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Cytojournal. 2010 Feb 10;7:1. doi: 10.4103/1742-6413.59887.

Two-color immunocytochemistry for evaluation of effusion fluids for metastatic adenocarcinoma.

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  • 1Department of Pathology, Medical College of Wisconsin, Milwaukee, Wisconsin, USA.



The evaluation of serous fluids by conventional one color immunocytochemistry is complex and challenging.


We selected and studied 37 serous fluid cytology specimens (23 pleural, 13 peritoneal, 1 pericardial), collected over a 4-year period. They were unequivocally positive for metastatic adenocarcinoma based on clinical correlation, cytomorphology, and one color immunocytochemistry on cell block sections. 3 mum serial sections of cell blocks were immunostained by a two chromogen method (peroxidase with brown chromogen followed by alkaline phosphatase with red chromogen). Combinations evaluated were: A- vimentin followed by cytokeratin (CK) 7; B- calretinin followed by BerEP4, C- calretinin followed by CK 20. Additionally, difficulty of interpretation was evaluated on a scale of 1(easy) to 5 (difficult). Cases demonstrating decreased or complete loss of immunoreactivity with alkaline phosphatase red chromogen system were also evaluated with routine one color immunostaining by alkaline phosphatase and peroxidase individually. The pretreatments for antigen retrieval and antibody dilutions were identical to those used for conventional one color immunostaining with respective immunomarker.


Combination 'A' showed correlation with the immunoreactivity pattern observed with one color immunostaining. However, the immunoreactivity of the second immunomarker was compromised in combinations B and C. In the latter group, the sections immunostained with one color alkaline phosphatase indicator system also showed weak immunoreactivity or complete loss of immunoreactivity for the corresponding second immunomarker. However, the peroxidase system showed proper immunoreactivity for those immunomarkers. Average difficulty of interpretation for the two color method was 1.06 (range- 1 to 2) as compared to 2.95 (range: 1 to 5) with the one color method. This difference was statistically significant (two-tailed P<.0001, paired t test). The higher scores of difficulty were observed in cases with a paucity of tumor cells and cases with predominance of isolated tumor cells.


Dual immunostaining facilitated identification of the foreign population of malignant cells in effusion fluids with objective, reproducible precision. However, due to relatively lower sensitivity of alkaline phosphatase as a second indicator system, immunoreactivity was diminished for BerEP4 and lost for CK 20.


Serous effusion; dual immunostaining; immunocytochemistry; immunohistochemistry; metastasis; serous cavity; serous fluid; two-colour immunostaining

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