Analytic Response Curves of Clinical Breast Cancer IHC Tests

Abstract

An important limitation in the field of immunohistochemistry (IHC) is the inability to correlate stain intensity with specific analyte concentrations. Clinical immunohistochemical tests are not described in terms of analytic response curves, namely, the analyte concentrations in a tissue sample at which an immunohistochemical stain (1) is first visible, (2) increases in proportion to the analyte concentration, and (3) ultimately approaches a maximum color intensity. Using a new immunostaining tool ( IHControls), we measured the analytic response curves of the major clinical immunohistochemical tests for human epidermal growth factor receptor type II (HER-2), estrogen receptor (ER), and progesterone receptor (PR). The IHControls comprise the analytes HER-2, ER, and PR at approximately log concentration intervals across the range of biological expression, from 100 to 1,000,000 molecules per test microbead. We stained IHControls of various concentrations using instruments, reagents, and protocols from three major IHC vendors. Stain intensity at each analyte concentration was measured, thereby generating an analytic response curve. We learned that for HER-2 and PR, there is significant variability in test results between clinical kits for samples with analyte concentrations of approximately 10 molecules/microbead. We propose that the characterization of immunostains is an important step toward standardization.

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

“…9,[15][16][17] For each analyte (HER2, ER, and PR), 5 different concentrations were manufactured at approximately log intervals, from 10 2 to 10 6 molecules per microbead. The IHControls are comprised of 2 different microbeads: analyte-coated glass test microbeads (7-8 lm diameter) and color standard microbeads (4.5-lm diameter).…”

“…The reason for using an MEF standard has been previously described. 9 Briefly, we quantify analyte concentration by attaching a fluorescein group to one end of each HER2, ER, or PR peptide during peptide synthesis. The fluorescein is incorporated onto a lysine, at its e-amino group.…”

“…9 In other laboratory disciplines, this would be analogous to verifying calibration. Short of that, the average laboratory can verify immunostain performance by using slides mounted with cell lines and tissue arrays, such as are available through national proficiency testing (PT) surveys.…”

“…A fluorochrome on the peptide provides traceability in establishing analyte concentration, in molecules per microbead. 9 The microbeads are suspended in a liquid that solidifies after pipetting onto a microscope slide, adhering the microbeads to the slide. The IHControl is pipetted onto a slide as an on-slide control, adjacent to a tissue sample, and accompanies the sample through all of the immunohistochemical staining steps.…”

“…We have previously described the product and technology. 9,[15][16][17] In this study, we evaluated the IHControls for potential use in a national survey of diagnostic IHC laboratories. We had 2 main goals.…”