Multiplex immunoassays of formalin-fixed, paraffin-embedded tissue lysates

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Multiplex immunoassays of formalin-fixed, paraffin-embedded tissue lysates

Geoffrey Baird, MD, PhD
Acting Assistant Professor
Department of Laboratory Medicine
University of Washington, Seattle

Introduction: Hormone-secreting pituitary adenomas are subclassified by the identity of the secreted hormone(s) to predict prognosis and guide therapy, either by pre-operative quantitative serum hormone testing or post-operative qualitative immunohistochemistry (IHC) of tumor tissue. In this study, pituitary hormones were quantitated in formalin-fixed, paraffin-embedded (FFPE) adenoma tissue extracts, to test the hypothesis that a quantitative immunoassay can generate results that correlate with standard pathologic IHC diagnosis. The entire assay, from pre-analytical specimen preparation to multiplex immunoassay analysis, is amenable to automation, indicating that selected currently manual anatomic pathology diagnostic practices could be performed in the automated clinical laboratory.
Methods: Proteins were extracted from archived FFPE pituitary adenoma tissue (qProteome, Qiagen), total extract protein concentrations were analyzed by the BCA method (Pierce), and extracts were analyzed with a commercial kit (HPT-66K, Millipore) on a multiplex bead-based immunoassay instrument (Luminex 200) to quantitate Growth Hormone (GH), Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH), Prolactin (PRL), Adrenocorticotropic Hormone (ACTH), and Thyroid Stimulating Hormone (TSH). Hormone concentrations were normalized to total protein content of each extract. IHC for each hormone was performed and interpreted using standard methods.
Results: Pituitary hormone concentrations were measured in extracts from two 10-micron thick FFPE sections each of IHC-classified ACTH-, LH-, GH-, and PRL-expressing adenomas as well as non-expressing (null) adenomas. IHC classification and extract hormone concentrations were 100% concordant, in that the concentration of a hormone was at least 4- to 322-fold higher in its appropriate tumor subclass (i.e. ACTH in IHC-classified ACTH-expressing adenoma) than in any other subclass. Likewise, IHC-classified null adenomas had significantly lower total hormone content than hormone-expressing adenomas (P=0.02), and some null adenomas had quantitative hormone results (detectable FSH and LH, but no other detectable hormone) which indicated that IHC may be too insensitive to detect physiologically meaningful hormone expression.
Conclusions: In contrast with histopathology and IHC, immunoassays are increasingly automated, do not generally require highly skilled labor, produce quantitative results with definable accuracy and precision, and can be easily interpreted by comparison of test results to reference ranges. This assay demonstrates that a multiplex, bead-based immunoassay can be used on FFPE tissue generated during pathologic diagnosis, and can generate quantitative information that correlates well with diagnosis. Additional studies are underway to analyze many more samples in order to develop hormone reference ranges and a diagnostic multivariate classifier of adenoma subtype. The application of this technique to other tissues and diagnoses is likewise under investigation. Automation of assays such as the one reported here will increase the precision, accuracy, and throughput of the solid tissue diagnostic process, while decreasing costs associated with labor.