QA for IHC and ISH USE.pdf

QUALITY ASSURANCE,
QUALITY CONTROL AND
EXTERNAL QUALITY
ASSESSMENT
Anthony Rhodes, PhD, FRCPath, FIBMS
(UK NEQAS Manager, 1992-2003)

SUMMARY
• Definitions of QA, QC and EQA
• Relevance to Pathology and in particular Immunohistochemistry (IHC) and
In Situ Hybridisation (ISH)
• QA and Laboratory Accreditation
• QC for IHC and ISH
• EQA for IHC and ISH
• What happens when you don’t have QA?
• National and International guidelines for assuring quality
• Technical and Clinical Validation
• Standardisation

QUALITY ASSURANCE
• Quality assurance (QA) encompasses all measures taken to ensure the
reliability of investigations, starting from satisfactory test sample selection,
analysing it appropriately, to recording the result accurately and reporting it
to the clinician for appropriate action, with all procedures being
documented for reference (UK NEQAS, 1998).
• In the clinical laboratory, QA is usually provided by participation in a
Laboratory Accreditation Programme.

LABORATORY ACCREDITATION
• Can be national or international, or both
• Involves registering with an approved accreditation programme
• Documentation of all the procedures in the laboratory, to include;
• Range of tests, SOP’s, turn around times, results of audits, adherence to good
laboratory practice and international guide lines (e.g. ASCO/CAP guidelines),
details of procurement, maintenance and servicing of all equipment, records
of QC, participation and performance in EQA/Performance Testing (PT) etc.
• Visit by inspectors that thoroughly inspect the lab, interview the staff,
review the documentation and record whether compliant or non-
compliant,
• Laboratory is given a time interval (e.g. months), to address areas of non-
compliance and provide evidence that it is now compliant.
• Laboratory is then give accreditation for a given time period

QUALITY ASSURANCE (QA)
Two of the main features of QA are;
• Internal Quality Control (IQC)
AND
• External Quality Assessment (EQA), sometimes named
Performance Testing (PT)

INTERNAL QUALITY CONTROL
(IQC)
• The set of procedures undertaken for the continual evaluation of the quality
of results on a day-to-day basis, in order to decide on whether they are
reliable enough to be released (WHO, 1981, Leblanc et al., 1985).

INTERNAL QUALITY CONTROL
Factors to consider;
• Fixation (pre-analytical)
• The assay (analysis)
• Evaluation of the results (post analysis)

CLASSIFICATION (HOPWOOD (2002) &
BAKER (1960)
1.Aldehydes e.g. formaldehyde, glutaraldehyde (all non-coagulant)
2.Oxidising agents: osmium tetroxide, potassium dichromate (non –
coagulant)
3.Protein-denaturing (coagulant): acetone, acetic acid, methyl alcohol,
ethyl alcohol
4.Physical: Heat, microwaves (coagulant)
5.Miscellaneous: Mercuric chloride, picric acid, (coagulants)

COAGULATED
PROTEIN
NON- COAGULATED
PROTEIN

RECOMMENDED FIXATION FOR CLINICAL
PATHOLOGY
Neutral Buffered Formalin (pH 7.0) for 24-48 hours, ensures suitable for IHC
and molecular techniques such as FISH.

TIME BEFORE FIXATION:
Warm and Cold Ischemic Time

DELAY BEFORE FIXATION
• There should be minimal delay following surgical removal
• Tissue sliced, to allow for even fixation, preservation of areas in the centre of
specimen
• Neutral Buffered Formalin (pH 7.0) for 24-48 hours, ensures suitable for IHC
and molecular techniques such as FISH.

0 hours
4 hours
8 hours
o/night
ER PR
0 hours
1 hours
4 hours
8 hours

IMPORTANCE OF FIXATION
FOR APPROPRIATE TIME.

Am J Clin Pathol 2003; 120: 66-92.

THE ASSAY
• Positive control; do we need a positive control slide?
• Placed on the same slide, or as additional slides?
• Fixation – Ideally the tissue used as a control should have undergone
identical fixation as the test case –otherwise there is potential for a false
negative result (if control fixed less than the test) or a false positive result (if
control fixed longer than the test)
• Negative controls; do we need them – Some labs use them, some do not!

IMMUNOHISTOCHEMISTRY (NEGATIVE
CONTROLS)
1) TISSUE FIXATION, EXPOSURE OF
EPITOPES (ANTIGEN RETRIEVAL)
Px
Px
Px
2) OMISSION OF PRIMARY ANTIBODY
3) SECONDARY ANTIBODY BINDING
4) BINDING OF TERTIARY LAYER
5) VISUALIZATION OF PEROXIDASE LABEL
EVALUATION
TECHNICAL ASPECTS OF ASSAY
Diagnostic interpretation of results
and/or scoring of slides.

FLUORESCENT IN SITU HYBRIDISATION (FISH)
OR CHROMOGENIC IN SITU HYBRIDISATION
(CISH)
C – G – A – G – T – C - T
G – C – T – C – A – G - A
Tissue section
Microscope slide
HER2 gene sequence
Probe with complimentary
base pair sequence
Chromogenic or Fluorescent signal

IDEAL ATTRIBUTES OF QUALITY CONTROL SLIDE
FOR IHC AND ISH
• Readily available and in limitless supply,
• Inexpensive
• Does not change over time – does not degrade, no loss of target
antigen/nucleic acid sequence
• Has undergone identical fixation to that of the test
• Undergoes exact same procedure as test case (e.g. on-slide control),
• Contains cells and tissues that are both positive and negative for the
antibody/probe (helps ensure appropriate specificity of the assay)
• Has a gradation of positivity i.e. strongly positive/medium/weakly positive
cells (helps ensure appropriate sensitivity of the assay)

QUALITY CONTROL FOR OESTROGEN
RECEPTORS

TISSUE MICROARRAYS
(TMA’S)
Kononen et al. Nature Medicine
1998; 4: 844-7.

USE OF TMA’S FOR CONTROLS:
• ADVANTAGE:
• Better use of scarce tissues i.e. can get a lot more control slides from one tissue
block
• DISADVANTAGES
• TMA technically difficult to build, cut and maintain
• Eventually, will run out
• Small amount of material/surface area
• To a certain extent it damages the donor block from which the recipient TMA
was formed.

USE OF CELL LINES FOR QUALITY
CONTROL
• ADVANTAGES:
• Suitable lines are commercially available,
• Potentially provide for a limitless supply of QC material
with the same level of gene amplification and antigen
expression,
• Allows for effective monitoring of assay sensitivity for
important predictive and prognostic markers that require
some degree of quantitative assessment e.g. ER, PR, HER-
2/neu.
• DISADVANTAGES
• Costly and technically difficult to produce
• There is no intervening connective tissue to assess
potential for background/non specific staining

HER-2 PLUS SLIDES
• A collaborative project between the European Collection
of Cell Cultures and University of the West of England, Bristol.
• Rhodes A, Jasani B, Couturier J, et al. A formalin fixed and
paraffin processed cell line standard for quality control of
immunohistochemical assay of HER-2/neu expression in breast
cancer. Am J Clin Pathol 2002; 117: 81-89

CONSENSUS REPORT ON A
STANDARD REFERENCE MATERIAL
(SRM) FOR HER2 TESTING
NCI/NIST/FDA Sponsored Workshop
HER2 Standard Needs Assessment
Gaithersburg, USA
April 30 2002
Reference: Hammond et al .Appl Immunohisto Mol Morph 2003; 11: 103-106

INTERNAL QUALITY CONTROL (IQC)
• Usefulness of IQC depends on pre-determined limits of acceptability.
• It will not detect poor quality if the threshold level of acceptability is set at a
low level!

VARIATION IN QUALITY OF IMMUNOHISTOCHEMISTRY BETWEEN
TWO DIFFERENT LABORATORIES
Oestrogen R, laboratory ‘X’ Oestrogen R, laboratory ‘Y’

EXTERNAL QUALITY ASSESSMENT (EQA)
• Detects differences of quality between laboratories and provides guidance
on how to achieve the standards deemed to be universally acceptable.
• “A system of retrospectively & objectively comparing the results from
different laboratories by an external agency,” WHO 1981

EQA PROGRAMMES IN EUROPE & US
• UK NEQAS for Immunohistochemistry and In Situ Hybridisation
• AFAQAP – France, also Groupe d’Etude des Facteurs
pronostiques Immunohistochimiques dans le Cancer du Sein
(GEFPICS)
• NORDIQC – Scandinavia
• QUALICONT – Hungary
• INQAT - Italy
• QUIP Ringversuche – Germany
• Austria
• SEAP – Spain
Outside Europe
• RCPA QA Programmes – Australia
• CAP - US

UK NEQAS FOR IMMUNOCYTOCHEMISTRY & ISH
HTTPS://UKNEQASICCISH.ORG/
• Founded in 1985 by Gerry Reynolds at Mount Vernon
Hospital, Middlesex., UK. In 1988 the scheme was
recognised by the UK Department of Health and from
that time it became known as the UK National External
Quality Assessment Scheme for Immunocytochemistry
(UK NEQAS-ICC).
• Moved to University College London in 1992.

UK NEQAS FOR IMMUNOCYTOCHEMISTRY
& ISH
• Currently UK NEQAS-ICC offers assessments in different
immunocytohistochemistry modules at approximately 3-monthly
intervals throughout the year.

THE EXTERNAL QUALITY ASSESSMENT CYCLE
Participating
Labs
(1) Test materials
Administrative
Centre
THE
EQA
CYCLE
(2) Labs test
slides
(3) Stained slides
returned to organising
centre.
(5) Analysis of
results, production
of reviews
(6) Reports, analysis and reviews
circulated to participants.
(4) Evaluation
of slides

EQA
• Ideally, an educational process where by we learn best practice from each
other e.g. which antibodies/staining platforms/methods give the best results.
• More recently, EQA has been used to monitor poor performance, where by
poor performing labs are assisted to improve but if fail to do so in a set
period of time their Laboratory Accreditation for performing designated tests
are suspended.

WHAT HAPPENS WHEN YOU DON’T HAVE A
STRINGENT QA PROGRAMME?

UK NEQAS FOR IMMUNOCYTOCHEMISTRY,
1997-2000
The ’writing on the wall’

DIFFICULTIES IN DETECTING LOW ER+VE TUMOURS
• Approx. 15% of tumour
nuclei stained for ER,
• LBA: cytosol 65fmol/mg
protein
• Patient outcome as
expected e.g. disease
free survival at 5-years.
Oestrogen R.
Rhodes A. et al. J Clin Pathol 2000; 53:125-130

PROPORTION OF TUMOUR NUCLEI
DEMONSTRATED IN A LOW ER
EXPRESSING IDC BY 200 LABS
No ER+v tumour
1-9% ER+ve tumour cells
>10% ER+v tumour cells
Background staining
31%
29%
36%
4%

METHODS OF EVALUATION FOR ER IN 200 LABS,
1997-2000
Threshold No %
10% 106 50.0
H Score 17 8.1
20 or 25% 13 6.1
5% 10 4.7
Quick Score 6 2.8
1% 3 1.4
Category Score 2 0.9
50% 2 0.9
Value known but each <0.9% 8 3.8
Unknown 45 21.2
TOTAL 212 100

CORRELATION OF RESULTS FOR ER ON EQA MATERIAL
AND ‘IN HOUSE’ TUMOURS
RHODES ET AL. J CLIN PATHOL 2000; 53: 292-301.
Organising Lab, ER UK NEQAS
tumour
Lab ‘X’, ER UK NEQAS tumour
Organising Lab, ER Tumour
from Lab ‘X’
Lab ‘X’, ER ‘in house’
tumour

ER & PR STATUS OF 4,053 BREAST CARCINOMAS
RHODES ET AL. J CLIN PATHOL 2000; 53: 688-696
Age (yrs) Er+vePr+ve Er-vePr-ve Er+vePr-ve ER-vePr+ve Total
21-30 18 (45%) 17 (43%) 3 (8%) 2 (5%) 40 (1%)
31-40 134 (50%) 97 (33%) 45 (15%) 16 (6%) 292 (7%)
41-45 195 (58%) 88 (26%) 35 (10%) 19 (6%) 337 (8%)
46-50 319 (59%) 116 (21%) 83 (15%) 26 (5%) 544 (13%)
51-55 278 (54%) 114 (22%) 112 (22%) 16 (3%) 520 (13%)
56-60 239 (51%) 102 (22%) 111 (24%) 16 (3%) 468 (12%)
61-65 271 (55%) 98 (20%) 113 (23%) 8 (1%) 490 (12%)
66-70 251 (57%) 85 (19%) 94 (21%) 9 (2%) 439 (11%)
71-75 194 (58%) 60 (18%) 76 (23%) 7 (2%) 337 (8%)
>75 323 (55%) 119 (20%) 132 (23%) 12 (2%) 586 (15%)
Total 2222 (55%) 896 (22%) 804 (20%) 131 (3%) 4053 (100%)

FREQUENCY OF RECEPTOR POSITIVITY IN 7016 BREAST
CARCINOMAS.
RHODES ET AL. J CLIN PATHOL 2000; 53: 688-696
EQA SCORE
20
18
16
14
12
10
8
6
4
FREQUENCY
(%)
100
90
80
70
60
50
40
30
20
EQASCORE
20
18
16
14
12
10
8
6
4
FREQUENCY
(%)
100
90
80
70
60
50
40
Oestrogen
receptors
Progesterone receptors

FIVE YEARS LATER (2005)
A DISTRESS CALL FROM NEWFOUNDLAND, CANADA!

ST JOHN’S, NEWFOUNDLAND, CANADA
Shipguy, Public domain, via Wikimedia Commons

ST JOHN’S HOSPITAL NEWFOUNDLAND BY VERNE EQUINOX
AT ENGLISH WIKIPEDIA, CC BY 3.0.

JNCI; VOL. 100, ISSUE 12 |
JUNE 18, 2008

SOME OF THE MAIN ISSUES AT THE ST. JOHN’S LAB.
• Newfoundland had no Laboratory Accreditation or sets standards for
conducting clinical laboratory tests (unlike other Canadian provinces)
• Rapid turn over of staff/shortage of pathologists (32% over 4 years)
• No one taking responsibility for overall QA of ER testing i.e. pathologist or
technician,
• Technical issues e.g. Inefficient antigen retrieval
• Lack of understanding/training (CME, CPD) e.g.
• Invasive lobular carcinomas are ER+ve
• High cut points (thresholds) for a positive ER result
• Erroneous reporting e.g. of on-slide control instead of the case
• Lack of sensitive controls e.g. negative/low +ve/high positive
• Use of internal controls (normal glands should have ER+ve nuclei)
• No participation in EQA

ASCO/CAP GUIDELINES FOR ER/PR AND
HER2
• Hammond EH, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, Fitzgibbons PL,
Frances G, Neal S. Goldstein NS, Hayes M, Hicks DG, Lester S, Love R, McShane L,
Miller K, Kent Osborne CK, Paik S, Perlmutter J, Rhodes A, Sasano H, Sweep FCG,
Taube S, Torlakovic EE, Valenstein P, Viale G, Visscher D, Wheeler T, Williams RB,
Wittliff JL, Schwartz JN, and Wolff AC. American Society of Clinical
Oncology/College of American Pathologists guideline recommendations for
immunohistochemical testing of estrogen/progesterone receptors in breast cancer.
J Clin Oncol 2010; 28: 2784-2795.
• Wolff AC, Hammond, MEH, Schwartz JN, Hagerty KL, Allred DC, Cote RJ, Dowsett
M, Fitzgibbons PL, PL, Hanna WM, Langer A, McShane, Paik LS, Pegram MD, Perez
EA, Press MF, Rhodes A, Sturgeon C, Taube SE, Tubbs R, Vance GH, van de Vijver
M, Wheeler TM, Hayes DF. American Society of Clinical Oncology/College of
American Pathologists guideline recommendations for HER2 testing in breast cancer.
J Clinical Oncol 2007; 25: 118-145

TECHNICAL & CLINICAL
VALIDATION

VALIDATION OF BIOMARKERS FOR CLINICAL USE
• The antibody suppliers, such as DAKO (Agilant) and Ventana (Roche)
instigate internal validation procedures for clinically used antibodies
• The checks by suppliers specializing in a wide range of predominantly
research antibodies e.g. Santa Cruz, traditionaly have not been tested as
stringently as antibodies produced by companies such as Roche/Dako etc,
supplying clinical hospital laboratories.
• Antibodies/Probes and kits for use in predictive testing go through more
stringent testing than those used to assist in diagnosis.

TECHNICAL AND CLINICAL VALIDATION
• Technical validation refers to the reproducibility of the antibody (IHC) or
probe. This ensures batch consistency, specificity (for the antigenic epitope)
and sensitivity (signal to noise ratio)
• Clinical validation, refers to the specificity and sensitivity of the marker for
clinical cases. If a new marker in research papers suggests it to be positive
for a specific cancer e.g. mesotheliomas -what % of previously diagnosed
mesothelioma’s are +ve (sensitivity), -what % of tumours are true positives
i.e. they are mesotheliomas and not adenocarcinomas (specificity).
• ASCO/CAP guidelines for HER2 recommend doing cross validation of
specificity and sensitivity using complimentary assays (IHC v FISH) on 100
cases –strictly speaking this would be only technical validation,
• Clinical validation would refer to the predictive value of HER2 and/or ER
assays in predicting a favourable response to targeted therapies
• e.g. clinical validation for ER would be as in the paper by Harvey et al (1999),
where it was shown that a 1% threshold included women likely respond to
tamoxifen.

STANDARDISATION OF IHC AND ISH
• This has been largely achieved by the use of automated systems (though
control slides are still not standardized).
• ADVANTAGE – ‘Helps’ ensure reproducibility on a day-to-day basis, which
is essential for markers such as HER2 and ER and any marker that assessed
in quantitative fashion and where this is predictive of likely response to
targeted therapy,
• DISADVANTAGES
• – IHC and ISH becomes a ‘black box’ where lab staff have less understanding
of the basic principles of the assay. This is OK until something goes wrong and
have to ‘trouble shoot’ or need to work up a new antibody.
• -Very expensive, suppliers frequently supply in a kit form, and rely on sales of
expensive consumables.

BIBLIOGRAPHY
• Rhodes A, Miller K. Internal Quality Control and
External Quality Assessment of
Immunocytochemistry, In: Bancroft and Gamble, 5th
Edn 2002.
• Wick MR, Swanson PE. Editorial: Targeted controls in
clinical immunohistochemistry; a useful approach to
quality assurance. Am J Clin Pathol 2002; 117: 7-8.
• Moskaluk CA. Editorial: Standardisation of clinical
immunohistochemistry: Why, how and by whom?
Am J Clin Pathol 2002; 118: 669-671
• Sompurama SR et al, A Novel Quality Control Slide
for Quantitative Immunohistochemistry Testing.
Journal of Histochemistry and Cytochemistry, Vol.
50, 1425-1434, November 2002.

BIBLIOGRAPHY (CONTINUED)
• Rhodes A. Invited Review: Developing a cell line standard for HER2. Cancer
Biomarkers 2005; 1: 229-232.
• Rhodes A, Borthwick D, Sykes R Al-Sam S, Paradiso A. The use of cell line standards
to reduce HER2 assay variation in multiple European cancer centres and the
potential of automated image analysis to provide for more accurate cut points
for predicting clinical response to trastuzumab. Am J Clin Pathol 2004; 122: 51-60.
• Rhodes A, Jasani B. Quality Assurance in Immunohistochemistry. Am J Surg Pathol
2003; 9: 1284-1285.
• Rhodes A, Jasani B, Anderson E, Dodson AR, Balaton AJ. Evaluation of HER-2/neu
immunohistochemical assay sensitivity and scoring on formalin fixed and paraffin
processed cell lines and breast carcinomas: A comparative study involving results
from laboratories in 21 countries. Am J Clin Pathol 2002; 118: 408-417.
• Rhodes A, Jasani B, Couturier J, McKinley MJ, Morgan JM, Dodson AR, Navabi H,
Miller KD, Balaton AJ. A formalin fixed and paraffin processed cell line standard for
quality control of immunohistochemical assay of HER-2/neu expression in breast
cancer. Am J Clin Pathol 2002; 117: 81-89.

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