IHC Analysis
If there is one image analysis question we hate, it is, “Can you perform image analysis on this badly overstained slide?”. The answer is frequently “No”. Although in some circumstances, image analysis can overcome issues of overstaining, nonspecific staining or just plain bad histology, there are definite limits. These issues usually greatly dimish the accuracy of quantitation. Finally improving the “look” of a digital image through electronic manipulation may have ethical issues, especially if the image is to be used for illustration or publication.
Generally in image analysis, less is more. The computer can pick up more subleties in color than the human eye, so understaining is preferable to overstaining. Unfortunately the computer is also very adept at picking up visual subleties that reflect staining errors and artifacts.
Here are some of the common sources of errors for IHC analysis, and how to avoid them:
False negatives
- Antibody is inappropriate, denatured or used at wrong concentration.
- Loss of antigen through poor fixation and/or diffusion.
- Presence of antigen at a density below level of detection.
False positives
- Cross-reactivity of antibody with unintended antigens.
- Nonspecific binding of the antibody to the tissue.
- Presence of endogenous peroxidase or avidity for the avidin-biotin complex.
- Entrapment of normal tissues by tumor cells.
- Leakage of protein from adjacent cells with subsequent absorption by target tumor cells
From an image analysis standpoint, the most difficult situations are where the biomarker is accurately stained, but is present in both target and non-target tissues. This is where histology pattern recognition can be very useful.
Some general guidelines for the analysis of slides from experimental studies
- Take care to assure immediate optimal fixation for all tissue samples. Uniformity of handling as well as fixation time is important.
- Staining procedures for all slides in a study need to be performed simultaneously in a single batch to assure uniformity of stain.
- Sampling must be strictly representational as well as consistent. Care must be taken to assure exact uniformity of analysis with respect to anatomical location (eg. Tissue trimming, sectioning)
- A preliminary evaluation of image analysis tools between some slides of varying stain intensities will help assure that analysis values are established optimally for all slides in the study
See our related post on how to improve whole slide scanning.
Flagship Biosciences provides high quality histology, immunohistochemistry and scanning services for pharmaceutical development research, medical device development, companion diagnostics, and academic research. If you are interested in Flagship’s histology services, you can learn more about them them here, contact us, or go directly to our Services Request Form.
