Role of Preanalytical Phase and Laboratory Process for Optimal Ancillary Testing in Cytopathology.

Background: Ancillary tests are increasingly important for primary cancer diagnosis, as well as predictive and prognostic information, and occasionally cytological samples are the only material readily available. Advanced ancillary testing, including immunohistochemistry (IHC), immunocytochemistry (ICC), in situ hybridization, and next-generation sequencing (NGS) should be carefully standardized and quality controlled in order to provide reliable results. The diversity of preanalytics in the cytological laboratory process poses challenges for quality management.

Summary: Paraffin-embedded cell blocks (CBs) from cytological samples that are collected in and fixed with formalin appear to be the easiest option for ancillary tests, the majority of which are developed for formalin-fixed and paraffin-embedded (FFPE) samples. They can be stained with the same IHC protocols and on-slide controls without additional validation. Fixation time of FFPE CB samples should be controlled since nucleic acid quality and quantity decrease in formalin in a time-dependent manner. Ethanol and methanol, the standard fixatives in cytology, alter the tertiary structure of proteins, thus impairing ICC staining. Depending on the antibody, staining signals can be weaker or even absent in alcohol-fixed cells. Nevertheless, air-dried or methanol-fixed cytospins, liquid-cytology samples, cell-free supernatants, and unstained or stained smears can successfully be used for ancillary testing, but this requires careful protocol optimization and validation. Appropriate on-slide controls are not easily available for ICC, but these can be prepared for example from cell lines or left-over patient samples. If polymerase chain reaction (PCR) or NGS-based testing is performed in-house, different cytological sample types can be validated for routine use.

Key messages: Ancillary tests like ICC need to be validated according to the up-to-date guidelines in order to use the optimal protocol for each sample and fixation type and to discover the possible limitations of the test. Appropriate controls that reflect the preanalytical conditions of the sample material ensure reliable and reproducible results. Cytological material suits well for molecular pathology and could be more widely exploited in diagnostics. European cytopathology laboratories need to recognize the requirements of the in vitro diagnostic (IVD) regulation especially in documenting validation, risk management, and clinical performance data of the laboratory-developed (in house) tests.