Selective laryngoscopy before thyroidectomy: a risk assessment.

BACKGROUND: Patients presenting for thyroidectomy may have an unrecognized pre-existing vocal cord palsy (VCP). This raises the danger of bilateral VCP if a patient sustains an injury to the RLN on the sole functioning side. Part of the rationale for routine preoperative laryngoscopy is to eliminate such a risk. This paper endeavours to quantify the relevant potential risk. METHODS: Patients who underwent laryngoscopy prior to thyroid or parathyroid surgery in an endocrine surgical unit over a 5 year period were identified. Literature review revealed four papers in which VCP prevalence in patients without risk factors was reported. Using our data, combined with that of these other authors, the background rate of pre-existing VCP was ascertained, and the subsequent risk of bilateral VCP estimated. RESULTS: Of our 632 patients who underwent preoperative laryngoscopy, there were four patients (0.63%) who were found to have a unilateral VCP, but all had voice symptoms or previous neck surgery. When patients with these risk factors are excluded, our data combined with the published data provides a pre-existing VCP rate of 0.2%. Calculations estimate that if preoperative laryngoscopy is omitted in patients with no risk factors, the risk of bilateral VCP, due to the nerve on the sole functioning side being injured, would be between 1/50000 and 1/150000, depending on an individual surgeon's level of experience. CONCLUSION: Selective use of laryngoscopy prior to thyroidectomy would result in an acceptably low statistical risk of bilateral VCP. Routine laryngoscopy for all patients is not necessary.

Clinical Utility of Genotyping Human Erythrocyte Antigens.

Traditional serological methods, which have been used for decades to evaluate the human erythrocyte antigen (HEA) composition of recipient and donor specimens, have some serious limitations. Specific reagent antisera are not available for all clinically relevant antigens (eg, V antigen). Reagent antisera are expensive, and serological testing is labor intensive. The results of serological testing are subjective and semiquantitative (eg, microscopic, weak, 1+, 2+, 3+, 4+), and may vary from one technologist to another. Further, in many clinical situations, serological testing may be difficult or impossible.Recent developments in nucleic acid-based testing (molecular diagnostics) have made it possible to genotype HEA in the clinical laboratory. Most allelic variations occur due to single nucleotide polymorphisms (SNPs), which can be detected and from which the phenotypes can be predicted. HEA genotyping offers several technical and clinical advantages compared with serological testing. The Immucor PreciseType HEA Test is the first and currently the only platform for clinical testing approved by the United States Food and Drug Administration (FDA), to our knowledge.

Documentation of quality control and operator training at point-of-care testing: a College of American Pathologists Q-Probes study of 106 institutions.

CONTEXT: Operator training, quality control, and proper follow-up for out-of-range quality control (QC) events are crucial steps that must be adequately performed and documented to ensure excellent patient care and regulatory compliance. OBJECTIVE: To examine point-of-care testing (POCT) personnel training and QC documentation/compliance. DESIGN: Participants in a POCT documentation study of the College of American Pathologists Q-Probes program collected data retrospectively for glucose and urine dipstick testing regarding test operators, operator competency assessment, and QC documentation. Documentation was assessed for participant adherence to 4 quality indicators: (1) whether test operator training was up to date, (2) whether the test operator names were noted in the test records, (3) whether QC was performed, and (4) whether out-of-range QC events were followed up. Data were analyzed for associations with institutional demographic and practice variables. RESULTS: The institutional median number of POCT personnel was 648 for blood glucose and 76 for urine dipstick testing, with a median number of 105 948 glucose tests and 9113 urine tests performed. Ninety-four percent (3830 of 4074) of the test operators completed training or competency assessment within the prior 12 months, 96.8% (21 603 of 22 317) of the test records documented the operator, and 95.7% (19 632 of 20 514) of the expected QC events (per institutional regulations) were documented. Approximately 3% (659 of 20 514) of the QC events were outside the designated range (an average of 6 out-of-range QC events were identified per institution [n = 106]). Of the out-of-range QC events, 92.6% (610 of 659) had documentation of appropriate follow-up. Most laboratories (176 of 179; 98.3%) violated specimen requirements by storing POCT urine specimens for less than 24 hours. CONCLUSIONS: There was greater than 90% compliance for POCT documentation and nearly 96% of expected QC events were properly documented.

Equivocal or ambiguous terminologies in pathology: focus of continuous quality improvement?

Ambiguous terminologies introduce uncertainty into pathology reports and may be misinterpreted by clinicians. Although absolute diagnostic certainty in all cases is not attainable, nevertheless, unbridled use of equivocal or ambiguous terminologies may lead to additional, sometimes unnecessary, tests and/or procedures directly or indirectly leading to increase in health care costs, as well as patient and clinician dissatisfaction. We evaluated the degree of certainty attributed to the commonly used ambiguous terminologies ("consistent with," "compatible with," "indicative of," "favor," "suggestive of," "suspicious for," "not excluded," "cannot exclude," "not ruled out," "not definite for," "not specific for," "indeterminate," "not identified") used in pathology reports by groups of attending physicians and their respective trainees using an online survey. There is no statistical difference in the interpretation of each terminology between attending pathologists and pathology trainees. There is also no significant difference between pathology and other attending groups for majority of the terminologies. However, there are significant differences between at least 2 of the 4 attending physician categories in the following pathology terminologies: "consistent with" (overall P=0.01), "compatible with" (P=0.02), "not excluded" (P=0.008), and "cannot exclude" (P=0.01). The pairwise comparisons among the 4 specialties show that there is significant difference in the interpretation of the degree of certainty between pathology and medicine in terms of "not excluded" (P=0.007) and "cannot exclude" (P=0.03). Focused peer review or monitoring of pathology reports with ambiguous terminologies may reduce their use and represent a worthwhile and achievable goal.

Utilization of stat test priority in the clinical laboratory: a College of American Pathologists q-probes study of 52 institutions.

CONTEXT: Utilization of stat testing priority is a balance between safe, efficient patient management and resource expenditure. OBJECTIVE: To determine the rate of stat testing, compare rates among institutions, and determine the distribution of turnaround time expectations for different turnaround time priorities. DESIGN: During a 7-day period, participants prospectively determined the total number of chemistry, hematology, and coagulation billable tests from inpatients and emergency department patients. Among these, the total numbers of billable tests performed stat were identified. Laboratories also reported the levels of test priority they offered and turnaround expectations for each level of test priority. RESULTS: Fifty institutions submitted data for the study, with 2 additional participants submitting partial results. Participants identified 639 589 chemistry, hematology, and coagulation billable tests, with 229 896 (35.9%) performed stat. The stat rate varied from 21.3% at the 10th percentile to 55.4% at the 90th percentile, with a median of 37.0% of participants' tests performed stat. Laboratories include a mean of 206 tests in chemistry, hematology, and coagulation test menus, with 67% of these tests offered stat. The fraction of the test menu offered stat varied from 29.0% at the 10th percentile to 97.8% at the 90th percentile, with a median of 73.3% of tests on the menu offered stat. The most common number of testing priorities offered by participating laboratories was 3 (44.2%). CONCLUSIONS: Among the 52 participating laboratories, the median stat testing rate was 37.0% and a median 73.3% of the test menu was offered stat.

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 method's 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.

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.

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.

OBJECTIVES: 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. STUDY DESIGN: We prospectively evaluated 14 HNSCC specimens, arising from patients undergoing chemoradiotherapy or radiotherapy alone with curative intent. A complete response was assessed by clinical evaluation with no evidence of gross tumor after a 2-year follow-up period. METHODS: Residual biopsy samples from eight complete responders (CR) and six nonresponders (NR) were evaluated by genome-wide gene expression profiling using HG-U133A 2.0 arrays. Univariate parametric t-tests with proportion of false discoveries controlled by multivariate permutation tests were used to identify genes with significantly different gene expression levels between CR and NR cases. Six different prediction algorithms were used to build gene predictor lists. Three representative genes showing 100% crossvalidation support after leave-one-out crossvalidation (LOOCV) were further validated using real-time QRT-PCR. RESULTS: We identified 167 significant probe sets that discriminate between the two classes, which were used to build gene predictor lists. Thus, 142 probe sets showed an accuracy of prediction ranging from 93% to 100% across all six prediction algorithms. The genes represented by these 142 probe sets were further classified into different functional networks that included cellular development, cellular movement, and cancer. CONCLUSIONS: The results presented herein offer encouraging preliminary data that may provide a basis for a more precise prognosis of HNSCC, as well as a molecular-based therapy decision for the management of these cancers.

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.

Assessing the impact of tissue devitalization time on genome-wide gene expression analysis in ovarian tumor samples.

The utilization of genome-wide gene expression microarray technology in tumor stratification has proven a powerful tool to identify gene expression signatures associated with cancer prognosis and is currently under evaluation in clinical laboratories. Standardized protocols, including tumor tissue postoperatively handling guidelines are yet to be defined. We aimed at assessing a systematic effect of devitalization in ovarian tumors' gene expression profiling, using high-density oligonucleotide microarrays, under a standardized protocol following strict quality control criteria. Residual tissue from the surgical pathology specimen was divided into 5 samples. Half of each was immediately snap frozen in liquid nitrogen. The remaining halves were kept at room temperature for 0, 15, 30, 60, and 120 minutes, at which time the tissue was snap frozen in liquid nitrogen, and stored at -80 degrees C until RNA extraction. The entire process from RNA extraction through feature intensity distribution was rigorously monitored for quality. Identification of altered gene expression among each pair of snap frozen and devitalized samples per ovarian tumor specimen was assessed by using the Significance score (S-score) method. We identified only 4 probe sets that seemed to correlate with devitalization time in one of the ovarian tumor specimens, suggesting that they are not likely to have an impact on gene expression profiling tumor stratification. Our study suggests that with proper sample handling and rigorous quality control procedures for RNA extraction and microarray analysis, tumor classification based on global gene expression data will not be adversely affected if devitalization times are kept within a 120-minute window.

A simple and rapid genotyping assay for simultaneous detection of two ADRB2 allelic variants using fluorescence resonance energy transfer probes and melting curve analysis.

Allelic variants at codons 16 and 27 of the beta(2)-adrenergic receptor gene (ADRB2) have shown clinical and pharmacological implications in asthma, hypertension, ischemic heart failure, diabetes, obesity, and cystic fibrosis. We have developed a simultaneous genotyping assay for the c.46A>G and c.79C>G allelic variants using hybridization probes and melting curve analysis. The assay was optimized on a panel of 30 DNA samples of known ADRB2 genotype as determined by sequencing with 100% concordance between the two techniques. Melting temperature (Tm) ranges for the different genotypes were obtained using data from three independent experiments. Single peaks for p.Arg16Arg (Tm = 57.76 degrees C +/- 0.10 degrees C) and p.Gly16Gly (Tm = 66.73 degrees C +/- 0.18 degrees C) and two melting peaks for p.Arg16Gly were obtained. Similarly, single peaks for p.Gln27Gln (Tm = 53.98 degrees C +/- 0.19 degrees C) and p.Glu27Glu (Tm = 64.93 degrees C +/- 0.16 degrees C) and two peaks for p.Gln27Glu were detected. Independent operators easily assigned genotypes in a sample set of 385 asthmatic patients. Haplotype and allele frequencies were in concordance with previously published data: Arg allele frequencies in children/adults were 0.34/0.30 in Caucasians and 0.45/0.52 in African Americans, and Gln allele frequencies were 0.58/0.52 in Caucasians and 0.82/0.84 in African Americans. Thus, the ADRB2 genotyping assay represents a highly reliable and rapid technique for routine clinical use in the simultaneous detection of ADRB2 variants.

Comparison of performance characteristics of three real-time reverse transcription-PCR test systems for detection and quantification of hepatitis C virus.

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 log(10) IU/ml with a linear dynamic range of 1.0 to 7.0 log(10) IU/ml. The Roche ASR in combination with the High Pure system (Roche ASR-HP) showed a sensitivity of 1.4 log(10) IU/ml with a linear dynamic range of 2.0 to 7.0 log(10) 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 (R(2) = 0.84 and R(2) = 0.93, respectively), with better agreement for the Abbott ASR-Q. However, correlation (R(2) = 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 log(10) 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.

Adequacy of pathology resident training for employment: a survey report from the Future of Pathology Task Group.

CONTEXT: The recent change in accreditation requirements for anatomic pathology and clinical pathology residency training from 5 to 4 years and the rapid advances in technologies for pathology services have sparked a renewed debate over the adequacy of pathology residency training. In particular, perceived deficiencies in training have been declared from a variety of sources, both in the form of recent editorial opinions and from surveys of community hospital pathologist employers in 1998, 2003, and 2005 by Dr Richard Horowitz. OBJECTIVE: To obtain more comprehensive data on the perceptions of strengths and weaknesses in pathology residency training. DESIGN: The College of American Pathologists conducted a survey of potential pathology employers (senior College of American Pathologists members, members designated as head of group, and members of the Association of Directors of Anatomic and Surgical Pathology). Also surveyed were recent graduates of pathology residency programs, who were identified as being junior members of the College of American Pathologists, were recent recipients of certification from the American Board of Pathology, or were contacted through their directors of pathology residency programs. RESULTS: There were 559 employer respondents, of whom 384 were responsible for hiring and/or supervising new pathologists. There were 247 recent graduates of pathology residency training programs who responded. From the employers' standpoint, the majority expressed overall satisfaction with recent graduates, but almost one third of employers indicated that new hires had a major deficiency in a critical area. Specific areas of deficiency were clinical laboratory management and judgment in ordering special stains and studies. In addition, one half of employers agreed that more guidance and support for newly trained pathologists is needed now than was required 10 years ago. Academic employers generally were more satisfied than private sector employers. Newly trained pathologists did not appear to be inappropriately overconfident in their abilities. In addition, their perceptions of those specific areas in which they are most and least prepared are very similar to the ratings provided by employers. On average, newly trained pathologists' ratings of their own preparedness are highest for specific aspects of general pathology and anatomic pathology, and lowest for specific aspects of clinical pathology and administration. In selecting new pathologists, employers perceived medical knowledge and interpersonal skills as the most important discriminating applicant characteristics. When new employees were asked why they thought they were offered their position, the discriminating qualifications cited most often were academic background and training, as well as completion of a fellowship and subspecialty training. CONCLUSIONS: It is our hope that the results of this survey can be used as input for further discussions and recommendations for training of pathology residents so as to further advance the ability of pathologists to provide quality patient care upon their graduation from training.

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.

Raman spectroscopic analysis of the enigmatic Comper pigments.

An early church decoration project carried out by Sir Ninian Comper in 1896-98, involving the rood screen and canopy in St. Mary's, Egmanton, is currently undergoing restoration. Despite the rather prolific works of this famous ecclesiastical architect, there is little information available about the actual pigments that he used in his projects that gave rise to the special nomenclature "Comper green" and "Comper red". Specimens of green, red, black, grey, white and blue paint from this work have been made available for Raman spectroscopic analysis, and their identification has been achieved for the first time. The characteristic red and green pigments used in Comper's work, Comper green and Comper red, are both seen to be mixtures; in the former, Raman bands from chrome yellow (lead(II) chromate) and Prussian blue are identified, and the latter is confirmed as being a mixture of vermilion (mercury(II) sulfide) and barytes (barium sulfate). The other colours are found to represent a rich diversity of palette and include haematite, lead tin yellow (type II), lamp black, gypsum, anhydrite, hydrocerussite and calcite. The information from this first Raman spectroscopic study of Comper's palette will assist the conservation and restoration of an important nineteenth century church decoration.

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.

Evaluation of quality-control criteria for microarray gene expression analysis.

BACKGROUND: Development of quality-control criteria to ensure reproducibility of microarray results for potential clinical application is still in its infancy. METHODS: In the present studies we developed quality-control criteria and evaluated their effect in microarray data analysis using total RNA from cell lines, frozen tumors, and a commercially available reference RNA. Quality-control criteria such as A(260)/A(280) ratios, percentage of rRNA, and median size of cDNA and cRNA synthesis products were evaluated for robustness in microarray analysis. Furthermore, precision studies using a reference material were performed on the Affymetrix HG-U133A high-density oligonucleotide microarrays. The same reference RNA sample was examined in 16 different chips run on 2 different days in the four different modules of the Affymetrix fluidics workstation. Fresh and frozen fragmented cRNAs were also compared. An ANOVA model was fit to identify the main sources of variation. RESULTS: Good-quality samples showed >30% rRNA in the electropherograms and cDNA and cRNA synthesis products with median sizes of 2.0 and 3.0 kb, respectively. Precision studies showed that the main source of variation was the day-to-day variability, minimally affecting hybridization exogenous control genes. Altogether, the results showed that the Affymetrix Genechip system is highly reproducible when RNA that meet the quality-control criteria are used (overall P >0.01). CONCLUSIONS: These results confirm the need to establish defined quality-control criteria for sample quality to distinguish between analytical and biological variability.

Evaluation of a linear amplification method for small samples used on high-density oligonucleotide microarray analysis.

High-density oligonucleotide microarray analysis has proven to be an excellent approach for gene expression profiling in human cancers. This technique assesses the expression of thousands of genes simultaneously, from at least 5 microg of total RNA per sample per experiment. This total RNA requirement poses a challenge when studying small, unique clinical samples, like biopsies. Recently, a new standardized protocol for small samples was released by Affymetrix, which includes a linear amplification step. To evaluate the impact of such amplification in the gene expression profiling of human ovarian cancer, we compared results obtained from 5 microg and 100 ng of total RNA from the same tumor sample, using the standard Affymetrix protocol and the new linear RNA amplification protocol, respectively. We identified a small bias in gene expression data caused by linear amplification, potentially due to shorter elongation products leading to misclassification of probe sets directed to the middle-5' region of the transcripts. Interestingly, the magnitude of the bias varied when different normalization and expression summary algorithms were used. However, this bias does not affect tumor gene expression profiling. Consequently, linear amplification may be of utility in cases of extremely low RNA recovery from critical and unique samples, such as small biopsies.