The updated College of American Pathologists principles of analytic validation of immunohistochemical assays: A step forward for cytopathology

Abstract

The Clinical Laboratory Improvement Amendments of 1988 (CLIA) require that the analytic validation of immunohistochemical (IHC) assays verify and document the performance characteristics of any clinical assay before its use in a clinical service.¹ The College of American Pathologists (CAP) published their original evidence-based clinical practice guideline in 2014 to address analytic validations of IHC assays.² More recently, the CAP Pathology and Laboratory Quality Center tasked a guideline update committee to perform a systematic review of the literature, published since, to provide updated evidence-based recommendations. Of relevance to the cytopathology community are a few updated recommendations that include specific guidelines for validating IHC assays performed on cytology specimens.³

The previous guideline had allowed laboratory medical directors to decide the specific details of validating IHC assays in cytology specimens, including the types of specimen preparations and the number of validation samples. The current updated guideline recommends that, for every new IHC assay that is introduced into clinical service, laboratories should perform a separate validation for cytology specimens if they are processed differently than the specimens used for the initial assay validation. Because most IHC validations are done on histologic specimens that are formalin-fixed and paraffin embedded (FFPE), based on these new guidelines, any cytology specimen that undergoes processing that deviates from the former would be subject to a separate validation. This would include cytology smears, cytospin preparations, liquid-based cytology preparations, as well as any cell block preparations that use alcohol-based fixatives, and all cytology specimens collected in nonformalin transport media (e.g., saline, CytoLyt, RPMI) before formalin fixation and paraffin embedding.

The rationale for this new conditional recommendation stems from the widely variable results reported in the literature that compare the performance of IHC assays on cytology specimens that are processed and/or fixed using alternate methods (e.g., ethanol, methanol), with or without postfixation in formalin.^4-12 With the increasing importance of predictive biomarkers in guiding therapeutic management that rely on reliable and accurate IHC assays, the analytic validation of IHC assays for cytology specimens cannot be overemphasized.

The recommendation has a significant impact on laboratories that perform IHC assays on cytology specimens, because it would likely apply to a majority of cytology specimens that will now require including alternatively fixed and/or processed cytologic preparations when validating any new analyte on FFPE histologic specimens. Importantly, the recommendation would apply only to new analytes that are being validated in a laboratory for clinical IHC assays and would not require any retrospective validation of the antibodies that are currently in use on cytology specimens.

The second guideline statement recommends that a minimum of 10 positive and 10 negative cytology specimens should be included in the validation of each new analyte, with a consideration for increasing the number of cases if predictive markers are being validated. This statement closely mirrors the recommendation of the minimum number of specimens that are included for any initial validation of an analyte (statements 3 and 4).³ Whereas the guideline acknowledges the limitation of finding the adequate number of recommended samples in cytology, especially when validating rare analytes, the panel determined that the recommendation of the minimum number of samples would be appropriate in most instances. However, for situations in which the recommended 20 cytology samples are not feasible, the laboratory medical director can document the rationale for the use of a smaller validation set.

The cytopathology community should view these recommendations not as an obstacle but, rather, as an opportunity to validate cytology specimen preparations for IHC assays that are increasingly being used not just for diagnostic clinical use but also for predictive biomarker testing. With these specific guideline recommendations for analytic validation of IHC assays on cytology specimens, one can hope for an increased use of cytology specimens through routine incorporation of these specimen preparations for testing that can guide therapeutic management.¹³ The majority of clinical trials exclude cytologic specimen preparations, especially non-FFPE cytology, from enrolling patients for biomarker-driven treatment protocols. This is largely related to a lack of awareness of non-FFPE cytologic substrates but also because of the lack of standardization of cytology specimen processing and the absence of specific guidelines for the validation of biomarker-directed clinical assays.^{14, 15} As the field of precision medicine expands, the cytopathology community must adapt and evolve to participate in this critical patient care. These new CAP guideline recommendations just may be the impetus needed to open the door to a wider adoption of cytology specimens into routine clinical practice for IHC-based assays that drive patient care and biomarker-driven therapeutics.

The author disclosed no conflicts of interest.