Validation and Quality Control for Immunohistochemistry - National

NSH SymposiumSeattle, WAValidation and Quality Control for IHC

September, 2010

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Validation and Quality Control for Immunohistochemistry

Tim Morken, B.A. HTL(ASCP)Univ. California San Francisco Medical Center

[email protected]

Patsy Ruegg, HT(ASCP)QIHCIHCtech, LLC., Aurora, CO

[email protected]

NSH Symposium, Sept 2010Seattle, Washington

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Presentation Goals

1. Explain the term "Quality Control"

2. Discuss QC in the context of a "Quality System“

3. Explain how proper validation leads to quality

4. Outline an IHC Quality System

5. Institute Quality Improvement using QC data.

Upon completion of this presentation, participants will be able to:

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Presentation Outline

Quality Management System

CAP / CLIA Requirements

Assay Design Specifications

Assay Validation

Quality Control

BREAK

ER / PR / Her2 Validation

Open discussion


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Wall Street Journal, January 2008

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New York Times, April 2010

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New York Times, July 2010


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"Bad Cancer Tests Drawing Scrutiny"

Her2• When locally-tested cases were re-tested at a central lab:

• 14-16% False Positive• 18 - 23% False Negative• Reddy JC, et. Al. Clinical Breast Cancer, Vol 7, No. 2, 153-157, 2006

Estrogen Receptor• When locally tested cases were re-tested at a central lab:

• 1.8% False Positive• 70% False Negative• Viale G, et. Al, J Clin Oncol, Vol 25 No. 25, 3846-3852

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Key References

Quality Assurance For Immuncytochemistry: Approved Guideline• Clinical Laboratory Standards Institute (formerly NCCLS) publication MM4-

A, Vol. 19, No. 26, 1999

Quality Assurance in Anatomic Pathology (book)• Nakhleh and Fitzgibbons, Ed., College of American Pathologists, 2005

College of American Pathologists (CAP)• General and Anatomic Pathology Accreditation Checklists• Current edition is dated June 15, 2009 (as of August 2010)

Reference handout

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Quality Management

Quality Management

• Definitions

• Interaction


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CAP Checklists: Quality Management

General Laboratory Checklist:• GEN.13806 - 20369

• Quality Management Program

• Quality Monitoring

Anatomic Pathology Checklist• ANP.10000

• Is Quality Management Program Defined and Documented?

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College of American Pathologists

Three Definitions concerning Quality Management:• 1) Quality Assurance (QA)• 2) Quality Control (QC)• 3) Quality Improvement (QI)

Together these constitute a "Quality System"• Interaction of these elements leads to better quality• System must be "worked" to succeed.

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CAP Quality Assurance Definition

1) Quality Assurance• The practice of assessing performance in

all steps of the laboratory testing cycle including pre-analytic, analytic and post-analytic phases to promote excellent outcomes in medical care.


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… Deconstructed

"…assessing performance…"• Requires a set of performance standards• Requires comparison of results against the

standards

"…to promote excellent outcomes…"• Requires action if standards are not met

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CAP Quality Control Definition

2) Quality Control• An integral component of quality assurance

consisting of the aggregate of processes and techniques to detect, reduce, and correct deficiencies in an analytical process.

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… Deconstructed

"…processes and techniques to detect, reduce, and correct deficiencies…"

• "…Detect…": • Requires procedures that anticipate possible non-conformances.

• "…Reduce and Correct…": • Leads to procedures that reduce or eliminate confounding results.


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CAP Quality Improvement Definition

3) Quality Improvement

• The practice of continuously assessing and

adjusting performance using statistically and

scientifically accepted procedures.

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… Deconstructed

"…continuously assessing and adjusting…"• Requires regular review of results data• Investigation of, and correction of root cause

"..statistically and scientifically…"• Data trends over a time period• Hypothesis of root cause• Eliminating variables• Testing solutions

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Quality Management System Summary

Quality Assurance• Describe the quality process• Set standards

Quality Control• Institute methods to identify problems• Record and report results

Quality Improvement• Track problem trends identified by QC results• Use trend information to correct problems


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Validation Process

Validation• Definitions• Processes

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Validation and Quality Control

Validation• Assay Design Specification

• ValidationVerificationOptimizationStandardization

Quality Control• Controls identified by validation• Standardized procedures• Identification of process deficiencies

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Validation – Optimization - Standardization

Verification

Optimization

Standardization


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CAP IHC Checklist

ANP.• 12425 ASR disclaimer for report• 21850 Positive and Negative controls for immunofluorescence• 22250 Procedure for each antibody• 22300 Documented modifications for fixation other than formalin• 22500 Buffer pH monitoring• 22550 Positive control use• 22570 Negative control use (tissue, reagent)• 22615 Avidin/Biotin blocking/controls• 22660 Review/Recording of IHC results• 22750 Validation of new antibody (except ER, PR, Her2)• 22760 New lot validation for Antibody and Detection system• 22800 Automated IHC staining instrument maintenance records• 22900 Quality of IHC stains• 22997 Her2 validation (IHC and ISH)

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Validation Definition (1)

Establishing documented evidence which provides a high degree of assurance that a specific process will consistently produce a result or product meeting it's predetermined specifications and quality attributes

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"Establishing documented evidence…"• Documentation of validation testing is readily available

“…which Provides a high degree of assurance…"• Studying an adequate number of samples to give

confidence that the new or changed process will work in your laboratory


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"…that a specific process will consistently produce…"• Identifying areas of actual or potential weaknesses so

improvements can be made prior to implementation.

"…a result or product meeting it's predeterminedspecifications and quality attributes."

• Establishing acceptance criteria before initiating the validation study.

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Introducing a New Antibody

We want to introduce a new antibody…• Who wants it?

• What is it to be used for?

• When will it be used?

• Where will it be used?

• Why do we need it?• How will it be implemented?

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Initial Assay Design Specification

Write a Specification:• The need for the assay

• The "why's"• Identify special requirements• Identify suppliers• Determine the expected results• Determine the validation procedure• Optimize the assay• Develop the standard protocol


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Final Assay Design Specification

Final outcome:• Validated reagent

• Controls specified

• Specify the expected results

• Specify rejection criteria (tolerance)

• "QC Data Sheet" to guide interpretation

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Total Test Validation

Pre-Analytic• Acquisition, Fixation, Processing, Sectioning, Storage

Analytic• Optimization (HIER, Dilution, time, detection, sensitivity, specificity)• Technologist training• Instruments

Post-Analytic• Controls • Interpretation• Reporting• Pathologist performance

(Adapted from: Goldstein NS, et..al., Appl Immunohistochem Mol Morph 15(2):124-133)

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Validation: Pre-Analytic

Pre-Analytic example:• Acquisition: Range of time before sample is put in fixative

• Fixation: Type and Range of time in fixative

• Processing: Several processing schedules

• Sectioning: Section thickness, drying, heating temperature

• Storage: Range of time periods for unstained slide storage (retention of antigenicity), temperature

• Range of block storage conditions


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Validation, Analytic Phase Terms (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.

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Validation: Analytical

Validate Accuracy with typical cases• Sensitivity:

• Expression range of Positive cases, low to high (10-15 cases)

• Specificity• Positive verses expected negative cases/tissues (10-15 cases)

• Determine best controls• Range of expression, similar to expected cases (normal or disease?)• Preferably acquired and processed in your institution

Determine Precision (Reproducibility)• Intra-run: 10 slides in one run• Inter-run: 10 slides, Ten different runs with one slide each• Should have similar staining pattern and intensity on all slides

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Validation Analytic Terms Applied to IHC

Accuracy:• Compare results with New antibody to a previously validated antibody

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

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

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

Reportable Range• Establish a scoring system• Definition of a positive result• Criteria for rejection of the test


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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!

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

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Dilution series for Sensitivity (1)

Dilution series to determine sensitivity

1:25 1:50

1:100 1:200

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Dilution series for Sensitivity (2)

Dilution series to determine sensitivity

1:400

1:800

NEG CONTL


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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

(adapted from: Theoretical and Practical Aspects of Test Performance, inImmunomicroscopy, Taylor & Cote, 2005)

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Optimization Specificity

“Non-specific” means background or staining irrelvant cells/structures

1:400

1:800

NEGATIVE

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Validation: IVD Class I

IVD Class I, FDA exempt reagentsAncillary to one or more other tests

• Confirm vendor specification• Determine optimal protocol (HIER, Dilution, etc)• Determine acceptable controls• Determine acceptable results• Test on a “sufficient” series of positive and

negative tissues (ANP.22750)


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Validation: Class II

IVD Class II FDA reagentsPredictive markers (ER, PR, Her2)

• Confirm vendor specification• Determine optimal protocol• Determine acceptable controls• Determine acceptable results• Validate on mix of 20-40 (ER, PR) or 25 to 100

(Her2) or more known positive and negative cases• Compare to previously validated tissue samples

(Her2: adapted from Arch Pathol Lab Med. 2007; 131:18-43)(ER, PR: adapted from Arch Pathol Lab Med. 2010; 134(6):930-935)

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Validation: ASR

ASR: Analyte Specific Reagent (ANP.12425)“Active ingredient” of a testNot validated by vendor

• Vendor cannot indicate protocol to customer• Vendor cannot specify expected results• Laboratory is responsible for entire validation

• Validation procedure (CAP checklist: Method Validation Gen.42020 - 42160)

• Expected results• Statistically valid test cohort (number, type of cases)• Comparison to similar test (i.e., another antibody to same target)• Documented results

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Validation: RUO

RUO: Research Use Only

FDA Says: Laboratories should not use RUO reagents.

CLIA says: CLIA-certificated laboratories may use any reagent as long as CLIA validation procedures are followed (CAP is the deemed accrediting agency for Anatomic Pathology under CLIA)

CAP says: Nothing in current checklist about RUO’s.

If your laboratory decides to use an RUO reagent:

• Document unsuccessful search for IVD/ASR reagent• Follow ASR validation procedures: Comprehensive validation.• CAP Method Validation checklist: Gen.42020 - 42160


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Positive Control Tissue ANP.22550

Ideally, same specimen type as case tested• Not always possible, so validation documents acceptable tissue

• Remember, some antigens are decreased in tumor, not elevated

• Normal tissue should have consistent level of antigen • ( However, is “normal” tissue really normal?)

Fixed / processed with same procedures as sampleBest practice: put on same slide as sample

• However, one control per run, per antibody is acceptable.

Internal positive controls may be used,• Document for which cases/antibodies it is acceptable

Low expressers are ideal to avoid false negativesMulti-tissue blocks with positive and negative tissues are ideal

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Negative Control Tissue ANP.22570

Tissue that is known to lack the antigen• Multi-tissue blocks, may have positive and negative

tissues

• Tissue elements within the positive control or test samples that should be negative

• Separate single negative tissue slide.

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Optimization Overview

Verify Vendor Specifications• “to ascertain the truth or correctness of, as by examination,

research, or comparison”Vendor literature is a starting point

• Review the datasheet• Review references given by vendor • Literature search adds to knowledge• Compare with other vendors, users

Verify/Optimize:• Antigen Retrieval• Dilution• Control tissue• Expected results


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Optimization Method

Optimization• Vendor-supplied protocols are recommendations• Ideally work it into your regular protocol• Antigen retrieval type (Try several)

• None• Digestion (two or more types)• HIER (range of pH, temperature, time)

• Dilution series (bracket vendor recommendation)• Detection efficiency• Chromogen efficiency• Staining method (Manual or Automated)

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Multi-expression tissue

LOW

MED

HIGH

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Optimization Result

Write an optimized procedure for each antibody, including:

• .22250: Test Procedure • reagents, dilution, HIER, etc

• .22300: Fixation• .22550: Positive Control• .22570: Negative Control• .22625: Avidin-biotin Block (if necessary)


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Standardization

Inter-Laboratory

Intra-Laboratory

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Inter-Laboratory Standardization

Effort to standardize procedures for IHC• Studies show large variations in procedures and results between

laboratories (CAP, UK-NEQAS, NordiQC)

• Long standing effort to promote standardization• Largely failed in US due to lack of consequences

• UK-NEQAS has had success - took 20 years to achieve

• Oncologists, due to variable Her2 results, are driving current efforts

• Recommendations for Improved Standardization of Immunohistochemistry• Appl Immunohistochem Mol Morph 2007 15(2);124-133

• Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer, American Society of Clinical Oncology/College of American Pathologists Guideline,

• Arch Pathol Lab Med V.131 Jan 2007, pp.18-43.

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Standardization within the Lab

Standardization• Research current literature for acceptable protocol

and "best" results (also UK-NEQAS, NordiQC)• Once validated and optimized the procedures must

be followed:• Every time!!!• By everyone!!!

• Record and report deviations from procedure• Helps with troubleshooting• Train your staff to accept that mistakes or variations will happen,

and admitting them is the first step of good troubleshooting.


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Validation: Post-Analytical

Controls• Define correct Scoring / Interpretation of staining• Define rejection criteria• Define reporting parameters• Document pathologist interpretation and performance• Determine variations from standard over time

• Reagent performance• Interpretation

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Quality Control

Processes and techniques to detect, reduce and correct deficiencies in an

analytic process.

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Quality Control Interpretation

Detect Deficiencies• Positive Control Tissue

• Negative Control Tissue

• Positive Control reagent (primary antibody)

• Negative Control Reagent


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QC: Pos / Neg Tissue Controls

Control Tissue• Positive Tissue Control

• If True Positive: Proves the system worksTests specific reactivity of primary antibodyRange of expression levels help determine sensitivityTests detection system

• If Negative: indicates some part of the system did not work No indication of what failed!

• Internal positive control elements• Indicates True Positive staining• Indicates tissue quality (fixation, processing, storage effects)

• Negative Tissue Control (negative for antibody)• Indicates specificity of primary antibody (cross-reactivity)• Indication of detection system problems (background, etc)

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QC: Positive Reagent Controls

Positive Reagent Control: Primary Antibody• On Positive Control tissue

• Indicates primary antibody reactivity, specificity

• Must be positive on the positive control tissueControls with range of expression will indicate sensitivityInternal control on patient tissue help determine tissue reactivity

• Negative control tissue must be negative (ANP.22570)Internal negative elements (Positive control or sample)Separate tissue block known to be negative for antibodyAt least one negative tissue control per antibody

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QC: Negaqtive Reagent Controls

Negative Reagent Control: (ANP.22570)• Replace positive primary antibody with:

Pre-immune serum from same animal (very rarely available)Isotype-specific negative control antibodyIrrelevant primary antibody from same species (expensive)Non-immune whole serum from same species (most common)Antibody diluent onlyWash buffer only

• Result: Must be negative on positive and negative tissuesIndicates specificity (cross reaction)Indicates problems with detection system (non-specific binding)Indicates problems with blocking reagents (not working?)Indicates patient tissue problems (fixation, processing, etc)


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Negative Control Reagent Exception

Special exception (comment to ANP.22570) :• If running:

• Two or more blocks from the same specimen,

• Received at the same time,

• Processed at the same time,

• For the same antibody:

• Only need to run a negative control reagent slide on one block of that specimen.

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Quality Improvement

Quality Control Feeds Quality Improvement QC Results:

• Record results

• Track trends in deficiencies

• Open a Quality Improvement investigation for deficiencies identified.

• Determine a course of action to reduce or correct deficiency identified.

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Quality Improvement: Reduce Deficiencies

Pre-Analytic• Standardized Acquisition, fixation, processing• Criteria for rejection of a specimen or sample

Analytical• Procedures detail optimal test protocol

• Must be followed by everyone to be effective

Post Analytical• Interpretation guidelines

• Acceptance criteria• Rejection criteria


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Quality Improvement: Correct Deficiencies

"Correction" entails (for example)• Do not perform test with inadequate specimen• Repeating the test• Determining what caused the failure• Testing different processes• Testing different reagents

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QC Trend

Example of QC Trend

0 2 4 6 8 10 12 14 16 18 20

False Negative

Tissue fell off

Background

Wrong tissue

Prob

lem

Typ

e

IHC Problems

Count 201062

False NegativeTissue fell offBackgroundWrong tissue

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Quality Improvement

Review trends at "defined" intervals• Monthly? Quarterly? (GEN.20262)

Identify most common and most serious issuesDetermine plan of action to identify root causeDetermine action to resolveTest solution(s)Determine if issue is resolved

Review Quality Assurance System (GEN.20369)• Review annually and determine if system works• Determine if improvements are needed


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Key Indicators

GEN:20316:Does the QM program include monitoring key indicators of quality?

• Some are defined in the CAP checklist• Patient/specimen ID accuracy

• Specimen Acceptability

• Some identified by trend analysis.• i.e. Failure/Repeat rate of a particular test

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Break

Break!

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Estrogen and Progesterone Receptor

ASCO/CAP Recommendations, ER, PR• VALIDATION of ER, PR:• Fitzgibbons, et. al., Arch Pathol Lab Med, June 2010

Vol. 134(6):930-935• Tissue requirements• Validation methodology

• TESTING OF ER, PR:• Hammond, MEH, et. Al., Arch Pathol Lab Med, June

2010, Vol 134(6):907-922• Tissue procurement• Fixation (6- 72 hours)• Testing • Interpretation and reporting


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Estrogen and Progesterone Receptor Validation (1)

ASCO/CAP Recommendations• Validate against previously validated tissue samples

• Another laboratory that has validated its assay against clinical outcomes• Validated tissue samples from another lab that uses an FDA-approved assay

and has validated the assay using ASCO/CAP testing requirements• Tissue samples validated using a separate assay (genetic, ligand binding

assay)• Tissues used in a proficiency testing program• Validated tissues provided by an established program

• ≥90% concordance with Positive validated samples• ≥95% concordance with Negative validated samples

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Estrogen and Progesterone Receptor Validation (2)

ASCO/CAP Recommendations• Initial test validation of FDA-cleared assays

• ≥20 positive specimens (≥ 5 must be weakly positive)• ≥20 negative specimens• ≤ samples tested in one run (test samples in multiple runs, mutliple operators)

• Or Follow verification procedures in the Assay insert • Test must be used unmodified from manufacturers instructions

• Initial test validation of Laboratory Modified Assays (LMA)• If the lab modifies the test in ANY way a more thorough validation is called for• ≥40 Positive specimens ( ≥10 must be weakly postive)• ≥40 Negative specimens

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CAP Her2 Validation (1)

ANP.22997- Her2 test validation• Wolff AC, et. al., Arch Pathol Lab Med 2007;131:18-43 • 25 - 100 cases, mix of:

• Variety of expression levels• Negative cases

• Compare to a validated alternative method (one or more)• Other antibody• FISH

• Fixation validation, if non-formalin• Validation of each change in methodology


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CAP Her2 Validation (2)

ANP.22998 Documented procedure for length of fixation

• 10% Neutral-buffered formalin (NBF)

• Minimum 6 hours, maximum 48 hours (under review)

• Record duration of fixation

• Qualify negative results of specimens fixed over 48 hours, consider confirmatory FISH testing

• Outside referral documentation of fixation duration

ANP.22999 Does lab use ASCO/CAP her2 scoring criteria?

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Other Controls for IHC

Tissue arraysCell Culture slidesPeptide spots

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Tissue Arrays as Controls

Tissue arrays allow extensive testing on one slide• Small arrays for particular antibodies

• Lung, thyroid (pos), tonsil (neg) for TTF-1• Pantomics Universal Array: 12 tissues: 90% IVD antibodies

Brain, tonsil, colon, lung, thyroid, uterus, prostate, breast ca, placenta, melanoma, thymus/thymoma, skeletal muscle

• Larger arrays for validation• "FDA" array with 33 normal tissues, 40 tumors

Biochain, Pantomics, others• 10's or 100's of Ca-types / mix for pos / neg validation


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Array Size variety

Variety of arrays sizes/composition

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6595

150

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Predictive Test Validated Arrays

Her2, ER, PR, Validated arrays: 3+, 2+, 1+, Neg

Courtesy: Pantomics, Ltd, www.pantomics.com

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Graduated Expression Arrays

Low Medium High

ER

PR

HER2

Courtesy: Pantomics, Ltd, www.pantomics.com


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Cell Culture Controls

Genetically engineered expression level• Her2

• Estrogen / Progesterone Receptor

• Human papilloma virus

Fixed, processed in formalin/paraffin

Excellent for validation of tissue controls

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Cell Culture Control Stain

Positive control cells using no AR has more background in serum than when AR used

POSITIVE HER2 CELLS W/O AR POSITIVE HER2 CELLS W/ AR

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Peptide Spots

Peptide Spots (concept)• Peptide simulates epitope of native antigen• Peptides can be blended with several epitopes of different

antibodies

• Peptides do not degrade during deparaffinization and HIER

• Peptides can be produced synthetically in infinite amounts, withidentical quality

• Can help detect changes in antibody dilution and HIER


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Peptide Spots image

Peptide spots are positive or negative

NEG AR NEG NO AR

+ AR + NO AR

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Peptide Spots vs Tissue Array

Top: Peptide spots (2 rows)

Middle: Cultured Cells

Bottom: Multi-tissue arrays with range of epressions

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Peptide Controls in Practice

Peptide controls included with CAP 2006 Her2-B proficiency testing survey

• 18.3% sub optimal staining as judged by peptide control• 35% due to HIER errors• 20% due to antibody or staining protocol• 45% due to combination of the two

Vani K, et.al., Arch Pathol Lab Med. 2008;132;211-216


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Review

Quality Control is only part of the picture• QC is part of the Quality Management System

Quality Control is dependent on proper Validation• Validation determines protocols and controls

Quality Control feeds data to Quality Improvement• Trend analysis identifies problems

Questions?

Thank you.

Validation of Primary Antibody

Antibody Validation 1-1-2010 Page 1 of 6

Date: __________________ Project Inputs and Overall Design(s)

Name of Reagent:

Clone: Labeling* IVD ASR RUO

Proposed by Approved by

IVD = in vitro Diagnostic Device, FDA Approved; ASR = Anylate Specific Reagent, FDA regulated, RUO = Research Use Only, not FDA approved or regulated

Intended Use Diagnostic (IVD, ASR required) Research Immunohistochemistry Immunofluorescence In situ hybridization Others:

This product is intended for : Description of reagent:

Expected Staining Pattern:

Positive Control: Others: Others:

Sources of Input References Date

Company: Labeled: IVD ASR RUO Market Information Clone/Animal host: Company: Labeled: IVD ASR RUO Market Information Clone/Animal host: Company: Labeled: IVD ASR RUO Market Information Clone/Animal host: Title: Reference: Scientific Literature Conclusion: Title: Reference: Scientific Literature Conclusion: Title: Reference: Scientific Literature Conclusion:



Validation Design Input Describe the validation requirements

Platform (circle one) Dako Autostainer Leica Bond Ventana Ultra Manual Antibody Antigen Retrieval method Blocking regime Primary Dilution recommendation (initial trial) Primary antibody incubation time Detection system Chromogen Reproducibility testing None Inter-run (# slides_____) Intra-run (# slides_____) Control tissues: Tissue Case Number Positive element Additional testing required:

Approved by IHC Lead Technologist Date Medical Director, Immunohistochemistry Date

Design Output: First Trial Evaluation of Antibody

Reagent Source Catalog number Lot Number Date

Test (IHC, ISH, IF) Date Pass Fail Comments

See attached test records Validation/Verification Results Reagent does / does not match criteria detailed in design specification

Describe results:

Approved by IHC Lead Technologist Date Date Medical Director, Immunohistochemistry Date



Optimization Instructions (First Pass) Step Modification Note Date

Optimizaton Results

Test (IHC, ISH, IF) Modification Date Pass Fail Comments

Optimization Instructions (Second Pass)

Step Modification Note Date

Optimizaton Results


Optimization Instructions (Third Pass)

Step Modification Note Date

Optimizaton Results




Optimized Procedure

Platform (circle one) Dako Autostainer Leica Bond Ventana Ultra Manual Antibody Antigen Retrieval method Blocking regime Primary Dilution recommendation (initial trial) Primary antibody incubation time Detection system Chromogen Control tissues: Tissue Case Number Positive element(s) Attach list if extra control tissue necessary

Approved by Medical Director, Immunohistochemistry Date IHC Lead Technologist Date



Reproducibility Intra-Run reproducibility: 5 to 10 identical slides within one run

Test Date Pass Fail Comments IHC IHC IHC

See attached test records Inter-run reproducibility: 5 to 10 identical slides on 5 to 10 separate runs

Test Date Pass Fail Comments IHC IHC IHC

See attached test records Reproducibility approval Reagent does / does not meet reproducibility criteria

Approved by IHC Lead Technologist Date Medical Director, Immunohistochemistry Date



Design Validation Validation criteria Y N N/A Do results of internal and / or external (consultants / pathologist) testing meet the requirements and specifications of the reagent?

Are test results on panel of normal and tumor tissues acceptable?

Are reproducibility tests acceptable? Validation report:

Does reagent meet specification criteria?

Positive staining criteria:

Rejection criteria:

Comments:

Final Approval Medical Director, Immunohistochemistry Date Date

CoPath Entry

Name Abbreviation Search terms Description Label Text Entered in Copath Date: By:

Validation and Quality Control for Immunohistochemistry NSH Symposium

Seattle, WA September, 2010

References

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Guidelines and Accreditation Checklists Quality Assurance For Immuncytochemistry: Approved Guideline, Clinical Laboratory Standards Institute (formerly NCCLS), Wayne PA, USA, publication MM4-A, Vol. 19, No. 26, 1999. www.clsi.org College of American Pathologists, Commission on Laboratory Accreditation. College of American Pathologists, Northfield, IL, USA, www.cap.org

September 27, 2007 Edition is the current edition in use (as of April, 2009) General Laboratory Checklist

Anatomic Pathology Checklist Clinical Laboratory Improvement Amendments (CLIA), Centers for Medicare and Medicaid Services, CMS-2226-F: 42 CFR 493 Interpretive Guidelines for Laboratories, Appendix C, Subpart K (Quality systems). www.cms.hs.gov/CLIA/03_interpretive_guidelines_for_laboratories.asp Books Quality Management in Immunohistochemistry, Brown RW, in Quality Management in Anatomic Pathology, Nakhleh RE, and Fitzgibbons PL, Eds. College of American Pathologists, Northfield, IL, USA, 2005. www.cap.org. Theoretical and Practical Aspects of Test Performance, in Immunohistology: A Diagnostic Tool for the Surgical Pathologist. 3rd. Ed., Volume 19 in Major Problems in Pathology, Taylor CR and Cote RJ, Eds., W.B Saunders, Philadelphia, 2005 Immunohistochemistry Quality Control, Hladik, CL and White, CL, in Theory and Practice of Histological Techniques, 4th Ed., Bancroft JD and Gamble M, Eds., Churchill Livingstone, 2007 Techniques of Immunohistochemistry: Principles, Pitfalls and Standardization, Taylor CR, et.al., in Diagnostic Immunohistochemistry, Dabbs, DJ, Ed., 2nd Edition, Churchill Livingstone, Selected Literature Validation and Standardization Tissue Preparation for Immunocytochemistry, Williams, JH, et.al., J Clin Pathol 1997;50:422-428 Conditional Epitopes: Is Your Antibody Always Specific?, Willingham, MC, J Histochem Cytochem, Vol. 47(10):1233-1235, 1999 Recommended Policies for Uses of Human Tissue in Research, Education, and Quality Control, Grizzle, W, et.al., Arch Pathol Lab Med, 1999; 123: 296-300



References

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Specificity Controls for Immunocytochemical Methods, Burry, RW, J Histochem Cytochem, Vol. 48(2): 163-165, 2000 A Practical Approach for Evaluating New Antibodies in the Clinical Immunohistochemistry Laboratory, Hsi, ED, Arc Pathol Lab Med. 2001; 125: 289-294 Immunohistochemistry for the Age of Molecular Morphology, Taylor, CR, editorial, Appl Immunohist Mol Morph, 9(1): 1-2, 2001 Standardization in Immunohistochemistry, O’Leary, TJ, Appl Immunohistochem Mol Morph, 9(1): 3-8, 2001 Immunohistochemical Detection of Interferon-γ: Fake of Fact?, Van der Loos, CM, et.al., J Histochem Cytochem 2001;49(6):699-709 Progesterone receptor by immunohistochemistry and clinical outcome in breast cancer: a validation study, Mohsin, SK, et.al., Mod Path (2004) 17, 1545-1554 Can a More Selective Application of Antigen Retrieval Facilitate Standardization in Immunohistochemistry?, Boenisch, T, Appl Immunohistochem Mol Morph, Vol. 12, No. 2, 172-176, June 2004 Technical Aspects of Immunohistochemistry, Ramos-Vara, JA, Vet Pathol 42:405-426 (20050 A Comparison of Immunohistochemical Stain Quality in Conventional and Rapid Microwave Processed Tissues, Emerson, LL, et.al., Am J Clin Pathol 2006; 125:176-183 Recommendations for Improved Standardization of Immunohistochemistry, Goldstein, NS, et.al., and members of Ad-Hoc Committee on Immunohistochemical Standardization, Appl Immunohistochem Mol Morph, 2007 15(2): 124-133 The use of positive controls in immunohistochemistry – Some indicators of what is appropriate and what is not, Immunocytochemistry 2007, vol. 6, Issue 1: 11-12, UK NEQAS for Immuncytochemistry and FISH, 2007 Suggested guidelines for Immunohistochemical techniques in veterinary diagnostic laboratories, Ramos-Vara, JA, et.al. J Vet Diagn Invest 20:393-413 (2008) Controls for IHC, NordiQC, www.nordiQC.org/techniques/controls_sections.htm, 2008 Sense and Sensitivity, Wikipedia, www.wikipedia.org/wiki/sensitivity_(tests) A good overview of the subject. Antibody Validation (Review). Bordeaux J, et. al., BioTechniques 48:197-209 (March 2010) Test Validation: A Brave new World for Anatomic Pathology, Brown, RW and Sharkey, FE, May 19, 2010, 2010 Laboratory Accreditation Program, College of American Pathologists (www.cap.org) Quality Assurance / Quality Control Interlaboratory quality assurance of Immunohistochemical procedures. Recommended practices for daily application, Rickert RR, Maliniak, RM, Arch Pathol Lab Med 1989 Jun;113(6):673-9. Comparative quality assessment in immunocytochemistry: pilot study of CD15 staining in paraffin wax embedded tissue in Hodgkin’s disease, Angel CA, et.al., J Clin Pathol 1989;42:1096-1100.

http://www.wikipedia.org/wiki/sensitivity_(tests



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Quality control in immunohistochemistry. Report of a workshop sponsored by the Biological Stain Commission, Elias JM, et.al., Am J Clin Pathol 1989 Dec;92(6): 836-43. The Taming of Immunohistochemistry: The New Era of Quality Control, Herman GE and Elfont EA, Biotech & Histochem, 1991;66(4):194-9 Quality control in immunohistochemistry: Experiences with the estrogen receptor assay, Bosmon FT, et.al., J Clin Pathol 1992;45:120-124 Audit and internal quality control in immunohistochemistry, Maxwell P and McCluggage WG, J Clin Pathol 2000; 53:929-932 The Total Test Approach to Standardization of Immunohistochemistry, Editorial, Taylor CR, Arch Pathol Lab Med – 2000;124:945-951 Recommendations for Quality Assurance and Improvement in Surgical Pathology and Autopsy Pathology, Association of Directors of Anatomic and Surgical Pathology, Am J Clin Path 2006;126:337-340 Technical variations in prostatic immunohistochemistry: need for standardization and stringent quality assurance in PSA and PSAP immunostaining, Varma M, et.al., J Clin Pathol 2004;57:687-690 Controls for Immunohistochemistry: Is “Brown” Good Enough?, Editorial, Ward JM, Toxicologic Pathology, 32:273-274, 2004. The Quality Systems Approach, Nevalainen DE, Arch Pathol Lab Med, 1999;123: 566-568 Evaluating Laboratory Performance on Quality Indicators With the Six Sigma Scale, Nevalainen DE, et.al., Arch Pathol Lab Med April 2000;124:516-519 Digital Image Documentation for Quality Assessment, Cruz D, et.al., Arch Pathol Lab Med Nov 2001; 125:1430-1435 Blinded Review as a Method for Quality Improvement in Surgical Pathology, Renshaw AA, et.al., Arch Pathol Lab Med, 2002;126:961-963 Quality Assurance in Immunohistochemistry, Results of an Interlaboratory Trial Involving 172 Pathologists, Rudigerf T, et.al., Am J Surg Pathol 26(7):873-882, 2002 Source of Error in Laboratory Medicine, Matlow AG, et.al, Lab Med June 2004, Vol. 35, No. 6: 331-334 Quality Systems – the Role of People, Passiment E., LabMed 2005;36(10): 630-632 Reducing Errors in the Practices of Pathology and Laboratory Medicine, Novis DA and Konstantakos G, Am J Clin Pathol 2006;126(Suppl):S30-S35 What is quality in surgical pathology?, Nakhleh RE, J Clin Pathol 2006;59:669-672 My approach to internal quality control in a clinical immunology laboratory, Lock RJ, J Clin Pathol 2006;59:681-684 Diagnostic Immunohistochemistry: What can Go Wrong?, Yaziji, H and Barry T, Adv Anat Pathol, Vol. 13, No. 5: 238-246, Sept 2006



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The Henry Ford Production System, Measures of Process Defects and Waste in Surgical Pathology as a Basis for Quality Improvement Initiatives, D’Angelo, R, et.al., Am J Clin Pathol 2007;128:423-429 Quality Management in Immunohistochemistry, Eisen RN, Diag Histopath 14(7):299-307 2008 External Quality Assessment in Immunohistochemistry – Is It the Solution to a Complex Problem?, Editorial, Reiner-Concin A, Breast Care 2008;3:78-79 Quality control in molecular immunohistochemistry, True LD, Histochem Cell Biol (2008) 130:473-480 Breast Carcinoma-specific QC Estrogen Receptor Reliability of Immunohistochemical demonstration of oestrogen receptors in routine practice: interlaboratory variance in the sensitivity of detection and evaluation of scoring systems, Rhodes A, et.al., J Clin Pathol 2000;53:125-30 The effect of fixation and processing on the sensitivity of oestrogen receptor assay by immunohistochemistry in breast carcinoma, Lee H, et.al., J Clin Pathol 2002;55:236-238 Minimum Formalin Fixation Time for Consistent Estrogen Receptor Immunohistochemical Staining of Invasive Breast Carcinoma. Goldstein NS, et. al., Am J Clin Pathol 2003;120:86-92 Estrogen Receptor Testing of Breast Cancer in Current Practice: What is the Question?, Schnitt SJ, J Clin Oncol, 2006;24(12):1797-1799 Frequency and reliability of oestrogen receptor, progesterone receptor and HER2 in breast carcinoma determined by immunohistochemistry in Australasia: results of the RCPA Quality Assurance Program, Francis GD, et.al., J Clin Pathol 2007;60:1277-1283 Effects of Fixation, Processing and Evaluation Criteria on Immunohistochemical Detection of Hormone Receptors in Breast Cancer, Oyama, T, et. al., Breast Cancer 14:182-188, 2007 Prognostic and Predictive Value of Centrally Reviewed Expression of Estrogen and Progesterone Receptors in a Randomized Trial by Comparing Letrozole and Tamoxifin Adjuvant Therapy for Postmenopausal Early Breast Cancer: BIG 1-98, Viale G, et.al., J Clin Oncol 25:3846-3852. 2007 Comparison of Evaluations for Hormone Receptors in Breast Carcinoma Using Two Manual and Three Automated Immunohistochemical Assays, Arihiro, K, et.al., Am J Clin Pathol 2007;127:356-365. Bad Cancer Tests Draw Scrutiny, Anne Wilde Mathews, Wall Street Journal, January 4, 2008. Commentary: Hormone Receptor Testing in Breast Cancer: A Distress Signal from Canada, Allred, DC, The Oncologist, 13; 1134-1136, Nov 2008 Consensus Recommendations on Estrogen Receptor Testing in Breast Cancer By Immunohistochemistry, Yaziji, H, et.al., including Members of the Standardization Ad-Hoc Consensus Committee, Appl Immunohistochem Mol Morphol, Vol. 16, No. 6, Dec 2008



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American Society of Clinical Oncology/College of American Pathologists Guideline Recommendations for Immunohistological Testing of Estrogen and Progesterone Receptors in Breast Cancer. = Recommendations for Validating Estrogen and Progesterone Receptor Immunohistochemistry Assays, Fitzgibbons PL, et. al., Arch Pathol Lab Med. 2010; 134(6): 930-935 Her2 Specific QC HER2 testing in breast cancer: NCCN Task Force report and recommendations, J Natl Compr Canc Netw. 2006 Jul;4 Suppl 3:S1-S22 Concordance between central and local laboratory HER2 testing from a community-based clinical study, Reddy JC, et.al., Clin Breast Cancer 2006 Jun;7(2):153-7 Effects of Fixative and Fixation protocols on Assessment of Her2/neu Oncogene Amplification Status by Fluorescence In Situ Hybridization, Willmore-Payne, C, et.al., Appl Immunohistochem Mol Morph 2007;15:84-87 American Society of Clinical Oncology / College of American Pathologists Guideline Recommendations for Human Epidermal Growth Factor Receptor 2 Testing in Breast Cancer, Wolff AC, et.al., Arch Pathol Lab Med 2007;131:18-43 The Need for a Quality Control or the Whole Process of Immunohistochemistry Human Epidermal Growth Factor Receptor 2/neu Determination: A United Kingdom National External Quality Network for Quality Assessment Service / Italian Network for Quality Assessment of Tumor Biomarkers Pilot Experience, Paradiso A, et.al., Letter to Editor, J Clin Pathol 2007;25(22) HER2 Testing in A Population-Based Study of Patients with Metastatic Breast Cancer Treated with Trastusumab, O’Malley FP, et.al., Arch Pathol Lab Med 2008;132:61-65 Implementation of American Society of Clinical Oncology/College of American Pathologists HER2 Guideline Recommendations in a Tertiary Care Facility Increases HER2 Immunohistochemistry and Fluorescence In Situ Hybridization Concordance and Decreases the Number of Inconclusive Cases. Middleton LP, et. al., Arch Pathol Lab Med. Vol. 133:775-780. Controls Tissue Array Controls The multi-tumor (sausage) tissue block. Novel method for immunohistochemical antibody testing. Battifora H, Lab Invest 1986;55:244-8 Tissue Array Technology for Testing Interlaboratory and Interobserver Reproduciblity of Immunohistochemical Estrogen Receptor Analysis in a Large Multicenter Trial, von Wasielewski R, et.al., Am J Clin Pathol 118(5):675-682 Assessment of Interlaboratory variation in the Immunohistochemical determination of estrogen receptor status using a breast cancer tissue microarray, Parker RL, et.al., Am J Clin Pathol 2002 May;117(5):723-8 Tissue microarrays for predictive molecular pathology, G Sauter and M Mirlacher, J Clin Pathol 2002;55:575-576



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Tissue microarrays: a new approach for quality control in immunohistochemistry, Packeisen J, et.al., J Clin Pathol 2002;55:613-615 Demystified: Tissue microarray technology, Packeisen, J, et.al., J Clin Pathol: Mol Pathol 2003;56:198-204 Miniature tissue microarrays for HercepTest® standardization and analysis, Gulmann C, et.al., J Clin Pathol 2004;57:1229-1231 The CD117 Immunohistochemistry Tissue Microarray Survey for Quality Assurance and Intralaboratory Comparison, Dorfman DM, et.al., A College of American Pathologists Cell Markers Committee Study, Arch Pathol Lab Med 2006;130:779-782 Clinical Validation of Breast Cancer Biomarkers Using Tissue Microarray Technology, Quraishi I, et.al., Appl Immunohistochem Mol Morph 2007;15:45-49



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Cell Culture Controls Her2 A formalin-fixed, paraffin-processed cell line standard for quality control of Immunohistochemical assay of HER-2/neu expression in breast cancer, Rhodes A, et.al., Am J Clin Pathol 2002 Jan;117(1):7-8 Available from Dako www.dakousa.com, slides (available in kits only?) Ventana Medical www.ventanamed.com, slides Invitrogen www.invitrogen.com, slides or block available QC Sciences www.qcsciences.com slides Estrogen and Progesterone Receptor Dako (available in kits only?) Invitrogen Human papilloma virus QC Sciences, slides Invitrogen, slides or block Peptide Controls Synthetic Peptides Identified from Phage-displayed Combinatorial Libraries as Immunodiagnostic Assay Surrogate Quality-Control Targets, Sompuram SR, et.al., Clin Chem 2002;48(3):410-420 A Novel Quality Control Slide for Quantitative Immunohistochemistry Testing, Sompuram SR, et.al., J Histochem Cytochem 2002; 50(11):1425-1433 National HER2 Proficiency Test Results Using Standardized Quantitative Controls, Vani, K, et.al., Arch Pathol Lab Med 2008;132:211-216 Quality Assessment Organizations College of American Pathologists (USA). www.cap.org, Several quality management tools including Q-probes, Anatomic Pathology Surveys/IHC, Anatomic Pathology Education programs with IHC, HistoQIP program. United Kingdom National External Quality Assessment Scheme (UK-NEQAS), www.ukneqasicc.ucl.as.uk, Of great interest are the downloadable pdf’s of the UK-NEQAS Immunocytochemistry Journal, which discusses in detail the results of the IHC surveys the UK-NEQAS conducts each year. www.ukneqasicc.ucl.as.uk/neqasicc.shtml Nordic Quality Control. www.nordiqc.org. A quality control survey program of the Nordic countries. Extensive reviews of antibodies, procedures and suggestions for IHC.

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Validation and Quality Control for Immunohistochemistry - National

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