[Histonet] New CAP question ANP.22760

Wed Jun 23 11:48:48 CDT 2010

Joe,

You wrote : The folks in the 'clinical' lab have been performing more comprehensive and complex validation procedures for a very long time ..."

Those were my thoughts exactly. While the person replying may or may not have specific histology experience she will have clinical lab experience (however, my guess is that she is exposed to histology regularly at CAP). Clinical labs have a bit of an easier time, actually, because they validate primarily to known concentration controls - analytical controls manufactured at a range of known concentrations for instance. The institution then adds in their normal controls for validation.

As far as the current question about validating a new lot of reagent the best practice is to run parallel tests on the same machine. If that is not easily possible on a particular manufacturer's instrument then the question should be asked of them: Why not? If this is a requirement the manufacturer should provide an easy path to meeting the requirement. However, if that is not the case then the institution simply writes a procedure to get around the inadequacies of the instrument (Maybe the vendor can help with that). Then follow the procedure. That should satisfy inspectors.

An "...appropriate panel of tissues..." is whatever the institution deems appropriate for the given antibody or reagent. This is a perfect place for tissue arrays. You can make your own or buy them.

IHC must meet CLIA validation guidelines but since IHC is generally qualitative the requirements must be understood and methods adapted to a qualitative scenario. Several IHC and Histotechnology books discuss the subject at length (Taylor, Dabbs, Bancroft for instance).

Below is a brief overview of how to do that. (for more in-depth info this was covered in an NSH teleconference I gave last year - PowerPoint, audio and references available from NSH-, and will be covered in a similar workshop at NSH in Seattle this year).

1)
CAP General Validation
CAP GEN.42020-42163 Test Method Validation Follows CLIA CFR Sec 493.1253 Does not apply well to IHC (IHC is usually qualitative)

But the general principle applies:
The laboratory must have data on each test's accuracy, precision, analytic sensitivity, interferences and reportable range.

Unmodified FDA-cleared or approved tests:  the lab may use manufacturer information or published reports but lab must verify outside data.

Non-FDA cleared: Lab MUST verify or establish analytic accuracy, precision, sensitivity, specificity and reportable range.

2) Validation includes:
Accuracy:
        Compare results with New antibody to a previously validated antibody    on the same tissues

Precision:
        Test samples with varying antigen expression
        Intra-run, Inter-run tests, 10 slides each (reproducibility)

Sensitivity:
        True Positive vs False Negative (higher % FN = less sensitive)

Interferences [Specificity]:
        True Negative vs False Positive (Higher % FP = less specific)
        Delineate what could interfere to give a false positive or false        negative result.

Reportable Range
        Establish a scoring system
        Provide the definition of a positive result

3)Sensitivity

Analytic Sensitivity:
        Lowest amount of substance detectable by the test
        Can only be done with controls of known concentration

Diagnostic Sensitivity:
        Ability of the test to determine true diagnostic positive verses false  negative (higher % FN = less sensitive)
        Requires comparison to a previously validated antibody

IHC Sensitivity:
        Extent to which an antibody can be diluted and still achieve target     recognition. NOTE: This is determined by antibody AND detection         system!

4) Specificity:

Analytic Specificity
        Accuracy on tests of known positive and negative controls
        Controls of known concentration
        Determine what could "Interfere" to confound the result

Diagnostic Specificity
        Ability of a test to determine true diagnostic negative verses false    positives (Higher % FP = less specific)
        Requires comparison to a previously validated antibody

IHC Specificity
        Ability of an antibody to bind exclusively to its particular antigen    in the absence of staining of other molecules
        Or, staining of other structures in addition to target  structures/cells

(Sensitivity and Specificity adapted from: Theoretical and Practical Aspects of Test Performance, in Immunomicroscopy, Taylor & Cote, 2005)

Tim Morken
Supervisor, Histology / IPOX
UCSF Medical Center
San Francisco, CA

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of JMyers1 <@t> aol.com
Sent: Tuesday, June 22, 2010 6:51 PM
To: tjasper <@t> copc.net
Cc: histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] New CAP question ANP.22760

Tom:

As much as I agree with your acknowledgment that its seems a bit odd for
the CAP to have a blood-banker responding to AP-related issue, I'm actually
not surprised.  The folks in the 'clinical' lab have been performing more
comprehensive and complex validation procedures for a very long time, and they
wonder why IHC isn't expected to follow the same requirements as chemistry,
immunology, etc. -- IHC is, after all, an awful lot like ELISA.  And
rightfully so, because IHC is, under CLIA (which supersedes CAP), considered
highly-complex, non-waived testing -- and is, therefore, subject to the same
Quality Systems regulations (in particular, 42CFR493.1252-1256, 1273, and 1281) as
the testing performed in other areas of the lab.

Could it be that, because AP produces qualitative results that are
interpreted by a pathologist and CP produces quantitative results that are
interpreted by an analyzer, we somehow think that CLIA rules don't apply to IHC?  I
certainly don't have the answer to that, but it make me wonder what the
future holds.  As witnessed by some of the newest CAP 'standards' (including the
question in question...no pun intended), e.g. ER/PR, where a minimum of 20
positive and 20 negative specimens must be tested, and where 10 of the
positives must be weakly positive -- an acknowledgment that validation specimens
must be carefully selected in order to obtain appropriate results), it
certainly doesn't appear that the regulation of IHC testing is going to become
more relaxed.

Joe Myers, M.S., CT(ASCP)

------------------------------

Message: 12
Date: Fri, 18 Jun 2010 12:38:07 -0700
From: "Thomas Jasper" <tjasper <@t> copc.net>
Subject: RE: [Histonet] New CAP question ANP.22760
To: "Mark Tarango" <marktarango <@t> gmail.com>
Cc: _histonet <@t> lists.utsouthwestern.edu_
(mailto:histonet <@t> lists.utsouthwestern.edu)

Mark,

Did you notice the credentials from this CAP representative? MT with a
Blood Bank specialty I believe.  What I glean from that is...more than
likely this person does not grasp the logistics of "contemporaneously"
staining identical Abs from separate lots.  She also likely does not
understand the logistical application for detection and automation
either.

I'm not trying to be overly critical of this person.  I'm sure she is
quite intelligent and would not have the MT/SBB if she wasn't
intelligent.  It comes down to a lack of understanding Anatomic
Pathology testing application re: automated IHC.  I believe this is a
common problem in and out of CAP. Many lab directors and other folks in
positions of authority without AP/Histology/Cytology backgrounds seem to
believe that broad clinical lab modalities apply to Anatomic Path
scenarios.  I used to refer to this in my former position as - "Trying
to put the yoke of clinical lab onto anatomic path."  We are
laboratorians, but in many instances do not fit the general clinical lab
mold.

It's unfortunate that CAP has put this person in the position to
respond.  It is apparent to me that she's not grasping the particulars
here.  She probably never will unless she decides to go into a working,
automated IHC "tissue" lab and take the time to ask questions and
understand (learn) what we're all about.

Thanks,
Tom Jasper

Thomas Jasper HT (ASCP) BAS
Histology Supervisor
Central Oregon Regional Pathology Services
Bend, OR 97701
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