Quantitative Assessment of Immunohistochemistry Laboratory Performance by Measuring Analytic Response Curves and Limits of Detection

Abstract

Context.-Numerous studies highlight interlaboratory performance variability in diagnostic immunohistochemistry (IHC) testing. Despite substantial improvements over the years, the inability to quantitatively and objectively assess immunostain sensitivity complicates interlaboratory standardization.Objective.-To quantitatively and objectively assess the sensitivity of the immunohistochemical stains for human epidermal growth factor receptor type 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR) across IHC laboratories in a proficiency testing format. We measure sensitivity with parameters that are new to the field of diagnostic IHC: analytic response curves and limits of detection.Design.-Thirty-nine diagnostic IHC laboratories stained a set of 3 slides, one each for HER2, ER, and PR. Each slide incorporated a positive tissue section and IHControls at 5 different concentrations. The IHControls comprise cell-sized clear microbeads coated with defined concentrations of analyte (HER2, ER, and/or PR). The laboratories identified the limits of detection and then mailed the slides for quantitative assessment.Results.-Each commercial immunostain demonstrated a characteristic analytic response curve, reflecting strong reproducibility among IHC laboratories using the same automation and reagents prepared per current Good Manufacturing Practices. However, when comparing different commercial vendors (using different reagents), the data reveal up to 100-fold differences in analytic sensitivity. For proficiency testing purposes, quantitative assessment using analytic response curves was superior to subjective interpretation of limits of detection.Conclusions.-Assessment of IHC laboratory performance by quantitative measurement of analytic response curves is a powerful, objective tool for identifying outlier IHC laboratories. It uniquely evaluates immunostain performance across a range of defined analyte concentrations.

Probing metabolic alterations in breast cancer in response to molecular inhibitors with Raman spectroscopy and validated with mass spectrometry

“…But IHC is mostly qualitative, suffers from inter-patient variability, and oen lacks accuracy when presented with heterogeneous tumors. 5,6 Clinical decisions based on change in tumor size in response to therapy are inherently slow and low-throughput as a decrease in tumor burden can take several weeks. Therefore, an unmet clinical need exists for rapid, accurate, and cost-effective diagnostic tools that can guide the best treatment choices at the earliest time point and reduce mortality due to ineffective cancer therapies.…”

Synthetic Antigen Gels as Practical Controls for Standardized and Quantitative Immunohistochemistry

“…The suitability of peptides as IHC controls has been studied in detail in Steven Bogen's laboratory, with the identification and immobilization of peptides recognized by clinically relevant antibodies to ER, PR, HER2, and others. [9][10][11][22][23][24]26,31 These and other authors [34][35][36] demonstrated the utility of short linear peptides coupled to solid supports as surrogates for full-length proteins in many antibody-mediated detection systems. Based on studies documenting the relative reactivity of peptide amino acid side chains with formaldehyde, and parameters including steric accessibility that affect formaldehyde reactivity, 27,28,40,43 we included amino acids and spacer sequences at both the N-and C-termini intended to favor formaldehyde-mediated crosslinking.…”

“…7,8 Work from Steven Bogen's laboratory using well-characterized peptide IHC epitopes bound to glass beads has shown that, even in the absence of tissue-specific preanalytic variables, the dynamic range and shape of analytic response curves vary with assay conditions. [9][10][11] At one extreme, assay conditions can produce a steplike response, with signal rising rapidly to a saturated level as antigen abundance increases above the lower LOD. With such conditions, antigen expression will appear nearly binary: absent below the detection threshold or fully present above it.…”

“…More recent valuable work by several investigators, notably those in the laboratory of Steven Bogen, has shown the utility of peptide epitopes or whole proteins chemically coupled to the surface of glass slides or to glass beads that replicate the three-dimensionality of cells in tissue. [9][10][11][22][23][24][25][26] These efforts address many of the same concerns we consider here, and some of these techniques have been offered as tools to standardize routine IHC lab work processes. 9,11,26 In this work, we reexplored the general concept demonstrated by Brandtzaeg,12 in which specific antigens are incorporated into carrier protein gels.…”

“…[9][10][11][22][23][24][25][26] These efforts address many of the same concerns we consider here, and some of these techniques have been offered as tools to standardize routine IHC lab work processes. 9,11,26 In this work, we reexplored the general concept demonstrated by Brandtzaeg,12 in which specific antigens are incorporated into carrier protein gels. We identified conditions that allow reproducible sample preparation at relatively low cost and using routine histology methods for a variety of antigens including whole proteins, recombinant protein domains, and small peptides.…”