[Histonet] antibody validation

Morken, Tim Timothy.Morken <@t> ucsfmedctr.org
Fri Sep 11 10:50:44 CDT 2009


We follow pretty much what you outline below. I believe following strict and comprehensive validation will save you headaches later on. You want to 1) convince yourself that the antibody works as advertised and as expected according to the literature and 2)Identify and solve any technical issues that may cause spurious results. You can't do that with a one-off test of one tissue sample. After a well-done validation you should have great confidence that it is being done right and works correctly. It is very useful to have the validation data in hand if anyone questions your test results.

Suggested references:

Recommendations for Improved Standardization of Immunohistochemistry, Goldstein, NS, et.al., and members of Ad-Hoc Committee on Immunohistochemical Standardization, Appl Immunohistochem Mol Morph, 2007 15(2): 124-133

A Practical Approach for Evaluating New Antibodies in the Clinical Immunohistochemistry Laboratory, Hsi, ED, Arc Pathol Lab Med. 2001; 125: 289-294

Quality Assurance For Immuncytochemistry: Approved Guideline, Clinical Laboratory Standards Institute (formerly NCCLS), Wayne PA, USA, publication MM4-A, Vol. 19, No. 26, 1999. www.clsi.org

Book chapters on IHC Validation and QC:

Quality Management in Immunohistochemistry, Brown RW, in Quality Management in Anatomic Pathology, Nakhleh RE, and Fitzgibbons PL, Eds. College of American Pathologists, Northfield, IL, USA, 2005. www.cap.org.

Theoretical and Practical Aspects of Test Performance, in Immunohistology: A Diagnostic Tool for the Surgical Pathologist. 3rd. Ed., Volume 19 in Major Problems in Pathology, Taylor CR and Cote RJ, Eds., W.B Saunders, Philadelphia, 2005 

Immunohistochemistry Quality Control, Hladik, CL and White, CL, in Theory and Practice of Histological Techniques, 4th Ed., Bancroft JD and Gamble M, Eds., Churchill Livingstone, 2007

Techniques of Immunohistochemistry:  Principles, Pitfalls and Standardization, Taylor CR, et.al., in Diagnostic Immunohistochemistry, Dabbs, DJ, Ed., 2nd Edition, Churchill Livingstone,

Tim Morken
Supervisor, Histology / IPOX
UCSF Medical Center
San Francisco, CA  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Akemi Allison-Tacha
Sent: Friday, September 11, 2009 8:21 AM
To: histonet
Subject: [Histonet] antibody validation

Good Morning and Happy Friday out there in Histo-Land!

I would like your assistance in an issue that I have just become aware of regarding antibody validation.  I have been requested to bring on-board approximately (7) new antibodies in the immediate future, and several antibodies later.

Although, many of you who know me realize I have worked in IHC R&D for a well known IHC company for many years, it has been a while since I have overseen a clinical IHC lab.  I would like your assistance and input on how you are validating new antibodies.   Not counting Her2-neu, it has been my understanding that it is recommended to test samples with varying antigen expression from 10-15 cases.  Furthermore, you should check for Sensitivity: expression range of Positive cases, low to high (10-15 cases).  Specificity: (+) vs expected (-) cases/tissues (10-15 cases) and Precision (Reproducibility): 

It has been my practice that any changes that are made to existing protocols, such as a new detection kit or antigen retrieval methodology, also requires re-validation.  

I am curious how you approach validation.  Also, do any of you just use a single known positive control for that antibody, and run it, and if it is positive, feel that it is satisfactory, and put it on-line for testing.

Thank you in advance for your input, and have a great weekend!

Akemi Allison-Tacha BS, HT(ASCP)HTL
Histology Manager
APMG Laboratories
105A Cooper Court, Los Gatos, CA 95032
Contact: 800.848.2764
V/M: 408.884.2718
Fax: 408.884.2758
Cell: 408.335.9994
(W) E-Mail: aallison-tacha <@t> apmglab.com
(P) E-Mail: akemiat3377 <@t> yahoo.com

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