[Histonet] IHC pos. & neg. control question

Tue May 24 23:12:37 CDT 2011

This is my understanding about this CAP checklist item - 

CAP is looking for 2 types of negative controls:

1. Negative reagent control - this is satisfied by running a negative control slide from the same patient block through the whole process without the primary antibody.

2.  Negative tissue control - this is satisfied by running a slide of multi tissue control containing tissues that are positive for the primary antibody and tissues that are negative for the primary antibody.  The negative tissue on the multi tissue control will serve as the negative tissue control  for the specific antibody.  If you look at the positive control and the patient tissue only the areas that contain the target antigen will stain.  There are other elements in the tissue that are known negative (e.g. smooth muscle in the small blood vessels will not stain with CD3 - then the smooth muscle in either the positive control and the patient tissue would serve as an internal negative tissue control for CD3).  This negative result on the tissue elements that are expected not to stain with a specific antibody should be negative - hence - negative tissue control and should be noted in the documentation (preferably in the final report when commenting on the results of IHC stains).

At Emory, we just had our CAP inspection (we only had a couple of deficiencies - xylene monitoring in the IHC area,  Space and one corrected on site - labeling of chemicals without manufacturer's supplied expiration dates).  Prior to inspection, we got clarification from CAP on this checklist item.  CAP prefers the use of multi tissue control (containing both positive and negative tissue elements), however using negative tissue elements in the positive control and patient tissue are acceptable as negative tissue control for the specific antibody and it should be noted / documented accordingly.  We document the negative control in our final reports when commenting on the results of IHC stains.

This is my two cents contribution to the discussion.

Godfrey

> From: jshea121 <@t> roadrunner.com
> To: histonet <@t> lists.utsouthwestern.edu
> Date: Thu, 19 May 2011 21:11:01 -0400
> Subject: [Histonet] IHC pos. & neg. control question
> 
> Curt is right, according to CAP ...
> 
> ANP.22570 QC - Antibodies Phase II
> 
> Appropriate negative controls are 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.
> 
> Evidence of Compliance:
> 
> ? Written procedure for the use of negative reagent and tissue controls for IHC AND
> 
> ? Patient reports or worksheet with control results
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