[Histonet] negative controls on immunos

Weems, Joyce JWeems <@t> sjha.org
Wed Mar 2 09:30:02 CST 2011

The CAP guidelines are pretty clear.  Copied from latest checklist.. Isn't this fun???????????!!!!!!!!! j:>)

ANP.22570       Phase II        N/A  YES  NO

Are appropriate negative controls used?

NOTE:  Negative controls must assess the presence of nonspecific staining in patient tissue as well as the specificity of each antibody. Results of controls must be documented, either in internal laboratory records, or in the patient report. A statement in the report such as, "All controls show appropriate reactivity" is sufficient.

A negative reagent control is used to assess nonspecific or aberrant staining in patient tissue related to the antigen retrieval conditions and/or detection system used.  A separate section of patient tissue is processed using the same reagent and epitope retrieval protocol as the patient test slide, except that the primary antibody is omitted, and replaced by any one of the following:

■       An unrelated antibody of the same isotype as the primary antibody (for monoclonal primary antibodies)
■       An unrelated antibody from the same animal species as the primary antibody (for polyclonal primary antibodies)
■       The negative control reagent included in the staining kit
■       The diluent/buffer solution in which the primary antibody is diluted

In general, a separate negative reagent control should be run for each block of patient tissue being immunostained; however, for cases in which there is simultaneous staining of multiple blocks from the same specimen with the same antibody (e.g., cytokeratin staining of multiple axillary sentinel lymph nodes), performing a single negative control on one of the blocks may be sufficient provided that all such blocks are fixed and processed identically.  This exception does not apply to stains on different types of tissues or those using different antigen retrieval protocols or antibody detection systems.  The laboratory director must determine which cases will have only one negative reagent control, and this must be specified in the department's procedure manual.

The negative reagent control would ideally control for each reagent protocol and antibody retrieval condition; however, large antibody panels often employ multiple antigen retrieval procedures. In such cases, a reasonable minimum control would be to perform the negative reagent control using the most aggressive retrieval procedure in the particular antibody panel.  Aggressiveness of antigen retrieval (in decreasing order) is as follows:  pressure cooker; enzyme digestion, boiling; microwave; steamer; water bath.  High pH retrieval should be considered more aggressive than comparable retrieval in citrate buffer at pH 6.0.

It is also important to assess the specificity of each antibody by a negative tissue control, which must show no staining of tissues known to lack the antigen.  The negative tissue control is processed using the same fixation, epitope retrieval and immunostaining protocols as the patient tissue. Unexpected positive staining of such tissues indicates that the test has lost specificity, perhaps because of improper antibody concentration or excessive antigen retrieval. Intrinsic properties of the test tissue may also be the cause of "non-specific" staining.  For example, tissues with high endogenous biotin activity such as liver or renal tubules may simulate positive staining when using a detection method based on biotin labeling.

A negative tissue control must be processed for each antibody in a given run.  Any of the following can serve as a negative tissue control:

1.      Multitissue blocks.  These can provide simultaneous positive and negative tissue controls, and are considered "best practice" (see below).
2.      The positive control slide or patient test slides, if these slides contain tissue elements that should not react with the antibody.
3.      A separate negative tissue control slide.

The type of negative tissue control used (i.e., separate sections, internal controls or multitissue blocks) should be specified in the laboratory manual (refer to ANP.22250).

Multitissue blocks may be considered best practice and can have a major role in maintaining quality.  When used as a combined positive and negative tissue control as mentioned above, they can serve as a permanent record documenting the sensitivity and specificity of every stain, particularly when mounted on the same slide as the patient tissue.  When the components are chosen appropriately, multitissue blocks may be used for many different primary antibodies, decreasing the number of different control blocks needed by the laboratory.  Multitissue blocks are also ideal for determining optimal titers of primary antibodies since they allow simultaneous evaluation of many different pieces of tissue.  Finally, they are a useful and efficient means to screen new antibodies for sensitivity and specificity or new lots of antibody for consistency, which should be done before putting any antibody into diagnostic use.

1)      Leong AS-Y, Cooper K, Leong FJW-M. Manual of Diagnostic Antibodies for Immunohistology. 2nd ed. London: Greenwich Medical Media; 2003
2)      Dabbs DJ. Diagnostic Immunohistochemistry. New York: Churchill Livingstone; 2002
3)      Burry RW. Specificity controls for immunocytochemical methods. J Histochem Cytochem 2000;48:163-166 

Joyce Weems 
Pathology Manager 
Saint Joseph's Hospital 
5665 Peachtree Dunwoody Rd NE 
Atlanta, GA 30342 
678-843-7376 - Phone 
678-843-7831 - Fax 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Greg Dobbin
Sent: Wednesday, March 02, 2011 09:15
To: Diana McCaig; histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] negative controls on immunos

Yes, it is always best to run every conceivable control available. Then you can be really, really, really sure. However, if you have practical issues that come into play such as cost of reagents, or extra controls taking up valuable space on the stainer then you might have to think about what you are trying to accomplish with each control and balance the cost vs benefit.    So what are we checking with the negative control? We are checking the reagents to ensure that there is no non-specific reaction arising from the detection kit or the buffers, etc. If you are running the same block tomorrow with the same detection kit (ie same lot) it is not necessary (in my humble opinion) to check it again with another negative control.    I suppose if you are worried that someone could be trying to sabotage your work by sneaking into your lab at night and contaminating your detection kit...then the simplest way to detect this sinister activity would be to run a negative control every time. Otherwise, I think one negative control slide per block per detection kit will be adequate.   Disclaimer: Please know that I have just tried to inject a little humor into this response. I am really not trying to be sarcastic in anyway. Cheers! Greg    Greg Dobbin, R.T. Chief Technologist, Anatomic Pathology Dept. of Laboratory Medicine, Queen Elizabeth Hospital, P.O. Box 6600 Charlottetown, PE    C1A 8T5 Phone: (902) 894-2337 Fax: (902) 894-2385   "I find that the harder I work, the  more luck I seem to have." - Thomas Jefferson   >>> "Diana McCaig" <dmccaig <@t> ckha.on.ca> 3/1/2011 2:07 PM >>> If you do a run of several immunos today and you run a negative control, and there is a request for additional immunos tomorrow, would you run another negative control with the additional slides.  They are being stained on a stainer and not manually,    diana  _______________________________________________ Histonet mailing list Histonet <@t> lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet 
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