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Published on: August 23, 2021
Integrated On-Slide Positive Controls for Immunocytochemistry on Cytology Slides
Irena Srebotnik Kirbiš1, Ruben Rodrigues Roque2,3, Margareta Strojan Fležar1
1Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Insights
This study presents a new, cost-effective method for creating integrated on-slide positive controls for immunocytochemistry (ICC) on methanol-fixed cytospins. The technique ensures reliable quality control for diagnostic cytology, improving efficiency and accuracy.
Area of Science:
- Cytopathology
- Immunocytochemistry
- Quality Assurance
Background:
- Integrated on-slide positive controls are crucial for quality assurance in immunohistochemistry.
- Standard protocols ensure identical analytical conditions for control and patient samples.
- A need existed for integrated controls in immunocytochemistry (ICC) on methanol-fixed cytospins.
Purpose of the Study:
- To develop a procedure for preparing integrated on-slide positive controls for ICC.
- To adapt existing methods for methanol-fixed cytospins.
- To ensure reliable quality control in diagnostic cytology.
Main Methods:
- Utilized leftover diagnostic cytology samples with confirmed marker expression (Calretinin, MOC31, TTF1, hormone receptors).
- Prepared cytospins, fixed in methanol, and covered with polyethylene glycol (PEG).
- Performed ICC on the Ventana/Roche automated platform ULTRA with validated protocols.
Main Results:
- 99% of integrated positive controls showed consistent, positive reactions for TTF-1, hormone receptors, and Calretinin/MOC31 double staining.
- ICC reactions on patient samples were interpretable.
- No significant cell carryover between control and patient samples was observed.
Conclusions:
- A novel, low-cost method for integrated on-slide positive controls in ICC on methanol-fixed cytospins was successfully developed.
- This approach ensures identical analytical conditions, enhancing quality control and reducing costs.
- The method is widely applicable in diagnostic cytology laboratories, offering time-efficient interpretation.
Introduction:
Integrated on-slide positive controls are a standard quality assurance and quality control measure for immunohistochemistry on formalin-fixed paraffin-embedded tissue sections. They ensure identical analytical conditions for the control and patient samples. Our aim was to develop a procedure for preparing integrated on-slide positive controls for immunocytochemistry (ICC) on methanol-fixed cytospins.
Methods:
Leftover diagnostic cytology samples with sufficient cells and confirmed expression of Calretinin, MOC31, TTF1, and hormone receptors were used as control samples. Cells from the control samples were deposited on the peripheral part of objective slides using standard cytocentrifuge equipment. Cytospins were immediately fixed in methanol for at least 30 min and then covered with polyethylene glycol (PEG). Completely dry and solid PEG was removed from the central part of the objective slides and stored at room temperature. Patient samples were subsequently added to the central part of a PEG-protected slide, with an appropriate positive control placed on the peripheral part, and then fixed in methanol. ICC was performed on the Ventana/Roche automated platform ULTRA, using optimized and validated protocols for TTF1, hormone receptors, and double immunostaining for Calretinin/MOC31. The quality of ICC reactions for both deposits on the same slide and potential cell carryover was evaluated retrospectively.
Results:
In the period from October 2021 to December 2023, the majority of integrated positive controls (364/368, 99%) consistently exhibited unequivocally positive reactions for TTF-1 (n = 93), hormone receptors (n = 84), and double staining for Calretinin/MOC31 (n = 191), with easily interpretable ICC reactions on corresponding patient samples. No obvious carryover of cells from the control sample to the patient sample was observed during this period.
Conclusion:
A novel approach developed for preparing integrated on-slide positive controls for ICC on methanol-fixed cytospins using standard cytocentrifugation is low-cost and can be widely applied in diagnostic cytology laboratories. Simultaneous ICC procedures for the control and patient samples on the same slide ensure identical analytical conditions for both samples, providing the highest level of quality control while reducing costs. Interpreting both ICC reactions on the same slide is time-efficient and convenient.
