Melissa Randolph

Background: The emergence of personalized therapy and molecular sequencing has significantly transformed the management of advanced non–small cell lung carcinoma (NSCLC). Supernatants obtained from fine needle aspiration (FNA) have been shown to be a reliable source of high‐quality nucleic acid for molecular testing. One pre‐analytic factor that may influence the success of molecular analysis is the collection medium used for the FNA rinse. Methods: This prospective study evaluated 31 FNA specimens collected in three different media—CytoRich Red, 10% neutral buffered formalin, or 10% bovine serum albumin (BSA)—to assess their impact on DNA yield and real‐time polymerase chain reaction (RT‐PCR) outcomes for KRAS ( Kirsten rat sarcoma viral oncogene homolog) and/or EGFR (epidermal growth factor receptor) mutations. DNA extraction was performed using the QIAamp DNA FFPE (formalin‐fixed paraffin‐embedded) Tissue Kit (Qiagen), and RT‐PCR was conducted using the QIAGEN therascreen EGFR or KRAS RGQ PCR kits. Concurrent direct smears or formalin‐fixed, paraffin‐embedded tissue from a cell block or core biopsy served as standard controls. Results: In the CytoRich Red group, 11 specimens (9 adenocarcinomas and 2 NSCLC) were analyzed. All 11 specimens generated results concordant with standard controls. One specimen required repeat DNA extraction due to initial pre‐test quality control failure. The formalin group included 12 specimens (7 squamous cell carcinomas [SCC], 3 adenocarcinomas, 1 NSCLC, and 1 large cell neuroendocrine carcinoma). Nine specimens produced concordant results with controls. Two specimens were negative for EGFR mutations, although the corresponding control tissues were not tested. Six required repeat DNA extraction because of initial pre‐test failure, and one specimen remained invalid after re‐extraction. Eight specimens were collected in BSA (5 adenocarcinomas, 2 NSCLC, and 1 SCC). Five produced concordant results with standard controls, while three specimens failed PCR pre‐tests despite repeat DNA extraction. Conclusion: In this small cohort, CytoRich Red demonstrated reliable performance for RT‐PCR‐based mutation testing. FNA supernatant represents a practical alternative specimen for lung cancer molecular analysis and may optimize specimen utilization while preserving cell block material.

Background: Anal cancer rates are rising among women, immunocompromised individuals, and Men who have sex with Men (MSM), independent of HIV status. While high‐resolution anoscopy remains the diagnostic standard, limited access has increased interest in alternative screening. HPV testing now complements or replaces cytology in cervical cancer prevention, but evidence for anal screening, especially using extended genotyping platforms like BD Onclarity, remains limited. Methods: This single center cross‐sectional study enrolled high‐risk individuals undergoing anal cancer screening from May 2024 to May 2025. Anal cytology and high‐risk HPV (HR‐HPV) DNA testing were performed using the BD Onclarity assay on the BD COR system. Demographics, cytology results, HRHPV genotypes, and available histopathology were analyzed. Results: Among 117 high‐risk participants (median age 42; 106 males, 8 females, 3 transgender women), 74.3% were ‐HIV positive, predominantly MSM‐. Abnormal cytology occurred in 41% of cases including ASC‐US ( n = 29; 60.4%), LSIL ( n = 16; 33.3%), ASC‐H ( n = 2; 4.1%), and HSIL ( n = 1; 2.1%). Valid BD Onclarity results were available for 104 individuals, with 61.5% testing HR‐HPV positive. Non‐16/18 HR‐HPV types were most common overall, while HPV16 predominated in abnormal cytology cases. Twelve individuals underwent biopsy, revealing no dysplasia ( n = 2), AIN1 ( n = 5), AIN2 ( n = 1), and AIN3 ( n = 4). Conclusions: Extended HR‐HPV genotyping using the BD Onclarity assay demonstrated a high prevalence of HR‐HPV in this high‐risk population, with multiple genotype infections commonly observed. While non‐HPV16/18 genotypes predominated overall, HPV16 was more frequently associated with abnormal cytologic and high‐grade histologic findings. Given the limited number of biopsy‐confirmed high‐grade lesions, these associations should be interpreted as descriptive and hypothesis‐generating. Cytology remains central to anal cancer screening, with extended HPV genotyping serving as a complementary adjunct rather than a standalone stratification tool. Larger longitudinal studies with systematic histopathologic follow‐up are needed to clarify the clinical significance of extended HPV genotyping in this population.

Introduction: Aspiration in infants is a diagnostic challenge. The lipid‐laden macrophage index (LLMI) developed in 1987 has been used as a supportive test; however, numerous recent studies have questioned its value and reproducibility. We evaluated a simplified LLMI in bronchoalveolar lavage (BAL) specimens from a pediatric cohort to assess its diagnostic utility. Methods: BALs from infants were prospectively collected over a 6‐month period for Oil Red O (ORO) staining to evaluate aspiration. BALs from adults with non‐aspiration pathologies were simultaneously collected for comparison. Clinical and demographic data were gathered to assess the diagnostic accuracy of the test. Only samples containing ≥ 100 evaluable macrophages and free of obscuring blood or inflammation were included. Positive staining was assessed at low magnification (10×), with only clearly positive cells (Colombo‐Hallberg scores 3 and 4) considered. A dichotomous threshold of < 50% or ≥ 50% positive macrophages was established through multidisciplinary consensus. To ensure consistency, a training session was conducted for the entire cytopathology division on the newly developed interpretation criteria. Results: 88/134 (66%) pediatric BAL samples with suspected aspiration and 63/75 (84%) adult samples with various non‐aspiration pathologies were adequate for analysis. Aspiration status in children was determined using multidisciplinary aerodigestive group evaluation (MAGE) and videofluoroscopic swallow study (VFSS). Test performance was assessed at various cutoffs. In the pediatric cohort (mean age 16.5 months, 58% male), aspiration was diagnosed in 47% by MAGE. Strong associations were seen with atopia/asthma (83%), functional dysphagia (64%), and congenital/developmental disorders (43%). A significant difference in ≥ 50% lipid‐laden macrophage involvement was observed between pediatric (12%) and adult (51%) samples ( p < 0.00001). Using MAGE and VFSS as gold standards, the test showed poor discriminatory power for detecting aspiration in infants (AUC 0.506–0.587). A 10% cutoff yielded the best performance (AUC 0.587, sensitivity 27%, specificity 93%), while a 50% cutoff offered practical advantages in workflow and reproducibility. Conclusions: The modified LLMI demonstrates limited diagnostic value for aspiration in infants. While a 10% cutoff offers slightly improved performance, the test may be phased out in favor of more reliable diagnostic methods.

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.

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.

Objectives: Fine-needle aspiration (FNA) is frequently used to diagnose metastatic melanoma. In this study, we validated the use of cell-transferred cytological smears for BRAF molecular testing. Study Design: We conducted a search of our laboratory information system for the period 2011-2013 in order to identify surgical pathology cases of either primary or metastatic melanomas in which BRAF mutation analyses had already been performed. Thirty FNA cases with diagnoses of metastatic melanoma from the same patients were identified. Direct smears from each FNA case were selected for mutation analyses using the cell transfer technique. Results: Mutation analyses were successfully performed on 28 of 30 FNA cases (93%) using the cell-transferred cytological smears. In 25 cases (8 BRAF mutations and 17 BRAF wild types), there was 100% agreement for the BRAF mutation between the cell-transferred cytological smears and the formalin-fixed paraffin-embedded tissues. Three FNA cases showed BRAF mutations that had not been detected in the correlated surgical specimens which were tested twice, and 2 cases failed to work. Conclusions: Cell-transferred cytological smears are a reliable and alternative resource for detecting BRAF mutations in metastatic melanoma.