Immunohistochemical validation studies in effusion cytology: A cautionary tale

Abstract

Precision medicine is driving the increased reliance of biomarker testing by immunohistochemistry (IHC) on cytological specimens. Although the original focus of IHC was to identify a target protein or analyte and deliver nominal results as a positive or negative signal, 1 it has now been expanded to include ordinal results in the form of scoring scales (eg, human epidermal growth factor receptor 2 [HER2] in breast cancer) or predefined positivity cutoffs (eg, a programmed death ligand 1 [PD-L1] assay). 2 The concept of validation as "demonstrating clinically relevant sensitivity and specificity" becomes even more important as IHC performed on limited samples takes on relevant roles not only for diagnosis but also for prognostic and therapeutic decisions. 3 Cell block preparations are the preferred type of cytological specimen for IHC of effusion fluids. In the United States, more than 10 methods are used for cell block preparation, the most common being plasma-thrombin, HistoGel (Richard-Allen Scientific, Kalamazoo, Michigan), and Cellient (Hologic, Marlborough, Massachusetts) 4 ; the first two use formalin, and the last one uses alcohol-based fixation and a unique processing method. Other cell block methods include simple sedimentation, collodion bags, and gelatin embedding. In an effort to boost cellular yields, variations of established methods have also been developed that include an alcohol pretreatment step before fixation in formalin. 5 IHC assays are typically validated with formalin-fixed, paraffin-embedded tissues. Laboratories need to determine whether cell blocks prepared with different fixation and/or processing protocols have equivalent immunoreactivity. 6 Multiple reports have documented decreased signal intensity or false-negative staining on alcohol-fixed cell blocks. Even the utilization of formalin postfixation has been reportedly plagued by decreased immunoreactivity. In a recent study of IHC on Cellient cell blocks, one-half of the antibodies tested failed the initial validation on IHC protocols optimized for formalin-fixed, paraffinembedded samples. 7 Alcohol fixation may also be introduced through the common practice of adding equal amounts of ethanol to effusion fluids at the time of collection. Fluids should ideally be received fresh by the laboratory and can be kept up to 14 days if refrigerated at 4°C with good preservation of morphology and immunogenicity. 8 One of the initial steps in the validation of IHC in cytological samples should be to test a limited selection of commonly used markers such as keratins, CD45, S100, and thyroid transcription factor (TTF1) in a set of cytological specimens to assess the influence of processing steps and specimen types on the assay results. A correlation with expected results in routinely processed tissue should be performed. 6

The utilization and utility of immunostains in body fluid cytology

“…A relatively frequent use of immunostains in body fluid cytology is the performance of predictive marker studies. Such studies can be reliably performed in malignant cells from effusions if the immunostains are thoroughly validated and the appropriate caveats regarding their use are kept in mind . In this study, we found a relative frequent use of estrogen and progesterone receptor (ER/PR) and Her2 stains for metastatic breast cancer.…”

Fluorescence in situ hybridization detection of chromosome 22 monosomy in pleural effusion cytology for the diagnosis of mesothelioma

“…Thoracentesis is a less invasive technique generally performed earlier than tissue biopsy 26 . However, cytologic diagnosis of mesothelioma can be challenging 27 . Cytomorphology is the fundamental basis for a diagnosis of mesothelioma based on cytology, but cytomorphologic features of mesothelioma cells may be observed in the reactive proliferation of mesothelial cells 26,28,29 .…”

“…26 However, cytologic diagnosis of mesothelioma can be challenging. 27 Cytomorphology is the fundamental basis for a diagnosis of mesothelioma based on cytology, but cytomorphologic features of mesothelioma cells may be observed in the Cancer Cytopathology July 2021 reactive proliferation of mesothelial cells. 26,28,29 In addition, identification of tumor cell invasion of surrounding tissues-the gold standard for diagnosis of MPM 4 cannot be achieved by cytologic evaluation.…”

Implementation of PD-L1 22C3 IHC pharmDx^TM in Cell Block Preparations of Lung Cancer: Concordance with Surgical Resections and Technical Validation of CytoLyt® Prefixation

“…Aside from analytical problems, preanalytical factors such as the frequent use of non-formalin fixatives with cytology samples pose challenges with IHC testing [32]. Technical validation of a satisfactory test performance is therefore required at the level of each laboratory [33].…”