[Histonet] RTU antibodies with 1+ year expiration date

[Morken, Timothy]

To the specific question of if lab-made predilutes will last longer than two weeks. Yes they can. But be sure you are using reasonably sterile technique so they don't become contaminated. Then validate how long they last.  I know of one lab in the "old" days of manual staining that kept their antibodies in Coplin jars in the fridge and used them for many weeks.  They just pulled out a particular antibody jar and added slides when that antibody was ordered. It made bulk manual staining much easier.

I agree with all Tony writes below. One observation from experience working for a vendor of antibodies... While vendors test for reasonable shelf life, the fairly standard 2-year expiration date is implemented more to limit a vendors liability than indicate actual shelf life. We tested ours for up to 4 months in a 60 C oven and I rarely saw one fail. Indeed, some, as Tony noted, became better with age and heat!

While those of us who have worked in IHC for many, many years know that most modern antibodies are very stable, and, if frozen, good for decades, the various inspection agencies are very focused on expiration dates. Part of that may be ease of enforcement (not having to look though hundreds of pages of revalidations) and part not trusting labs to do good revalidations. And many labs would much rather not revalidate since it takes work to do and if you have many antibodies, it could be a lot of work (we have over 250 on our menu). Much easier to get new ones. We don't revalidate, but we also make every effort to only have on hand that which will not expire before used. We have very few that have so few orders that the vial lasts 2 years. And we still dilute a fair number that are not available as predilutes from our instrument vendor.



And thanks for all the references!


Tim Morken
Pathology Site Manager, Parnassus 
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center




-----Original Message-----
From: Tony Henwood (SCHN) via Histonet [mailto:histonet at lists.utsouthwestern.edu] 
Sent: Monday, December 05, 2016 2:44 PM
To: Allan Wang
Cc: histonet at lists.utsouthwestern.edu
Subject: Re: [Histonet] RTU antibodies with 1+ year expiration date

Hi Alan,

Not every source states that diluted antibodies only last for a few weeks, in fact my experience as well as those of the commercial suppliers (see their data sheets) indicates otherwise, for example, I just re-validated an antibody (CD45RO)  that was prepared 8 years ago (but unfortunately rarely used) and it worked very well, in fact we had to re-titre it since it seems our detection system has improved its sensitivity (we have a Bond 3). Since it is not routinely used (last time was at least 4 years ago) we have retired it.

The following is an extract from the handout of a workshop we gave on an Immunohistochemistry Validation workshop we gave this year:

Expired Antibodies
Usually when a new concentrated antibody is received it will have an expiry date of around 2 years from receipt, but most of us will find that we can continue to use antibodies well past this expiry date. So where are we at?
Recent discussions on Histonet reveal:
?	At least five labs admit to routinely discarding expired antibodies.
?	Why don’t companies extend expiry dates?
?	Why not freeze aliquots of antibody to extend the shelf life?
?	If controls continue to stain appropriately, then why can’t expiry be extended?
An expiration date labelled on a vial merely reflects the time span tested by the manufacturer during which performance of a reagent has remained stable. It does not imply per se, that the reagent will not function properly beyond the indicated date. In fact, because it is unrealistic for a manufacturer to test an antibody for aging during decades before putting it on the market, most expiration spans for primary antibodies are set arbitrarily within 6 to 24 months (Balaton et al 1999).

Dr Hadi Yaziji explains: Vendors do stability testing on their antibodies, where they leave them at room temperature for an extended time (one month, six months, etc.), or they do what's referred to as 'accelerated' testing, where they put them in a microwave which is supposed to accelerate the degradation of the protein. That's usually how they decide to assign an expiration date.

Dr Hadi Yaziji also suggests that if you aliquot the antibody before its expiration date and store it in the -20oC freezer, the clock is in effect "frozen" too. For example, if there are 6 months left before the antibody expires and you freeze the aliquots for 5 years, then if you thaw one of the frozen aliquots, the thawed aliquot will still be good for 6 months. He also recommends that you make sure you don't thaw and re-freeze the aliquot. Unfortunately, some antibody data sheets state “do not freeze”. One Histotechnologist reported that their CAP inspector told them it was not acceptable, so had to allow him to watch them throw all the frozen aliquots away.

Both Dr Terry Marshall (UK) and Dr Richard Cartun suggest that if the antibody continues to stain control sections appropriately, with no loss of sensitivity and no increase in non-specific staining then its use should be continued. If positive control samples are deemed unsatisfactory, even if the antibody is within the manufacturer’s printed expiration date, evaluation of the clinical specimen is aborted and the test deemed invalid. The quality of the primary antibody is therefore not based on an expiration date, but rather on its performance on a case-by-case basis with appropriate positive and negative control samples (Savage & DeYoung 2010).

Savage & DeYoung (2010) had previously examined the staining patterns of 26 recently acquired primary antibodies and their expired counterparts. Two reviewers examined sequential sections of formalin-fixed, paraffin-embedded tissue samples for staining intensity and percentage of positivity. Appropriate positive and negative control studies were performed. Of the 26 antibodies, 20 exhibited no difference in percentage of positivity or staining intensity. Of the remaining 6, 3 showed better performance with the expired cohort and 3 with non-expired antibodies. However, no antibody staining characteristics varied by more than 1 step and in no case was positive staining lost after antibody expiration. Negligible differences exist in immunostaining between outdated and current antibodies. They concluded that exemption for primary antibodies from existing regulations would conserve resources without adversely impacting patient care. 

Balaton et al (1999) compared 55 antibodies that had expired 6 to 134 months previously and found there was no significant difference observed in the specificity, sensitivity, and pattern of staining between old versus new antibodies.
Recently Maria Argentieri et al (2013) investigated whether the shelf-life of diagnostic antibodies was longer than the expiry date on the label. They had four independent laboratories test a small number of diagnostic antibodies kept at +4°C for 12?26 years, and found them to work perfectly on routine histology sections. Monoclonal antibodies originally supplied as culture supernatants or as ascites (neat or diluted), of all isotypes, as well as all of the polyclonal antibodies, produced satisfactory staining irrespective of their age. Notable exceptions were ammonium-precipitated, IgM or conjugated antibodies.

Drachenberg et al (2001) suggest that instead of a mechanical adherence to the manufacturers’ recommendations, a rational quality assurance system would be preferable.

References:
Argentieri, M. C., Pilla, D., Vanzati, A., Lonardi, S., Facchetti, F., Doglioni, C., ... & Cattoretti, G. (2013). Antibodies are forever: a study using 12?26‐year‐old expired antibodies. Histopathology, 63(6), 869-876.
Balaton, A. J., Drachenberg, C. B., Rucker, C., Vaury, P., & Papadimitriou, J. C. (1999). Satisfactory performance of primary antibodies beyond manufacturers' recommended expiration dates. Applied Immunohistochemistry & Molecular Morphology, 7(3), 221.
Drachenberg, C. B., Papadimitriou, J. C., Balaton, A. J., & Vaury, P. (2001). The total test approach to standardization of immunohistochemistry. Archives of pathology & laboratory medicine, 125(4), 471-471.
Savage, E. C., & DeYoung, B. R. (2010). Antibody Expiration in the Context of Resource Limitation What Is the Evidence Basis?. American journal of clinical pathology, 134(1), 60-64.


Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA) Principal Scientist, the Children’s Hospital at Westmead Adjunct Fellow, School of Medicine, University of Western Sydney
Tel: 612 9845 3306
Fax: 612 9845 3318
Pathology Department
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead Locked Bag 4001, Westmead NSW 2145, AUSTRALIA 

-----Original Message-----
From: Allan Wang via Histonet [mailto:histonet at lists.utsouthwestern.edu]
Sent: Tuesday, 6 December 2016 7:42 AM
To: histonet at lists.utsouthwestern.edu
Subject: [Histonet] RTU antibodies with 1+ year expiration date

Hello list,

Thanks for your responses to my previous dehydration question. I pretty much got conflicting responses about going straight to 100% alcohol, but I think I will just start them out in 80% alcohol to be safe.

Something that's been bugging me: Every source states that diluted antibodies shouldn't be kept for more than a few weeks. Yet Ventana dispensers of pre-diluted antibodies have expiration dates of 1 - 1.5 years, plus they sit out at room temperature for 5 hours at a time whenever they're used. Ventana provides user-fillable dispensers, but if we dilute antibodies ourselves and can't use them for more than a few weeks, it would be kind of pointless for low-volume needs.

Their specs say: "The antibody is diluted in 0.08 M PBS with 3% carrier protein and 0.05% ProClin 300, a preservative."
Isn't this pretty much the same stuff that antibody diluent is composed of, except with sodium azide instead of ProClin 300 (I think they're equivalent).

I'm already planning on doing some long-term testing of my own over a year with HER-2 and Ki67, but hopefully someone can provide some insight.

Thanks,
Allan Wang
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