Re: Revalidating immuno reagents / Joseph Nocito

From:Andre Balaton <>

Dear Joe,

Please find below a letter we sent recently to Archives of Pathology and
Laboratory Medicine concerning the use of antibodies beyond manufacturer's
Test Approach to Standardization of Immunohistochemistry (Letter). Arch
Pathol Lab Med 2001; 125: 471). We've been using "outdated" antibodies for
years without any problem. A known positive control is always run with the
test case.

" To the Editor-We read with great interest the excellent and much needed
article by Clive Taylor, MD, PhD, on standardization of immunohistochemistry
(1).  In several parts of the article, Dr Taylor described the necessity to
use immunohistochemistry in a cost-efficient manner, from the selection of
antibodies applied to a specific tumor to the choice of an automated system.

With respect to cost-efficiency measures, we would like to add some thoughts
concerning the existing guidelines and, more specifically, the potential
decision to approve the use of reagents beyond the manufacturers'
recommended expiration dates. A previous study sponsored by the College of
American Pathologists Cell Markers Committee, based on results from 221
laboratories performing staining protocols with 4 different antibodies,
recommended a ruling on extending the useful reagent shelf life of
antibodies (2).  Motivated by the same idea, we performed a study evaluating
65 primary antibodies for performance at various times after the
manufacturers' expiration dates. These times ranged from 6 to 131 months
postexpiration (mean 32.7 months). Similar to the study reported by Tubbs et
al (3) we found overwhelming evidence that "expired" antibodies perform
satisfactorily. These results indicate that instead of a mechanical
adherence to the manufacturers' recommendations, a rational quality
assurance system would be preferable.

The setting of guidelines by the regulating entities such as the Health Care
Financing Administration are absolutely necessary to assure the best
possible quality of service by medical laboratories. These regulations are
particularly useful in the application of newer methods, and without any
doubt have been of crucial importance for the rational development and
appropriate use of immunohistochemical stains. On the other hand, with the
significant financial strains that medical institutions and particularly
academic centers have suffered in recent years due to the reorganization of
health care financing and the increasing restrictions for reimbursement from
insurance companies, it may be time to re-evaluate some of the established
guidelines in order to avoid unnecessary expenses. It is a fact that
depending on the size of an immunohistochemistry laboratory, antibodies
costing several hundreds or thousands of dollars are discarded unnecessarily
every month. The excellent guidelines for quality assurance that have been
provided by the regulating organizations, such as the College of American
Pathologists, have been generally accepted and are widely practiced in
immunohistochemistry laboratories. The application of these guidelines to
the issue of expiration dates for primary antibodies could continue to
assure the best results while at the same time provide for the rational use
of resources.


1.  Taylor, CR. The total test approach to standardization of
immunohistochemistry. Arch Pathol Lab Med 2000;124:945-951.
2.  Tubbs, RR, R Nagle, and K Leslie. et al,. for the Cell Markers Committee
of the College of American Pathologists. Extension of useful reagent shelf
life beyond manufacturers' recommendations. Arch Pathol Lab Med
3.  Balaton, AJ, CB Drachenberg, C Rucker, P Vaury, and JC. Papadimitriou.
Satisfactory performance of primary antibodies beyond manufacturers'
recommended expiration dates. Appl Immunohistochem Mol Morphol
1999;7:221-225.   "

Here follows Clive Taylor's reply (Arch Pathol Lab Med 2001; 125: 472):

" In Reply.-The letter by Drachenberg et al is exactly the type of dialogue
that pathologists must initiate to ensure proper utilization of
immunohistochemical testing. The importance of this issue is emphasized
further by the expectation of continuing increases in demand for
immunohistochemistry, as well as a rapid growth of in situ hybridization and
related molecular methods as applied to tissue sections. These latter
methods are directly comparable to immunohistochemistry and will require
analogous attention to issues of reproducibility and quality control. "

Hope this helps.

Have a nice day,


Andre J. Balaton
Centre de Pathologie

----- Message d'origine -----
De : "HistoNet Server" <>
À : "HistoNet Server" <>
Envoyé : vendredi 6 avril 2001 08:43
Objet : Daily Digest

> Date: 5 Apr 2001 07:11:16 -0600
> From: "Nocito, Joseph" <>
> Subject: Revalidating immuno reagents
> Good morning all,
> During the last two days, we have been inspected by CLIA. The
> inspector found an expired immuno reagent in the refrigerator (I know,
> on me).  I explained to this person that although the reagent is expired,
> always run a known positive control with each run.  If the control didn't
> work, the case is repeated with another lot number. This was not good
> for this person. She wanted proof.  Short of performing a short immuno run
> in front of this person, I couldn't site any references.
> Does any one have a procedure that can be referenced dealing with
> validaing and re-validating reagents?  I'll also mention that each primary
> antibody gets titered upon receipt and each new detection kit gets tested
> and the results recorded before it is put into use.
> Thanks in advance.
> Joe Nocito, B.S., HT(ASCP)QIHC
> Histology Supervisor
> Christus Santa Rosa Hospitals
> San Antonio, Texas
> ---------------------------------------------------------------------->

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