CytoLyt fixation significantly inhibits MIB1 immunoreactivity whereas alternative Ki‐67 clone 30‐9 is not susceptible to the inhibition: Critical diagnostic implications

Buonocore, Darren J.; Konno, Fumiko; Jungbluth, Achim A.; Frosina, Denise; Fayad, Mariam; Edelweiss, Marcia; Lin, Oscar; Rekhtman, Natasha

doi:10.1002/cncy.22170

Cited by 32 publications

(37 citation statements)

References 24 publications

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“…Other studies have PD-L1 Immunostaining in Cytology/Koomen et al shown a detrimental effect of CytoLyt fixation on IHC detection of some antigens, such as thyroid transcription factor-1 (TTF-1) 18 and Ki-67. 21 A study by Lloyd et al 30 demonstrated a similar effect on PD-L1 staining, with CytoLyt fixation showing poor PD-L1 immunostaining results. Gosney at al 31 observed no effect of alcohol-based fixatives, including CytoLyt, on PD-L1 immunostaining with the 22C3 pharmDx assay in cytology specimens.…”

Section: Discussionmentioning

confidence: 91%

Formalin fixation for optimal concordance of programmed death‐ligand 1 immunostaining between cytologic and histologic specimens from patients with non–small cell lung cancer

Koomen

Starre-Gaal

Vonk

et al. 2020

Cancer Cytopathology

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BACKGROUND: Immunohistochemical staining of programmed death-ligand 1 (PD-L1) is used to determine which patients with non-small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD-L1 immunostaining between cytology cell blocks and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD-L1 immunoreactivity was studied. METHODS: Paired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine-needle aspiration on pneumonectomy/lobectomy specimens. Formalin-fixed, agar-based or CytoLyt/PreservCyt-fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory-developed test) antibodies. PD-L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD-L1-expressing human cell lines fixed in various fixatives at increasing increments in fixation duration. RESULTS: Agar cell blocks and tissue blocks showed substantial agreement (κ = 0.70 and κ = 0.67, respectively), whereas fair-to-moderate agreement was found between Cellient cell blocks and histology (κ = 0.28 and κ = 0.49, respectively). Cell lines fixed in various alcohol-based fixatives showed less PD-L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory-developed test and the 22C3 pharmDx assay. CONCLUSIONS: Performing PD-L1 staining on cytologic specimens fixed in alcohol-based fixatives could result in false-negative immunostaining results, whereas fixation in formalin leads to higher and more histology-concordant PD-L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin. Cancer Cytopathol 2020;0:1-14.

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Section: Discussionmentioning

confidence: 91%

Formalin fixation for optimal concordance of programmed death‐ligand 1 immunostaining between cytologic and histologic specimens from patients with non–small cell lung cancer

Koomen

Starre-Gaal

Vonk

et al. 2020

Cancer Cytopathology

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“…7 The type of fixative can cause decreased antigenicity of certain antibodies, including other nuclear markers such as MIB1 and ER as well as cytoplasmic stains such as S100. [16][17][18] This decrease in antigenicity might be attributed to differences in the effect of each fixative on the cells obtained. Formalin fixes specimens through the formation of intramolecular and intermolecular cross-links, whereas alcohol removes water from the free carboxyl, hydroxyl, amino, amido, and imino groups of the proteins, resulting in protein coagulation and tissue shrinkage.…”

Section: Discussionmentioning

confidence: 99%

“…Although immunostains are generally validated using formalin‐fixed, paraffin‐embedded tissue, several cytology laboratories use material previously fixed in an alcohol‐based medium before cell block processing . The type of fixative can cause decreased antigenicity of certain antibodies, including other nuclear markers such as MIB1 and ER as well as cytoplasmic stains such as S100 . This decrease in antigenicity might be attributed to differences in the effect of each fixative on the cells obtained.…”

Section: Discussionmentioning

confidence: 99%

“…Patient Demographics tumor staining and staining intensity were carefully estimated, as is routinely done in our clinical practice, by the same cytopathologist to ensure consistency.Chart review of all patients was performed retrospectively to determine the patient's smoking and alcohol use, how they were treated, and clinical outcomes. Direct evaluation of HR-HPV using in situ hybridization (clones16,18, 26, 31, 33, 35, 39, 41, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, 82, and E6/E7 messenger RNA; Ready to Use; Advanced Cell Diagnostics) targeting the transcripts of E6 and E7 messenger RNA was performed in all cases retrospectively.…”

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confidence: 99%

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Cytologic evaluation of p16 staining in head and neck squamous cell carcinoma in CytoLyt versus formalin‐fixed material

Buonocore

Fowle

Lin

et al. 2019

Cancer Cytopathology

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Background The management of high‐risk human papillomavirus (HR‐HPV)–related oropharyngeal head and neck squamous cell carcinomas (HNSCCs) are distinct from HNSCC linked to smoking and alcohol use. HR‐HPV–positive HNSCC frequently presents as a cervical lymph node metastasis. Because fine‐needle aspiration (FNA) is often the initial diagnostic procedure, evaluating HR‐HPV status in cytology specimens is important. The overexpression of p16 is a surrogate for HR‐HPV; however, the evaluation of p16 in FNAs remains controversial. Methods From September 2015 to December 2016, cytopathologists performed 25 FNAs of neck lymph nodes that were suspicious for HR‐HPV–positive HNSCC. Initial passes produced smears for on‐site evaluation and CytoLyt material. Additional passes were formalin‐fixed. A CytoLyt cell block (CCB) and a formalin‐fixed cell block (FFCB) were prepared, and p16 immunocytochemistry was performed. Results In 24 of 25 cases, the FFCB had diffuse (≥70% of cells), strong nuclear/cytoplasmic p16 staining. In all 24 of these cases, HR‐HPV was detected by in situ hybridization. The corresponding CCB had weak‐to‐moderate p16 staining in <70% of cells (range, 5%‐60% of cells) in 17 cases, 4 had weak‐to‐moderate diffuse staining, and 4 were acellular. The percentage of p16‐positive cells was significantly higher with FFCB than with CCB (formalin: 94% ± 2%; CytoLyt, 38% ± 7%; 2‐tailed, paired Student t test; P < .001; Fisher exact test, P < .001). Conclusions The fixative used had a drastic impact on p16 staining, which explained the staining variability reported in the literature. FFCBs show a diffuse staining pattern, which correlates with HR‐HPV status, whereas CCBs show a weaker and inconsistent staining pattern, which is more difficult to interpret.

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“…Many laboratories use different fixatives and have different methods of cellblock preparations or indications to create a cellblock. The effects of fixation in downstream analysis, has become a well‐documented fact in diagnostic cytology 6,7 . There are still questions among cytopathologists on whether a cell block should be part of cytology preparations.…”

Section: 20164 Category Criteria Category Criteriamentioning

confidence: 99%

Evolution of guidelines for respiratory cytology by the Papanicolaou Society of Cytopathology

Moreira

2020

Diagnostic Cytopathology

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Cytology serves a fundamental role in the evaluation of the lower respiratory tract. Cytological specimens are often the first diagnostic attempt for the evaluation of radiographic alterations. The categorization of the pathological process as infectious, inflammatory or neoplastic is critical in guiding further clinical management of the affected patient. Therefore it is imperative that cytopathologists use a standardized approach to sample handling and reporting in order to establish clear communication with the treating physician. The Papanicolaou Society of cytopathology has been an active leader in this field and has proposed several guidelines for sampling, handling, and reporting of lower respiratory tract cytology. These guidelines have been updated to incorporate new emerging concepts and technologies in the field. Respiratory medicine is a fast growing area with constant new challenges, thus requiring an ever demanding adaptation from cytopathologists and cytology specific guidelines.

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CytoLyt fixation significantly inhibits MIB1 immunoreactivity whereas alternative Ki‐67 clone 30‐9 is not susceptible to the inhibition: Critical diagnostic implications

Cited by 32 publications

References 24 publications

Formalin fixation for optimal concordance of programmed death‐ligand 1 immunostaining between cytologic and histologic specimens from patients with non–small cell lung cancer

Formalin fixation for optimal concordance of programmed death‐ligand 1 immunostaining between cytologic and histologic specimens from patients with non–small cell lung cancer

Cytologic evaluation of p16 staining in head and neck squamous cell carcinoma in CytoLyt versus formalin‐fixed material

Evolution of guidelines for respiratory cytology by the Papanicolaou Society of Cytopathology

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