Optimized Processing of Fine-Needle Lymph Node Biopsies for Automated Immunostaining

Abstract

A straightforward, reliable technique for postcollection processing and evaluation of cytologic specimens for antigen detection using an automated immunostainer was developed. Visual assessment of cell suspension turbidity was used in parallel with light microscopic examination of concentrated cytospin preparations to verify the diagnostic utility of samples for immunocytochemical staining. Fine-needle lymph node biopsies from 81 dogs with lymphadenomegally and a cytologic or histologic diagnosis of lymphoma were introduced into ethylenediamine tetra-acetic acid tubes containing standardized storage media. Cell suspension turbidity was assessed to estimate cell concentration and resultant volume required for cytospin preparations with optimal cellularity. Preliminary cytospin preparations (using estimated volumes based upon turbidity) were stained using modified Wright stain and examined microscopically for intact neoplastic cell concentration. Once an optimal volume for cytospin preparations was established, additional concentrated slides were prepared for immunophenotyping, using an automated immunostainer and antibodies specific for cluster of differentiation (CD)79a and CD3e. All cell suspension samples with adequate gross turbidity had ample intact neoplastic cell concentration for immunocytochemical staining. Based on CD79a and CD3e expression, 51 (63%) B cell, 19 (23%) T cell, 3 mixed T and B cells (4%), and 3 non-T- and non-B-cell lymphomas (4%), as well as 5 (6%) nondiagnostic samples were identified. Three out of 5 of the nondiagnostic samples were submitted early in the investigation prior to the establishment of gross specimen turbidity guidelines. Immunocytochemical staining results were in complete agreement with all 6 available immunohistochemical correlates. The ability to visually assess sample adequacy prior to sample submission may encourage more widespread use of immunocytochemical techniques.