaction for RNA extraction quality control as RNaseP is recommended.

Evaluation of nCoV-QS (MiCo BioMed) for RT-qPCR detection of SARS-CoV-2 from nasopharyngeal samples using CDC FDA EUA qPCR kit as a gold standard: an example of the need of validation studies

BackgroundSeveral qPCR kits are available for SARS-CoV-2 diagnosis, mostly lacking of evaluation due to covid19 emergency.ObjectiveWe evaluated nCoV-QS (MiCo BioMed) kit using CDC kit as gold standard.ResultsWe found limitations for nCoV-QS: 1) lower sensitivity 2) lack of RNA quality control probe 3) no capacity to quantify viral load.ConclussionsValidation studies should be implemented for any SARS-CoV-2 RT-qPCR commercial kit to prevent unreliable diagnosis.

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus

We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338–BHHTEGST–Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.

Impact of 2013 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines on HER2 fluorescent in situ hybridization (FISH) testing in breast cancers: Experience from a national reference laboratory.

e23188 Background: Accurate HER2 testing is expected to identify patients who will benefit from HER2 directed therapy (HDT) minimizing false positive and negatives. Like many biologic processes, HER2 protein expression and gene copy numbers are a continuum, however these tests are expected to be reported dichotomously. In 2013, ASCO/CAP revised the guidelines in an attempt to decrease false negative results and reverted to FDA approved ratio of ≥ 2.0 for HER2 positivity. We reviewed HER2 FISH testing results after the implementation of 2013 ASCO/CAP guidelines to determine the effects on HER2 reporting from our national reference laboratory. Methods: HER2 FISH testing performed between 5/2015-4/2016 at ARUP Labs following current 2013 ASCO/CAP guidelines was included. HER2 to control probe ratios, mean HER2 and control probe copy numbers were used to reassign HER2 status using 2007 ASCO/CAP, and FDA guidelines for each case. Results: HER2 FISH results were available in 2,017 cases. 342 (17.0%) cases were amplified, 301 (14.9%) were equivocal, and 1374 (68.1%) were non-amplified. After additional testing with alternate probe, 93 (31.2%) of the equivocal cases were reclassified as amplified increasing amplified cases to 21.6%. All of the equivocal cases which were reinterpreted as amplified with alternate probe showed low level amplification (range: 2.0-3.6; mean: 2.3). HER2 positivity rates following 2013 ASCO/CAP guidelines, both at initial testing and after additional testing to resolve equivocal results were significantly higher compared to 2007 ASCO/CAP guidelines and FDA criteria. Conclusions: 2013 ASCO/CAP guidelines lead to higher number of HER2 FISH positive and equivocal cases. In a reference laboratory setting where alternative control probe was used to resolves equivocal FISH cases, 31.2% of patients with initial equivocal results become HER2 positive. However, it is not known if these patients benefit from targeted therapies as they would not be included in the original adjuvant or metastatic trials.

RAI1 Alternate Probe Identifies Additional Breast Cancer Cases as Amplified Following Equivocal HER2 Fluorescence In Situ Hybridization Testing: Experience From a National Reference Laboratory

Context.—In 2013 the American Society of Clinical Oncology and College of American Pathologists updated the HER2 guidelines and changed the equivocal category for HER2 in situ hybridization testing to an average HER2 copy number of 4.0 to 5.9 with a HER2:CEP17 ratio of less than 2.0 and proposed retesting, with an option of using another control probe to avoid false-negative results. RAI1, located at band position 17p11.2, is a popular alternate probe locus for retesting equivocal changes. Objective.—To review experience with the RAI1 alternate probe in HER2 fluorescence in situ hybridization equivocal breast cancers. Design.—Primary and metastatic breast cancers with equivocal HER2 fluorescence in situ hybridization, retested with an alternate (RAI1) probe, were identified. HER2, RAI1, and CEP17 copy numbers, HER2 to control probe ratios, and genetic heterogeneity were recorded. Hematoxylin-eosin–stained slides were reviewed for type and grade of cancer. Results.—Of 876 cases tested with CEP17 as the reference probe, 97 (11.1%) had equivocal HER2 fluorescence in situ hybridization results. Additional testing with the RAI1 probe classified 39.2% cases (38 of 97) as amplified with a HER2:RAI1 ratio ranging from 2.0 to 3.2 (mean, 2.37); 3.1% (3 of 97) were still unclassifiable because of a deletion of RAI1. Conclusions.—RAI1 identified close to 40% of original HER2 fluorescence in situ hybridization equivocal cases as amplified, making these patients eligible for targeted therapies. It is not known whether guidelines for US Food and Drug Administration–approved probes can be extrapolated to alternate probes when an alternate control probe shows losses or gains. Because of the lack of guidelines for reporting HER2 status with alternate probes, laboratories face challenges in interpreting results.

Low Level Amplification (Duplication) of 1q21 in Myeloma and Prognosis; the Role of CKS1B.

Introduction: Molecular cytogenetic studies have revealed that, to a great extent, the heterogeneity of myeloma is largely dictated by the underlying genetic and cytogenetic aberrations present in the clonal plasma cells. Most recently the group from the University of Arkansas identified strong prognostic associations with an increased level of gene expression of a cell cycle associated gene, CKS1B. CKS1B favors cell cycle progression by promoting degradation of p27 with release of the cyclin dependent kinases and entry into mitosis. Patients and methods: To further test this hypothesis we studied: a) via FISH for CKS1B amplification in a cohort of patients treated at the Mayo Clinic with high dose chemotherapy and stem cell support, as well as a group of patients with cytogenetically defined hypodiploidy, and b) a cohort of myeloma patients that were studied by gene expression profiling. Gene expression analysis was performed on CD138-enriched plasma-cell RNA using Affymetrix U133A chips (Affymetrix, Santa Clara, CA). Results: Of 159 patients studied 46 exhibited FISH abnormalities consistent with increase number of signals for CKS1B (30%): amplification was marginal or low in 44 cases, and in only two cases the ratio between CKS1B and control probe was greater than 2.0. Therefore, the predominant pattern is one of gene duplication rather than amplification where gene copy usually exceeds 10 and ratio with control probe exceeds 5 (e.g. HER-2/neu amplification). Patients with CKS1B duplication had a higher prevalence of chromosome 13 deletion (72%), and t(4;14) (29%). The presence of CKS1B duplication was more frequent (53%) among 19 patients with hypodiploidy. Using gene expression data for CKS1B, we found a positive association with t(14;16)(q32;q23). The level of CKS1B gene expression positively correlated with the PCLI (p<0.0001) but there was heterogeneity in this relationship (r2 = 0.34). We found no correlation between the expression level of CKS1B and p27 (r2 = 0.008, p = 0.37). While CKS1B gene duplication had a negative effect on survival this effect was weak (29.9 versus 38 months, p = 0.124. Median follow-up is 55 months) and disappeared when the variable was entered into the multivariate model including PCLI, B2-microglobulin and all the major genetic abnormalities by FISH. Likewise the level of expression of CKS1B determined by gene expression only carried prognostic significance when extreme levels of expression were utilized as a prognostic variable. Discussion: In this study we show that increase copy number of CKS1B is present in one third of patients with MM (majority of these being gene duplication) and seem to be associated with a shorter overall survival, but the net effect of this is rather weak in our series. Further study is needed to understand its potential role as a progression factor in the plasma cell neoplasms.