[Histonet] IHC pos. & neg. control question

[Shea's]

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