David S. Wilkinson

Genome-wide detection of LOH in prostate cancer using human SNP microarray technology.

Loss of heterozygosity (LOH) of chromosomal regions is crucial in tumor progression. In this study we assessed the potential of the Affymetrix GeneChip HuSNP mapping assay for detecting genome-wide LOH in prostate tumors. We analyzed two human prostate cell lines, P69SV40Tag (P69) and its tumorigenic subline, M12, and 11 prostate cancer cases. The M12 cells showed LOH in chromosomes 3p12.1-p22.1, 11q22.1-q24.2, 19p13.12, and 19q13.42. All of the prostate cases with informative single-nucleotide polymorphism (SNP) markers showed LOH in 1p31.2, 10q11.21, 12p13.1, 16q23.1-q23.2, 17p13.3, 17q21.31, and 21q21.2. Additionally, a high percentage of cases showed LOH at 6p25.1-p25.3 (75%), 8p22-p23.2, and 10q22.1 (70%). Several tumor suppressor genes (TSGs) have been mapped in these loci. These results demonstrate that the HuSNP mapping assay can serve as an alternative to comparative genomic hybridization for assessing genome-wide LOH and can identify chromosomal regions harboring candidate TSGs implicated in prostate cancer.

Assessment monitoring of laboratory critical values: A College of American Pathologists Q-tracks study of 180 institutions

CONTEXT Critical laboratory value reporting is a highly visible and essential key activity for clinical laboratories. OBJECTIVE To measure critical laboratory value reporting in multiple institutions over time and to examine the practice patterns and demographic factors associated with sustained improvement in critical value reporting. DESIGN A longitudinal cohort study of 180 clinical laboratories that provided quarterly critical values reporting data for 2 to 16 quarters was conducted using a uniform definition of successful caregiver notification. Mixed linear model analysis of the 2001 through 2004 dataset was performed. RESULTS A decrease in total and inpatient rates of undocumented critical values per 1000 results was associated with (1) the American Association of Blood Banks inspection within the past 2 years (P = .01, for both total and inpatient rates); (2) unit secretary/clerical staff not authorized to accept inpatient critical value notification (P = .004 [total] and .001 [inpatient]); and (3) the mandatory practice of requiring notification of health care providers when handling inpatients known to have results repeatedly in the critical range (P = .01, for both total and inpatient rates). Continued participation in the Q-Tracks monitoring program was associated with significant and progressive improvement in total, inpatient, and outpatient critical value reporting (P = .02, .01, and .003, respectively). CONCLUSIONS Critical value reporting improved as the duration of participation in the Q-Tracks monitoring program increased. Improved total and inpatient critical value reporting was associated with factors that may be markers for institutions with priorities of quality management and enhanced communication with responsible caregivers.

Blood bank safety practices: mislabeled samples and wrong blood in tube--a Q-Probes analysis of 122 clinical laboratories.

CONTEXT Although a rare occurrence, ABO incompatible transfusions can cause patient morbidity and mortality. Up to 20% of all mistransfusions are traced to patient misidentification and/or sample mislabeling errors that occur before a sample arrives in the laboratory. Laboratories play a significant role in preventing mistransfusion by identifying wrong blood in tube and rejecting mislabeled samples. OBJECTIVES To determine the rates of mislabeled samples and wrong blood in tube for samples submitted for ABO typing and to survey patient identification and sample labeling practices and sample acceptance policies for ABO typing samples across a variety of US institutions. DESIGN One hundred twenty-two institutions prospectively reviewed inpatient and outpatient samples submitted for ABO typing for 30 days. Labeling error rates were calculated for each participant and tested for associations with institutional demographic and practice variable information. Wrong-blood-in-tube rates were calculated for the 30-day period and for a retrospective 12-month period. A concurrent survey collected institution-specific sample labeling requirements and institutional policies regarding the fate of mislabeled samples. RESULTS For all institutions combined, the aggregate mislabeled sample rate was 1.12%. The annual and 30-day wrong-blood-in-tube aggregate rates were both 0.04%. Patient first name, last name, and unique identification number were required on the sample by more than 90% of participating institutions; however, other requirements varied more widely. CONCLUSIONS The rates of mislabeled samples and wrong blood in tube for US participants in this study were comparable to those reported for most European countries. The survey of patient identification and sample labeling practices and sample acceptance policies for ABO typing samples revealed both practice uniformity and variability as well as significant opportunity for improvement.

Genes involved in radiation therapy response in head and neck cancers.

This is a pilot study designed to identify gene expression profiles able to stratify head and neck squamous cell carcinoma (HNSCC) tumors that may or may not respond to chemoradiation or radiation therapy.

Clinical utility of a quantitative polymerase chain reaction for diagnosis of cytomegalovirus disease in solid organ transplant patients.

BACKGROUND Accurate and rapid diagnosis of human cytomegalovirus (HCMV) disease in solid organ transplant patients remains a challenge. We evaluated the clinical utility of a quantitative polymerase chain reaction (QPCR) method to diagnose transplant patients with HCMV disease. METHODS A total of 429 plasma samples from 121 solid organ transplant patients were prospectively collected and evaluated for HCMV using a QPCR assay. To enhance the sensitivity of the QPCR assay, plasma samples were centrifuged in a manner designed to concentrate the virions before nucleic acid extraction. Quantitation was achieved by co-amplifying an internal quantitative standard (IS) that contained the same primer sequences as for HCMV. Polymerase chain reaction products were detected by hybridization to 96-well microtiter plates coated with either a HCMV- or an IS-specific probe. RESULTS A total of 103 patients had all samples negative by QPCR. None of the 103 patients developed HCMV disease during the study. In contrast, 18 patients showed at least 1 sample positive by the QPCR assay, but only 8 of these developed HCMV disease. The mean viral load value for patients without HCMV disease was 93 viral copies (vc) per ml of plasma (range: 35-325 vc/ml plasma) and for the 8 patients with HCMV disease was 67,686 vc/ml plasma (range: 167-1,325,000 vc/ml plasma) (P<0.001). Using a cut-off value of 100 vc/ml plasma and clinical diagnosis of HCMV disease, the QPCR assay showed a sensitivity of 100% and specificity of 99.1%. CONCLUSION HCMV viral load may be useful in the diagnosis of HCMV disease in solid organ transplant patients.

Comparison of Performance Characteristics of Three Real-Time Reverse Transcription-PCR Test Systems for Detection and Quantification of Hepatitis C Virus

ABSTRACT We evaluated the performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus (HCV) and performed a direct comparison of the systems on the same clinical specimens. Commercial HCV panels (genotype 1b) were used to evaluate linear range, sensitivity, and precision. The Roche COBAS TaqMan HCV test for research use only (RUO) with samples processed on the MagNA Pure LC instrument (Roche RUO-MPLC) and Abbott analyte-specific reagents (ASR) with QIAGEN sample processing (Abbott ASR-Q) showed a sensitivity of 1.0 log10 IU/ml with a linear dynamic range of 1.0 to 7.0 log10 IU/ml. The Roche ASR in combination with the High Pure system (Roche ASR-HP) showed a sensitivity of 1.4 log10 IU/ml with a linear dynamic range of 2.0 to 7.0 log10 IU/ml. All of the systems showed acceptable reproducibility, the Abbott ASR-Q being the most reproducible of the three systems. Seventy-six clinical specimens (50 with detectable levels of HCV RNA and various titers and genotypes) were tested, and results were compared to those of the COBAS Amplicor HCV Monitor v2.0. Good correlation was obtained for the Roche RUO-MPLC and Abbott ASR-Q (R2 = 0.84 and R2 = 0.93, respectively), with better agreement for the Abbott ASR-Q. However, correlation (R2 = 0.79) and agreement were poor for Roche ASR-HP, with bias relative to concentration and genotype. Roche ASR-HP underestimated HCV RNA for genotypes 3 and 4 as much as 2.19 log10 IU/ml. Our study demonstrates that Roche RUO-MPLC and Abbott ASR-Q provided acceptable results and agreed sufficiently with the COBAS Amplicor HCV Monitor v2.0.

Clinical Verification of the Performance of the Pathwork Tissue of Origin Test Utility and Limitations

Gene expression-based assays have been introduced into the clinical arena to assist in the diagnosis of poorly differentiated or undifferentiated tumors. The US Food and Drug Administration has cleared the microarray-based Pathwork Tissue of Origin (TOO) Test (Pathwork Diagnostics, Sunnyvale, CA) for the molecular characterization of such challenging specimens. We aimed at verifying the analytic and clinical performance of this test on 43 poorly differentiated and undifferentiated tumor samples, including 6 off-panel cases and 7 cancers of unknown primary (CUP). Our results showed 97% (95% confidence interval, 80.4%-99.8%) agreement between the Pathwork TOO Test result and the complete diagnosis, which included clinical correlations and immunohistochemical staining, after the original diagnosis. We concluded that for off-panel and CUP samples, the tissue type and the cell type may be confounded by the Pathwork TOO Test and that careful clinicopathologic assessment is needed when interpreting results from this helpful ancillary tool for pathologists.

Staffing Benchmarks for Clinical Laboratories A College of American Pathologists Q-Probes Study of Staffing at 151 Institutions

CONTEXT Inadequate staffing of clinical laboratories may compromise quality and throughput, whereas excess staff unnecessarily increases the cost of testing. OBJECTIVES To measure productivity of technical staff and management span of control in a large number of laboratories and to determine factors associated with favorable staffing ratios. DESIGN A total of 151 clinical laboratories provided information about technical and management staffing and output (workload) for 4 laboratory sections: anatomic pathology, chemistry/hematology/immunology, microbiology, and transfusion medicine. RESULTS For each laboratory section, there was wide variation in labor productivity (output per nonmanagement full-time equivalent) and in management span of control (nonmanagement full-time equivalent per manager). Productivity ratios for the 10th- and 90th-percentile laboratories varied more than 3-fold. Except in histology, laboratory sections with higher test volumes had higher labor productivity (P < .001 for cytology, chemistry/hematology, and transfusion medicine; P = .003 for microbiology). Even within peer groups composed of sections with similar volume, there was wide variation in labor productivity. A number of variables other than test volume were associated with labor productivity and management span of control. Staffing ratios for each laboratory section and for sections of different sizes are presented. CONCLUSIONS Despite standardization of testing methods in the clinical laboratory industry, there is wide variation in staffing level among institutions. This variation suggests opportunities to improve staff productivity in many facilities.

The Rate of Manual Microscopic Examination of Urine Sediment: A College of American Pathologists Q-Probes Study of 11 243 Urinalysis Tests From 88 Institutions

CONTEXT The manual microscopic examination (MME) of the urine sediment is an imprecise and labor-intensive procedure. Many laboratories have developed rules from clinical parameters or urinalysis results to limit the number of these examinations. OBJECTIVE To determine the rate of urinalysis specimens on which an MME of the urine sediment was performed, document how various rules influence this rate, and determine whether any new information was learned from the MME. DESIGN Participants selected 10 random urinalysis tests received during each traditional shift and determined if an MME was performed until a total of 50 urinalysis tests with an MME were reviewed. Participants recorded the rules that elicited an MME and any new information learned from such an examination. RESULTS The MME rate for the median institution was 62.5%. An MME of urine was most frequently done for an abnormal urinalysis result and often resulted in new information being learned, irrespective of the rule that elicited the MME. The median institution learned new information as a result of the manual examination 66% of the time. The use of an automated microscopic analyzer was associated with fewer manual examinations (P = .005), whereas the ability of a clinician to order a manual examination was associated with more manual examinations (P = .004). CONCLUSIONS The use of an automated microscopic analyzer may decrease the number of MMEs. An MME when triggered by an abnormal macroscopic appearance of urine, a physician request, or virtually any positive urinalysis result often resulted in new information.

Performance Evaluation of Two Methods Using Commercially Available Reagents for PCR-Based Detection of FMR1 Mutation

The current workflow for clinical Fragile X testing is time consuming and labor intensive. Recently developed PCR-based methods simplify workflow, amplify full mutation alleles, and improve sensitivity for detecting low-level mosaicism. We evaluated the performance characteristics and workflow of two methods using commercially available reagents for determining FMR1 mutation status. We also tested each methods ability to detect mosaicism (range, 100% to 1% for males; 50% to 1% for females). One method used reagents from Asuragen (AmplideX FMR1 PCR, research use only). The second method used analyte specific reagents from Abbott Molecular, including FMR1 Primer 1 (for repeat sizing) and FMR1 Primer 2 (for screening of expanded alleles). Each reaction was evaluated for accuracy, precision, correlation with previous results, and workflow. Both methods performed equally well in accuracy and precision studies using NIST standards and previously characterized Coriell samples. Both methods showed 100% concordance with results from a previous consensus study and for previously analyzed patient samples. The Asuragen reagents were able to detect full mutation mosaicism down to 5% and premutation mosaicism to 1%. The Abbott Molecular Primer 2 reagents were able to detect both full mutation and premutation mosaicism down to 25%. Both PCR-based methods for the determination of FMR1 mutation status performed well, with expected results in their final diagnoses, and differed significantly only in their workflow.