Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

Choi, Ung Su; Kim, D. Y.

doi:10.1111/j.1365-2303.2010.00756.x

Cited by 6 publications

(8 citation statements)

References 18 publications

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“…One veterinary report described using anti‐cytokeratin to stain a cytologic smear previously stained with a methanolic Romanowsky stain, but the technical information regarding this procedure was not included . Another veterinary report demonstrated success with an anti‐Ki67 on canine aqueous Romanowsky‐stained slides . In this study, the previously‐stained slides were microwaved in an oven and heated to a boil in a citrate buffer at pH 6 for two 5‐minute periods followed by cooling to room temperature for 20 minutes.…”

Section: Discussionmentioning

confidence: 99%

“…Human medicine has investigated the use of a variety of immunomarkers on previously stained slides, but many of the reports involve the Papanicolaou staining method with infrequent reference to Romanowsky‐stained materials . However, only rare reports have evaluated using ICC on prestained or formalin‐fixed cytologic slides in veterinary medicine, and all studies focused on one to two immunomarkers . Signal detection using immunofluorescent antibodies against CD79a or CD3 were shown to be unsuccessful in smears that had been previously stained with Giemsa .…”

Section: Introductionmentioning

confidence: 99%

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Optimized immunocytochemistry using leukocyte and tissue markers on Romanowsky‐stained slides from dogs and cats

Raskin

Vickers²,

Ward³

et al. 2019

Veterinary Clinical Pathol

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Background: Romanowsky staining is often the initial method used to stain hematologic and cytologic materials. While immunocytochemistry (ICC) is a well-established method on air-dried smears, there are rare veterinary reports of ICC involving Romanowsky-stained slides.Objectives: This study aimed to compare immunoreactivity of unstained vs Romanowsky-stained specimens, evaluate reactions over time, and assess ICC associations with confirmatory tests of 50 lymphoma cases. Another goal aimed to optimize manual ICC protocols with cellular and tissue immunomarkers to detect CD3ε, CD20, Pax5, MHCII, lysozyme, MUM1, vimentin, cytokeratin, and Melan-A antigens on Romanowsky-stained specimens. Materials and methods:Cytologic specimens from cases of lymphoid and nonlymphoid neoplasms were stained with a methanolic Romanowsky method. Additional unstained slides from these cases were used for comparison with the stained materials. Antigen retrieval involved a citrate buffer pH6 or Tris/EDTA pH9 at 95°C for 25 minutes in a decloaking chamber. Immunocytochemistry used known positive and secondary antibody-only negative cytologic controls. Immunoreactivity of unstained and prestained lymphoma slides was graded by the intensity and percent of stained cells. Signal grading was monitored over time for diagnostic differences.Results: Unstained and Romanowsky-stained slides had similar membrane/cytoplasm graded reactions, but unstained slides produced stronger signals. Romanowskystained blood films from B-cell and T-cell leukemias showed minimal loss of signal when monitored over 20 weeks. Signal differences did not change the diagnosis. There was a significant association between ICC and confirmatory tests. Optimization involved antibody dilution and antigen retrieval methodology for each antibody tested. Conclusions:Immunocytochemistry of Romanowsky-stained material can be successfully performed using antibodies against CD3ε, CD20, cytokeratin, lysozyme, Melan-A, MHCII, MUM1, Pax5, and vimentin. K E Y W O R D Santigen retrieval, CD20, CD3, lymphoma, lysozyme, Melan-A, MHCII, Pax5 | 89 RASKIN et Al.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Optimized immunocytochemistry using leukocyte and tissue markers on Romanowsky‐stained slides from dogs and cats

Raskin

Vickers²,

Ward³

et al. 2019

Veterinary Clinical Pathol

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“…Only a few investigators have performed direct immunolabeling of nuclear, cytoplasmic, and membranous antigens in neoplastic tissue smears previously stained with Papanicolaou, May‐Grünwald Giemsa, and Diff‐Quick stains that were not subjected to stain removal procedures . Romanowsky stains bind to cell components by electrostatic forces without causing conformational changes in the target epitopes .…”

Section: Discussionmentioning

confidence: 99%

“…Historically, PSICC methodology was tailored to individual cases. Therefore, systematic studies on the reproducibility, efficacy, and reliability of immunostaining are rare . Hence, the present study aimed to fill this gap by establishing reproducible guidelines for PSICC labeling of selected cellular and viral antigens in tissue imprints, FNA, and cytocentrifuged fluids with the focus specifically on antigen preservation and retrieval.…”

Section: Introductionmentioning

confidence: 99%

Antigens under cover—The preservation and demasking of selected antigens for successful poststaining immunocytochemistry of effusion, brain smears, and lymph node aspirates

Dörfelt¹,

Matiasek²,

Felten³

et al. 2019

Veterinary Clinical Pathol

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Background:In clinical cytology, the applicability of an ancillary test such as immunocytochemistry is too often limited by low sample volume, poor cell representation, and sample preservation. Diagnosticians often read Romanowsky-stained cytology, although specific techniques such as immunocytochemistry are often essential for a definitive diagnosis. Objectives:The goal of the present study aimed to investigate if immunocytochemistry on previously-stained cytologic specimens was possible. Different pretreatments were examined to determine which treatment preserved antigenicity best. Methods:One hundred and twenty-two impression smears and 64 fine-needle aspirate preparations of brain and lymph nodes were processed and evaluated microscopically. The impact of staining cytologic preparations with a modified Wright's stain, using a destaining method, performing a coverslipping and decoverslipping process, and subjecting smears to a microwave treatment (MWT) were examined for the immunolabeling of selected nuclear, cytoplasmic, and plasmalemmal antigens, as well as intracellular feline coronavirus (FCoV). Biotinylated secondary antibodies were used, and the bound primary antibody was visualized using an ABC amplification kit.Results: Cellular antigens were reliably detected with immunocytochemistry after smears were stained with a Romansky stain and were coverslipped early after staining and stayed coverslipped until immediately before immunolabeling. The staining intensity reached the same levels as that of the controls if the films underwent MWT in citrate buffer. In contrast, FCoV antigen detection was abolished after any physicochemical interference. Conclusions:Poststaining immunocytochemistry represents a practical tool for additional investigations on prestained cytologic specimens when searching for cellular antigens. Paired untreated samples should be kept in case the workup requires testing for more vulnerable viral antigens. K E Y W O R D Scoverslip, destaining, feline infectious peritonitis, microwave, neurocytology

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“…Recently, an article from Choi et al . [18] has described a protocol for Ki-67 staining on Diff-Quick-stained smears from breast tumors in dogs. The aim of this study is to develop a robust protocol for ICC and ISH, which could work on air-dried MGG- and Diff-Quick-stained smears.…”

Section: Introductionmentioning

confidence: 99%

Establishing a protocol for immunocytochemical staining and chromogenicin situhybridization of Giemsa and Diff-Quick prestained cytological smears

2012

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Background:Protocols for immunocytochemical staining (ICC) andin situhybridization (ISH) of air-dried Diff-Quick or May-Grünwald Giemsa (MGG)-stained smears have been difficult to establish. An increasing need to be able to use prestained slides for ICC and ISH in specific cases led to this study, aiming at finding a robust protocol for both methods.Materials and Methods:The material consisted of MGG- and Diff-Quick-stained smears. After diagnosis, one to two diagnostic smears were stored in the department. Any additional smear(s) containing diagnostic material were used for this study. The majority were fine needle aspirates (FNAC) from the breast, comprising materials from fibroadenomas, fibrocystic disease, and carcinomas. A few were metastatic lesions (carcinomas and malignant melanomas). There were 64 prestained smears. Ten smears were Diff-Quick stained, and 54 were MGG stained. The antibodies used for testing ICC were Ki-67, ER, and PgR, CK MNF116 (pancytokeratin) and E-cadherin. HER-2 Dual SISH was used to test ISH. Citrate, TRS, and TE buffers at pH6 and pH9 were tested, as well as, different heating times, microwave powers and antibody concentrations. The ICC was done on the Dako Autostainer (Dako®, Glostrup, Denmark), and HER-2 Dual SISH was done on the Ventana XT-machine (Ventana / Roche®, Strasbourg, France).Results:Optimal results were obtained with the TE buffer at pH 9, for both ICC and ISH. Antibody concentrations generally had to be higher than in the immunohistochemistry (IHC). The optimal microwave heat treatment included an initial high power boiling followed by low power boiling. No post fixation was necessary for ICC, whereas, 20 minutes post fixation in formalin (4%) was necessary for ISH.Conclusions:Microwave heat treatment, with initial boiling at high power followed by boiling at low power and TE buffer at pH 9 were the key steps in the procedure. Antibody concentrations has to be adapted for each ICC marker. Post fixation in formalin is necessary for ISH.

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Immunocytochemical detection of Ki‐67 in Diff‐Quik‐stained cytological smears of canine mammary gland tumours

Abstract: Diff-Quik-stained cytology smears can be used to detect the presence of Ki-67 antigen when histology sections are not available.

Cited by 6 publications

References 18 publications

Optimized immunocytochemistry using leukocyte and tissue markers on Romanowsky‐stained slides from dogs and cats

Optimized immunocytochemistry using leukocyte and tissue markers on Romanowsky‐stained slides from dogs and cats

Antigens under cover—The preservation and demasking of selected antigens for successful poststaining immunocytochemistry of effusion, brain smears, and lymph node aspirates

Establishing a protocol for immunocytochemical staining and chromogenicin situhybridization of Giemsa and Diff-Quick prestained cytological smears

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