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Updated: Jun 13, 2026
Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma
Published on: January 9, 2019
Optimized processing of fine-needle lymph node biopsies for automated immunostaining
Adam D Aulbach1, Cheryl L Swenson, Matti Kiupel
1MPI Research Inc., 54943 North Main Street, Mattawan, MI 49071-9399, USA. adam.aulbach@mpiresearch.com
Insights
A new method uses visual assessment of cell suspension turbidity to ensure adequate cytologic samples for canine lymphoma immunophenotyping. This reliable technique streamlines processing and improves diagnostic accuracy for antigen detection.
Area of Science:
- Veterinary Pathology
- Immunocytochemistry
- Canine Lymphoma Diagnostics
Background:
- Accurate immunophenotyping of canine lymphoma is crucial for diagnosis and treatment.
- Current methods for assessing sample adequacy for immunocytochemistry can be time-consuming.
- A need exists for a straightforward, reliable technique for pre-analysis evaluation of cytologic specimens.
Purpose of the Study:
- To develop and validate a technique for postcollection processing and evaluation of cytologic specimens for antigen detection.
- To assess the utility of visual estimation of cell suspension turbidity for determining sample adequacy for immunocytochemical staining.
- To establish a reliable method for immunophenotyping canine lymphoma using automated immunostaining.
Main Methods:
- Developed a technique using visual assessment of cell suspension turbidity and light microscopy of cytospin preparations.
- Collected fine-needle lymph node aspirates from 81 dogs with lymphoma.
- Processed samples using standardized media and automated immunostaining with CD79a and CD3e antibodies.
Main Results:
- Visual assessment of turbidity reliably predicted adequate cell concentration for immunocytochemistry.
- Identified 63% B cell, 23% T cell, 4% mixed T and B cell, and 4% non-T/non-B cell lymphomas.
- Achieved 94% diagnostic yield, with nondiagnostic samples primarily from early in the study before turbidity guidelines were established.
- Immunocytochemistry results showed complete agreement with available immunohistochemical correlates.
Conclusions:
- Visual assessment of cell suspension turbidity is a straightforward and reliable method for evaluating sample adequacy for canine lymphoma immunophenotyping.
- This technique simplifies postcollection processing and enhances the diagnostic utility of immunocytochemical staining.
- The method promotes wider adoption of immunocytochemical techniques in veterinary diagnostics.
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.
