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Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies
Published on: September 29, 2009
Multiplex Cryoimmunostaining in Fine-Needle Aspiration Biopsies and Effusion Samples: Preliminary Results
Simona Miceska1, Ivana Kholová2,3,4, Veronika Kloboves Prevodnik5,6
1Department of Cytopathology, Institute of Oncology Ljubljana, Ljubljana, Slovenia.
Insights
Multiplex cryoimmunostaining (mCryo) allows multiple markers on one slide, improving diagnostics for low-cellularity samples. Further validation is needed for routine cytopathology implementation.
Area of Science:
- Cytopathology
- Immunohistochemistry
- Biomarker Analysis
Background:
- Standard immunocytochemistry (ICC) is limited to single markers per slide, impacting diagnostic accuracy in low-cellularity samples.
- Multiplex cryoimmunostaining (mCryo) offers a potential solution by enabling simultaneous marker evaluation.
Purpose of the Study:
- To evaluate the applicability of mCryo compared to standard ICC on routinely prepared cytospins.
- To assess mCryo's diagnostic potential for paucicellular samples in cytopathology.
Main Methods:
- Residual fine-needle aspiration biopsy (FNAB) and effusion samples were used.
- Methanol-fixed cytospins underwent mCryo staining with a panel of antibodies (EpCAM, CK7, CD56, TTF-1, p40, CD45, calretinin).
- Standard ICC served as the control, with all slides reviewed blindly.
Main Results:
- mCryo demonstrated preserved morphology and minimal background staining.
- High concordance with ICC was observed for EpCAM, CK7, p40, and TTF-1 in carcinomas.
- Unexpected positivity for CK7 and CD56 in melanomas and carcinomas by mCryo warrants further investigation into specificity.
Conclusions:
- mCryo enables simultaneous multi-marker evaluation on routine cytospins, offering diagnostic advantages for paucicellular specimens.
- Further optimization of the mCryo method and antibody panels is essential for its integration into routine cytopathology practice.
Abstract:
Introduction: Immunocytochemistry (ICC) on cytospins and cell blocks is usually limited to a single marker per slide, often restricting diagnostic accuracy when sample cellularity is low. Multiplex cryoimmunostaining (mCryo), though not yet used in a routine cytopathology practice, may help overcome these limitations. Our study evaluated the applicability of mCryo compared with standard ICC on routinely prepared cytospins.
Methods:
Residual fine-needle aspiration biopsy and effusion samples from various diseases were used. mCryo staining with antibody panel consisting of EpCAM, CK7, CD56, TTF-1, p40, CD45, and calretinin (X-ZELL, Singapore) was tested on methanol-fixed cytospins, corresponding ICC-stained cytospins served as controls. All slides were reviewed blindly.
Results:
Forty-two samples were included in the analysis (21 carcinomas, eight lymphomas, five melanomas, eight non-neoplastic). mCryo revealed preserved morphology and no background staining in most of the cases. A 100% of true positivity was observed for EpCAM (21/21), CK7 (21/21), p40 (3/3), and TTF-1 (1/1) in carcinomas matching ICC results. TTF-1 was also true-positive in 2/2 benign thyroid nodule samples. CD45 was detected in all samples with both methods, labeling lymphocytes. Calretinin was true-positive only in 2/6 effusion samples. CK7 positivity was unexpectedly detected in all melanomas (5/5) by mCryo but not by ICC. Moreover, CD56 was also positive in all carcinomas and melanomas by mCryo (26/26), while only 9 cases were positive by ICC, raising the question of unspecific staining, antibody cross-reactivity, or spectral overlap of fluorochromes.
Conclusions:
mCryo enables simultaneous evaluation of multiple markers per slide on routinely prepared cytospins, offering potential diagnostic advantages for paucicellular samples. However, further method and panel improvement, and validation are essential for accurate diagnostic implementation into routine practice.
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