Performance of Rapid On-Site Evaluation in Breast Fine-Needle Aspiration Biopsies: Identifying Areas of Diagnostic Challenge.

INTRODUCTION: Fine-needle aspiration (FNA) is a well-established method for sampling breast lesions with high accuracy and positive predictive value. Despite its decline in recent years relative to the use of core needle biopsies, there are several advantages to FNA which include cost-effectiveness, low complication rate, and the ability to perform rapid on-site evaluation (ROSE). The aim of this study was to evaluate breast FNAs with ROSE to identify diagnostic challenges during ROSE. MATERIALS AND METHODS: We identified all breast FNAs with ROSE performed at Massachusetts General Hospital from January 2014 to December 2019. From the electronic medical record, clinical, radiological, and follow-up pathology results were recorded. Comparison between the rapid and final cytological diagnosis was made. All discrepancies were documented with major discrepancy defined as a malignant rapid interpretation not confirmed on final diagnosis or a negative rapid interpretation upgraded to suspicious or positive on final diagnosis. RESULTS: The study cohort consisted of 483 breast FNAs with ROSE. The rapid and final cytological interpretations showed good correlation, with only 6 (1.2%) major discrepancies. Problematic areas included low-grade, lobular, and fibroepithelial lesions with low cellularity being a contributory factor to misclassification. CONCLUSIONS: FNA remains a highly accurate method for the evaluation of breast lesions with ROSE.

Smears are important for adequate cytologic diagnosis of kidney lesions.

INTRODUCTION: Traditionally at our institution, smears with or without liquid-based cytology (LBC) and core biopsies (CBs) have been obtained by radiologists performing image-guided fine-needle aspiration biopsies (FNABs) of deep organs. Since 2015, however, there has been a shift to providing cytology with samples for LBC only when obtaining CBs. The impression among our institution's cytologists is that LBC alone is less often adequate for diagnosis compared with smears and LBC together. We examined a series of kidney FNABs pre- and post-"LBC only" to evaluate this impression. MATERIALS AND METHODS: With institutional review board approval, we compared all kidney FNABs from 2012 to those from 2015. We recorded the type(s) of cytology preparation(s), the number of cytology slides, the cytology diagnosis, the concurrent CB diagnosis, and whether there was a subsequent excision and the excision diagnosis. We examined cytology and CB slides as needed. RESULTS: In 2012, 105 patients underwent 111 kidney biopsies, 109 with smears made. In 2015, 58 patients underwent 62 kidney biopsies, 7 with smears made. In 2012, there were 13 (12%) nondiagnostic (ND) cytology cases and 19 (17%) cases where the cytology and CB diagnoses were discrepant. By comparison, in 2015, there were 20 (32%) ND cytology cases and 21 (33%) discrepant cases. CONCLUSIONS: There were more cytology slides per case and fewer ND diagnoses in 2012 compared with 2015 (12% versus 32%, respectively, P = 0.001). Concordance was also better in 2012 (83% versus 67%, P = 0.015). We believe that our metrics would improve if we returned to the procedures followed in 2012.

Automated Extraction of Formalin-Fixed, Paraffin-Embedded Tissue for High-Risk Human Papillomavirus Testing of Head and Neck Squamous Cell Carcinomas Using the Roche Cobas 4800 System.

CONTEXT: -Testing for high-risk human papillomavirus (HR-HPV) in head and neck squamous cell carcinomas (HNSCCs) is important for both prognostication and clinical management. Several testing platforms are available for HR-HPV; however, effective alternative automated approaches are needed. OBJECTIVE: -To assess the performance of the automated Roche cobas 4800 HPV real-time polymerase chain reaction-based system on formalin-fixed, paraffin-embedded HNSCC specimens and compare results with standard methods of in situ hybridization (ISH) and p16 immunohistochemistry. DESIGN: -Formalin-fixed, paraffin-embedded samples of HNSCC were collected from archival specimens in the Department of Pathology, Massachusetts General Hospital (Boston), and prepared using the automated system by deparaffinization and dehydration followed by tissue lysis. Samples were integrated into routine cervical cytology testing runs by cobas. Corresponding formalin-fixed, paraffin-embedded samples were evaluated for HR-HPV by ISH and p16 by immunohistochemistry. Discrepant cases were adjudicated by polymerase chain reaction. RESULTS: -Sixty-two HNSCC samples were analyzed using the automated cobas system, ISH, and immunohistochemistry. Fifty-two percent (n = 32 of 62) of formalin-fixed, paraffin-embedded tumors were positive for HR-HPV by cobas. Eighty-eight percent (n = 28 of 32) of cases were the HPV 16 subtype and 12% (n = 4 of 32) were other HR-HPV subtypes. Corresponding testing with ISH was concordant in 92% (n = 57 of 62) of cases. Compared with the adjudication polymerase chain reaction standard, there were 3 false-positive cases by cobas. CONCLUSIONS: -Concordance in HNSCC HR-HPV status between cobas and ISH was more than 90%. The cobas demonstrated a sensitivity of 100% and a specificity of 91% for detection of HR-HPV. Advantages favoring cobas include its automation, cost efficiency, objective results, and ease of performance.

A superior method for cell block preparation for fine-needle aspiration biopsies.

BACKGROUND: Cell block (CB) techniques for fine-needle aspiration biopsies (FNABs) vary. A direct comparison of CB techniques with statistical validation was performed to identify the best method. METHODS: Three CB techniques were compared: 1) FNAB rinsed in saline and clotted with plasma and thrombin (SPT); 2) FNAB rinsed in formalin and clotted with HistoGel (HG); and 3) FNAB rinsed in formalin, centrifuged, and the pellet captured in a collodion bag (ColB). FNAB was performed on 35 random surgical specimens for smears and each CB technique. A randomized blinded review of hematoxylin and eosin-stained CB slides was performed and each case was scored on a scale of 1 to 3 for cellularity, preservation, and architecture and the overall best CB was identified. Significance was determined by the Mann-Whitney U test for nonparametric ordinal data. RESULTS: The mean cellularity score was 1.71 for SPT (standard deviation [SD], 0.89), 1.68 for HG (SD, 0.67), and 3.0 for ColB (SD, 0). The mean preservation score was 1.31 for SPT (SD, 0.58), 1.54 for HG (SD, 0.70), and 2.91 for ColB (SD, 0.37). The mean architecture score was 1.45 for SPT (SD, 0.70), 1.43 for HG (SD, 0.60), and 2.71 for ColB (SD, 0.57). There was no statistical significance noted between SPT or HG when compared for each category. ColB was found to be superior to both SPT and HG when compared for each category (P<.05). The overall best CB was obtained with ColB in 33 of 35 cases (94%), with SPT proving superior in 1 of 35 cases (3%) and HG superior in 1 of 35 cases (3%). CONCLUSIONS: ColB appears to be a superior technique for CB, yielding greater cellularity, preservation, and architecture in the majority of cases. Cancer Cytopathol 2016;124:508-18. © 2016 American Cancer Society.

Next-Generation Sequencing and Fluorescence in Situ Hybridization Have Comparable Performance Characteristics in the Analysis of Pancreaticobiliary Brushings for Malignancy.

Cytological evaluation of pancreatic or biliary duct brushings is a specific, but insensitive, test for malignancy. We compared adjunctive molecular testing with next-generation sequencing (NGS) relative to fluorescence in situ hybridization (FISH) for detection of high-risk neoplasia or malignancy. Bile duct brushings from 81 specimens were subjected to cytological analysis, FISH using the UroVysion probe set, and targeted NGS. Specimens were placed into negative/atypical (negative) or suspicious/positive (positive) categories depending on cytology and negative or positive categories on the basis of FISH and NGS results. Performance characteristics for each diagnostic modality were calculated on the basis of clinicopathologic follow-up and compared in a receiver operating characteristic analysis. There were 33 high-risk neoplasia/malignant strictures (41%) and 48 benign (59%). NGS revealed driver mutations in 24 cases (30%), including KRAS (21 of 24 cases), TP53 (14 of 24 cases), SMAD4 (6 of 24 cases), and CDKN2A (4 of 24 cases). Cytology had a sensitivity of 67% (95% CI, 48%-82%) and a specificity of 98% (95% CI, 89%-100%). When added to cytology, NGS increased the sensitivity to 85% (95% CI, 68%-95%), leading to a significant increase in the area under the curve in a receiver operating characteristic analysis (P = 0.03). FISH increased the sensitivity to 76% (95% CI, 58%-89%), without significantly increasing the area under the curve. These results suggest that ancillary NGS testing offers advantages over FISH, although studies with larger cohorts are needed to verify these findings.

Young investigator challenge: Comparison of 2 different methods of manual slide screening in semiautomated gynecologic cytology.

BACKGROUND: Negative gynecologic cytology cases (ie, those diagnosed as negative for intraepithelial lesion or malignancy) are manually reviewed by 2 methods using semiautomated screening: 1) immediate full slide review (FSR) after fields-of-view analysis (FOV) (FOV + FSR), and 2) quality-assurance/high-risk, quintile-directed full manual review (FMR). Data supporting current guidelines were limited. The authors investigated FMR, FOV + FSR, and the review process in general. METHODS: Gynecologic cytology cases from 2009 to 2014 at Massachusetts General Hospital were analyzed. The data comprised 93,169 patients, 194,656 specimens, and 49,979 human papillomavirus (HPV) tests. RESULTS: In patients who underwent FMR, the epithelial cell abnormality (ECA) rate was correlated with the HPV-positive rate (correlation coefficient [r(2) ] = 0.82; Y = 0.19X + 0.02), and both rates decreased with age. For patients who underwent FOV + FSR, the ECA rate was also related to the HPV-positive rate (r(2) = 0.86; Y = 0.39X + 0.11), and both rates decreased with age. The FMR group had similar HPV-positive rates compared to the FOV + FSR group (2%-52% vs 9%-68%, respectively). HPV-positive patients had a higher risk of ECA than HPV-negative patients (40% vs 8%; P < .0001). Currently, manual review allocates resources inefficiently, to older, HPV-negative patients as compared to younger, HPV-positive patients. Additionally, too many patients who have HPV-positive specimens with ECA proceed from primary screening to FMR. This imbalance can be observed in the slope of the line comparing the FMR ECA rate with the HPV-positive rate (which might serve as a surrogate marker for adequacy of FOV + FSR screening). Therefore, laboratories should try to reduce the number of HPV-positive cases that reach FMR. CONCLUSIONS: Current review procedures devote too few resources to younger patients. FMR should be assigned according to HPV status and age. The slope of the line comparing the FMR ECA rate with the FMR HPV-positive rate might be a useful surrogate marker of screening accuracy.

Performance of the Roche cobas 4800 high-risk human papillomavirus test in cytologic preparations of squamous cell carcinoma of the head and neck.

BACKGROUND: Determining high-risk human papillomavirus (HR-HPV) status of head and neck squamous cell carcinoma (HNSCC) defines a tumor subset with important clinical implications. Cytologic sampling often provides the sentinel or sole diagnostic specimen. The authors assessed the performance characteristics for the Roche cobas 4800 HPV real-time polymerase chain reaction (PCR)-based system (cobas) on cytologic specimens of HNSCC compared with standard methods of in situ hybridization (ISH) for HR-HPV and immunohistochemistry (IHC) for p16 on formalin-fixed, paraffin-embedded (FFPE) tissue. METHODS: Samples of HNSCC were collected by fine-needle aspiration and from surgical biopsies or resections, fixed, and processed with the cobas system. Available corresponding FFPE samples were synchronously evaluated for HR-HPV using ISH and IHC. Discrepant cases underwent additional PCR studies for adjudication. RESULTS: Thirty-six samples from 33 patients were analyzed. Forty-two percent (n = 15) of tumors were positive for HR-HPV according to cobas. Corresponding histology with ISH (n = 30) was concordant in 91% of samples. Compared with the adjudication PCR standard, there were 3 false-positive cases according to cobas. Ninety-two percent (n = 12) of cases were the HPV16 subtype. The overall sensitivity for the cobas system was 100%, and the specificity was 86%. CONCLUSIONS: Concordance in HNSCC HR-HPV status between cobas and ISH/IHC was > 90%, and cobas demonstrated a sensitivity of 100% and a specificity of 86%, broadening options for HR-HPV testing of fine-needle aspiration samples. Advantages for this system include subtyping of HR-HPV and the ability to discern HR-HPV status earlier in a patient's treatment course.

Effects on cervical cytology screening productivity associated with implementation of the BD FocalPoint™ Guided Screener Imaging System.

OBJECTIVES: Automated screening will become important due to an aging workforce, declining numbers of new cytotechnologists, and the need for increased screening sensitivity in the vaccine era, where high-grade abnormalities will decline. This study documents workload in gynecologic cytology throughput before and after the implementation of the BD FocalPoint™ Guided Screener (GS) System. STUDY DESIGN: We collected daily screening data from 3 time periods: the 12 months prior to GS implementation, the 6 months immediately after implementation, and the ensuing 7-18 months after implementation. Data was tabulated at the individual and total laboratory levels. RESULTS: In the 6-month period immediately following implementation, productivity increased in 3 of 5 cytotechnologists, as compared to the figures 12 months before implementation. The laboratory increased productivity slightly (+2.4%), with individual changes ranging from -6.9 to +14.7%. In the 7- to 18-month 'mature' period after implementation, productivity increased in all 5 cytotechnologists with an average of +15.4%. Individual increases ranged from +6.1 to +26.9%. CONCLUSIONS: Overall productivity increased in the period beyond 6 months, and this increase was eventually noted in all personnel. Increased productivity was associated with a short period of learning in which the magnitude of the effect was less than in the mature period.

Comparison of BD Surepath and ThinPrep Pap systems in the processing of mucus-rich specimens.

BACKGROUND: Excessive mucus, as well as blood and inflammation, can be problematic in the processing and screening of liquid-based cervical Pap preparations by interfering in the process of cell retrieval onto specimen filters or slides. This study compares the capacity of the BD SurePath and ThinPrep liquid-based Papanicolaou (Pap) tests to handle mucus-laden specimens. METHODS: A 100 µL volume of pooled cervical epithelial cells was added to BD SurePath and ThinPrep liquid Pap test vials. Aliquots of cervical mucus that had been collected and pooled from previously processed mucus-rich Pap specimens were added to each series of test vials in amounts of 250 µL, 500 µL, 1000 µL, and 2000 µL. The vials were then routinely processed on their respective instruments and the test slides evaluated for cellularity of the squamous cell component. RESULTS: The BD SurePath test specimens showed no reduction of the squamous cell component resulting from the addition of any of the aliquots of mucus. The ThinPrep test specimens showed a marked loss of cellularity upon the addition of the first aliquot of mucus, with large areas of the filters showing nearly complete absence of squamous cells. CONCLUSIONS: Excessive mucus remains a potentially limiting factor in the ThinPrep Pap system. This appears to be the result of direct obstruction of the filtration membrane causing a markedly diminished retrieval of squamous epithelial cells and potentially impacting disease detection. The BD SurePath system, conversely, manages mucus by a cell enrichment process, which results in undiminished cell recovery as observed in this study's test conditions.

Simultaneous quantification of amphetamine and methamphetamine in meconium using ISOLUTE HM-N-supported liquid extraction columns and GC-MS.

A procedure is described for the rapid extraction and quantification of amphetamine and methamphetamine from meconium using ISOLUTE HM-N-supported liquid extraction columns and gas chromatography-mass spectrometry (GC-MS). Because of the matrix complexity of meconium samples, extraction and sample preparation prior to instrumental analysis can prove difficult and time-consuming. The present study introduces a novel sample preparation technique for the simultaneous quantification of amphetamine and methamphetamine in meconium using GC-MS. Extraction of both analytes was achieved using ISOLUTE HM-N-supported liquid extraction columns containing a modified form of diatomaceous earth. Limits of detection for both analytes were 30 ng/g and the lower limit of quantitation was 75 ng/g. Linearity was achieved over the range 75-3000 ng/g. The methodology showed excellent intrarun precision with %CV values ranging from 2 to 8% for both analytes. Interrun precision experiments produced %CV values between 7 and 10% for both analytes. The reported methodology proved suitable for the accurate quantification of amphetamine and methamphetamine in meconium samples and greatly reduced the sample preparation time normally required for traditional solid-phase extraction. The development and validation of such analytical methodologies will prove beneficial for the identification of prenatal substance abuse, which is an ongoing concern across socioeconomic lines.

Comparison of the effectiveness of two liquid-based Papanicolaou systems in the handling of adverse limiting factors, such as excessive blood.

BACKGROUND: Excessive blood may compromise gynecologic Papanicolaou (Pap) smears. Liquid-based cytologic techniques have been developed in part to address this problem. In the current study, conditions of excessive blood were simulated to compare the ability of two liquid-based systems, ThinPrep and SurePath, to satisfactorily process specimens in the presence of this potentially limiting factor. METHODS: Equal volumes of washed epithelial cells derived from pooled residues of liquid Pap vials were added to a series of ThinPrep and SurePath vials. Increasing volumes of freshly drawn, packed erythrocytes were added to the vials in progressive amounts from 50 microL or 100 microL up to 3000 microL. The vials were processed on their respective instruments according to U.S. Food and Drug Administration-approved procedures for a total of six test runs. The cellularity of the slides was measured by averaging epithelial cell counts in a total of five 40x fields. RESULTS: SurePath preparations were uncompromised by blood until aliquots from 1000 microL to 3000 microL were reached. The ThinPrep system invariably was overwhelmed by the first 50-microL or 100-microL aliquot of blood, with epithelial cell counts dropping immediately to near zero. CONCLUSIONS: The cell enrichment process of the SurePath system capably handled significantly greater amounts of potentially obscuring blood than the membrane filtration method of the ThinPrep system, which was compromised by as little as

Total versus inhalable sampler comparison study for the determination of asphalt fume exposures within the road paving industry.

Exposure to asphalt fumes has a threshold limit value (TLV of 0.5 mg m(-3) (benzene extractable inhalable particulate) as recommended by the American Conference of Governmental Industrial Hygienists (ACGIH). This reflects a recent change (2000) whereby two variables are different from the previous recommendation. First is a 10-fold reduction in quantity from 5 mg m(-3) to 0.5 mg m(-3). Secondly, the new TLV specifies the "inhalable" fraction as compared to what is presumed to be total particulate. To assess the impact of these changes, this study compares the differences between measurements of paving asphalt fume exposure in the field using an "inhalable" instrument versus the historically used 'total' sampler. Particle size is also examined to assist in the understanding of the aerodynamic collection differences as related to asphalt fumes and confounders. Results show that when exposures are limited to asphalt fumes, a 1:1 relationship exists between samplers, showing no statistically significant differences in benzene soluble matter (BSM). This means that for the asphalt fume ACGIH TLV, the 'total' 37-mm sampler is an equivalent method to the "inhalable" method, referred to as IOM (Institute of Occupational Medicine), and should be acceptable for use against the TLV. However, the study found that when confounders (dust or old asphalt millings) are present in the workplace, there can be significant differences between the two samplers' reported exposure. The ratio of IOM/Total was 1.37 for milling asphalt sites, 1.41 for asphalt paving over granular base, and 1.02 for asphalt over asphalt pavements.