[Histonet] validation

[Fulton Regan]

Hello Nancy, 
 
Given your interest in best practices for validations,  I’d like to offer some recent literature references that may be of some use.
 
Your use of 20 positives and 20 negatives exceeds the minimum standard, from CAP Guidance. For purely diagnostic markers. (10+ and 10- are suggested in such cases), while 20+ and 20- are suggested as a minimum for predictive markers, such as ER, PR, etc… Your practice exceeds the minimum, but is certainly an excellent approach for better control of your assays!  
 
Patrick, L. F., Linda, A. B., Lisa, A. F., Alsabeh, R., Regan, S. F., Jeffrey, D. G., … Swanson, P. E. (2014). Principles of analytic validation of immunohistochemical assays: Guideline from the College of American Pathologists Pathology and Laboratory Quality Center. Archives of Pathology and Laboratory Medicine, 138(11), 1432–1443. https://doi.org/10.5858/arpa.2013-0610-CP
 
With respect to negative controls, there is another good reference, below:
 
Torlakovic, E. E., Francis, G., Garratt, J., Gilks, B., Hyjek, E., Ibrahim, M., … Vyberg, M. (2014). Standardization of Negative Controls in Diagnostic Immunohistochemistry. Applied Immunohistochemistry & Molecular Morphology, 22(4), 241–252. https://doi.org/10.1097/PAI.0000000000000069
 
Internal negative control elements can definitely be part of the validation plan and can be used in specificity calculations.  Of course, it is important to confirm that these internal negatives are in fact negative!  However, it is also best practice to design validations as “fit for purpose”. That is,  does the assay reliably distinguish among the differential diagnostic  considerations?  So, for specificity, a collection of “pertinent negatives” should also be included in the validation.  As an example: in the case of Beta-catenin for the diagnosis of fibromatosis, one should include some cases that resemble fibromatosis.  These might include, GIST, SCHWANNOMA, LEIOMYOMA, NEUROFIBROMA, and others, as your question clearly anticipated. 
 
I hope this of some use to you. 
 
Feel free to contact me if I can be of any further help. 
 
Regan



Regan Fulton, MD/PhD
 
CEO
Array Science, LLC
Tissue Microarray Technologies
475 Gate 5 Road, #102
Sausalito, CA 94965

fulton at ArrayScience.com