The value of mutational profiling of the cytocentrifugation supernatant fluid from fine-needle aspiration of pancreatic solid mass lesions.

Fine-needle aspiration (FNA) of pancreatic solid masses can be significantly impacted by sampling variation. Molecular analysis of tumor DNA can be an aid for more definitive diagnosis. The aim of this study was to evaluate how molecular analysis of the cell-free cytocentrifugation supernatant DNA can help reduce sampling variability and increase diagnostic yield. Twenty-three FNA smears from pancreatic solid masses were performed. Remaining aspirates were rinsed for preparation of cytocentrifuged slides or cell blocks. DNA was extracted from supernatant fluid and assessed for DNA quantity spectrophotometrically and for amplifiability by quantitative PCR (qPCR). Supernatants with adequate DNA were analyzed for mutations using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, microsatellite fragment analysis for loss of heterozygosity (LOH) of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q). In selected cases, microdissection of stained cytology smears and/or cytocentrifugation cellular slides were analyzed and compared. In all, 5/23 samples cytologically confirmed as adenocarcinoma showed detectable mutations both in the microdissected slide-based cytology cells and in the cytocentrifugation supernatant. While most mutations detected were present in both microdissected slides and supernatant fluid specimens, the latter showed additional mutations supporting greater sensitivity for detecting relevant DNA damage. Clonality for individual marker mutations was higher in the supernatant fluid than in microdissected cells. Cytocentrifugation supernatant fluid contains levels of amplifiable DNA suitable for mutation detection and characterization. The finding of additional detectable mutations at higher clonality indicates that supernatant fluid may be enriched with tumor DNA. Molecular analysis of the supernatant fluid could serve as an adjunct method to reduce sampling variability and increase diagnostic yield, especially in cases with a high clinical suspicion for malignancy and limited number of atypical cells in the smears.

Pitfalls in the diagnosis of herpes simplex infection in respiratory cytology.

OBJECTIVE: To evaluate the prevalence and potential pitfalls in making an accurate diagnosis of respiratory herpetic infection. STUDY DESIGN: Eighteen cases with the diagnosis of herpes simplex virus (HSV) infection were identified from a total of 7,501 (0.24%) respiratory specimens. All cases were evaluated for classic cytomorphologic features of HSV infection and associated cytologic findings. The parameters studied included number of cells with HSV cytopathic effect, intranuclear inclusions, multinucleation, presence of atypical squamous cells, reparative changes, presence and degree of inflammation and associated obscuring factors. RESULTS: Only a minority of cases (28%) had numerous cells with classic viral cytopathic change. Four (22%) of 18 cases showed atypical squamous cells, and 5 (28%) revealed reparative changes. The majority of the cases were associated with inflammation, which was severe in 4 cases (22%). Blood and degenerative changes obscured the cytologic findings in 3 cases (17%). One case showed a necrotic background. CONCLUSION: Due to the low prevalence of HSV infection in respiratory cytology, a high index of suspicion is necessary for an HSV diagnosis. Pitfalls for a false negative diagnosis include limited number of cells with viral cytopathic change, only mononuclear cells with viral changes and obscuring inflammation or blood. Pitfalls for a false positive diagnosis of malignancy include atypical keratinized squamous cells, atypical repair, cellular degeneration and necrotic background.