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Cell Block Preparation from Cytology Specimen with Predominance of Individually Scattered Cells
Published on: July 21, 2009
Young investigator challenge: Validation and optimization of immunohistochemistry protocols for use on cellient cell
Jennifer L Sauter1, Karen L Grogg1, Julie A Vrana1
1Division of Anatomic Pathology, Mayo Clinic, Rochester, Minnesota.
Insights
Validating immunohistochemistry (IHC) protocols for Cellient cell blocks (CCB) is crucial. While many antibodies validated after optimization, some required further refinement for reliable results on CCBs.
Area of Science:
- Anatomic Pathology
- Immunohistochemistry
- Cytopathology
Background:
- Immunohistochemistry (IHC) is vital for accurate diagnosis in surgical pathology.
- The Cellient cell block (CCB) system offers advantages for processing cytology samples.
- Standardizing IHC protocols for CCBs is necessary for reliable diagnostic interpretation.
Purpose of the Study:
- To establish and validate a robust process for IHC protocol validation on the Cellient cell block (CCB) system.
- To assess the performance of various antibodies using IHC on CCBs.
- To determine the concordance of IHC staining between CCBs and traditional formalin-fixed, paraffin-embedded (FFPE) tissues.
Main Methods:
- Thirty antibodies were initially tested on CCBs using established FFPE IHC protocols.
- Cytology samples were processed into both thrombin cell blocks (TCB) and CCBs for parallel IHC analysis.
- Antibody immunoreactivity was scored, and concordance between TCBs and CCBs was determined based on predefined criteria.
Main Results:
- Over half (57%) of the tested antibodies initially failed validation criteria on CCBs.
- Eight of the thirteen initially unvalidated antibodies were successfully optimized and validated for CCB use.
- Three antibodies (Ber-EP4, D2-40, PAX8) could not be validated even after protocol optimization.
Conclusions:
- A significant proportion of antibodies require protocol optimization for successful IHC on CCBs.
- Validation of IHC protocols on alcohol-fixed CCB material is essential before clinical application.
- Further optimization of IHC conditions may be necessary to achieve adequate immunoreactivity for specific antibodies on CCBs.
Background:
The objective of the current study was to establish a process for validating immunohistochemistry (IHC) protocols for use on the Cellient cell block (CCB) system.
Methods:
Thirty antibodies were initially tested on CCBs using IHC protocols previously validated on formalin-fixed, paraffin-embedded tissue (FFPE). Cytology samples were split to generate thrombin cell blocks (TCB) and CCBs. IHC was performed in parallel. Antibody immunoreactivity was scored, and concordance or discordance in immunoreactivity between the TCBs and CCBs for each sample was determined. Criteria for validation of an antibody were defined as concordant staining in expected positive and negative cells, in at least 5 samples each, and concordance in at least 90% of the samples total. Antibodies that failed initial validation were retested after alterations in IHC conditions.
Results:
Thirteen of the 30 antibodies (43%) did not meet initial validation criteria. Of those, 8 antibodies (calretinin, clusters of differentiation [CD] 3, CD20, CDX2, cytokeratin 20, estrogen receptor, MOC-31, and p16) were optimized for CCBs and subsequently validated. Despite several alterations in conditions, 3 antibodies (Ber-EP4, D2-40, and paired box gene 8 [PAX8]) were not successfully validated.
Conclusions:
Nearly one-half of the antibodies tested in the current study failed initial validation using IHC conditions that were established in the study laboratory for FFPE material. Although some antibodies subsequently met validation criteria after optimization of conditions, a few continued to demonstrate inadequate immunoreactivity. These findings emphasize the importance of validating IHC protocols for methanol-fixed tissue before clinical use and suggest that optimization for alcohol fixation may be needed to obtain adequate immunoreactivity on CCBs.
