Background: A simple and efficient slide preparation procedure was developed for immunocytochemical (ICC) detection of diagnostic and prognostic factors on cytological samples where required numbers of cytospins fixed and stored in methanol were prepared from samples suspended in a cell medium.
Aim: To determine the reliability of ICC detection of different diagnostic and prognostic markers on cytological samples processed according to our slide preparation procedure.
Methods: Cytospins for all ICC reactions were prepared from samples suspended in buffered based cell medium. Preservation of immunoreactivity after fixation and storage of cytospins in methanol as well as optimal conditions for reliable ICC detection were first determined for estrogen receptors (ER) on a human breast cancer cell line (MCF-7) and then confirmed on fine needle aspiration biopsies (FNAB) of breast cancer. The same fixation and storage procedure was then evaluated on cytospins prepared from different cytological samples for CD markers (CD3, CD10, CD20, CD30, CD31, CD45, CD56, CD68, CD117, CD138), cytokeratins (MNF116, AE1/AE1, CK 7, CK 19, CK 20), intermediate filaments (desmin, smooth muscle actin, vimentin) and 20 other diagnostic markers (α1-fetoprotein, calcitonin, calretinin, chromogranin, HMB-45, HCC, Ki67, MelanA, MOC31, p53 in p16, progesterone receptors, P504S, PSA, RCC, S100, synaptophysin, thyroglobulin, TTF-1, WT-1). The reliability of ICC reactions was determined evaluating the concordance of ICC reactions between cytospins prepared from diagnostic cytological samples, cytological samples prepared from fresh biopsies, and immunohistochemical (IHC) reactions on corresponding formalin fixed paraffin embedded (FFPE) tissue sections. Differences in percentages of ER positive MCF-7 cells after different fixation and storage procedures were analysed with Duncan test, while Cohen kappa test (κ) was used for analysis of concordance between ICC and IHC reactions.
Results: The highest percentage of ER positive MCF-7 cells with the lowest variability (72 ± 5 %) and complete concordance between ICC and IHC assessment of ER on cytological and corresponding FFPE samples (52/52, 100 %, κ = 1,00) was found on cytospins fixed and stored in methanol. Fixation of cytospins in methanol also preserved immunoreactivity of all other 38 markers evaluated. High concordance between ICC results on methanol fixed cytospins prepared from diagnostic cytological samples and cytological samples prepared from fresh biopsies with IHC results on corresponding FFPE tissue sections was found in both tested groups (141/146, 97 %, κ = 0,93 in 81/86, 94 %, κ = 0,88).
Conclusion: The cytological sample processing procedure for ICC evaluated in this study is simple, efficient and yield reliable results.