Laboratory-Developed L1 Sequencing and Type-Specific, Real-Time Polymerase Chain Reaction for the Detection and Typing of Human Papillomaviruses in Formalin-Fixed, Paraffin-Embedded Tissues

2013 ◽  
Vol 137 (1) ◽  
pp. 50-54 ◽  
Author(s):  
Anne Mills ◽  
Rajeev Balasubramaniam ◽  
Teri A. Longacre ◽  
Christina S. Kong ◽  
Benjamin A. Pinsky

Context.—The detection and typing of high-risk and low-risk human papillomavirus (HPV) in archival formalin-fixed, paraffin-embedded tissues by nucleic acid amplification testing is an important adjunct to immunohistochemical staining in evaluation of squamous cell proliferations of the oropharynx, larynx, and anal canal. Objective.—To evaluate semiautomated, xylene-free extraction from formalin-fixed, paraffin-embedded tissues combined with laboratory-developed HPV L1 sequencing and type-specific HPV 6, 11, 16, and 18 real-time polymerase chain reaction for identification and typing of HPV in the clinical laboratory. Design.—We evaluated the adequacy of extraction using β-globin amplification and compared L1 sequencing and real-time polymerase chain reaction methods for typing accuracy using 68 formalin-fixed, paraffin-embedded tissues, including 56 anorectal biopsy or surgical resection specimens and 12 laryngeal papilloma specimens from patients with recurrent respiratory papillomatosis. Results.—Adequate DNA was obtained from 68 of 68 specimens analyzed and all were HPV positive. In 47 cases where L1 sequencing demonstrated that the predominant HPV type was 6, 11, 16, or 18, type-specific, real-time polymerase chain reaction provided concordant results. Sequencing revealed additional low-risk (HPV 40) and high-risk HPV types (HPV 31, 33, 56, and 58) in anorectal specimens, whereas HPV 6 or 11 were the types found in laryngeal papillomas. Conclusion.—Both L1 sequencing and type-specific, real-time polymerase chain reaction are suitable methods for routine HPV testing of formalin-fixed, paraffin-embedded tissues in a clinical laboratory setting.

2016 ◽  
Vol 140 (8) ◽  
pp. 844-848 ◽  
Author(s):  
Darcy A. Kerr ◽  
Brenda Sweeney ◽  
Ronald N. Arpin ◽  
Melissa Ring ◽  
Martha B. Pitman ◽  
...  

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.


2019 ◽  
Vol 152 (6) ◽  
pp. 799-807 ◽  
Author(s):  
Andrew P Norgan ◽  
Lynne M Sloan ◽  
Bobbi S Pritt

Abstract Objectives Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. Methods FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. Results FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. Conclusions While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.


Author(s):  
Hamza Tariq ◽  
Preethi D. Menon ◽  
Hongxin Fan ◽  
Kumari V. Vadlamudi ◽  
Sri Lakshmi Pandeswara ◽  
...  

Context.— Associations between granulomatous lobular mastitis (GLM) and Corynebacterium kroppenstedtii have been reported since 2002, but large scale studies to assess the actual prevalence of this bacterium in GLM have not been performed. Objective.— To assess the prevalence of C kroppenstedtii in GLM using real-time polymerase chain reaction and Sanger sequencing. Design.— We analyzed formalin-fixed, paraffin-embedded tissues from 67 cases of GLM by sequential DNA amplification and sequencing to assess the rate of C kroppenstedtii detection in GLM. A retrospective analysis including patient demographics, history of pregnancy and lactation, clinical signs and symptoms, radiographic findings, histologic pattern, Gram stain results, and microbial cultures was performed on 67 cases of GLM. In addition, 10 cases of nongranulomatous breast abscess were included as controls. Results.— C kroppenstedtii 16S rRNA SYBR real-time polymerase chain reaction was positive on formalin-fixed, paraffin-embedded tissues from 46 of 67 (68.7%) GLM cases, while all control cases were negative. Among the positive cases, the majority showed features of cystic neutrophilic granulomatous mastitis. Conclusions.— C kroppenstedtii was highly prevalent in GLM cases and was not found to be associated with nongranulomatous breast abscess in our study (P < .001).


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