[Histonet] negative controls on immunos

Angela Bitting akbitting <@t> geisinger.edu
Wed Mar 2 10:42:45 CST 2011

Fun no, impractical yes.

>>> "Weems, Joyce" <JWeems <@t> sjha.org> 3/2/2011 10:30 AM >>>
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

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

1.      Multitissue blocks.  These can provide simultaneous positive
and negative tissue controls, and are considered "best practice" (see
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;
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
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 
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