Quality metrics in non-gynecologic cytology: results from the 2022 American Society of Cytopathology survey.

INTRODUCTION: Rapid advancements in minimally invasive techniques and the discovery of molecular biomarkers have resulted in major changes in the practice of non-gynecologic cytology and have highlighted a need for novel quality assurance (QA) metrics. MATERIALS AND METHODS: To obtain data regarding the current and desired usage, methods of collection, and barriers to the implementation of non-gynecologic cytopathology QA, an 18-question survey was constructed by the Clinical Practice Committee of the American Society for Cytopathology. RESULTS: A total of 206 responses were received. Respondents included 112 (54.4%) cytopathologists, 81 (39.3%) cytotechnologists, and 13 others. Almost all (97%) acknowledged the value of assessing QA metrics in cytology. The most commonly used QA metrics were cytotechnologist-pathologist diagnostic agreement and pathologist amendment rates. The desire to implement non-gynecologic QA metrics was significantly higher among academic hospitals, relative to nonacademic facilities. A combined manual and electronic approach to collect QA data was generally used (70% of institutions). QA metrics were more often collected by the cytology laboratory supervisors (59.5%), while the evaluation was most often performed by the cytology laboratory director (76.5%). Limited staffing and laboratory information system (LIS) capabilities were cited as major challenges in the implementation of novel QA metrics. CONCLUSIONS: While the collection of quality data might be perceived as an onerous task, a thoughtful selection of quality indicators, with an inbuilt search option in LIS, can contribute to the successful implementation of non-gynecologic QA metrics.

Cell blocks in cytology: review of preparation methods, advantages, and limitations.

Cell blocks are cytologic preparations that are processed as paraffin embedded blocks in a manner comparable to formalin-fixed paraffin-embedded tissue in surgical pathology. In addition to serving as an adjunct to other cytologic preparations for morphologic diagnosis, cell blocks play an increasingly important role as they yield tissue sections that can be utilized for ancillary testing such as immunohistochemical stains and molecular studies. While essentially universally viewed as playing a pivotal role in cytopathology practice, there are various factors that limit their use in practice and contribute to dissatisfaction with cell block quality. Cell block preparation, as opposed to tissue processing in surgical pathology, is more variable with many different protocols in use today. This review explores the most commonly used cell block preparation techniques currently in use with review of the unique advantages and limitations each method presents. The goal of this work is to serve as a resource that can aid in making more informed decisions about which cell block protocol may work best for individual laboratories.

Results from the 2019 American Society of Cytopathology survey on rapid onsite evaluation (ROSE)-part 2: subjective views among the cytopathology community.

INTRODUCTION: This study aims to improve understanding of the cytopathology community's perspective regarding the value of rapid onsite evaluation (ROSE) in clinical practice. MATERIALS AND METHODS: The American Society of Cytopathology membership was surveyed in 2019 to obtain subjective data on the cytopathology community's perceptions regarding ROSE. Comments were categorized by major themes and attitudes and analyzed by respondent's role in laboratory, practice size, and practice setting (Fisher's exact and χ2 tests). RESULTS: A total of 541 responses were received from 255 cytopathologists/pathologists, 261 cytotechnologists, 19 trainees, and 6 others (as previously reported). Reasons for which cytopathology personnel provide this service aligned with their perceptions of why clinicians request ROSE. A minority of respondents, disproportionally from high volume centers, felt ROSE is unnecessary. Overall attitude regarding ROSE was generally positive. There were no significant differences in attitude regarding ROSE according to role in laboratory or practice size, but respondents from academic centers provided a significantly higher percentage of positive comments than those in private or community practice. Although survey respondents generally felt that ROSE is valuable to patient care, they also highlighted several challenges, including staffing, time commitment, and inadequate reimbursement. Implementation of telecytology was felt to potentially alleviate some of these challenges. CONCLUSIONS: Survey results show that the cytology community views ROSE favorably, practices vary considerably, and there is a perceived need for improved reimbursement. Data from this study may be used to identify areas that warrant additional research to clarify the clinical value of ROSE.

Results from the 2019 American Society of Cytopathology survey on rapid on-site evaluation-Part 1: objective practice patterns.

INTRODUCTION: Rapid on-site evaluation (ROSE) is a service provided by cytologists that helps ensure specimen adequacy and appropriate triage for ancillary testing. However, data on the current usage patterns across different practice settings have been lacking. MATERIALS AND METHODS: To obtain an accurate and timely assessment of the current state of practice of ROSE, a 14-question online survey was constructed by the Clinical Practice Committee of the American Society for Cytopathology. The survey was available to the membership of the American Society for Cytopathology for a 3-week period in early 2019. RESULTS: A total of 541 responses were received, including from 255 cytopathologists/pathologists, 261 cytotechnologists, 19 cytology resident/fellow trainees, and 6 others. ROSE was offered as a clinical service by 95.4% of the respondents, with telecytology for ROSE used in 21.9% of the practices. Endobronchial ultrasound-guided transbronchial needle aspiration was the procedure most frequently reported to use ROSE (mean, 59.1%; median, 70%). Cytotechnologists were involved in ROSE in most practices. The number of daily ROSE procedures correlated with the annual nongynecologic cytology volumes. Approximately 70% of ROSE procedures were reported to require >30 minutes, on average, for the cytologist. CONCLUSIONS: The results from our survey of cytologists have shown that the reported practice patterns for the usage of ROSE vary considerably. The presented data can help inform future guideline recommendations and the implementation of ROSE in different clinical settings.

Risk of malignancy and neoplasia predicted by three molecular testing platforms in indeterminate thyroid nodules on fine-needle aspiration.

BACKGROUND: The management of thyroid nodules with indeterminate cytology is challenging. Recently, molecular testing on fine-needle aspirates (FNAs) has been advocated to determine whether clinical follow-up or surgery is warranted for patients. Three different testing platforms were performed on aspirates from our institution (Afirma Thyroid FNA Analysis, RosettaGX Reveal, and Interpace ThyGenX/ThyraMIR). This study compares their diagnostic efficacy. METHODS: We conducted a retrospective analysis of indeterminate thyroid FNAs with correlating molecular testing over 4 years (2015-2018). The aspirates included diagnoses of follicular lesion of undetermined significance, follicular neoplasm, or suspicious for malignancy (SM). Based on cases that underwent surgical resection (Afirma, n = 37; Rosetta, n = 19; Interpace, n = 14), we calculated sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) for risk of malignancy and neoplasia. RESULTS: The three tests performed similarly when predicting risk of malignancy. They showed high sensitivity (80-100%) and NPV (90-100%) but lower specificity (10-64%) and PPV (21-44%). When assessing their value to predict neoplasia, each test had a high PPV (76-89%) but low NPV (20-33%). The sensitivity for neoplasm was intermediate to high (50-93%), and the specificity remained extremely variable (11-67%). CONCLUSION: Overall, these molecular platforms performed similarly, displaying high NPV but low to intermediate PPV for malignancy and low NPV but high PPV for neoplasm. The risk of neoplasm is a good index for surgery, and we argue that many of the neoplasms are low-risk tumors. We endorse conservative treatment with lobectomy for cases that are indeterminate at FNA but suspicious by molecular testing.

Molecular Testing for EGFR Mutations and ALK Rearrangements in the Cytological Specimens From the Patients With Non-Small Cell Lung Cancer.

OBJECTIVE: The aim of this study was to investigate epidermal growth factor receptor (EGFR) gene mutations and anaplastic lymphoma kinase (ALK) gene rearrangements using cytological specimens from the patients with a diagnosis of primary or metastatic lung non-small cell carcinoma. MATERIALS AND METHODS: A total 307 cases were submitted for EGFR mutational analysis and 265 cases for ALK analysis. The cytological specimen sources included lung, lymph node, liver, bone, adrenal gland, mesentery mass, and body fluids/bronchial brushing. EGFR mutations in the exons 18 to 21 were analyzed with Qiagen EGFR Pyro Kits. Fluorescence in situ hybridization (FISH) studies for ALK rearrangement inv(2)(p21; p23) were performed on the paraffin-embedded cell block sections utilizing dual-color Vysis LSI ALK Break Apart Probe Kit. RESULTS: Among 307 fine needle aspirate cases for EGFR analysis, 302 cases (269 from cell blocks, 33 from direct smears) had sufficient material for EGFR test. Five cases failed due to inadequate cellularity. Twenty six of 302 (8.6%) cases were positive for EGFR mutations. A total of 265 cases submitted for ALK analysis included 240 cases of fine needle aspirate, 25 cases of pleural fluid/pericardial fluid/bronchial washings. Eight cases failed because of low cellularity, whereas 257 of 265 cases had sufficient material for ALK FISH study. Nine of 257 cases (3.5%) revealed ALK rearrangement by FISH. CONCLUSIONS: The current study demonstrates that cytological specimens can yield sufficient material for EGFR mutations and ALK rearrangement test. Our study reveals that 8.6% of EGFR mutation rate and 3.5% of ALK rearrangement rate in the cytology specimens from the patients with primary or metastatic lung non-small cell carcinoma.

A retrospective analysis of the performance of the RosettaGX® Reveal™ thyroid miRNA and the Afirma Gene Expression Classifiers in a cohort of cytologically indeterminate thyroid nodules.

BACKGROUND: Molecular tests are increasingly used to triage cytologically indeterminate thyroid nodules for surgery and/or follow-up. We retrospectively compared the performance of the Afirma Gene Expression Classifier (AGEC) with that of the more recently developed RosettaGX® Reveal™ miRNA Classifier (Reveal) in a cohort of Bethesda III-V thyroid FNAs with surgical follow-up. DESIGN: Eighty-one samples (54 Bethesda III, 26 Bethesda IV, 1 Bethesda V) with available AGEC (74 AGEC-SUSP and 7 AGEC-BENIGN) and surgical pathology results were studied from three academic centers. Reveal was performed in a blinded fashion. RESULTS: The final diagnoses were benign/NIFTP (n = 63) and malignant (n = 18). The overall "correct" rate was 64.2% for Reveal and 28.4% for AGEC (P = 1.4e-6). The specificity of Reveal was 60.3%, compared with 9.5% for AGEC (P = 2.1e-9). Among the 18 malignant cases, 77.8% and 94.4% were correctly classified as suspicious by Reveal and AGEC, respectively (P = 0.2). In the FLUS and the FN group, the specificity of AGEC was lower than the specificity of Reveal. Whether the 7 NIFTP in our study were considered benign or malignant, specificity and PPV of Reveal were higher than those of AGEC. Reveal also outperformed AGEC in correctly classifying the 26 benign Hürthle lesions studied (P = 7.6e-5). CONCLUSION: Reveal outperformed AGEC in this cohort, whether NIFTP is considered benign or malignant, and in Hürthle lesions. Reveal has the potential to reduce the number of unnecessary resections in patients with indeterminate thyroid cytology. Based on our findings and the practical advantages offered by Reveal methodology, large prospective studies are warranted. Diagn. Cytopathol.

Utilization of direct smears of thyroid fine-needle aspirates for ancillary molecular testing: A comparison of two proprietary testing platforms.

BACKGROUND: Ancillary molecular testing has been recommended for thyroid fine-needle aspirates (FNA) with indeterminate cytologic diagnoses. Rosetta Genomics and Interpace Diagnostics have developed assays that can utilize direct smears as the testing substrate. METHODS: A retrospective study of indeterminate thyroid FNAs with known histologic follow-up was performed. One Diff-Quik-stained smear and one Papanicolaou-stained smear with similar cellularity (at least 60-100 lesional cells) from each case were sent to Rosetta and Interpace, respectively, for analysis. The results were directly compared and correlated with the final histopathology. Neither company was aware of the follow-up histologic findings in these cases. RESULTS: A total of 10 thyroid FNAs were identified from our 2015 files. The cytologic diagnoses included follicular lesion of undetermined significance (FLUS, n = 5), follicular neoplasm/suspicious for follicular neoplasm (FN/SFN, n = 4), and suspicious for malignancy (SM, n = 1). Of the seven cases with benign histology, six smears were classified as benign by the RosettaGX microRNA classifier, and one case was designated as suspicious. Five cases were negative by both ThyGenX oncogene panel and ThyraMIR microRNA classifier. One case was negative by ThyGenX and positive on follow-up ThyraMIR, and one case was positive for KRAS mutation and positive on ThyraMIR. Both the RosettaGX and ThyGenX/ThyraMIR tests demonstrated positive results for the three histologically malignant cases. CONCLUSION: This study demonstrates that two molecular testing platforms performed equally well using our stained direct smears. Both molecular tests revealed a 100% negative predictive rate. RosettaGX showed a 75% positive predictive value in comparison to 60% for ThyGenX/ThyraMIR.

Improvements in cell block processing: The Cell-Gel method.

BACKGROUND: The ability to produce adequate cell blocks profoundly impacts the diagnostic usefulness of cytology specimens. Cell blocks are routinely processed from fine-needle aspiration specimens or concentrated fluid samples. Obtaining directed passes for the sole purpose of producing a cell block is common practice, particularly when the cytopathologist anticipates the need for ancillary immunocytochemical stains and/or molecular studies. METHODS: The authors developed an effective and inexpensive process for producing cell blocks that consistently yields abundant cellular material, which they have termed the Cell-Gel method. This method can be simplified into 3 main steps: 1) preparing the sample; 2) constructing the cell block; and 3) processing the cell block. Highlights of the protocol include using a hemolytic fixative for sample preparation and disposable base molds for cell block construction. RESULTS: The cell block failure rate in the current study decreased from 18% with the HistoGel Tube method (January 2014-December 2014) to 6% with the Cell-Gel method (January 2015-December 2016). The authors evaluated 110 cell blocks processed with the HistoGel Tube method and 110 cell blocks processed with the Cell-Gel method, for a total evaluation of 220 cell blocks. CONCLUSIONS: The authors have developed an effective and inexpensive protocol for producing cell blocks that consistently yields abundant cellular material. The Cell-Gel method uses a hemolytic fixative and disposable base molds to produce adequate cell blocks. When the method was implemented, the cell block failure rate of the study laboratory decreased by approximately 67%. Cancer Cytopathol 2017;125:267-276. © 2016 American Cancer Society.

Anaplastic Lymphoma Kinase Immunocytochemistry on Cell-Transferred Cytologic Smears of Lung Adenocarcinoma.

BACKGROUND: Anaplastic lymphoma kinase (ALK) immunohistochemical staining on formalin-fixed paraffin-embedded tissue or cell blocks (CB) has been reported as an effective alternative to fluorescence hybridization in situ (FISH) for the detection of ALK gene rearrangement. However, CB frequently lack adequate cellularity even when the direct smears are cellular. This study aims to assess the utility of ALK immunocytochemical (ICC) staining on direct smears using the cell transfer (CT) technique for the detection of ALK rearrangement. METHODS: Fine-needle aspiration (FNA) cases of lung adenocarcinoma in which the ALK status had been determined by FISH on CB or a concurrent biopsy were identified. ICC staining for ALK was performed on alcohol-fixed Papanicolaou-stained direct smears using the CT technique. ALK immunoreactivity was evaluated using a modified semiquantitative scale. Results were compared with those of FISH. RESULTS: A total of 47 FNA specimens were included. Five of 7 FISH-positive cases showed positive ALK ICC staining (71.4%), and 39 of 40 FISH-negative cases were negative on ALK ICC staining (97.5%). The overall correlation between ALK ICC and FISH was 93.6%. CONCLUSION: ICC performed on FNA smears using the CT technique is an alternative method for the assessment of ALK rearrangement, especially when CB lack adequate cellularity.

Reprocessing unsatisfactory ThinPrep papanicolaou tests using a modified SurePath preparation technique.

BACKGROUND: The frequency of unsatisfactory gynecologic specimens has increased in the study laboratory over the last few years due to the advent of personal lubricants. Similarly, lysed blood, protein, and necrotic debris present a challenge in terms of negative cell transference caused by a clogged filter. In the current study, the authors evaluated the potential use of a modified SurePath reprocessing technique to decrease the frequency of unsatisfactory specimens. METHODS: An aliquot for human papillomavirus testing was set aside and the remaining specimen in the ThinPrep vial was submitted for sedimentation. A methanol wash was performed using preservative that was added to the remaining sediment. The specimen was vortexed and a protein wash of Tris-buffered deionized water was added before processing. The specimens were stained using the ThinPrep staining protocol. Both the original specimen and the reprocessed specimens were manually reviewed by 1 cytotechnologist and 2 pathologists to determine specimen adequacy. RESULTS: A total of 1937 ThinPrep Papanicolaou tests were reprocessed and examined. Of these, 1093 (56%) specimens were satisfactory, 624 of which (57%) demonstrated evidence of a transformation zone component. Epithelial cell abnormalities were identified in 116 specimens (10.6%), including 11 high-grade squamous intraepithelial lesion specimens (1.0%); 5 specimens with a classification of atypical squamous cells, cannot rule out a high-grade lesion (0.5%); 21 low-grade squamous intraepithelial lesion specimens (1.9%); and 79 specimens classified as atypical squamous cells of undetermined significance (7.2%). CONCLUSIONS: The modified SurePath processing technique was adept at handling nearly all of the challenges that biological and environmental conditions (blood, protein, lubricant, etc) present in liquid-based filter preparations. A total of 1093 (56%) of 1937 unsatisfactory ThinPrep Papanicolaou tests were converted to a satisfactory state, resulting in 116 abnormalities (10.6%) being diagnosed that otherwise would have gone undetected

Utilization of cell-transferred cytologic smears in detection of EGFR and KRAS mutation on adenocarcinoma of lung.

Cell-transfer technique has been proven useful for performing immunocytochemistry on fine-needle aspiration smears. However, its utility for EGFR and KRAS molecular testing has not been validated. Molecular testing was performed using the cell-transfer technique on both Papanicolaou-stained ethanol-fixed and Hema 3-stained air-dried smears from 32 fine-needle aspiration samples that had diagnoses of adenocarcinoma of the lung, and then was compared to the results of the corresponding formalin-fixed paraffin-embedded tissues. The molecular testing was successfully performed on 32 of 32 ethanol-fixed and 31 of 32 air-dried samples. The molecular results on ethanol-fixed and air-dried smears showed 100% agreement. There is 100% (32/32) agreement for the EGFR and 97% (31/32) agreement for the KRAS between the cell-transfer technique and formalin-fixed paraffin-embedded tissues. One discrepant case was due to low percentage of tumor cells on the smears. Cell-transfer technique is a reliable alternative method for EGFR and KRAS testing if the cell blocks lack adequate cellularity.

Endoscopic ultrasound-guided fine-needle aspiration cytology diagnosis of intraductal papillary mucinous neoplasm of the pancreas is highly predictive of pancreatic neoplasia.

Intraductal papillary mucinous neoplasms (IPMN) have been considered difficult to diagnose by fine-needle aspiration (FNA) cytology. We identified 57 cases diagnosed as IPMN or consistent with IPMN by endoscopic ultrasound (EUS)-guided FNA over a 9-yr period. Histologic follow-up was available for 20 patients (35%). Pancreatic neoplasia was demonstrated in 18 of these cases (90%). The histologic diagnoses were IPMN (16 cases), pancreatic intraductal neoplasia (grade 1b, 1 case), invasive mucin-producing adenocarcinoma (1 case), and chronic pancreatitis with a pseudocyst (2 cases). Sixty-two cases of IPMN without coexisting adenocarcinoma were diagnosed by histology during this time period. Of these, 35 (56%) had a preceding EUS-guided FNA. The diagnosis made by EUS-guided FNA in these 35 cases was negative or nondiagnostic (6 cases), benign cyst (1 case), chronic pancreatitis (2 cases), atypical ductal cells (2 cases), adenocarcinoma or suspicious for adenocarcinoma (3 cases), consistent with mucinous cystic neoplasm (4 cases), and IPMN or consistent with IPMN (16 cases). An EUS FNA diagnosis of probable or definite neoplasia was, therefore, made in 71% of cases of histologically proven IPMN.