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

Abstract

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.

Needle rinse cell blocks as an ancillary technique: Diagnostic and clinical utility in gastrointestinal neoplasia

“…As the NRCB is formalin‐fixed and follows paraffin embedding similar to a small biopsy, immunohistochemistry protocols are the same as those validated for histopathologic material (similar antibody dilutions, antigen recovery, and controls) 10,11 . For immunocytochemistry, a large antibody panel can be more difficult to perform, and specific immunostaining procedures must be validated with adequate negative and positive controls for the cytologic samples 17 …”

Doing more with less: multiple uses of a single slide in veterinary cytology. A practical approach

“…Several fixatives and protocols have been proposed for ICC in cytopathology (Suthipintawong et al 1996 ). Consequently, marked variability in protocols still prevails among diagnostic laboratories of human and veterinary pathology (Fischer et al 2014 ; Kirbis et al 2020 ; Raskin et al 2019 ; Valli et al 2009 ).…”

“…Moreover, cellularity and cell preservation often vary between smears, and the inability to assess the quality of the smear prior to ICC may be a considerable limitation, leading to low diagnostic yield (Priest et al 2017 ). This can be prevented by using previously stained slides (Raskin et al 2019 ). With microscopic screening, only slides with representative or good quality material will be used for ICC (i.e., adequate cellularity, and without high hemodilution or ruptured cells that will produce heavy background).…”

A rapid multiple immunofluorescence method for the simultaneous detection of CD20 and CD3 in canine and feline cytological samples

“…CD20 is a well-known B-cell marker, and its expression is noted in the pre-B-cell stage up to the activated B-cell stage [ 2 ]. Furthermore, it has been widely used as a reliable marker for immunohistochemical and immunocytochemical diagnosis of canine and feline B-cell lymphoma [ 4 , 8 , 10 , 11 , 12 ]. However, whether it could be used for diagnosis with the RMIF method has not yet been evaluated.…”