Quality Control Methods for Optimal BCR-ABL1 Clinical Testing in Human Whole Blood Samples

Reliable breakpoint cluster region (BCR)–Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and RNA input in the reverse transcription (RT) reaction on BCR-ABL1 and β-glucuronidase (GUSB) cDNA yield were assessed in whole blood samples mixed with K562 cells. BCR-ABL1 was measured relative to GUSB to control for sample loading, and each gene was measured relative to known numbers of respective internal standard molecules to control for variation in quality and quantity of reagents, thermal cycler conditions, and presence of PCR inhibitors. Clinical sample and reference material measurements with this test were concordant with results reported by other laboratories. BCR-ABL1 per 103 GUSB values were significantly reduced (P = 0.004) after 48-hour storage. Gene-specific primers yielded more BCR-ABL1 cDNA than random hexamers at each RNA input. In addition, increasing RNA inhibited the RT reaction with random hexamers but not with gene-specific primers. Consequently, the yield of BCR-ABL1 was higher with gene-specific RT primers at all RNA inputs tested, increasing to as much as 158-fold. We conclude that optimal measurement of BCR-ABL1 per 103 GUSB in whole blood is obtained when gene-specific primers are used in RT and samples are analyzed within 24 hours after blood collection

Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/147795/1/his13758.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/147795/2/his13758_am.pd

Neurofilament is superior to cytokeratin 20 in supporting cutaneous origin for neuroendocrine carcinoma.

AIM: Primary cutaneous neuroendocrine carcinoma, or Merkel cell carcinoma (MCC), cannot be distinguished morphologically from small-cell neuroendocrine carcinomas (SmCC) from other sites. Immunohistochemistry is required to confirm cutaneous origin, and is also used for detection of sentinel lymph node (SLN) metastases of MCC. Cytokeratin 20 (CK20) expression is commonly used for these purposes, but is negative in some MCC cases, and has unclear specificity. We evaluated immunohistochemistry for neurofilament and CK20 in MCC compared with SmCC from other sites. METHODS AND RESULTS: We evaluated neurofilament expression in 55 MCC specimens from 39 unique patients, including nine CK20-negative MCC tumours. Neurofilament expression was observed in 42 of 55 (76.4%) MCC cases, including seven of nine (77.8%) CK20-negative MCC cases. Neurofilament was expressed in nine of 12 (75%) Merkel cell polyomavirus-positive tumours and five of 10 (50%) virus-negative tumours. Compared to a standard immunohistochemical panel (cytokeratin cocktail and CK20), neurofilament was 87.5% sensitive for detecting SLN metastases. Neurofilament and CK20 expression was also assessed in 61 extracutaneous SmCC from 60 unique patients, with primary sites including lung (27), bladder (18), cervix (3), gastrointestinal tract (3), sinonasal tract (2) and other sites (7). The specificity of neurofilament and CK20 for MCC versus non-cutaneous SmCC was 96.7% and 59.0%, respectively. CONCLUSIONS: Neurofilament has superior specificity to CK20 in distinguishing MCC from non-cutaneous SmCC. Neurofilament is frequently expressed in CK20- and virus-negative MCC tumours. Limitations of neurofilament immunohistochemistry include lower sensitivity than CK20 and subtle staining in some tumours. However, our findings indicate that neurofilament is useful for excluding non-cutaneous SmCC

A New StaRT-PCR Approach to Detect and Quantify Fish Viral Hemorrhagic Septicemia Virus (VHSv): Enhanced Quality Control with Internal Standards

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world\u27s most important finfish diseases, killing \u3e80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR® green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR®green real time qRT-PCR tests, and 47–70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0 × 100–1.2 × 105 VHSv/106actb1molecules in wild caught fishes and 1.0 × 100–8.4 × 105 molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean = 1.9 × 104) than those without (1.1 × 103) (p \u3c 0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv

A New StaRT-PCR Approach to Detect and Quantify Fish Viral Hemorrhagic Septicemia Virus (VHSv): Enhanced Quality Control with Internal Standards

Viral Hemorrhagic Septicemia virus (VHSv) causes one of the world\u27s most important finfish diseases, killing \u3e80 species across Eurasia and North America. A new and especially virulent strain (IVb) emerged in the North American Great Lakes in 2003, threatening fisheries, baitfish, and aquaculture industries. Weeks-long and costly cell culture is the OIE and USDA-APHIS approved diagnostic. A new Standardized Reverse Transcriptase Polymerase Chain Reaction (StaRT-PCR) assay that uniquely incorporates internal standards to improve accuracy and prevent false negatives was developed and evaluated for its ability to detect and quantify VHSv. Results from StaRT-PCR, SYBR® green real time qRT-PCR, and cell culture were compared, as well as the effects of potential PCR inhibitors (EDTA and high RNA). Findings show that StaRT-PCR is sensitive, detecting a single molecule, with 100% accuracy at six molecules, and had no false negatives. In comparison, false negatives ranged from 14 to 47% in SYBR®green real time qRT-PCR tests, and 47–70% with cell culture. StaRT-PCR uniquely controlled for EDTA and RNA interference. Range of VHSv quantitation by StaRT-PCR was 1.0 × 100–1.2 × 105 VHSv/106actb1molecules in wild caught fishes and 1.0 × 100–8.4 × 105 molecules in laboratory challenged specimens. In the latter experiments, muskellunge with skin lesions had significantly more viral molecules (mean = 1.9 × 104) than those without (1.1 × 103) (p \u3c 0.04). VHSv infection was detected earlier in injection than in immersion challenged yellow perch (two versus three days), with molecule numbers in both being comparable and relatively consistent over the remaining course of the experiment. Our results show that the StaRT-PCR test accurately and reliably detects and quantifies VHSv

Sequences of primers, probes, and standard templates for two-color fluorometric real-time measurement.

<p>NT: native template. IS: internal standard. BHQ: black hole quencher. Bold: modified nucleotides. Underline: probe binding site.</p

Targeted RNA-Sequencing with Competitive Multiplex-PCR Amplicon Libraries

<div><p>Whole transcriptome RNA-sequencing is a powerful tool, but is costly and yields complex data sets that limit its utility in molecular diagnostic testing. A targeted quantitative RNA-sequencing method that is reproducible and reduces the number of sequencing reads required to measure transcripts over the full range of expression would be better suited to diagnostic testing. Toward this goal, we developed a competitive multiplex PCR-based amplicon sequencing library preparation method that a) targets only the sequences of interest and b) controls for inter-target variation in PCR amplification during library preparation by measuring each transcript native template relative to a known number of synthetic competitive template internal standard copies. To determine the utility of this method, we intentionally selected PCR conditions that would cause transcript amplification products (amplicons) to converge toward equimolar concentrations (normalization) during library preparation. We then tested whether this approach would enable accurate and reproducible quantification of each transcript across multiple library preparations, and at the same time reduce (through normalization) total sequencing reads required for quantification of transcript targets across a large range of expression. We demonstrate excellent reproducibility (R² = 0.997) with 97% accuracy to detect 2-fold change using External RNA Controls Consortium (ERCC) reference materials; high inter-day, inter-site and inter-library concordance (R² = 0.97–0.99) using FDA Sequencing Quality Control (SEQC) reference materials; and cross-platform concordance with both TaqMan qPCR (R² = 0.96) and whole transcriptome RNA-sequencing following “traditional” library preparation using Illumina NGS kits (R² = 0.94). Using this method, sequencing reads required to accurately quantify more than 100 targeted transcripts expressed over a 10⁷-fold range was reduced more than 10,000-fold, from 2.3×10⁹ to 1.4×10⁵ sequencing reads. These studies demonstrate that the competitive multiplex-PCR amplicon library preparation method presented here provides the quality control, reproducibility, and reduced sequencing reads necessary for development and implementation of targeted quantitative RNA-sequencing biomarkers in molecular diagnostic testing.</p></div