Establishing a protocol for immunocytochemical staining and chromogenicin situhybridization of Giemsa and Diff-Quick prestained cytological smears

Abstract

<jats:sec id="st1"><jats:title>Background:</jats:title><jats:p>Protocols for immunocytochemical staining (ICC) and<jats:italic>in situ</jats:italic>hybridization (ISH) of air-dried Diff-Quick or May-Grünwald Giemsa (MGG)-stained smears have been difficult to establish. An increasing need to be able to use prestained slides for ICC and ISH in specific cases led to this study, aiming at finding a robust protocol for both methods.</jats:p></jats:sec><jats:sec id="st2"><jats:title>Materials and Methods:</jats:title><jats:p>The material consisted of MGG- and Diff-Quick-stained smears. After diagnosis, one to two diagnostic smears were stored in the department. Any additional smear(s) containing diagnostic material were used for this study. The majority were fine needle aspirates (FNAC) from the breast, comprising materials from fibroadenomas, fibrocystic disease, and carcinomas. A few were metastatic lesions (carcinomas and malignant melanomas). There were 64 prestained smears. Ten smears were Diff-Quick stained, and 54 were MGG stained. The antibodies used for testing ICC were Ki-67, ER, and PgR, CK MNF116 (pancytokeratin) and E-cadherin. HER-2 Dual SISH was used to test ISH. Citrate, TRS, and TE buffers at pH6 and pH9 were tested, as well as, different heating times, microwave powers and antibody concentrations. The ICC was done on the Dako Autostainer (Dako<jats:sup>®</jats:sup>, Glostrup, Denmark), and HER-2 Dual SISH was done on the Ventana XT-machine (Ventana / Roche<jats:sup>®</jats:sup>, Strasbourg, France).</jats:p></jats:sec><jats:sec id="st3"><jats:title>Results:</jats:title><jats:p>Optimal results were obtained with the TE buffer at pH 9, for both ICC and ISH. Antibody concentrations generally had to be higher than in the immunohistochemistry (IHC). The optimal microwave heat treatment included an initial high power boiling followed by low power boiling. No post fixation was necessary for ICC, whereas, 20 minutes post fixation in formalin (4%) was necessary for ISH.</jats:p></jats:sec><jats:sec id="st4"><jats:title>Conclusions:</jats:title><jats:p>Microwave heat treatment, with initial boiling at high power followed by boiling at low power and TE buffer at pH 9 were the key steps in the procedure. Antibody concentrations has to be adapted for each ICC marker. Post fixation in formalin is necessary for ISH.</jats:p></jats:sec>

Antigens under cover—The preservation and demasking of selected antigens for successful poststaining immunocytochemistry of effusion, brain smears, and lymph node aspirates

“…According to the ROSE concept (Rapid On‐Site Evaluation), cytologic examination is a valuable tool for establishing a definitive tissue diagnosis in practice and can complement histologic studies by revealing previously unrecognized features . There is, however, an enormous demand for more specific diagnoses to confirm or specify atypical and rare infections or undifferentiated neoplasia .…”

“…Previous studies and case reports have demonstrated the feasibility of subjecting cytologic, histologic, and histochemically stained slides, and immunocytochemistry and in situ hybridization procedures, to second analyses . These studies were mainly carried out after the removal of previous dyes by alcohol, bleaching with hydrogen chloride, or applying MWTs in citrate buffer and distilled water …”

“…Only a few investigators have performed direct immunolabeling of nuclear, cytoplasmic, and membranous antigens in neoplastic tissue smears previously stained with Papanicolaou, May‐Grünwald Giemsa, and Diff‐Quick stains that were not subjected to stain removal procedures . Romanowsky stains bind to cell components by electrostatic forces without causing conformational changes in the target epitopes .…”

“…Investigation of cytologic specimens obtained by fine‐needle aspiration (FNA) is the least invasive approach to obtain a diagnosis of disease . Cytomorphology, however, can provide only limited insights into the biology of a neoplastic disorder or the etiology of an infectious process.…”

“…Historically, PSICC methodology was tailored to individual cases. Therefore, systematic studies on the reproducibility, efficacy, and reliability of immunostaining are rare . Hence, the present study aimed to fill this gap by establishing reproducible guidelines for PSICC labeling of selected cellular and viral antigens in tissue imprints, FNA, and cytocentrifuged fluids with the focus specifically on antigen preservation and retrieval.…”