Clinical staging of cervical lymph nodes from patients with squamous cell carcinoma of the head and neck (SCCHN) has only 50% accuracy compared with definitive pathologic assessment. Consequently, both clinically positive and clinically negative patients frequently undergo neck dissections that may not be necessary. To address this potential overtreatment, sentinel lymph node (SLN) biopsy is currently being evaluated to provide better staging of the neck. However, to fully realize the potential improvement in patient care afforded by the SLN procedure, a rapid and accurate SLN analysis is necessary. We used quantitative reverse transcription--PCR (QRT-PCR) to screen 40 potential markers for their ability to detect SCCHN metastases to cervical lymph nodes. Seven markers were identified with good characteristics for identifying metastatic disease, and these were validated using a set of 26 primary tumors, 19 histologically positive lymph nodes, and 21 benign nodes from patients without cancer. Four markers discriminated between positive and benign nodes with accuracy > >97% but only one marker, pemphigus vulgaris antigen (PVA), discriminated with 100% accuracy in both the observed data and a statistical bootstrap analysis. A rapid QRT-PCR assay for PVA was then developed and incorporated into a prototype instrument capable of performing fully automated RNA isolation and QRT-PCR. The automated analysis with PVA provided perfect discrimination between histologically positive and benign lymph nodes and correctly identified two lymph nodes with micrometastatic tumor deposits. These assays were completed ( from tissue to result) in f f30 minutes, thus demonstrating the feasibility of intraoperative staging of SCCHN SLNs by QRT-PCR. (Cancer Res 2005; 65(6): 2147-56)
Background: PCR-based assays can improve clinical care, but they remain technically demanding and labor-intensive. We describe a new instrument, the GeneXpert ® , that performs automated nucleic acid isolation, reverse transcription, and fluorescence-based quantitative PCR in ϳ35 min. Methods: Yield and integrity of RNA isolated on the GeneXpert were compared with Qiagen-based extraction for parallel samples (5-m frozen tissue sections). The reproducibility of automated RNA isolation, reverse transcription, and quantitative PCR was determined by replicate (n ؍ 10) analysis of 10 tissues, using duplex (target and endogenous control) reverse transcription-PCR reactions for two gene combinations. The GeneXpert was then used to perform rapid analysis of lymph nodes from melanoma, breast cancer, and lung cancer patients and analysis of melanoma metastatic to the lung, primary lung adenocarcinoma, and healthy lung tissue. Results: On the GeneXpert, RNA was recovered in slightly over 6 min, and the yield was ϳ70% of that from parallel Qiagen reactions. The RNA integrity was comparable to that of Qiagen-isolated RNA as determined by gel electrophoresis. For the melanoma samples, the 95% prediction interval for the ⌬Ct for a new measurement was ؎1.54 cycles, and for breast cancer samples, the interval for a newly observed ⌬Ct was ؎1.40 cycles.
A rapid, fully automated QRT-PCR assay definitively characterizes breast cancer SLN with accuracy equal to conventional pathology. This approach is superior to intraoperative SLN analysis and can provide standardized, objective results to assist in pathologic diagnosis.
Microfluidic cassettes that perform integrated biological sample preparation and DNA analysis require fluidic control and transport mechanisms built into the device. In this study, pneumatically actuated diaphragm pumps and valves were employed to achieve precise fluidic manipulation and enabled the execution of several sample-processing steps within a single cassette. However, the design of the microfluidic cassette to accomplish this multi-step fluidic protocol required a complex three-dimensional fluid path through valves, bends, various sized passageways and a porous filter for cell capture. In order to understand the fluidic behavior in such a device, measurements were taken of the pneumatic pressure delivered to the diaphragm pump as it pushed sample through the complicated fluidic pathway. Simultaneously monitored were the resulting volumetric flow rate, and the corresponding pre- and post-filter fluid pressures. The data enabled the construction of a model that simulated the fluidic action through the device using established fluid mechanics theory that closely matched flow rate and pressure data. The ability to simulate the behavior of diaphragm pumping and resulting fluidic movements in complex microfluidic devices provides a greater comprehension of this phenomenon and a useful tool in the application to future devices for biochemical analysis.
Infections with DNA viruses are frequent causes of morbidity and mortality in transplant recipients.
We have previously reported that a quantitative reverse transcription (QRT)-PCR assay accurately analyzes sentinel lymph nodes (SLNs) from breast cancer patients. The aim of this study was to assess a completely automated , cartridge-based version of the assay for accuracy , predictive value , and reproducibility. The triplex (two markers ؉ control) QRT-PCR assay was incorporated into a single-use cartridge for point-of-care use on the GeneXpert system. Three academic centers participated equally. Twenty-nine positive lymph nodes and 30 negative lymph nodes were analyzed to establish classification rules. SLNs from 120 patients were subsequently analyzed by QRT-PCR and histology (including immunohistochemistry) , and the predetermined decision rules were used to classify the SLNs; 112 SLN specimens produced an informative result by both QRT-PCR and histology. By histological analysis , 21 SLNs were positive and 91 SLNs were negative for metastasis. QRT-PCR characterization produced a classification with 100% sensitivity , 97.8% specificity , and 98.2% accuracy compared with histology (91.3% positive predictive value and 100% negative predictive value). Interlaboratory reproducibility analyses demonstrated that a 95% prediction interval for a new measurement (⌬Ct) ranged between 0.403 and 0.956. This fully automated QRT-PCR assay accurately characterizes breast cancer SLNs for the presence of metastasis. Furthermore , the assay is not dependent on subjective interpretation , is reproducible across three clinical environments , and is rapid enough to allow intraoperative decision making. (J Mol Diagn
Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants can potentially guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here, we present the development and validation of a rapid COVID-19 variant DETECTR assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene.
Laboratory tests for the accurate and rapid identification of SARS-CoV-2 variants have the potential to guide the treatment of COVID-19 patients and inform infection control and public health surveillance efforts. Here we present the development and validation of a COVID-19 variant DETECTR® assay incorporating loop-mediated isothermal amplification (LAMP) followed by CRISPR-Cas12 based identification of single nucleotide polymorphism (SNP) mutations in the SARS-CoV-2 spike (S) gene. This assay targets the L452R, E484K, and N501Y mutations associated with nearly all circulating viral lineages. In a comparison of three different Cas12 enzymes, only the newly identified enzyme CasDx1 was able to accurately identify all three targeted SNP mutations. We developed a data analysis pipeline for CRISPR-based SNP identification using the assay from 91 clinical samples (Ct < 30), yielding an overall SNP concordance and agreement with SARS-CoV-2 lineage classification of 100% compared to viral whole-genome sequencing. These findings highlight the potential utility of CRISPR-based mutation detection for clinical and public health diagnostics.
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