Experimental validation of peptide immunohistochemistry controls

Abstract

Peptide immunohistochemistry (IHC) controls are a new quality control format for verifying proper IHC assay performance, offering advantages in high throughput automated manufacture and standardization. We previously demonstrated that formalin-fixed peptide epitopes, covalently attached to glass microscope slides, behaved (immunochemically) in a similar fashion to the native protein in tissue sections. To convert this promising idea into a practical clinical laboratory quality control tool, we tested the hypothesis that the quality assurance information provided by peptide IHC controls accurately reflects IHC staining performance among a diverse group of clinical laboratories. To test the hypothesis, we first designed and built an instrument for reproducibly printing the controls on microscope slides and a simple software program to measure the color intensity of stained controls. Automated printing of peptide spots was reproducible, with coefficients of variation of 4% to 8%. Moreover, the peptide controls were stable at <or=4 degrees C for at least 7 months, the longest time duration we tested. A national study of 109 participating clinical laboratories demonstrated a good correlation between a laboratory's ability to properly stain formalin-fixed peptide controls to their ability in properly staining a 3+ HER-2 formalin-fixed tissue section mounted on the same slide (r=0.87). Therefore, peptide IHC controls accurately reflect the analytical component of an IHC stain, including antigen retrieval. Besides its use in proficiency survey testing, we also demonstrate the feasibility of applying peptide IHC controls for verifying intralaboratory IHC staining consistency, using Levy-Jennings charting.

Figure 1

Examples of stained peptide control spots and their respective numerical scores. The y axis is expressed as mean pixel intensity, in relative units, and is not calibrated against a color intensity reference standard.

Figure 2

Software program screen for measuring stained peptide spot intensity. After subtracting the background level of intensity over irrelevant areas of the slide (not shown), color intensity is measured over the stained peptide spots (contained within the yellow circles). The value, in mean pixel intensity, is depicted in the higher magnification inset. The peptide control spots shown in the inset are not duplicates of each other, but rather serial dilutions. Duplicate peptide controls were printed with a horizontal orientation on the slide.

Figure 3

Photograph of the prototype slide printer, with a higher magnification of the printer head (inset), showing eight nozzles, each of which dispense microliter-sized droplets of peptide onto passing slides. As the slides proceed towards the right, they pass onto a heated platen, which accelerates the peptide coupling reaction to the activated glass surface.

Figure 4

Consistency of slide-to-slide printing of peptide controls for each of the eight printer nozzles. The tight SD for each printer head nozzle demonstrates the high reproducibility in printing. Two separate experiments are shown.

Figure 5

Stability of peptide controls, for HER-2 (top panel), progesterone receptor (PR) 636 monoclonal antibody (middle panel), and estrogen receptor (ER) 1D5 monoclonal antibody (bottom panel). The dotted line represents the immunoreactivity of peptide controls stored at room temperature. The slight dip in immunoreactivity at the 3 month time interval is related to a slight inconsistency in the staining protocol (immunohistochemical detection) rather than an actual decrease in stability. Each time point is the mean ± SD of four replicate peptide controls.

Figure 6

Relationship of the HER-2 staining intensity of a 3+ tissue biopsy section to the staining intensity of a 50 micromolar HER-2 peptide control spot on a glass microscope slide. The peptide spot intensities (x axis) are measured in units of mean pixel intensity (MPI). When error bars are shown, then the value represents the mean ± SD of more than one participating laboratory for that level of HER-2 peptide staining. The absence of an error bar means that only one laboratory returned a HER-2 peptide control value at that particular level of staining (expressed as “mean pixel intensity”, on the x axis). We fit the data with a best-fitting curve using a linear regression line.

Figure 7

Image intensity comparison between peptide IHC controls (50, 10, 2, and 0.4 μM concentration, whose intensities are shown at the right by the arrows) and HER-2 cell lines previously calibrated to 0, 1+, 2+, and 3+ staining intensity. The regression line has a correlation coefficient (r) = 0.95. The absorbance units on the y axis are relative units generated by the iCys laser scanning Cytometer.

Figure 8

Levy-Jennings charting for estrogen and progesterone receptor immunohistochemistry, over a two month period. The mean and ± 2SD boundaries are also shown. No staining failures occurred in this time frame.