Immunocytochemistry - UK NEQAS for Immunocytochemistry ...

In This Issue:
Volume 3 Issue 4
123 EDITORIAL
UK NEQAS
124 INTER-LAB SURVEY OF
TECHNICAL VARIATIONS
IN PROSTATIC IMMUNO-
HISTOCHEMISTRY: B A S A L
CELL MARKERS
(Murali Varma et al)
128 REPORT OF RESULT S
F ROM THE UK NEQAS-ICC
QUESTIONNAIRE
(Roger Martin et al)
135 I N T RODUCTION TO
RUN 65 REVIEWS
136 GENERAL PATHOLOGY
MODULE
(Pan Cytokeratin,Thyroglobulin)
142 BREAST HORMONAL
R E C E P TOR MODULE
(Oestrogen receptors (ER))
147 BREAST HER-2
MODULE
(HER-2)
151 LYMPHOMA MODULE
(CD3 & bcl-2)
158 NEURO PATHOLOGY
MODULE
(Synaptophysin and Cytokeratin)
163 C Y TOLOGY MODULE
(Melanoma markers, Cytokeratins)
168 THE ALIMENTA RY T R AC T
(PILOT) MODULE
(CD117)
172 INSTRUCTIONS
FOR AUTHORS
Cover Photo: Optimal demonstration
of thyroglobulin in thyroid follicular cells
Immunocytochemistry
The official Journal of the UK National Quality Assessment Scheme for Immunocytochemistry
and In-Situ Hybridisation
UK NEQAS-ICC
QUESTIONNAIRE RESULTS
UK NEQAS reviews of run 65

General Information
The Journal is open to the publication of original
papers and review articles on Immunocytochemistry.
All articles, papers and letters should be submitted to:
Mr. Andrew Dodson. Editor-in-Chief,
Immunocytochemistry,
Royal Liverpool University Hospital, Liverpool. L69 3GA.
E-mail: dodson@liv.ac.uk
For further information of the UK NEQAS-ICC scheme,
immunocytochemistry EQA enquiries, including slide
returns, or to request further copies of this journal
please contact:
Dr Merdol Ibrahim, Scheme Manager.
UK NEQAS-ICC Office.
Suite 3/22 Hamilton House, Mabledon Place,
London. WC1H 9BB.
United Kingdom.
Tel: (44) 207 554 8679 E-mail: merdol.ibrahim@ucl.ac.uk
Immunocytochemistry — General Information
Immunocytochemistry is the recognised publication of the UK National External Quality Assessment Scheme for
Immunocytochemistry and In-Situ Hybridisation (UK NEQAS- ICC). It is published quarterly and has a current distribution of
over 1,800 to medical, technical and research staff working within the scientific area of Immunocytochemistry.
121
For information on Training issues, Meetings, Courses,
please contact:
Mr Keith Miller. UK NEQAS-ICC Histopathology,
21 University Street, University College London,
London, WC1E 6JJ. United Kingdom.
Tel: (44) 207 679 6048 E-mail: k.miller@ucl.ac.uk
For advertising opportunities please contact:
KT Advertising: Tel: (44) 709 230 6006
E-mail: info@KT-Group.co.uk
UK NEQAS-ICC wish to thank all advertisers and sponsors
for their support, but point out that any product or service
advertised in this Journal does not necessarily denote
an endorsement by UK NEQAS.
UK NEQAS-ICC reserves the right to refuse any article
or advertisement without question.
JOURNAL EDITOR ORGANISER MANAGER
Mr A Dodson Mr K Miller Dr M Ibrahim
ASSESSORS
Dr S Al-Sam, Essex, UK Dr E Anderson, Manchester, UK Dr N Anderson, Belfast, UK
Dr M Arends, Cambridge, UK Dr M Ashton-Key, Southampton, UK Dr A Balaton, Bievres, France
Dr D Barnes, London, UK Ms S Barnett, London, UK Dr E Baslev, Herlev, Denmark
Mr D Blythe, Leeds, UK Dr L Bobrow, Cambridge, UK Dr J Bulmer, Newcastle, UK
Dr J Cabecadas, Lisbon, Portugal Mr KK Chan, Cambridge, UK Dr C D'Arrigo, London, UK
Mr A Dodson, Liverpool, UK Mr D Fish, Reading, UK Mrs. S Forrest, Liverpool, UK
Ms J Freeman, London, UK Dr C Gillett, London, Mr J Gregory, Birmingham, UK
Ms J Gorst, Bucks, UK Prof. A Hanby, Leeds, UK Ms L Happerfield, Cambridge, UK
Dr R Hunt, Stockport, UK Dr M Ibrahim, London, UK Mr P Jackson, Leeds, UK
Prof. B Jasani, Cardiff, UK Ms S Jordan, London, UK Prof. E Kaye, Dublin, Ireland
Mrs I Kirbis, Ljubljana, Slovenia Dr G King, Aberdeen, UK Dr T Krenacs, Szeged, Hungary
Dr JM MacKenzie, Aberdeen, UK Mr. C. Marsh, Newcastle, UK Dr P Maxwell, Belfast, UK
Mr K McAllister, Dublin, Ireland Mrs J McGloin, London, UK Dr S McQuaid, Belfast, UK
Mr B Mepham, Southampton, UK Mr K Miller, London, UK Ms J Moorhead, London, UK
Ms P Munson, London, UK Dr M Myskow, New Zealand Mr S Nielsen, Aalborg, Denmark
Dr G Orchard, London, UK Dr S Pinder, Cambridge, UK Mrs F Rae, Edinburgh, UK
Dr B Rasmussen, Roskilde, Denmark Dr A Riley, Stirling, UK Mr J Ronan, Nottingham, UK
Dr V Save, Cambridge, UK Dr F Schmitt, Porto, Portugal Ms D Steele, London, UK
Dr M Thom, London, UK Mrs B Totty, Cambridge, UK Mrs. R Van Wijk, Cape Town, South Africa
Dr M Vyberg, Aalborg, Denmark Mrs J Williams, Portsmouth, UK Mrs S Wise, London, UK
Dr C Wong, Hong Kong Ms S Wozniak, Cardiff, UK
Office Staff Technical Data Input Steering committee Chairman
Mrs AL Rhodes Mr D Fish (Techniques in Cellular Pathology)
Mrs M Perez Mrs Barbara Totty
Accounts Publishing Manager Typesetting
Ms S Slaymark Mr R Martin Medical Informatics Unit, University of Oxford
Published by The KT Group © 2005 UK NEQAS-ICC /KTG/MIU/2000/09-05

Run 65 Editorial
123
Immunocytochemistry — Editorial
Articles for Immunocytochemistry Journal are like buses (or good English cricketers), you wait for
ages then two or three come along at once. I am in the happy position of having more articles to
publish than can reasonably be accommodated in one issue of the journal. This issue brings you a
paper from Varma et al concerning prostate immunohistochemistry, and a report from Martin,
Dodson and Ibrahim on the results of the UK NEQAS-ICC user satisfaction survey; you will have to
wait until the Run 66 issue comes out to see what didn’t make the ‘cut’.
August 2005
Andy Dodson
Department of Pathology
Royal Liverpool University Hospital
For the most up to date
information check out
The UK NEQAS-ICC website
at:
wwwwwwwwwwww....uuuukkkknnnneeeeqqqqaaaassssiiiicccccccc....uuuuccccllll....aaaacccc....uuuukkkk

Immunocytochemistry
Immunocytochemistry 2005; 3: 124 – 127
© UK NEQAS for Immunocytochemistry, 2005
Report
Inter-laboratory survey of technical variations in prostatic
immunohistochemistry: basal cell markers
Murali Varma 1
, Daniel M Berney 2
, Bharat Jasani 1
, Anthony Rhodes 3
1. University Hospital of Wales, Cardiff, UK; 2. St Bartholomew’s Hospital, London, UK; 3. University of the West of England, Bristol, UK
Correspondence to: Dr M Varma, Department of Histopathology, University Hospital of Wales, Heath Park, Cardiff CF14 4XN Wales, UK
Tel: +44-2920745316; Fax: +44-2920742701; E-mail: Murali.Varma@cardiffandvale.wales.nhs.uk
Summary
Aims: A survey of the extent of variation in the use of basal cell markers in prostate immunohistochemistry.
Methods: A questionnaire was sent to all laboratories registered with the United Kingdom National
External Quality Assurance Scheme for Immunocytochemistry enquiring about the immunohistochemical
methods routinely used for the diagnosis of prostate cancer.
Results: Responses were received from 220 (68%) of laboratories. Basal cell marker immunohistochemistry
was performed by 115 (87%) of 133 responding UK laboratories. Most (60%) of these laboratories used a
single basal cell marker. The most commonly used markers were high-molecular weight cytokeratin antibody
34βE12 (77%), cytokeratin 5/6 (42%) and LP34 (26%). The more recently described basal cell marker, p63
was available in only 4% of laboratories. All the basal cell markers were consistently used after pre-treatment,
with heat induced epitope retrieval the most commonly used method used by UK laboratories for all the
markers.
Conclusions: There is considerable variation in the choice of basal cell markers used by UK laboratories
to distinguish benign prostate glands from prostate cancer, with most centres using only a single marker.
Since none of these markers react with all benign glands, use of a combination of basal cell markers is
suggested to help resolve this important differential diagnosis.
© UK NEQAS for Immunocytochemistry, 2005
INTRODUCTION
The absence of basal cells in malignant glands as
opposed to their ubiquitous presence in benign
prostate glands, a finding first described by Totten et al in
1953, is a well-established criterion for diagnosis of
prostate cancer 1 . However, it is often not possible to reliably
distinguish basal cells from stromal fibroblasts on routine
histology and so immunostaining with basal cell markers
is widely used as an aid to diagnosis in morphologically
difficult cases.
A variety of basal cell markers are available, but there
is little information on the range and rate of use of these
antibodies.
All the commonly used basal cell markers are monoclonal
antibodies that generally require pre-treatment when
124
used in paraffin embedded material. Predigestion
using enzymes such as proteases and pronases as
well as heat induced epitope retrieval (HIER) using
microwaving or steam are commonly used pre-treatment
methods.
It is recognised that the pre-treatment method may
impact the sensitivity and specificity of immunostaining
but the frequency of use of different pre-treatment
methods with each basal cell marker in routine diagnostic
practice is unknown.
We therefore conducted a postal survey of immunohistochemical
methods used in the diagnosis of prostate
cancer in laboratories registered with the United
Kingdom National External Quality Assurance Scheme
for Immunocytochemistry (UK NEQAS-ICC).

METHODS
• A short questionnaire was circulated to all laboratories
subscribing to the UK NEQAS-ICC scheme in 2003
(Figure 1). The use of the recently developed, promising
positive marker for prostate cancer, alpha-methylacyl-
CoA racemase (AMACR) 2 was not studied as it had
been commercially available only for a few months
at the time of the survey.
• In order to maximise the response rate, the questionnaire
along with a stamped and addressed envelope
was circulated with the EQA slides in two successive
runs of the scheme. Duplicated responses were
identified and excluded.
• From the replies received, the frequency of use of
various basal cell markers and the pre-treatment
methods used for them were determined.
• The survey findings related to prostate specific antigen
and prostate specific acid phosphatase immunohistochemistry
have been discussed in a separate report 3 .
Figure 1. Questionnaire circulated to all subscribers to the UK
NEQAS-ICC Scheme.
RESULTS
The questionnaire was circulated to 394 laboratories,
323 of which were known to subscribe to the prostate
cancer immunohistochemistry EQA module. Of the
latter 323 centres, 196 represented UK laboratories.
125
Immunocytochemistry
After excluding duplicates, 220 (68%) replies were
received; 133 from the UK and 87 from outside the UK
(13 from The Republic of Ireland, 58 from continental
Europe and 16 from elsewhere). The response rate for
UK laboratories was therefore calculated to be 68%
(133/196).
Since virtually all UK laboratories practicing diagnostic
prostate immunohistochemistry are known to participate
in the UK NEQAS-ICC scheme, the response received
from them can be considered to be representative. The
situation regarding UK NEQAS-ICC participation by
laboratories outside the UK is not known so the responses
from these non-UK laboratories may not be representative
of the practice in these countries. These data were
therefore considered separately from those from UK
laboratories.
Table 1. Number of basal cell markers used in each laboratory.
Table 2. Choice of basal cell marker (percentage figures are
percentage of laboratories that used basal cell markers).
The frequency of use of various basal cell markers is
summarised in Tables 1 and 2. Over half the responding
laboratories used only a single basal cell marker, with
high-molecular weight cytokeratin antibody clone
34βE12 being the most widely used.
The pre-treatment methods used for the basal cell
markers are summarised in Tables 3 and 4. All the basal
cell markers used were of the monoclonal antibody
type and were used after pre-treatment, with HIER
being the most commonly applied method.

Immunocytochemistry
Table 3. Pre-treatment methods used for basal cell markers in UK Laboratories (percentage figures are percentage of laboratories
that used that particular basal cell marker).
Table 4. Pre-treatment methods used for basal cell markers in Non-UK Laboratories (percentage figures are percentage of
laboratories that used that particular basal cell marker).
DISCUSSION
Immunohistochemistry using markers specific for basal
cells of the prostate gland is widely used as an adjunct
to the diagnosis of prostate cancer 4 . In our survey, 87%
of laboratories in which prostate marker immunohistochemistry
was available used at least one basal cell
marker. It is possible that some of the respondents that
did not use basal cell markers used PSA and/or PSAP only
to establish the prostatic origin of metastasis and were
presumably not concerned with the primary diagnosis
of prostate cancer.
A number of prostatic basal cell markers are currently
available. In the UK, the most widely used basal cell
marker appears to be the high-molecular weight cytokeratin
antibody clone 34βE12 (77%). Cytokeratin 5/6 and
LP34 were used by a significant minority of respondents
(41% and 25% respectively) while p63 was routinely
employed by only 4% of centres. The popularity of
34βE12 is probably related to the fact that its utility has
been extensively studied and validated in the literature,
particularly in the USA. In contrast, the infrequently used
p63 has only recently been established as a basal cell
marker.
126
The wide variation in the use of basal cell markers
would reflect the paucity of studies comparing them
and providing guidance regarding their relative usefulness
and efficacy. A few small studies have reported
cytokeratin 5/6 and p63 to be marginally more sensitive
than 34βE12 5,6,7 . Freeman et al found LP34 to be slightly
more sensitive than cytokeratin 5/6 and cytokeratin 14
but the panel of markers studied did not include
34βE12 8 . Moreover, as the authors commented, unlike
other basal cell markers, LP34 generally reacts with the
secretory cells of the prostate gland, albeit less intensely
than basal cells, making interpretation sometimes more
difficult.
Unlike most other immunohistochemical markers, basal
cell markers are used to distinguish benign prostate
glands from prostate cancer and the diagnosis of
malignancy is confirmed by a negative immunoreaction,
so it would be important to make every effort to maximise
the sensitivity of immunostaining with these markers.
However, it is generally recognised that none of the
basal cell markers are absolutely sensitive as a small
proportion of benign glands are negative with each of

these markers 5,6,9 . Using a combination of basal cell
markers has been shown to increase the sensitivity of
immunostaining 10 . In this context, it is noteworthy that
currently less than half of UK laboratories (40%) appear
to use more than one basal cell marker.
The ideal combination of basal cell markers for use in
diagnostic immunohistochemistry practice is uncertain.
34βE12, cytokeratin 5/6 and LP34 are similar in that they
are directed against high-molecular weight cytokeratins
and the immunoreactivity is membranous/intracytoplasmic.
In contrast, p63, a homologue of p53, displays
nuclear immunoreactivity. Hence, using one of the
high-molecular weight cytokeratin antibodies in tandem
with p63 as suggested by Zhou et al may be more
advantageous 10 .
The optimal use of basal cell markers may be in
combination with AMACR, which has been reported
to be consistently positive in prostate cancer. The use of
such a panel would help exclude false negative
immunonegativity with basal cell markers due to technical
factors in immunostaining or cautery effect in
transurethral resection specimens. However, AMACR
immunoreactivity should always be interpreted in
conjunction with basal cell immunohistochemistry, as
high-grade PIN often expresses AMACR 11 .
It is recognised that high-molecular weight cytokeratin
antibodies are sensitive to formalin fixation and require
pre-treatment when used on paraffin embedded
material 12 . A few studies using monoclonal antibody clone
34βE12 have found high-molecular weight cytokeratin
expression in prostate cancer after the use of heat
pre-treatment but not after enzyme predigestion 12,13,14 .
This immunoreactivity was generally restricted to rare
tumour cells. This potential pitfall in prostate cancer
diagnosis is significant, as most centres in the UK appear
to use heat pre-treatment for high-molecular weight
cytokeratin antibodies, LP34, 34βE12 and cytokeratin 5/6.
In conclusion, our study confirms that there is a wide
variation in the immunohistochemical methods used to
identify basal cells of the prostate gland. This variation
probably reflects the fact that none of the available
markers have been clearly shown to be better than the
others. In view of the clinical importance of basal cell
immunohistochemistry in confirming the diagnosis of
adenocarcinoma and the lack of a completely sensitive
marker, use of a combination of at least two basal cell
markers is recommended.
127
REFERENCES
Immunocytochemistry
1. Totten RS, Heineman MW, Hudson PB, et al. Microscopic differential
diagnosis of latent carcinoma of the prostate. Arch Pathol 1953; 55:
131-141
2. Jiang Z, Woda BA, Rock KL, et al. P504S: A new molecular marker for the
detection of prostate carcinoma. Am J Surg Pathol 2001; 25: 1397-1404
3. Varma M, Berney DM, Jasani B, Rhodes A. Technical variations in
prostatic immunohistochemistry: need for standardisation and stringent
quality assurance in PSA and PSAP immunostaining. J Clin Pathol
2004; 57: 687-690
4. Wojno KJ, Epstein JI. The utility of basal cell-specific anti-cytokeratin
antibody in the diagnosis of prostate cancer. Am J Surg Pathol 1995;
19: 251-260
5. Abrahams NA, Ormsby AH, Brainard J. Validation of cytokeratin 5/6 as
an effective substitute for keratin 903 in the differentiation of benign
from malignant glands in prostate needle biopsies. Histopathology
2002; 41: 35-41
6. Shah RB, Zhou M, LeBlanc M, et al. Comparison of the basal cell-specific
markers, 34betaE12 and p63, in the diagnosis of prostate cancer.
Am J Surg Pathol 2002; 26: 1161-1168
7. Weinstein MH, Signoretti S, Loda M. Diagnostic utility of immunohistochemical
staining for p63, a sensitive marker of prostatic basal cells.
Mod Pathol 2002; 15: 1302-1308
8. Freeman A, Treunicht K, Munson P, et al. A comparison of basal cell
markers used in the prostate. Histopathology 2002; 40: 492-494 (letter)
9. Varma M, Amin MB, Linden MD and Zarbo RJ. Discriminant staining
pattern of small glandular and preneoplastic lesions of the prostate
using high molecular weight cytokeratin antibody. Mod Pathol 1997;
10: 93A (abstract)
10. Zhou M, Shah R, Shen R, et al. Basal cell cocktail (34betaE12 and p63)
improves the detection of prostate basal cells. Am J Surg Pathol 2003;
27: 365-371
11. Wu CL, Yang XJ, Tretiakova M, et al. Analysis of alpha-methylacyl-CoA
racemase (P504S) expression in high-grade prostatic intraepithelial
neoplasia. Hum Pathol 2004; 35: 1008-1013
12. Varma M, Linden MD, Amin MB. Effect of formalin fixation and
epitope retrieval techniques on antibody 34betaE12 immunostaining
of prostatic tissues. Mod Pathol 1999; 12: 472-478
13. Lindemann N, Weidner N. Immunohistochemical profile of prostatic
and urothelial carcinoma: Impact of heat-induced epitope retrieval
and presentation of tumors with intermediate features. Appl
Immunohistochem 1996; 4: 264-275
14. Bennett CJ, Hicks JL, Gage WR, et al. “False positive” immunostaining
of 34BE12 in prostate cancer: Systematic study of the effect of antigen
retrieval conditions using high density tissue microarrays. Mod Pathol
2003; 1: 141A (abstract)

Immunocytochemistry
Immunocytochemistry 2005; 3: 128 – 136
© UK NEQAS for Immunocytochemistry, 2005
Article
Report of results from the UK NEQAS-ICC questionnaire
Roger J Martin 1 , Andrew Dodson 2 , Merdol Ibrahim 3
1: Consult Business Development Ltd. 2: Department of Pathology, Royal Liverpool University Hospital. 3: UK-NEQAS-ICC
Correspondence to: Dr. Merdol Ibrahim, UK NEQAS-ICC Office, Suite 3/22 Hamilton House, Mabledon Place, London WC1H 9BB UK
E-mail: merdol.ibrahim@ucl.ac.uk; Tel: +44(0)207 554 8679; Fax: +44(0)207 554 8685
Summary
In 2004 a questionnaire was circulated to all UK NEQAS-ICC participants. The aims the survey were: firstly
to assess user-satisfaction with various aspects of the scheme as it is now; and, secondly to encourage
suggestions for improvements to the scheme, including new antibodies to be included. Forty-eight
completed questionnaires were returned, representing a response-rate of over 10%. The following report
describes the results in detail.
UK NEQAS-ICC would like to thank all those participants who took the trouble to fill-in and return their
questionnaires: your feedback is greatly appreciated.
© UK NEQAS for Immunocytochemistry, 2005
INTRODUCTION
The UK NEQAS-ICC Scheme is run primarily for the benefit
of its users. It is intended that it should be a tool which
participants can use to help them maintain and
improve the quality of their immunocytochemistry. To
be effective it must serve its users’ needs.
Opportunities for the management team at UK NEQAS-
ICC to hear participants’ views with regard to the service
it provides are relatively limited. It was therefore decided
that a more proactive and structured approach was
required, and a short questionnaire was formulated
covering all the major areas of activity. Users were
asked to grade their response to various questions as
Very satisfied, Satisfied, or Dissatisfied, and space was
given for those who wished to elaborate on their
responses to do so. In addition, information regarding
UK NEQAS-ICC organised meetings was sought, and
suggestions for antibodies not currently covered, which
might be included in future runs were also asked for.
THE QUESTIONS
The questionnaire comprised twelve questions. The first
six dealt with various aspects of the assessment process
itself:
128
Question 1. How satisified are you with the way you
receive your EQA samples?
Question 2. How satisfied are you with the time you are
given to stain the samples?
Question 3. How satisfied are you with the procedure
for returning the samples?
Question 4. How satisfied are you with the time taken to
receive your results?
Question 5. How satisfied are you with the format in
which you receive your results?
Question 6. How satisfied are you with the comments
which accompany the results?
The seventh question concerned the Immunocytochemistry
Journal:
Question 7. How satisfied are you with the Immunocytochemistry
newsletter?
Question 8 was about enquiries and complaints:
Question 8. Have you ever had cause to contact the
UK NEQAS-ICC office with a question or complaint?
If yes – how satisfied were you with the way this was
handled?
The final four questions were centred around the area
of meetings:
Question 9. Have you attended a UK NEQAS-ICC meeting
in the last 12 months?
If yes – how satisfied were you with this meeting?

Question 10. How satisfied are you with the number and
locations of UK NEQAS-ICC meetings?
Question 11. How satisfied are you with the opportunities
you have to discuss technical or logistical issues at
meetings?
Question 12. Do you intend to attend any UK NEQAS-ICC
meetings next year?
RESULTS
Question 1. How satisfied are you with the way you
receive your EQA samples?
Very satisfied Satisfied Dissatisfied
24/47 (51%) 21/47 (45%) 2/47 (4%)
One returnee did not answer this question.
Comments received relating to this question:
• ‘Sometimes feel EQA samples stain more weakly than
in-house samples. May have been cut and stored for
some time’
• ‘I would like to know when the samples will arrive, for
example by e-mail’
• ‘It would be useful to receive the forms each time by
e-mail’
• ‘Two sections do not allow for much experimentation
to give the best possible stain’
• ‘More UK NEQAS sections could be supplied so staining
could be…optimised’
Question 2. How satisfied are you with the time you are
given to stain the samples?
Very satisfied Satisfied Dissatisfied
21/47 (45%) 23/47 (49%) 3/47 (6%)
129
Immunocytochemistry
One returnee did not answer this question.
Comments received relating to this question:
• ‘The time period is too short to assess the previous
results, make improvements and stain the next run…’
• ‘The time is sometimes a little short’
• ‘Should always allow a full calendar month’
Question 3. How satisfied are you with the procedure
for returning the samples?
Very satisfied Satisfied Dissatisfied
25/48 (52%) 17/48 (35%) 6/48 (13%)
Comments received relating to this question:
• ‘Perhaps paperwork could be simplified/reduced’
• ‘Forms are clumsy and out-of-date…Can they be
streamlined and generally easier/quicker to fill-in?’
• ‘Space too small on forms. We wish we could fill-out
the form through the internet’
• ‘No pre-typed labels for addresses return provided’
• ‘It is rather monotonous writing the same information
on numerous sheets’

Immunocytochemistry
Question 4. How satisfied are you with the time taken to
receive your results?
Very satisfied Satisfied Dissatisfied
10/47 (21%) 32/47 (68%) 5/47 (11%)
One returnee did not answer this question.
In total, ten comments were received relating to this
question, all were variations on the following:
• ‘If the results came earlier it would be better, but we
understand that it takes time to review all slides’
Question 5. How satisfied are you with the format in
which you receive your results?
Very satisfied Satisfied Dissatisfied
23/48 (48%) 22/48 (46%) 3/48 (6%)
No comments were received relating to this question.
Question 6. How satisfied are you with the comments
which accompany the results?
Very satisfied Satisfied Dissatisfied
9/46 (20%) 25/46 (54%) 12/46 (26%)
130
Two returnees did not answer this question.
This question attracted the most comments of any in the
survey, with a total of seventeen. The range is covered
in the following examples:
• ‘Could have more depth in comments’
• ‘Breakdown of where exactly marks have been
deducted and why’
• ‘Could not the marking be out-of-10, with one overall
set of comments?’
• ‘If there are bad results, please give information
about the reasons if you can…fixation, antibody
dilution etc’
• ‘Inconsistency between marker’
• ‘They don’t always help improve staining – not
specific enough’
• ‘More detail in comments section. Photograph of top
scoring section to accompany results’
• ‘An average result for all members of the scheme
included with results’
Question 7. How satisfied are you with the Immunocytochemistry
newsletter?
Very satisfied Satisfied Dissatisfied
26/48 (54%) 22/48 (46%) 0/48 (0%)
Comments received relating to this question:
• ‘Trends in detection methods which are giving the
best results. Can we standardise, should we try – stats
may be of help’

• ‘More information on meetings’
• ‘A mean of each stain from all participants would be
helpful with the graphs for statistical interpretation
purposes’
Question 8. Have you ever had cause to contact the
UK NEQAS-ICC office with a question or complaint?
31/48 (65%) of returnees had contacted the UK NEQAS-
ICC office, 17/48 (35%) had not.
Question 8 (follow-up question). If yes – how satisfied
were you with the way this was handled?
Very satisfied Satisfied Dissatisfied
16/30 (53%) 12/30 (40%) 2/30 (7%)
Of the 31 returnees to whom this question applied, one
did not answer.
One comment was received relating to this question:
• ‘Two times. The first time we got no answer. In the
other case we got a satisfactory answer’
Question 9. Have you attended a UK NEQAS-ICC meeting
in the last 12 months?
15/48 (31%) of returnees had attended a meeting, 33/48
(69%) had not.
Question 9 (follow-up question). If yes – how satisfied
were you with this meeting?
Very satisfied Satisfied Dissatisfied
8/15 (53%) 7/15 (47%) 0/15 (0%)
131
Immunocytochemistry
The graphical representation of these results:
Question 10. How satisfied are you with the number and
locations of UK NEQAS-ICC meetings?
Very satisfied Satisfied Dissatisfied
3/33 (9%) 22/33 (67%) 8/33 (24%)
Fifteen returnees did not answer this question.
Comments relating to questions 10 – 12 have been
grouped together at the end of this section.
Question 11. How satisfied are you with the opportunities
you have to discuss technical or logistical issues at
meetings?
Very satisfied Satisfied Dissatisfied
2/24 (8%) 17/24 (71%) 5/24 (21%)
Twenty-four returnees did not answer this question.
The graphical representation of these results is shown at
top of next page.

Immunocytochemistry
Question 12. Do you intend to attend any UK NEQAS-
ICC meetings next year?
41/45 (91%) of returnees were planning on attending a
meeting, 4/45 (9%) were not.
132
Three returnees did not answer this question.
Comments relating to meetings:
• ‘It would be nice to have the occasional meeting in
other parts of the country, and not just in London’
• ‘For overseas participants it is difficult to attend
meetings. A summary of the main topics discussed…would
help’
• ‘Perhaps devote a session to questions and answers
for each module’
• ‘Dublin is too far away with no provision for attending
1 of the 3 days, which makes it too expensive and
too time consuming’
• ‘More focus on the technical parts’
• ‘Come to Northern England’
Results Summary Chart. Numerical values were assigned to the responses as follows: Very satisfied = +1, Satisfied = 0,
Dissatisfied = −1. A user satisfaction index was calculated for each question by multiplying the assigned values by the
proportions in each category, and summing the resulting figures. The results are depicted in the chart above. A value of
+1 would indicate that 100% of returnees are very satisfied, while a value of −1 would mean that 100% were dissatisfied.

SUGGESTIONS FOR NEW ANTIBODIES TO BE
INCLUDED
A number of suggestions for new antibodies that might be
looked at were received. These were mainly applicable
to the General Module or to the Lymphoma Module (the
most frequently requested are listed below).
General Module: Cytokeratins 7 and 20, Thyroid
Transcription Factor-1, AMACR (P504S), p63.
Lymphoma Module: bcl6, CD5, CD10, CD21, CD23.
There were also a number of requests for CD117 (c-KIT)
to be included (since this survey was conducted UK
NEQAS-ICC has announced the launch of the
Alimentary Tract Module which looks at this antigen).
DISCUSSION
This survey has been of great value, and while much of
the feedback it has provided has been very positive it
has also served to highlighted a number of areas
where improvements can be made. The UK NEQAS-ICC
management team have made, and are continuing to
make changes to a number of operational aspects to
address these; a number of which are discussed below.
BETTER COMMUNICATION AND FEEDBACK
The Scheme is moving towards e-mail to communicate
in a more efficient and time saving manner with all
participants. A recent example has been notifications
with respect to UK NEQAS-ICC workshops, which have
been sent out electronically. It is intended that e-mail
notifications will be implemented for all aspects of UK
NEQAS-ICC activities, and for this reason all participants
who have not already done so, are urged to submit
their current e-mail address. E-Mail addresses may be
sent to the UK NEQAS-ICC office using either of
the following addresses: rmkdalr@ucl.ac.uk or
m.perez@ucl.ac.uk
During each assessment there are approximately 5000
slides that are assessed in a three-to-four week period.
This means that there are a great deal of scores and
comments to be collated and sent-out, however, it is
still very important to get the results to all participants as
soon as possible. In a move to speed-up this operation
assessors now log their scores directly into laptops
(previously paper score sheets were used, with scores
being transferred later to a central database). In
addition to improvements in speed, this also allows
assessors the opportunity to type in an individual comment
if the prefigured ones are not appropriate for any given
slide. This is something which could not be accommodated
by the paper-based system, and it is hoped that
it will be of considerable benefit.
133
Immunocytochemistry
EDUCATIONAL REMIT OF THE SCHEME
The Scheme aims to educate and help all participants.
In particular it aims to help participants to improve and
adapt their protocols where it is felt that improvements
can be made. ‘Best Methods’ have been published in
the Immunocytochemistry Journal for every antibody
used in the Scheme. This resource should be extremely
valuable for participants wishing to make changes to
their own methods when they prove sub-optimal.
It is gratifying to see that the Immunocytochemistry
Journal performed well, with this aspect of the
Scheme’s activities achieving the highest satisfaction
rating of any in the survey. Even so, the management
team at UK NEQAS-ICC are not resting on their laurels
with respect to the Journal. It is intended that improvements
to layout will be made in the next few months,
with individual Module reports having a more logical
and consistent layout.
Discussions are currently underway concerning additional
statistical analysis that can be undertaken on the data
produced at each Run, which we hope will be of further
benefit to participants.
TIME ALLOWED FOR SLIDE STAINING
It is felt that a turnaround of about three weeks to stain
the UK NEQAS-ICC and in-house control slides is adequate.
We believe that the UK NEQAS-ICC samples should fit in
with the normal days run for any particular laboratory.
With regard to the question of only two slides being
provided per antibody request, it should be noted that
any increase in the number of slides would have an
enormous effect on workload for the laboratory providing
this material, and very probably a considerable cost
implication. It is not envisaged that this will be changed
for these reasons, however, if any participant does
require further slides to test out their protocols these
can normally be provided on an ad hoc basis upon
request.
IMPROVED WEB-BASED COMMUNICATION
With respect to procedure for returning samples; a move
towards an internet-based form submission procedure is
currently underway. Forms will be adapted to make
their completion easier and participants will be able to
submit via the website. At present an interim procedure
has been put in place, whereby participants may
download and then e-mail the data sheets. This facility
can be accessed from the website:
www.ukneqasicc.ucl.ac.uk/runXXdatasheets
(just replace the ‘XX’ with the Run number).

Immunocytochemistry
Behind the scenes the website is also undergoing a
transformation and we hope that soon all participants
will be able to access results, view more images, and
download and submit new versions of datasheets.
Furthermore a user-group and messaging system will be
put in place to allow participants to communicate with
one another. A special acknowledgment should be
made to Rifat Hamoudi who has been working
extremely hard to put this in place.
MEETING / WORKSHOPS / DISCUSSIONS
The view that UK NEQAS-ICC meetings should occur
around the UK and not only in London, which was
expressed by a number of participants in their survey
responses has been noted. In 2005, workshops and
lectures have taken place in London, Wolverhampton
and Northumbria, it is hoped that this means that UK
NEQAS-ICC has ‘come to Northern England’!
The next major conference will be in Dublin (July 2006).
This city was found to offer both exemplary conference
facilities and excellent social opportunities when the UK
NEQAS-ICC conference was previously held there. The
conference organisers will be looking into having further
technical sessions during this, and future meetings.
It is also envisaged that the setting-up of a user-group
and messaging system on the website will allow individuals
participants to resolve issues by a system of self-help in
a way that meetings or the Immunocytochemistry
Journal may not be able to do.
CONCLUSION
UK NEQAS-ICC would like to once again thank those
participants who responded to the survey. It is hoped
that they will agree with us that this has been a most
worthwhile exercise. If any participant has additional
comments on this survey, or indeed any aspect of the
Scheme the team at UK NEQAS-ICC headquarters
would be delighted to hear from them.
134

UK NEQAS-ICC REVIEWS OF RUN 65
INTRODUCTION
135
Immunocytochemistry — Introduction to Reviews
The UK National External Quality Assessment Scheme for Immunocytochemistry (UK NEQAS-ICC) assessments takes place
at approximately three-monthly intervals, throughout the fiscal year. Currently laboratories are able to participate in up
to 6 different modules, depending on their service commitments and specialised areas of interest. These modules are as
follows:
1. The general pathology module; catering for routine markers used by the majority of pathology departments
offering a routine immunocytochemistry service.
2. The breast hormonal receptor module; catering for laboratories routinely demonstrating oestrogen and
progesterone receptors on paraffin processed tissues.
3. The breast HER-2 module; catering for laboratories routinely testing for HER-2 on paraffin processed tissues.
4. The lymphoma module; catering for the markers used by laboratories with a specialised interest in lymphoid
pathology.
5. The neuropathology module; catering for the markers common to most neuropathology departments.
6. The cytology module; catering for markers commonly requested on cytological preparations.
7. Alimentary tract pathology (pilot) module; catering for laboratories specialising in this area or pathology.
What happens at assessment?
At each assessment, laboratories are sent formalin fixed and paraffin processed sections (alcohol fixed cytospins for
the cytology scheme) along with instruction sheets for the schemes to which they have subscribed. They are requested
to demonstrate two different antigens (one for each of the breast schemes) on the slides provided and return the
best for each antigen for assessment along with their usual In House control slide stained with the same marker(s).
For most of the modules, one of the antigens requested is repeated from one assessment to the next for a period of
twelve months, and serves as a ‘gold standard’. This allows participants to implement recommended changes if
their quality of immunostaining is found to be sub-standard and to test out their improved technique at the next
assessment. They are also requested to complete a short questionnaire giving brief details of the antibody and
method they have employed. The slides bearing each participant’s unique code number (to ensure anonymity) are
then marked by an expert panel consisting of senior biomedical or clinical scientists and usually one consultant
histopathologist.
Interpretation of the scores achieved at assessment
Each of the four assessors awards a mark from 1 to 5 using the guidelines issued for each antigen. The marks are then
added together to give a score out of 20. An acceptable level of staining attracts marks greater than 12/20. A
borderline mark in the range 10/20 – 12/20 indicates that whilst some information can be obtained from the slide,
the staining is sub-optimal. Lastly, a score of less than 10/20 is given for poor immunocytochemistry that has failed to
clearly demonstrate the required components.
Journal contents
The following pages summarises the results of Assessment Run 62 and provide; guidelines as to how the assessors
marked the immunostaining, brief reviews for each antigen assessed, colour photomicrographs where appropriate,
best methods, distribution of results and tabulation of technical data.
Best methods
Included with the reviews are examples of methods employed by participants who achieved some of the best
immuno-staining at this assessment. We have selected techniques, which employ primary antibodies used by the
majority of participants. Examples of the quality of staining that was achieved using these methods are shown in the
colour plates.
Technical data
All the basic technical data collated from this assessment is in tabulated form in order to be economic with the space
available. This is restricted to the sections provided by UK NEQAS-ICC i.e. the standard material.

Immunocytochemistry — General Pathology
The General Pathology Module
Julie Williams
Antigens assessed: Pan Cytokeratin*, Thyroglobulin
*The small number of laboratories not stocking an
antibody to pan-cytokeratin were requested to carry-out
staining for high-molecular weight cytokeratins with, for
example LP34, and for low-molecular weight cytokeratins
with, for example CAM5.2, on two separate sections.
Tissue sections circulated: composite block of tonsil and
gut (pan-cytokeratin), thyroid (thyroglobulin).
Number of participating laboratories: 396 (pancytokeratin),
330 (thyroglobulin)
General guidelines used in the assessment of slides:
SCORE STAINING PATTERN
0 No returns.
1 Little or no staining of the antigen in question.
2 Very weak demonstration of cells expected to
stain or many of these cells were not demonstrated.
3 Weak demonstration of cells expected to stain.
4 Good demonstration of all cells expected to stain.
5 Excellent demonstration of all cells expected to
stain, with little or no background.
NB. These are only very general guidelines and marks were
deducted for such things as poor localisation of staining or diffuse
staining, inappropriate staining of certain cell types, excessive
background staining, excessive counter-stain, uneven staining
or other factors which made interpretation difficult.
The four individual scores for each slide were added together
to give a mark out of 20.
Pan-Cytokeratin
Features of optimal immunostaining (Plates 1 & 2)
• Intense cytoplasmic staining of the complete epithelial
layer in both the tonsil and gut. There should be no
staining of lymphocytes although occasional staining
of plasma cells by some cytokeratin antibodies has
been reported (Wotherspoon et al).
• The tissue morphology should be relatively
untouched by retrieval.
Features of sub-optimal immunostaining (Plates 3 & 4)
• Generalised weak or incomplete staining of epithelium.
• Excessive proteolysis of supporting connective tissue
resulting in poor morphology.
• Inappropriate staining of some cells (lymphocytes)
or cell components (nuclei), usually the result of
excessive heat-induced epitope retrieval (HIER).
• Excessive background staining of connective tissue
and lymphocytes.
136
Recommended antigen retrieval system for
Pan-Cytokeratin
According to data provided by the manufacturers,
both proteolytic enzyme digestion and HIER are suitable
for most clones. With proteolytic digestion it is important
to ensure that the digestion time is tailored to the length
of fixation. It is the experience gained from assessment
of slides submitted to this scheme that the use of
enzymes such as freshly prepared 0.1% chymotrypsin in
0.1% calcium chloride (pH adjusted to 7.8) at 37 o C is
very effective for pan-cytokeratin staining. Good results
were obtained by participants using both proteolytic
enzyme digestion and HIER (see Table 3).
A significant improvement in performance
There was a significant improvement in the scoring for
pan-cytokeratin in this run compared to the last three
runs (Runs 62-64). At this run 73% of participants
achieved a score of >12/20 on the UK NEQAS-ICC
sections, this compares to only 49% on the previous
one. The main cause of low marks was again the
incomplete staining of the epithelium in both tonsil and
gut, which is clearly demonstrated in Plates 3 and 4.
Participants should be aware that, in order to obtain a
pass mark with pan cytokeratin the full depth of epithelium
must be stained. Scoring on the laboratories own tissue
was again very good as 92% achieved a score of
>12/20. There does not appear to be any obvious reason
for the improvement in the scores on the UK NEQAS-ICC
sections this time except that laboratories may have
taken the advise to adjust their retrieval times to suit the
fixation of the UK NEQAS-ICC sections. Please note that
participants had marks deducted for using non-pan
cytokeratin antibodies such as CAM 5.2 and highmolecular
weight cytokeratins, unless they were used in
conjunction with other cytokeratins as stipulated on the
datasheet.
Notes on cytokeratins and their antibodies
Cytokeratins belong to a group of proteins known as
intermediate filaments that constitute the cytoskeletal
structure in all epithelial cells. It is very important when
making your choice of antibody to choose a marker
which will demonstrate a wide range of cytokeratins
such as the clones AE1/AE3 or MNF116 (the two main
clones used by participants). Both from personal experience
and from published studies (Goddard et al),
AE1/AE3 is a better overall cytokeratin marker than
MNF116 as it is made up of a cocktail of more high and
low molecular weight cytokeratins: AE1 – Cytokeratins
10, 13, 14, 15, 16 & 19; and AE3 – Cytokeratins 1, 2, 3, 4,
5, 6, 7 & 8. In contrast, MNF116 demonstrates
Cytokeratins 5, 6, 8, 17 and probably 19. From the UK

NEQAS-ICC results the proportion of participants
achieving acceptable staining was the same for both
of the main clones.
Thyroglobulin
Main features of optimal results with thyroglobulin
(Plate 5)
• Intense staining of thyroid follicular cells or thyrocytes,
with some staining of the colloid.
Main features of sub-optimal results with thyroglobulin
(Plates 6 & 7)
• Weak staining or no staining of the thyroid follicular
cells or thyrocytes, regardless of the fact that colloid
staining was present in some of these sections.
• Presence of ‘corkscrew’ artifact, probably
caused by over pre-treatment.
Choice of antibody and recommended antigen
retrieval for thyroglobulin
Of the two most common thyroglobulin antibodies used
by participants (see Table 2) the polyclonal antibody
with no antigen retrieval gave the best overall staining.
The main monoclonal antibody used scored lower (30%
scored >12/20) than the polyclonal (68% scored >12/20).
A study by De Micco et al suggested that some monoclonal
thyroglobulin clones did not stain as well as the
polyclonal antibodies especially in thyroid tumours.
Notes on thyroglobulin
Thyroglobulin is a large glycoprotein possessing different
molecular forms depending on its metabolic rate i.e.
native, intracellular etc. which is found predominately in
its 660 kD form. Thyroglobulin is synthesied by thyrocytes
and transported to the apical surface where it is secreted
into the lumen of thyroid follicles and stored as a major
Plate 1. Optimal staining of pan-cytokeratin in tonsil (UK NEQAS-ICC
section). Please note complete depth of epithelium is stained. This
slide scored 20/20.
137
Immunocytochemistry — General Pathology
component of colloid. Antibodies to thyroglobulin react
with normal, hyperplastic and neoplastic thyroid tissue
and are most useful for the identification of papillary and
follicular thyroid carcinomas. They are also commonly
used to identify metastatic tumours from the thyroid. It
has been reported that the level of staining depends on
the degree of differentiation. Generally more poorly
differentiated tumours contain less thyrogobulin than
well differentiated (DeLellis and Shin). Antibodies to
thyroglobulin do not react with epithelial cells of the
lung, breast, gastrointestinal tract or kidney, nor with the
carcinomas of these organs. It must be noted that
thyroglobulin may diffuse in to other cells in the tissue
including tumour cells of medullary and anaplastic
carcinomas (LiVolsi) and sinus histiocytes in lymph nodes
producing false positive results (Venkatraman et al).
REFERENCES
1. Wotherspoon AC, Norton AJ, Isaacson PG. Immunoreactive
cytokeratins in plasmacytomas. Histopathology 1989; 14(2): 141-50.
2. Goddard MJ, Wilson B, Grant JW. Comparison of commercially
available cytokeratin antibodies in normal and neoplastic adult
epithelial and non-epithelial tissues. J Clin Pathol 1991; 44: 660-
663
3. De Micco C, et al. Thyroglobulin in Medullary Thyroid Carcinomas.
Human Pathology 1993; 24 (3): 256-262.
4. DeLellis RA & Shin S J. Diagnostic Immunohistochemistry of
Endocrine Tumours. In Diagnostic Immunohistochemistry. Dabbs D.
(ed). Churchill Livingstone, Philadelphia 2002 ; 215.
5. LiVolsi VA. Surgical Pathology of the Thyroid. Volume 22 in the Series
Major Problems in Pathology. J.L. Bennington (ed). W.B. Saunders
Company, Philadelphia 1990; 392.
6. Venkatraman V, Maxwell P, McCluggage WG. Thyroglobulin
immunoreactivity in lymph node histiocytes: a potential diagnostic
pitfall. J Clin Pathol 2001; 54: 314-316
Plate 2 . Optimal demonstration of pan-cytokeratin in section of
gut (UK NEQAS-ICC section). Please note that all epithelial cells
are stained to the full depths of the crypts. This slide scored 20/20.

Immunocytochemistry — General Pathology
Plate 3. Poor demonstration of pan-cytokeratin in sections of tonsil
(UK NEQAS-ICC section). Please note that the complete depth of
epithelium is not staining, this should be compared to the optimal
staining in Plate 1. This slide scored 8/20.
Plate 5. Optimal staining of thyroglobulin (UK NEQAS-ICC section)
as demonstrated by strong staining of the follicular cells and some
staining in the colloid in the normal thyroid. This slide scored 20/20.
Plate 7. ‘Corkscrew’ artifact, commonly seen in a number of the
thyroid sections and probably caused by excessive pre-treatment.
138
Plate 4. Weak demonstration of pan cytokeratin in sections of gut
(UK NEQAS-ICC section). Please note that the complete depth
of epithelium within the crypts is not staining, this should be
compared to the optimal staining in Plate 2. This slide scored 8/20.
Plate 6. Poor demonstration of thyroglobulin (UK NEQAS-ICC section).
Please note strong staining in the colloid only. It is important to note
that for an acceptable score follicular cells should also be stained as
in Plate 5.

frequency of returns
Distribution of scores
139
Immunocytochemistry — General Pathology
Fig 1. Run 65A Cytokeratin (UK NEQAS-ICC sections) Fig 2. Run 65B Cytokeratin (In House sections)
frequency of returns
Run 65A Cytokeratin on UK NEQAS Sections
Summary
Scores >12/20 287(73%)
Scores 10-12/20 69(17%)
Scores 12/20 148(45%)
Scores 10-12/20 69(21%)
Scores 12/20 381(92%)
Scores 10-12/20 24(6%)
Scores 12/20 165(48%)
Scores 10-12/20 83(24%)
Scores

Immunocytochemistry — General Pathology
Pan-Cytokeratin
Participant scored 19/20 (UK NEQAS-ICC slide) and 19/20
(In House slide) using this method.
Method: Ventana iView DAB Kit
Automation: Ventana NexES system
Buffer and pH: Ventana buffer
Blockade Type: Ventana blockade system
Antigen Retrieval: Ventana Protease I for 20 minutes
Primary Antibody: DakoCytomation M0821 (Clone MNF116)
dilution 1:75 for 30 minutes at 37 o C
Chromogen: Ventana DAB for 8 minutes
140
Thyroglobulin
Participant scored 20/20 (UK NEQAS-ICC slide) and 16/20
(In House slide) using this method.
Method: Vector Elite
Automation: LabVision Autostainer
Buffer and pH: PBS
Blockade Type: Hydrogen peroxide and serum blocks
Antigen Retrieval: None
Primary Antibody: DakoCytomation A0251 dilution 1:2000
for 40 minutes at room temperature
Chromogen: DakoCytomation K3466 DAB, for 10
minutes
MAIN TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE GENERAL PATHOLOGY MODULE
The following tables record the number of participants (N) using each system. The percentage (%) refers to the
proportion of these participants achieving acceptable staining (a score >12/20). For example, 15 participants
used A. Menarini MU071 (clones AE1/AE3) of whom 73% achieved acceptable staining.
Table 1. Primary antibody Cytokeratin markers [CK]
Antibody Details N (%)
A. Menarini MU071UC (clone AE1/AE3) 15 73
Becton Dickinson 349205 (Clone CAM 5..2) 4 25
Boehinger Mannheim 1124 (Clone AE1/AE3) 1 100
Chemicon MAB 3412 (Clone AE1/36+2B11) 2 100
DakoCytomation M0821 (Clone MNF116) 171 79
DakoCytomation M3515 (Clone AE1/AE3) 102 74
DakoCytomation M0630 HMWCK (Clone 34BE12) 1 100
Home made cocktails 6 67
Immunotech 1918 (Clone KL1) 7 43
Incomplete data 21 81
Novocastra NCL-PAN CK 16 69
Other 12 50
Ventana 250 2595 AE1/AE3 15 73
Zymed 08 1132 (Clone AE1/AE3) 11 64
Table 2. Primary antibody Thyroglobulin
Antibody Details N (%)
Biogenex MU032 UC 4 0
Biomedia 9833 Polyclonal 1 100
Binding Site PH 068 1 100
CMA 113 (Clone 2H11/6E1) 2 50
DakoCytomation M0781 (Clone DAK IG6 ) 119 30
DakoCytomation A0251 139 68
DakoCytomation N1565 3 33
Novocastra NCL-THY 22 91
Neomarkers MS 206 Polyclonal 2 100
ID Labs BP 54p 1 0
Incomplete data 17 41
Immunon 492020 2 100
Other 4 50
Ventana 250-2740 and 760-2671(Clone 2H11/6E1) 6 0
Zymed 18-0134 (Clone 2H11/6E1) 4 0

Table 3. Pre-treatment
Pan-Cytokeratins Thyroglobulin
Pre-treatment N % N %
Biogenex Protease XXIV EK002 5K 22 68 1 0
Biogenex Protease HK056-5K 1 0 1 0
Becton Dickinson Trypsin 215240 1 0 - -
DakoCytomation Pronase 52013 4 50 - -
DakoCytomation Protease K 53020 16 81 1 0
DakoCytomation K2375 5 80 1 0
Difco Trypsin 215230 18 89 8 12
Enzyme digestion + HMAR 17 76 1 0
ICN 150213 Trypsin 20 90 3 0
Incomplete data 9 78 7 14
Microwave Oven (MW) 62 60 56 23
None 2 50 180 67
Other 17 59 1 100
PC in MW 23 87 13 8
Pressure Cooker (PC) 66 82 32 22
Sigma Pepsin P7000 1 0 - -
Sigma C4129 Chymotrypsin 16 88 1 0
Sigma P8038 Protease 8 100 2 0
Sigma T7409 Trypsin 1 0 2 0
Sigma T8128 Trypsin 1 0 - -
Sigma P5147 Protease 7 43 - -
Water bath 95-98oC 13 38 8 25
Ventana Benchmark 9 44 6 16
Ventana Protease 1 45 82 2 0
Table 5. Detection system
Pan-Cytokeratins Thyroglobulin
Type, supplier & product code N (%) N (%)
Biogenex Super Sensitive Multi link/
HRP LP000-UL
12 75 8 50
Biogenex HK 330 9K 1 100 1 0
Biogenex HK 519-06K HRP 1 100 1 0
Biocarta BCA HP 504 US 1 100 - -
Chemicom HP1000 2 100 2 50
DakoCytomation ChemMate K5005 1 0 1 100
DakoCytomation ChemMate K5001 78 72 61 61
Dakocytomation ChemMate
Envision K5007 DAB
54 81 45 56
DakoCytomation Duet St.ABC K0492 7 71 7 28
DakoCytomation Envision Plus systems 27 70 26 50
DakoCytomation K0690 3 33 2 0
DakoCytomation LSAB Kit/HRP K0675 12 33 8 25
DakoCytomation St.ABC/HRP K0377 2 100 2 50
LabVision TS 125 14 64 13 62
Incomplete data 17 59 13 46
Other 15 60 9 44
Power Vision DPVB 999 HRP 1 100 1 100
Vector 7000 series 18 83 18 39
Vector Elite ABC PK 6102 1 100 1 100
Vector Elite ABC PK 6100 9 67 7 14
Vector Elite Universal ABC PK6200 29 90 25 56
Ventana basic system 13 92 12 0
Ventana iVIEW system 60 73 49 29
VisionBiosystem D59404 2 100 2 50
Zymed 87 8143 2 0 2 0
Zymed 85 9043 2 50 2 0
141
Immunocytochemistry — General Pathology
Table 4. Chromogen
Pan-Cytokeratins Thyroglobulin
Chromogen & Supplier N (%) N (%)
Biogenex HK-153-5K DAB 19 84 14 36
DakoCytomation Envision Plus kits 20 80 19 58
DakoCytomation K3466 DAB 18 83 15 47
DakoCytomation K3468 DAB 37 54 32 44
DakoCytomation ChemMate
K5001 DAB
77 73 61 62
Dakocytomation ChemMate
K5005 Alk phos
1 0 1 100
Dakocytomation S3000 DAB 3 100 3 100
Dakocytomation ChemMate
Envision K5007 DAB
52 81 44 52
Incomplete data 24 67 19 26
KEM-EN-TEC 4170 DAB 6 83 5 40
LabVision TA 125 HD 9 44 9 44
Other 17 65 10 30
Sigma D5635 DAB 1 100 1 100
Sigma D5637 DAB 7 71 7 100
Sigma D5905 DAB 4 100 4 50
VisionBiosystem Bond x DAB 2 100 2 50
Vector SK4100 DAB 8 63 6 17
Ventana iVIEW DAB 68 79 56 23
Zymed 87 8143 6 50 6 17
Table 6. Automation
Pan-Cytokeratins Thyroglobulin
Instrument & Supplier N (%) N (%)
Biogenex Optimax 23 78 22 36
Biogenex Genon MX 6000i 13 100 10 60
DakoCytomation Autostainer 78 72 71 55
DakoCytomation TechMate 500 33 73 24 58
DakoCytomation TechMate Horizon 6 50 4 75
Incomplete data 4 75 3 33
Leica Histostainer 1 100 - -
LabVision Autostainer 30 77 28 50
None 100 67 78 46
Shandon Cadenza 1 100 1 0
Shandon Sequenza 24 71 17 53
Ventana ES 4 75 4 25
Ventana Benchmark 27 70 21 19
Ventana Gen 11 2 50 2 0
Ventana NexES 36 86 31 26

Immunocytochemistry — Breast Hormonal Receptor
The Breast Hormonal Receptor Module
Keith Miller & Merdol Ibrahim
Antigens assessed: Oestrogen receptors (ER).
Tissue sections circulated: Sections from a composite
block comprising three infiltrating ductal carcinomas
(IDC’s) with differing levels of receptor expression (see
Table below).
IHC Staining characteristics of invasive tumour nuclei*
Tumour Approx. % Main Intensity
(A). IDC 90-95 High (see Plate 8)
(B). IDC 50-75 Medium – High (see Plate 9)
(C). IDC 0 Negative (see Plate 10)
* Refers to the staining characteristics observed when the
tumours were immunostained by the organising laboratory
using the Novocastra 6F11 clone.
Instructions at Assessment:
Participants were requested to demonstrate ER on
the slides provided.
Number of participating laboratories: 343
Assessment of staining of In House tumours:
Participants were also requested to stain their own In
House control with ER and submit at least 2 unstained
sections from the same tissue block, thus allowing the
organising laboratory to stain the same case. The subsequent
comparison of the staining achieved by the
participant and organising laboratories on the In House
sections, allowed for a more accurate assessment of
the staining quality present.
Introduction
Oestrogen and progesterone receptors are localised in
nuclei of epithelial cells. Nuclei of approximately 7% of
these cells are immunoreactive to current day oestrogen
receptor antibodies in normal resting breast tissue,
with a higher proportion in lobular than in ductal cells
(Petersen et al). When breast carcinoma arises
demonstrating the presence of oestrogen receptors in
the malignant cells, as we all know, is very important as
they provide a target for certain therapies, such as
Tamoxifen and, more recently, Anastrazole (Thurlimann
et al). Nearly 66% of breast cancers from premenopausal
women and 75% from post-menopausal
women contain immuno-detectable oestrogen receptors
(Mayor). If however, a particular case is negative, the
inclusion of positive normal glands is important as they
can often give an indication as to reliability of the
immunostain performed on the slide.
142
Guidelines used in the evaluation of oestrogen and
progesterone receptor assays: Four assessors marked the
slides independently; each awarding scores from 1 to 5
using the guidelines shown. The 4 individual scores for
each slide were added together to give a mark out of 20.
Those sections showing the expected standard of
immunostaining achieved marks in the 13/20 – 20/20 range.
SCORE STAINING PATTERN
0 No slides returned.
1 & 2 Staining of considerably less nuclei than expected
in either, or both of the two positive tumours.
3 Staining of 10% or greater of tumour nuclei in each
of the two positive tumours, though substantially
less than expected to stain, or staining is weaker
than expected.
4 & 5 Demonstration of the proportion of nuclei of
invasive tumours expected to stain, with roughly
the expected staining intensity.
NB. These are only general guidelines and marks were deducted
for excessive cytoplasmic staining, excessive background staining,
diffuse nuclear staining, excessive counter-stain, uneven staining
or other factors that made interpretation difficult.
The four individual scores for each slide were added
together to give a mark out of 20.
Distribution of scores
frequency of returns
Run 65E Oestrogen Receptors on UK NEQAS Sections
Summary
Scores >12/20 116 (36%)
Scores 10-12/20 77 (23%)
Scores 12/20 253(78%)
Scores 10-12/20 53(17%)
Scores

Features of optimal immunostaining (See Plates 8, 9 and
10)
• Staining of the expected proportion of invasive
tumour nuclei with the anticipated staining intensity
(see Plates 11, 12, and 13)
• Intense nuclear staining of the appropriate distribution
in normal glands
• Cytoplasmic staining not excessive
• No background staining of connective tissues or
inappropriate localised staining
Features of sub-optimal results (see Plates 11, 12, 13, 14
and 15)
• Relatively weak nuclear staining of the receptor
positive tumours
• Excessive cytoplasmic staining
• Excessive background staining of connective tissue
elements
• Inappropriate staining of some cells e.g. lymphocytes,
fibroblasts
The importance of good fixation!
One of the most important factors often ignored when
dealing with breast tissue, because of pressure from
those looking after the patients, is good fixation. It cannot
be stressed enough that good fixation is vital to preserve
ER protein. Also, good fixation is required to preserve the
degree of morphological detail needed to diagnose
borderline lesions. Small specimens may be fixed whole
but larger ones really need to be examined and
sliced within a few hours of excision to allow optimum
penetration of fixative. One of the added benefits of
well-processed breast tissue is that sectioning is easier
and retention of section on the slides is better, the latter
is especially significant when employing aggressive
heat retrieval methods.
Antigen retrieval
Participants achieving poor results for hormonal receptors
should note that inefficient antigen retrieval is frequently
the main factor responsible for weak staining and
attention to this has on previous occasions resulted in
significant improvement (Rhodes et al).
Immunocytochemistry — Breast Hormonal Receptor
143
Performance and sections supplied by UK NEQAS-ICC.
The performance on UK NEQAS-ICC sections on this
occasion was very disappointing. Only 36% of participants
achieved a score of more than 12/20. This again is most
likely due to the extended fixation of the tissue. This once
again indicates that there appears to be a problem with
the tissue being fixed for longer than necessary.
However, the extended fixation is making more and
more laboratories review their retrieval protocols and this
may provide some benefit when staining their local
cases. The extended fixation has also enabled the
identification of the more efficient retrieval systems.
Participants employing pressure-cooking retrieval
together with citrate buffer at pH6.0 are, for the most
part achieving the desired results.
For those concerned with poor performance please be
assured that performance on In House sections, which
was very good indeed with 78% scoring more than
12/20, will be taken into account.
REFERENCES
1. Petersen OW, Hoyer PE, van Duers B. Frequency and distribution
of estrogen receptor-positive cells in normal, non-lactating
human breast tissue. Cancer Res 1987; 47: 5748-5751
2. Thurlimann B, Hess D, Koberle D, et al. Anastrozole ('Arimidex')
versus tamoxifen as first-line therapy in postmenopausal
women with advanced breast cancer: results of the doubleblind
cross-over SAKK trial 21/95 — a sub-study of the TARGET
(Tamoxifen or 'Arimidex' Randomized Group Efficacy and
Tolerability) trial. Breast Cancer Res Treat 2004; 85(3): 247-54.
3. Mayor S. News Roundup: UK survey finds variation in
oestrogen receptor testing. BMJ 2001; 323: 713
4. Rhodes A, Jasani B, Balaton A, et al. Study of interlaboratory
reliability and reproducibility of estrogen and progesterone
receptor assays in Europe: documentation of poor reliability
and identification of insufficient microwave antigen retrieval
time as a major contributory element of unreliable assays.
Am J Clin Pathol 2001; 115: 44-58

Immunocytochemistry — Breast Hormonal Receptor
Plate 8. UK NEQAS-ICC high expressor section (Section A) optimally
stained. The smaller figure shows an enlarged section from the
main image showing a clean crisp staining of tumour nuclei.
Plate 10. UK NEQAS-ICC non-expressor section (Section C) optimally
stained. The smaller magnified image shows an example of strong
positivity in the normal glands.
Plate 12. UK NEQAS-ICC medium - high expressor section (Section
B) sub-optimally stained. The tumour nuclei are virtually negative.
The excess counterstain further masks any ER positivity which may
be present.
144
Plate 9. UK NEQAS-ICC medium – high expressor section
(Section B) optimally stained. The smaller figure shows closer
detail of antibody localisation.
Plate 11. UK NEQAS-ICC high expressor section (Section A)
sub-optimally stained. Very poor demonstration of tumour nuclei
with the tumour remaining largely unstained.
Plate 13. In House section. Staining result produced by the
participant indicates low expression of ER.

Plate 14. Section from same In House case as shown in Plate 13.
Staining by the UK NEQAS-ICC reference laboratory shows staining
in a greater proportion of nuclei, and with a greater intensity than
that produced by the participant.
Plate 16. Section from same In House case as shown in Plate 15.
Staining by the UK NEQAS-ICC reference laboratory shows that this
tumour is in fact ER positive, a result which the participant has missed
altogether.
BEST METHODS
Oestrogen Receptor
Participant scored 20/20 (UK NEQAS-ICC slide) and
18/20 (In House slide) using this method.
Method: DakoCytomation ChemMate K5001
Automation: None
Buffer and pH: DakoCytomation ChemMate K5001
buffer
Blockade Type: DakoCytomation ChemMate K5001
blocking solution
Antigen Retrieval: Tefal pressure cooker to heat 1600 ml of
Vector unmasking solution pH 6.0, for
2.5 minutes at full pressure
Primary Antibody: LabVision RM-9101-S dilution 1:100 for
60 minutes at room temperature
Chromogen: DakoCytomation ChemMate K5001
DAB for 5 minutes
Immunocytochemistry — Breast Hormonal Receptor
145
Plate 15. In House section. Staining result produced by the participant
indicates that this tumour was negative for ER expression.
Oestrogen Receptor
Participant Scored 19/20 (UK NEQAS-ICC slide) and
20/20 (In House slide) using this method.
Method: Neomarkers Ultravision LP TL-012 HDN
Automation: DakoCytomation Autostainer
Buffer and pH: Tris buffered saline pH7.6
Blockade Type: Hydrogen peroxide blockade, serum
Antigen Retrieval: Dakocytomation Water bath GIR109
using 250 ml of citrate buffer pH 6.0 for 40
minutes at 98 o C
Primary Antibody: Neomarkers RM-9101-S dilution 1:80 for
30 minutes at room temperature
Chromogen: DakoCytomation K3468 DAB for 5
minutes

Immunocytochemistry — Breast Hormonal Receptor
MAIN TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE BREAST PATHOLOGY
HORMONAL RECEPTOR MODULE
The following tables record the number of participants (N) using each system. The percentage (%) refers to the proportion of these
participants achieving acceptable staining (a score >12/20). For example, in the first table 2 participants used A.Menarini MU 272
antibody (clone 1D5) neither of whom achieved acceptable staining.
Table 7. Primary Antibody Oestrogen receptors (ER)
Antibody details N %
A.Menarini MU 272 (Clone 1D5) 2 0
Biocarta CM 093C (Clone 6F11) 1 0
DakoCytomation M7047 ER (clone 1D5) 101 23
Immunotech 1545 (Clone ID5) 1 100
Incomplete data 5 0
LabVision RM 9101-S (Clone SP1) 30 67
Novocastra NCL-ER 6F11 ER (Clone 6F11) 131 42
Other 1 0
Ventana 250-2596 ER (Clone 6F11) 25 40
Zymed 08 1149 (Clone 1D5) 3 33
Table 9. Detection System
Type, Supplier & Product Code N (%)
Biogenex LP000-UL 11 45
Biogenex HK 330 9K 1 0
Chemicom HP 1000 2 50
DakoCytomation ChemMate L.St.Av/HRP K5001 58 38
DakoCytomation ChemMate Envision K5007 DAB 46 35
DakoCytomation Duet St.ABC K0492 7 43
DakoCytomation Envision 14 21
DakoCytomation LSAB Kit/HRP K0675 7 0
DakoCytomation St.ABC/HRP K0377 3 33
Incomplete data 15 26
Others 9 33
LabVision TS 125 HR 13 23
Vector Elite ABC PK6100 7 57
Vector Elite Universal ABC PK6200 23 43
Vector Elite ABC PK7200 14 57
Ventana iView System 51 39
Ventana System 250-2001 8 63
Vision Biosystems D59404 2 0
Zymed 87 8143 1 100
Table 11. Automation
Instrument & Supplier N (%)
Biogenex Optimax 20 20
Biogenex GENON MX i6000 13 38
DakoCytomation Techmate 500 26 46
DakoCytomation Techmate Horizon 4 25
DakoCytomation Autostainer 62 39
Incomplete data 2 50
LabVision Autostainer 23 39
None 72 35
Shandon Sequenza 17 35
Shandon Cadenza 2 0
Ventana Benchmark 23 35
Ventana ES 3 100
Ventana NexES 28 43
Ventana GEN 11 2 0
Vision Biosystems Bond X 3 0
146
Table 8. Pre-treatment
Pre-treatment N (%)
Incomplete data 6 33
Microwave Oven 80 25
Other 1 0
PC inside MW oven 46 48
Pressure Cooker 129 44
Water bath 95-98oC 14 14
Ventana benchmark 24 29
Table 10. Chromogen
Chromogen & Supplier N (%)
Biogenex HK-153-5K DAB 16 38
DakoCytomation K3466 DAB 10 40
DakoCytomation K3468 DAB 31 35
DakoCytomation K5001 DAB 58 38
DakoCytomation K5005 Alk Phos 1 0
DakoCytomation S3000 DAB 1 100
DakoCytomation K5007 DAB 43 37
DakoCytomation Envision 7 43
KEM-EN-TEC -4170 DAB 7 43
Other 12 8
Incomplete data 24 21
LabVision TS 125 HR 8 25
Sigma D5637 DAB 6 33
Sigma D5905 DAB 5 40
Vector SK4100 DAB 5 60
Ventana DAB 55 44
Vision Biosystems DAB 2 0
Zymed kits DAB 4 25

The Breast HER-2 Module
Keith Miller & Merdol Ibrahim
Antigens assessed: HER-2
Number of participating laboratories: 161
Sections circulated: Formalin fixed and paraffin processed
sections from a composite block comprising human breast carcinoma
cell lines; MDA-MB-453, BT-20, MCF-7 and the ovarian
carcinoma cell line SKOV-3. In a previous study, FISH analysis on
these cell lines showed the SKOV-3 and MDA-MB-453 cell lines to
have HER-2/neu gene amplification, whilst the cell lines BT-20
and MCF-7 did not (Rhodes A, et al Ref 1). Briefly, the ratio of
HER-2/neu to chromosome 17 signals were 4.2 and 3 for the
SKOV-3 and MDA-MB-453 cell lines and 0.9 and 1.0 for the BT-20
and MCF-7 cell lines, respectively. The sections were positioned
on the microscope slide as indicated in Figure 7.
Fig. 7. Position of cell lines on the microscope slide and the most appropriate scores.
E
Run 64E
(A)
SKOV-3 (3+)
(B)
MDA-MB-453 (2+)
(C)
BT-20 (0 or 1+)
(D)
MCF-7 (0)
Assessment of slides: The immunohistochemical results were
evaluated by UK NEQAS-ICC assessors scoring independently
using the method of evaluation initially devised for the Clinical
Trials Assay. The scoring was as follows:
SCORE STAINING PATTERN
0 No staining at all or very slight partial membrane
staining in less than 10% of tumour cells.
1+ Faint barely perceptible membrane staining in more
than 10% of tumour cells; the cells are only stained in
part of their membrane.
2+ Weak to moderate complete membrane staining
observed in more than 10% of tumour cells.
3+ Strong complete membrane staining in more than 10%
of tumour cells.
Features of optimal immunostaining (see Plates 17,
18 and 19)
• Staining of the expected proportion of membranes
in the given cell lines as set up in the assessment
guidelines above
• Cytoplasmic staining is not excessive.
• Counterstain is not excessive
Features of sub-optimal immunostaining (see Plates 20,
21 and 22)
• Damaged cell morphology
• Excessive cytoplasmic staining making membrane
staining difficult to interpret
• Excessive counterstain masking immunostaining of
membrane
147
Immunocytochemistry — Breast HER-2
Staining of the MDA-MB-453 cell line
Previous analysis by FISH showed the cell lines SKOV-3
and MDA-MB-453 to have HER-2 gene amplification,
whilst the cell lines BT-20 and MDA-MB-453 did not
(Rhodes A, et al Ref 2). When testing these cell lines and
reviewing the results of over 90 laboratories from last
year, it became apparent that the MDA-MB-453 could
not be reliably immunostained with greater than 2+
staining intensity, without over-staining the BT-20 and
MCF-7 cell lines i.e. these cell lines typically stain 2+ and
1+ respectively, if 3+ staining occurs on the MDA-MB-453
cell line. Consequently, 2+ staining is the most appropriate
result for this cell line. This was considered by the
assessment panel to be the ‘safest’ level of sensitivity to
achieve on the MDA-MB-453 cell line, providing that
guidelines are followed which recommend that all 2+
staining on tumours should be regarded as an equivocal
result which require further analysis by FISH (Ellis IO, et al).
An assay sensitivity level that equates to 1+ staining on
the MDA-MB-453 cell line might have better correlation
with FISH results, as many 2+ tumours have been shown
by FISH not to have HER-2 gene amplification (Tubbs RR,
et al). However, by staining this cell line and tumours with
similar amounts of HER-2 expression as 2+ it ensures that
these tumours are further analysed by FISH to identify
those having HER-2 gene amplification and those having
no amplification. This should therefore in turn ensure that
patients with 2+ tumours with gene amplification and
patients with 2+ tumours without gene amplification both
receive appropriate therapy.
Choice of antibody and methodology
Once again, as in previous assessments the
DakoCytomation HercepTest kit seems to out perform
most other methods according to the data (see Tables
12 and 14).
However, reports continue to suggest that the
DakoCytomation HercepTest kits vary slightly from
batch to batch with regard to the 1+ staining, and this
was again confirmed at assessment. Nevertheless, the
evidence still points to the HercepTest kit being the most
reliable if immunocytochemistry is the method of choice
for determining levels of HER-2 protein expression.
AN IMPORTANT NOTE REGARDING ANTIGEN
RETRIEVAL: Those intending to persevere with their
own in house methods should note the data on
retrieval methods in Table 13. The data on this
occasion are overwhelmingly in favour of water-bath
retrieval, those using the water bath methodology
are significantly more successful than those using
alternative retrieval methods. 10mM citrate buffer
at pH6.0 would appear to be the solution of choice
when retrieving using the water-bath.

Immunocytochemistry — Breast HER-2
Poor performance concerns
The data shown in the Tables indicate that many
laboratories are attempting to use their own specially
devised In House methods for HER-2 testing without
success. EQA is only a small snapshot of performance
and may not always give a true reflection of the quality
of immunocytochemistry being produced by a particular
laboratory. With this in mind and the performance
issues this assessment run raises, it is recommend that
those laboratories who are struggling to perform to the
standard set by the UK NEQAS-ICC assessors review
their HER-2 testing. Auditing of results will in most cases
reveal that 25-30% of breast cancer cases should exhibit
over-expression of the HER-2 protein (3+). According to
Nichols et al this should correlate well with HER-2 gene
amplification. If a particular laboratory finds that their
results do not meet these criteria then it is recommended
that some cases be sent to a recognised specialist centre
so that a valid correlation can be made. For those who
are unsure of the type of immunostaining required,
please refer to the images given in this report. As far as In
House staining was concerned, a number of laboratories
Plate 17. Good demonstration of 3+ staining of the UK NEQAS-ICC
SKOV-3 cell line
Plate 19. Expected faint and/or incomplete membrane (1+) staining
of the UK NEQAS-ICC BT-20 cell line
148
are submitting samples where the normal glands are
stained. It is recommended that if this occurs, that the
test should either be repeated using a validated
method or, if appropriate, perhaps FISH testing for gene
amplification should be considered.
REFERENCES
1. Rhodes A, Jasani B, Couturier J, et al. A formalin fixed, paraffin processed cell
line standard for quality control of immunohistochemical assay of HER-2neu
expression in breast cancer. Am J Clin Pathol. 2002; 117: 81–89.
2. Ellis IO, Dowsett M, Bartlett J, et al. Recommendations for HER2 testing in
the UK. J Clin Pathol 2000; 53: 890–892.
3. Tubbs RR, Pettay JD, Roche PC, et al. Discrepancies in clinical laboratory
testing of eligibility for trastuzumab therapy: apparent immunohistochemical
false-positives do not get the message. J Clin Oncol 2001; 19: 2714–272
4. Rhodes A, Jasani B, Anderson E, et al. 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
5. Nichols DW, Wolff DJ, Self S, et al. A testing algorithm for determination of
HER2 status in patients with breast cancer. Ann Clin Lab Sci. 2002 Winter;
32 (1): 3-11
Plate 18. Optimal demonstration of 2+ staining of the UK NEQAS-ICC
MDA-MB-453 cell line
Plate 20. Poor demonstration of staining of the UK NEQAS-ICC SKOV-3
cell line (expected to be 3+). Morphology of cells is disrupted and
membrane localisation of immunostain is poor

Plate 21. Poor demonstration of staining of the UK NEQAS-ICC MDA-
MB-453 cell line (expected to be 2+). Membrane localisation of
immunostain is poor and haematoxylin counterstain is excessive
Plate 23. Good demonstration of 3+ staining in the SK-BR-3 cell line
from a DakoCytomation control slide
Plate 25. Good demonstration of 1+ staining in the MDA-175 cell
line from a DakoCytomation control slide
149
Immunocytochemistry — Breast HER-2
Plate 22. Excessive haematoxylin staining masks any membrane
staining which may be present in the UK NEQAS-ICC BT-20 (1+) cell
line
Plate 24. SK-BR-3 (3+) cell line from a DakoCytomation control
slide, showing excessive cytoplasmic staining rendering the cell
membranes difficult to interpret
Plate 26. Excessive cytoplasmic staining in the DakoCytomation
MDA-175 (1+) cell line

Immunocytochemistry — Breast HER-2
BEST METHODS Both the DakoCytomation HercepTest and Ventana Pathway system are recognised methods of choice to
demonstrate the Her 2 receptor. Those intending to employ these systems should contact the relevant companies for the exact
protocol for each of the above methods.
The following method was obtained from a participant who achieved the appropriate result on UK NEQAS-ICC material
without using one of the above-recognised methods. However, it should be borne in mind that although some laboratories
have been successful using microwave and pressure cooker retrieval methods for HER-2 immunostaining, UK NEQAS-ICC
data shows that water-bath retrieval is more reliable.
Her-2
Method: DakoCytomation Labelled St.Av K0675 system
Automation: A. Menarini Optimax plus
Buffer and pH: Optimax buffer
Blockade Type: None stated
Antigen Retrieval: Pressure cooker to heat 2L of 10mM
citrate buffer ph6.0, for 5.5 minutes at
full pressure
Primary Antibody: DakoCytomation A0485 polyclonal
dilution 1:1000 for 60 minutes
Chromogen: DakoCytomation K3468 for 10 minutes
MAIN TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE HER-2 MODULE
The following tables record the number of participants (N) using each system. The percentage (%) refers to the
proportion of these participants achieving appropriate staining. For example, in the first table 50 participants used
DakoCytomation Hercep Test Kit K5204 of whom 56% achieved acceptable staining.
Table 12. Primary antibody (HER-2)
Antibody details N %
A.Menarini MU 1344 UC (Clone CB11) 1 0
Cell Marque CMA 601 (Clone CB11) 1 0
DakoCytomation HerCep Kit K5204 50 56
DakoCytomation HerCep Kit K5205 13 62
DakoCytomation HerCep Kit K5206 9 67
DakoCytomation A0485 c-erbB2 (Polyclonal) 32 19
Neomarkers MS 730 1 0
Novocastra NCL-CB11 (clone CB11) 11 27
Incomplete data 4 25
Immunotech 1591 (clone 3B5) 1 0
Oncogene OP15 (clone 3B5) 1 0
Ventana 760 2694 CB11 (Pathway) 7 14
Zymed 28 003 or 18-107 7 0
Table 14. Detection System
Type, Supplier & Product Code N (%)
Biogenex Super Sentitive Multi link/HRP LP000-UL 3 0
DakoCytomation Hercep test K5204 49 57
DakoCytomation Hercep test K5205 13 62
DakoCytomation Hercep test K5206 9 67
DakoCytomation ChemMate L.St.Av/HRP K5001 10 10
DakoCytomation Duet St.ABC K0492 1 0
DakoCytomation Envision Plus 9 44
DakoCytomation ChemMate Envision K5007 5 40
DakoCytomation LSAB Kit/HRP K0675 3 33
DakoCytomation K5005 1 0
Incomplete data 3 0
Lab Vision TA125ML 5 20
Other 1 0
Power Vision DPVB999 HRP 1 0
Vector Elite Universal ABC PK6200 2 0
Vector PK7200 (ready to use) 3 33
Ventana System 19 5
Zymed 87 8143 1 0
150
Table 13. Pre-treatment
Pre-treatment N (%)
Incomplete data 6 0
Microwave Oven 17 18
None 3 33
Pressure cooker 19 21
Pressure cooker inside microwave oven 4 25
Ventana benchmark 9 0
Water bath 95-98 o C for 40 minutes - 80 55
and 20 minutes cooling
Table 15. Automation
Instrument & Supplier N (%)
Biogenex Optimax 1 100
Biogenex Genon i6000 4 0
DakoCytomation TechMate 500 10 30
DakoCytomation Autostainer 19 68
DakoCytomation TechMate Horizon 3 33
LabVision Autostainer 19 68
None 72 43
Shandon Sequenza 7 43
Ventana Benchmark 8 0
Ventana ES 3 0
Ventana NexES 6 0

The Lymphoma Module
Tibor Krenacs
Antigens assessed: CD3 & bcl-2
Tissue section circulated: reactive tonsil (CD3 and bcl-2)
Number of participating laboratories: 205 (CD3),
204 (Bcl-2)
CD3
Introduction
The pentameric CD3 molecule is non-covalently
associated with the dimeric T-cell receptor (TCR) and
both are localised in the T-lymphocyte membrane
(Griesser and Mak). CD3 is thought to mediate signal
transduction though the membrane upon TCR activation.
Distribution of scores
frequency of returns
frequency of returns
Run 65L CD3 on UK NEQAS Sections
Summary
Scores >12/20 143(69%)
Scores 10-12/20 30(15%)
Scores 12/20 130(64%)
Scores 10-12/20 43(21%)
Scores 12/20 168(82%)
Scores 10-12/20 24(12%)
Scores 12/20 151(74%)
Scores 10-12/20 40(20%)
Scores

Immunocytochemistry — Lymphoma
Guidelines used in the assessment CD3 reactions
SCORE STAINING PATTERN
0 No returns.
1 Little or no staining of T-cells
2 Very weak demonstration of T-cells with many of those
expected to stain, not demonstrated
3 Weak demonstration of T- cells with fewer stained than
expected
4 Good demonstration of the vast majority of T-cells
expected to stain, with good localisation of CD3 on the
cytoplasmic membrane.
5 Very intense staining of most of the T-cells expected to
stain, with crisp localisation of CD3 on the cytoplasmic
membrane, crisp morphology and no background
staining
NB. These are only very general guidelines and marks were deducted for
such things as poor localisation of staining or diffuse staining, inappropriate
staining of certain cell types, excessive background staining, excessive
counter-stain, uneven staining or other factors which made interpretation
difficult.
The four individual scores for each slide were added
together to give a mark out of 20.
Features of optimal immunostaining (see Plates 27 and 28)
The following features should be seen in a section of
reactive tonsil well stained for CD3:
• Crisp cell membrane staining of T lymphocytes in all
expected compartments (see Introduction above)
• Moderate cytoplasmic staining is a normal feature
but is insufficient without the obvious cell membrane
labelling (please consider that the rim of cytoplasm is
usually narrow)
• Extracellular appearance of the antigen should be
minimal or lacking
Features of sub-optimal immunostaining (see Plates 29,
30, 31 and 32)
Most low scoring slides were marked down because they
displayed:
• Weak, uneven, partially missing or incomplete staining
of T lymphocytes.
• Diffuse or extracellular localisation of the antigen.
• High background or non-specific staining.
Please note:
Most of the sub-optimal features criticised above
were most probably due to insufficient HIER (weak,
uneven staining – see Plates 30), the overuse of HIER
(strong staining with soft margins, and possibly tissue
damage – see Plates 29), the inappropriate dilution
of primary antibody under the conditions used
(high background, non-specific staining – see Plate
31 and 32), or the possible combinations of these
factors.
152
The best indicators of staining quality are single positive
T-cells found e.g. in the germinal centre (see Plate
28). Close apposition of extra-follicular T-cells may
hinder the clear definition of their membranes.
Summary of findings at this CD3 Assessment
In tonsil sections provided by UK NEQAS-ICC the number
of laboratories passing CD3 staining was lower (69% vs.
75%), while the number of failing laboratories was higher
(16% vs. 11%) than those in Run 64. This was most probably
due to a longer duration of formalin fixation of the tonsil
tissues used in Run 65 than those used in Run 64. This notion
is supported by the fact that there was no difference
between the results achieved on the In House sections
across the two runs (80% vs. 82% passes, and the same 6%
failures in both runs).
The results of those using the rabbit polyclonal CD3
antibody (DakoCytomation A0452, 73%) still surpass
those using monoclonal CD3 antibodies (NovoCastra
NCL-CD3-PS1, 66% pass; DakoCytomation M7452, 65 %
pass rate) (see Table 16).
Heat pre-treatment using a pressure cooker either metal or
a microwavable plastic one allowed the highest passing
rates (76 and 78% respectively) (see Table 18). The rank of
detection systems based on the passing rates achieved
by laboratories were ChemMate-PO (Dakocytomation,
86%); Vector Elite Universal (Vector, 86%); EnVision
(DakoCytomation, 80%); and ChemMate Envision
(DakoCytomation, 78%) (see Table 19). Using standardised
DAB chromogen kits (K5001; EnVision DAB; K3466; and
K5007; all from DakoCytomation) allowed higher (80% or
above) pass rates than the home-prepared versions of
developers (see Table 20). Some automated immunostainers
produced clearly better results than manual
immunostaining, allowing 86% (A. Menarini Optimax,
DakoCytomation Autostainer), 81% (DakoCytomation
TechMate 500), 72% (Ventana NexES) and 69 % (Ventana
Benchmark) passing rates versus the 59% achieved with
manual procedures (see Table 21).
Bcl-2
Introduction
The bcl-2 molecule is an oncogene product (oncoprotein)
blocking apoptotic cell death (Hockenberry et al). In
lymphoid tissues bcl-2 is expressed by mantle zone B-cells
and all effector T-cells, while germinal centre B-cells
lack this molecule. bcl-2 immunoreaction results in an
eccentric cytoplasmic signal since it is localised in
the nuclear envelope, endoplasmic reticulum and
mitochondrial membrane in positive cells.
The upregulation of bcl-2 expression in lymphoid nodular
structures is a specific feature of most follicular lymphomas
resulted from of a non-random chromosomal translocation
t(14;18), when the bcl-2 gene is inserted into juxtaposition
to the highly active IgH gene (Ngan et al).

Guidelines used in the assessment of bcl-2 immunostaining
SCORE STAINING PATTERN
0 No returns.
1 Little or no staining of bcl-2 in the cells expected to stain
(see text above)
2 Very weak demonstration of bcl-2 in most cells expected
to stain, or many of these cells were not demonstrated
3 Weak demonstration of most cells expected to stain.
4 Good demonstration of all the cells expected to stain,
with clear signs of eccentric sub-cellular distribution and
a dynamic range of expression levels of bcl-2
5 Very intense staining in most of the relevant cells with
crisp eccentric localisation of bcl-2 in most cells, crisp
morphology and no background staining
NB. These are only very general guidelines and marks were deducted
for such things as poor localisation of staining or diffuse staining, inappropriate
staining of certain cell types, excessive background staining, excessive
counter-stain, uneven staining or other factors which made interpretation
difficult.
The four individual scores for each slide were added
together to give a mark out of 20.
Features of optimal immunostaining (see Plates 33, 34, 35
and 36)
• Strong cytoplasmic labelling of most lymphocytes
except germinal centre B-cells
• Eccentric ring-like appearance of immunostaining
within individual cells with a wide range of expression
levels in adjacent cells resulting in a dynamic staining
pattern
• Extracellular appearance of the antigen should be
minimal or lacking
Features of sub-optimal immunostaining (see Plates 37,
38 and 39)
• Weak, uneven, partially missing staining of relevant cells.
• High background or non-specific staining of cell types
not expected to stain
Plate 27. CD3 antibody staining T-cells in reactive tonsil (UK NEQAS-ICC
section). Strong labelling of peri-follicular T-cell region and dispersed intrafollicular
T-lymphocytes (score20/20). All Plates here with the exception of
Plate 29 are of immunostaining reactions using DAB as a chromogen.
153
Immunocytochemistry — Lymphoma
Please note: The reasons for poor results most probably
were insufficient HIER (weak, missing staining – see Plates
37 and 38), overused/inappropriate HIER and/or the
inappropriate dilution of primary antibody under the
conditions used (non-specific staining – see Plate 39).
The sensitivity of bcl-2 staining is best assessed on the
demonstration of germinal centre T cells (see Plates 34,
35 and 36).
Summary of bcl-2 results from this assessment
Antibody clone 124 (DakoCytomation M0887) is still the
best choice allowing the highest pass rate (68%) when
demonstrating bcl-2 antigen (see Table 17). Concerning
correlations between the results (pass rates) and other
technical details, most of the general notes made at the
summary of CD3 results (see above) are valid here also.
Some exceptions include lower pass rates for bcl-2
compared to CD3 detection when using Vector Elite
Universal ABC kit PK6200 (26% vs. 86%, see Table 19),
Ventana iView DAB chromogen-substrate (38% vs. 68%,
see Table 20), Ventana NexES (56% vs. 72%) or Ventana
Benchmark (25% vs. 69%, see Table 21) automated
immunostainers. These discrepancies were most probably
due to using primary antibodies different from clone 124,
which were found less successful (see Table 17). The 100%
passing rates achieved with using A. Menarini Optimax
immunostainer is also worth mentioning, though the
number of participants using this option is relatively low.
REFERENCES
1. Griesser H, Mak TW. The T-cell receptor-structure, function, and clinical
application. Hematol Pathol. 1994; 8: 1-23
2. Hockenberry DM, Zutter M, Hickey W et al. Bcl-2 protein is topographically
restricted in tissues characterised by apoptotic cell
death. Proc Natl Acad Sci (USA) 88:6961-6965.
3. Mason DY, Cordell J, Brown M et al. Detection of T cells in paraffin
wax embedded tissue using antibodies against a peptide
sequence from the CD3 antigen. J Clin Pathol 1989; 42:1194-1200.
4. Ngan B-Y, Chen-Levy Z, Weiss LM et al. Expression in non-Hodgkin’s
lymphoma of the bcl-2 protein is associated with the t(14;18)
chromosomal translocation. New Engl J Med 318:1638-1644.
Plate 28. CD3 antibody staining T-cells in reactive tonsil (UK NEQAS-ICC
section). Higher power image of the same reaction as that on Plate 27. All
T-cells show clear cell membrane staining within and around the lymphoid
follicle, (score 20/20).

Immunocytochemistry — Lymphoma
Plate 29. CD3 staining on reactive tonsil (UK NEQAS-ICC section). Strong
but slightly diffuse staining of T-cells particularly in the peri-follicular
region. Immunostained using an alkaline phosphatase labelled detection
system and New Fuchsin as chromogen (score 14/20).
Plate 31. CD3 staining on reactive tonsil (UK NEQAS-ICC section).
Specific T-cell staining accompanied by excessive background
(score 12/20).
Plate 33. bcl-2 antibody staining in an In House section of reactive lymph
node. Low power view shows excellent staining of areas expected to be
positive, e.g. peri-follicular T-cell regions and mantle zone B-cell regions
(score 20/20).
154
Plate 30. CD3 staining on reactive tonsil (UK NEQAS-ICC section).
Moderate and incomplete staining of T-cells (score 12/20).
Plate 32. CD3 staining on reactive tonsil. Strong non-specific staining in
the germinal centre obscures specific T-cell staining (score 8/20).
Plate 34. bcl-2 staining in reactive tonsil (UK NEQAS-ICC section).
Peri- and intra-follicular T-cells and mantle B-cells are strongly positive
(score 20/20).

Plate 35. bcl-2 staining in reactive tonsil (UK NEQAS-ICC section). Higher
power view of the same reaction as on Plate 34 shows eccentric staining
of individual cells and a wide range of bcl-2 expression levels among
positive cells.
Plate 37. bcl-2 staining in reactive tonsil (UK NEQAS-ICC section). The
same area as that seen in Plates 36 and 38. Moderate but unequivocal
staining in most cells expected to stain (score 14/20).
Plate 39. bcl-2 staining in reactive tonsil (UK NEQAS-ICC section).
Strong non-specific staining in the nuclei of germinal centre B-cells
obscure bcl-2 reaction (score 8/20).
155
Immunocytochemistry — Lymphoma
Plate 36. bcl-2 staining in reactive tonsil (UK NEQAS-ICC section). The
same area as that seen in Plates 37 and 38. Strong cytoplasmic staining
in all lymphocytes expected to stain including germinal centre T-cells
(score 20/20).
Plate 38. bcl-2 staining in reactive tonsil (UK NEQAS-ICC section). The same
follicular area as that seen in Plates 36 and 37. Very weak and incomplete
staining of peri-follicular lymphocytes. The germinal centre-T cells, which
are expected to stain with bcl-2 are not demonstrated (score 8/20).

Immunocytochemistry — Lymphoma
MAIN TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE LYMPHOMA MODULE
The following tables record the number of participants (N) using each system. The percentage (%) refers to the
proportion of these participants achieving acceptable staining (a score >12/20). For example in the first table,
15 participants used LabVision CD3 code RM-9017 of whom 67% achieved acceptable staining.
Table 16. Primary Antibody (CD3)
Antibody Details N %
DakoCytomation A0452 34 73
DakoCytomation M7254 65 65
DakoCytomation M 7193 2 0
Cell Marque CMC 364 3 67
ID Labs BP517 1 100
Incomplete data 3 100
LabVision RM-9017 15 67
Novocastra NCL-CD3-PS1 68 66
Neomarkers MS 401 (Clone PS1 ) 1 100
Other 2 100
Ventana 790 2921 9 78
Zymed 18 0102 1 100
Table 18. Pre-treatment
CD3 bcl-2
Pre-treatment N % N %
Incomplete data 1 100 1 100
Microwave Oven 61 70 61 61
Other 5 40 5 100
Pressure Cooker 85 76 84 77
Pressure Cooker inside Microwave 18 78 18 78
Water bath 95-98 o C 15 40 14 36
Ventana Benchmark 18 67 18 22
156
Table 17. Primary Antibody (bcl-2)
Antibody Details N %
Biogenex MU 287-UC 4 50
DakoCytomation M0887 173 68
Neomarkers MS 123 PO 1 0
Novocastra NCL-BCL-2 13 46
Incomplete data 5 60
Ventana 760-2693 (Clone P2D11F11) 6 50
Table 19. Detection system
CD3 bcl-2
Type, supplier & product code N % N %
A.Menarini Multi link/HRP LP000-UL 9 67 9 56
Chemicon HP 1000 1 0 1 0
DakoCytomation ChemMate - 42 86 42 88
L.St.Av/HRP K5001
DakoCytomation ChemMate - 27 78 26 81
EnVision K5007 DAB
DakoCytomation Duet St.ABC K0492 3 67 3 100
DakoCytomation Envision 15 80 15 67
DakoCytomation LSAB Kit/HRP K0675 6 17 5 20
DakoCytomation St.ABC/HRP K0377 1 100 1 100
Incomplete data 5 80 5 60
Other 9 56 9 56
LabVision TS 125 HR 6 33 6 33
Power vision DPVB 999 HRP 1 0 1 0
Vector Elite ABC PK6102 1 0 1 100
Vector Elite Universal ABC PK6200 14 86 14 26
Vector Elite ABC PK6100 4 75 4 100
Vector Elite ABC PK7200 RTU kit 10 60 10 50
Ventana basic system 8 63 8 63
Ventana iView system 37 68 37 35
Zymed 87 8143 1 0 1 0
Zymed 85 9043 1 0 1 0

Table 20. Chromogen
CD3 bcl-2
Chromogen & Supplier N % N %
A. Menarini HK 153 5K DAB 12 67 12 58
DakoCytomation K5001 DAB 43 84 43 88
DakoCytomation K3466 DAB 5 80 5 80
DakoCytomation K3468 DAB 23 56 21 56
DakoCytomation S3000 DAB 2 100 2 100
DakoCytomation Envision DAB 11 82 11 82
DakoCytomation ChemMate
EnVision K5007
27 78 27 78
Incomplete data 11 73 11 64
KEM-EN-TEC 4170 DAB 2 50 2 50
LabVision TA 125 HD DAB 4 25 4 25
Other 7 71 7 71
Sigma D5637 DAB 3 33 3 67
Sigma D5905 DAB 2 50 2 50
Vector SK4100 DAB 4 25 4 50
Ventana iView DAB 40 68 40 38
Zymed 00 2020 DAB 3 33 3 0
BEST METHODS
CD3
Participant Scored 20/20 (UK NEQAS-ICC slide) and
20/20 (In house slide) using this method.
Method: DakoCytomation ChemMate kit K5001
Automation: DakoCytomation TechMate 500 plus
Buffer and pH: DakoCytomation ChemMate EnVision
K5001 buffer
Blockade Type: DakoCytomation ChemMate EnVision
K5001 blocking solution
Antigen Retrieval: Pressure cooker to heat 3L of EDTA buffer
pH 8.0 for 1.5 minutes at full pressure
Primary Antibody: Novocastra NCL-CD3-PS1 1:100 dilution,
for 50 minutes at room temperature
Chromogen: DakoCytomation ChemMate K5001 DAB,
for 15 minutes
Bcl-2
Participant scored 19/20 (UK NEQAS-ICC slide) and
20/20 (In House slide) using this method.
Method: DakoCytomation ChemMate kit K5001
Automation: DakoCytomation TechMate 500 plus
Buffer and pH: DakoCytomation ChemMate kit K5001
Blockade Type: DakoCytomation ChemMate kit K5001
Antigen Retrieval: Prestige pressure cooker to heat 3L of
EDTA buffer pH 8.0 at full pressure for
1.5 minutes
Primary Antibody: DakoCytomation M0887 (clone 124),
dilution 1:300 for 50 minutes at room
temperature
Chromogen: DakoCytomation ChemMate kit K5001
DAB, time not stated
157
Immunocytochemistry — Lymphoma
Table 21. Automation
CD3 bcl-2
Instrument & Supplier N % N %
A. Menarini Optimax 14 86 14 100
A. Menarini Genon MX i6000 8 88 8 63
Dakocytomation Autostainer 35 86 35 77
Dakocytomation TechMate Horizon 1 100 1 100
Dakocytomation TechMate 500 27 81 27 85
Incomplete data 2 100 2 100
Lab Vision Autostainer 16 56 16 50
None 49 59 48 65
Shandon Sequenza 10 40 9 56
Shandon Cadenza 1 0 1 0
Ventana ES 5 60 5 0
Ventana NexES 18 72 18 56
Ventana Gen11 1 0 1 0
Ventana Benchmark 16 69 16 25
CD3
Participant Scored 20/20 (UK NEQAS-ICC slide) and
17/20 (In house slide) using this method.
Method: Vector Elite Universal kit PK6200
Automation: None
Buffer and pH: Tris buffer pH7.6
Blockade Type: Hydrogen peroxide solution
Antigen Retrieval: Kenwood Pressure Cooker, to heat 1.5L of
DakoCytomation Target Retrieval solution
S3308, pH10.0 for 2 minutes at full pressure
Primary Antibody: Novocastra NCL-CD3-PS1 dilution 1:100
for 30 minutes at room temperature
Chromogen: DakoCytomation K3466 DAB, pH7.5
for 5 minutes
Bcl-2
Participant scored 19/20 (UK NEQAS-ICC slide) and
20/20 (In House slide) using this method.
Method: Vector Elite Universal PK6200
Ventana DAB kit 253-2198
Automation: Ventana NexES
Buffer and pH: Phosphate buffered saline
Blockade Type: Hydrogen peroxide and serum
Antigen Retrieval: Kenwood Pressure cooker, to heat 1.5L of
citrate buffer pH 6.0 for 2 minutes at full
pressure
Primary Antibody: DakoCytomation M0887 (clone 124),
dilution 1:40 at 42 o C for 30 minutes
Chromogen: DakoCytomation K3466 DAB pH7.5
for 5 minutes

Immunocytochemistry — Neuropathology
The Neuropathology Module
Kwok Kee Chan
Antigens assessed: Synaptophysin and Cytokeratin
Tissue sections circulated: a block with a medulloblastoma
(synaptophysin); a block with a secondary
carcinoma in brain (cytokeratin)
Number of participating laboratories:
57 (synaptophysin), 56 (cytokeratin)
Distribution of scores
frequency of returns
Figure 12. Run 65G: Synaptophysin (UK NEQAS-ICC Sections)
frequency of returns
Run 65G Synaptophysin on UK NEQAS Sections
Summary
Scores > 12/20 48(84%)
Scores 10-12/20 7(12%)
Scores 12/10 42(75%)
Scores 10-12/20 11(20%)
Scores 12/20 56(96%)
Scores 10-12/20 1( 2%)
Scores 12/20 56(100%)
scores scores
Figure 14. Run 65J: Pan-cytokeratin (UK NEQAS-ICC Sections) Figure 15. Run 65K: Pan-cytokeratin (In House Sections)
frequency of returns

Synaptophysin
In this run, 84% of the participants achieved satisfactory
to good results with the UK NEQAS-ICC sections. The
demonstration of synaptophysin in medulloblastoma is
often requested for the confirmation of the neuronal
origin of the tumour. Moreover, the tumour may appear as
bi-phasic with islands of strongly stained, neurogenically
more matured cells. Synaptophysin is a fixation sensitive
antigen, i.e. prolonged fixation in formalin may require a
correspondingly more extraneous antigen retrieval
regime to recover the reactivity of the antigen. As to the
tissue that was used in this run, it was moderately fixed,
so such regimes may not have been required in order to
produce strong positive staining. On the In House sections,
96% of participants achieving an acceptable result.
Features of optimal immunostaining (Plates 40 and 41)
• Medulloblastoma, intense and specific staining in
the perikarya of the tumour cells.
Plate 40. Optimal demonstration of synaptophysin on UK
NEQAS-ICC section of tumour.
Plate 42. Sub-optimal demonstration of synaptophysin on UK NEQAS-
ICC section of tumour. Staining is very weak (compare with Plate 40).
159
Immunocytochemistry — Neuropathology
Features of sub-optimal immunostaining (Plates 42 and 43)
• Very weak staining in the perikarya of the tumour cells.
Cytokeratin
About 75% of the laboratories achieved satisfactory to
good staining on the UK NEQAS-ICC sections of a
secondary carcinoma in the central nervous system
and 100% on the In House sections. Anti-cytokeratins is
often incorporated into the panel of antibodies for the
differential diagnosis of secondary tumours. Hence, a
broad-spectrum cytokeratin antibody is essential for
such purpose as to ensure successful detection of
carcinoma of various origins.
Features of optimal immunostaining (Plates 44 and 45)
• Clear demonstration of tumour cells in the metastatic
carcinoma.
Features of sub-optimal immunostaining (Plates 46 and 47)
• Very weak staining of the tumour cells.
Plate 41. Higher power view of same section as shown in Plate 40.
Plate 43. Higher power view of same section as shown in Plate 42.

Immunocytochemistry — Neuropathology
Plate 44. Optimal demonstration of cytokeratins on UK NEQAS-ICC
section of tumour.
Plate 46. Poor demonstration of cytokeratins on UK NEQAS-ICC
section of tumour. Staining is weak and uneven.
Plate 48. Optimal result with cytokeratin on a participant’s In
House control. The full depth of squamous epithelium is well
stained.
160
Plate 45. Higher power view of same section as shown in Plate 42.
Plate 47. Higher power view of same section as shown in Plate 46.
Plate 49. Optimal result with cytokeratin on a participant’s In House
control. The full depth of crypt epithelium is well stained in this gut
section.

BEST METHODS
Synaptophysin
Participant scored 20/20 (UK NEQAS-ICC slide) and
16/20 (In House slide) using this method.
Method: Ventana iView DAB kit
Automation: Ventana NexES
Buffer and pH: Ventana APK was buffer
Blockade Type: Ventana solution
Antigen Retrieval: Water-bath to heat 200 ml of EDTA buffer
pH 8.0 for 23 minutes at 98 o C
Primary Antibody: DakoCytomation N1566
pre-diluted antibody for 30 minutes
Chromogen: Ventana DAB for 8 minutes
Pan-cytokeratin
Participant scored 20/20 (UK NEQAS-ICC slides) and
19/20 (In House Sections) using this method.
Method: DakoCytomation Envision K4007 kit
Automation: LabVision Autostainer
Buffer and pH LabVision TBS TA-125-TB buffer with
0.05% Tween-20
Blockade Type: Hydrogen peroxide solution
Antigen Retrieval: Sigma Chymotrypsin C-4129
(concentration not stated), pH7.8 for
10 minutes at 37 o C
Primary Antibody: DakoCytomation M3515 (clones AE1/AE3)
dilution 1:85 for 30 minutes room
temperature
Chromogen: DakoCytomation EnVision DAB K4007
for 5 minutes
161
Immunocytochemistry — Neuropathology
Synaptophysin
Participant scored 20/20 (UK NEQAS-ICC slide) and
17/20 (In House slide) using this method.
Method: DakoCytomation ChemMate K5001 kit
Automation: DakoCytomation TechMate 500 plus
Buffer and pH: DakoCytomation ChemMate K5001
buffer
Blockade Type: DakoCytomation ChemMate K5001
solution
Antigen Retrieval: Samsung 800W Microwave oven to
heat 400 ml of citrate buffer pH6.0 for
20 minutes
Primary Antibody: DakoCytomation A0010 polyclonal,
dilution 1:40 for 50 minutes at room
temperature
Chromogen: DakoCytomation DAB K5001 for 15
minutes at room temperature
Pan-cytokeratin
Participant scored 20/20 (UK NEQAS-ICC slides) and
20/20 (In House Sections) using this method.
Method: Vector Elite RTU kit PK7200
Automation: DakoCytomation Autostainer plus
Buffer and pH Tris buffer saline pH 7.6
Blockade Type: 3% Hydrogen peroxide, and horse serum
Antigen Retrieval: Kenwood PC400 Pressure cooker to heat
1.6L of Vector unmasking solution pH 6.0
for 4 minutes at full pressure. Then 2
seconds in Sigma Protease (concentra
tion not stated) Type XXIV P5147 at pH 7.4
at room temperature
Primary Antibody: DakoCytomation M0821 (clone MNF116)
dilution 1:100 for 30 minutes at room
temperature
Chromogen: A. Menarini HK-153-5K DAB for 10 minutes

Immunocytochemistry — Neuropathology
MAIN TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE NEUROPATHOLOGY MODULE
The following tables record the number of participants (N) using each system. The percentage (%) refers to the
proportion of these participants achieving acceptable staining (a score >12/20). For example in the 1st table, 20
participants used DakoCytomation Synaptophysin (code A0100), 70% of whom achieved acceptable staining.
Table 22. Primary Antibody. [Synaptophysin]
Antibody Details N %
A. Menarini MU 363 1 100
Boehninger 902314 (Clone SY38 ) 1 100
DakoCytomation M0776 (Clone SY38) 19 84
DakoCytomation A0010 20 70
DakoCytomation N1566 1 100
Incomplete data 1 100
Lab Vision RM 9111 SO 2 50
Novocastra NCL-SYNAP 9 100
Other 1 100
Zymed 18-0130 3 100
Table 24. Pre-treatment
SYNAPTOPHYSIN PAN-CK
Pre-treatment N % N %
DakoCytomation Protease K S3020 - - 1 100
Difco Trypsin 215240 - - 6 67
Heat and Enzyme - - 4 100
ICN Trypsin - - 2 50
Incomplete data 1 0 1 100
None 3 67 - -
Microwave Oven 27 89 13 69
Other - - 3 100
Pressure Cooker 15 80 10 70
Pressure Cooker inside Microwave Oven 6 100 2 100
Sigma Pepsin P7000 - - 2 100
Sigma Protease P5147 1 0 1 0
Sigma Protease P8038 - - 1 100
Sigma Chymotrypsin C-4129 - - 3 100
Ventana benchmark 2 100 - -
Ventana Protease 1 - - 7 57
Water bath 98oC for 40 minutes 3 67 - -
Table 26. Chromogen
SYNAPTOPHYSIN PAN-CK
Chromogen & Supplier N % N %
Biogenex HK-153-5K DAB 2 50 2 50
DakoCytomation K3468 5 60 5 60
DakoCytomation K5001 DAB 12 100 12 83
DakoCytomation S3000 1 100 1 0
DakoCytomation Envision 4 50 4 75
Dakocytomation K5007
ChemMate Envision
9 100 9 78
Dakocytomation K3219 DAB 1 100 1 100
Merk New Fuchsin 4041 1 100 1 100
Sigma A5754 AEC 1 0 1 0
Sigma D3466 1 100 - -
Sigma D5905 1 100 1 100
Sigma D5635 DAB 1 100 1 100
Sigma D5637 DAB 3 100 3 67
Vision Biosystem DAB 1 100 1 100
Ventana iView DAB 7 100 6 67
Zymed DAB kit 1 100 1 100
Table 25. Detection System
SYNAPTOPHYSIN PAN-CK
Type, Supplier & Product Code N % N %
A.Menarini Multi link/HRP LP000-UL 1 100 1 0
DakoCytomation ChemMate
L.St.Av/HRP K5001
12 100 12 83
DakoCytomation Duet St.ABC K0492 1 100 1 100
DakoCytomation Envision Kits 7 57 7 43
DakoCytomation St.ABC/HRP K0377 1 100 1 100
DakoCytomation K0675 2 50 1 100
DakoCytomation K5007 ChemMate Envision 9 100 9 78
DakoCytomation K0391 1 100 1 100
LabVision TS 125 HRP 1 100 1 100
Incomplete data 2 50 2 100
Vector Elite ABC PK 6100 2 50 2 50
Vector Elite Universal ABC PK6200 5 80 5 60
Vector PK7200 RTU 4 50 4 100
Ventana basic system 4 100 4 75
Ventana iView System 3 100 2 50
Vision Biosystem D59404 1 100 1 100
Zymed 85 9043 1 0 1 100
Zymed 87 8143 1 100 1 100
Table 27. Automation
SYNAPTOPHYSIN PAN-CK
Instrument & Supplier N % N %
162
Table 23. Primary antibody. [Cytokeratin]
Antibody Details N %
Becton Dickinson 349205 (CAM 5.2)* 4 100
DakoCytomation M0821 (Clone MNF116 ) 25 80
DakoCytomation M3515 (Clone AE1/AE3 ) 14 57
DakoCytomation H7118 (Clone 34βE12)* 1 100
Home made cocktails 2 100
Novocastra NCL-AE1/AE3 1 100
Sigma C2562 1 100
Ventana 760 2595 (Clone AE1/AE3 ) 4 25
Zymed 28001 (cocktail) 2 100
Zymed 18-0132 (Clone AE1/AE3) 1 100
*Although CAM5.2 and 34βE12 are not pan-cytokeratin antibodies participants who
used them were not penalized at this assessment.
A. Menarini Optimax 1 100 1 100
DakoCytomation Autostainer 12 83 12 67
DakoCytomation TechMate 500 11 100 11 91
LabVision Autostainer 2 50 2 100
Incomplete data 2 50 2 100
None 19 79 18 61
Shandon Sequenza 3 33 3 100
Ventana NexES 3 100 3 100
Ventana Benchmark 2 100 1 0
Ventana ES 2 100 2 50
Vision Biosystem Bond X 1 100 1 100

The Cytology Module
Dr Perry Maxwell
Antigens Assessed: Melanoma Markers. Cytokeratins
Material circulated: cell line from a metastatic
melanoma (melanoma markers), clinical sample from
adenocarcinoma in pleural fluid (cytokeratins)
Number of participating laboratories: 87 (melanoma
markers), 87 (cytokeratins)
Guidelines used in the assessment of slides:
SCORE STAINING PATTERN
0 No returns
1 Little or no staining of the antigen in question.
2 Very weak demonstration of the cells expected to
stain or majority of these cells were not demonstrated.
Inappropriate staining of other cells which
may lead to problems in a diagnostic setting.
3 Weak demonstration of most of the cells expected
to stain.
4 Good demonstration of all the cells expected to
stain
5 Excellent demonstration of all the cells expected
to stain
NB. These are only very general guidelines and marks were deducted for
such things as poor localisation of staining or diffuse staining, inappropriate
staining of certain cell types, excessive background staining, excessive
counter-stain, uneven staining or other factors which made interpretation
difficult.
Distribution of scores
frequency of returns
frequency of returns
Run 65R Melanoma Markers on UK NEQAS cytospins
Summary
Scores> 12/20 61(70%)
Scores 10-12/20 18(21%)
Scores 12/20 75(89%)
Scores 10-12/20 9(11%)
Figure 17. Run 65R: Melanoma markers (UK NEQAS-ICC cytospins) Figure 18. Run 65S: Melanoma markers (In House Slides)
Run 65T Cytokeratin on UK NEQAS Cytospins
Summary
Scores >12/20 83(96%)
Scores 10-12/20 3( 3%)
Scores 12/20 81(93%)
Scores 10-12/20 5( 6%)
Scores< 10/20 1( 1%)
scores scores
Figure 19. Run 65T: Cytokeratin (UK NEQAS-ICC cytospins) Figure 20. Run 65U: Cytokeratin (In House Slides)
frequency of returns
frequency of returns

Immunocytochemistry — Cytology
Plate 50. Melanoma marker (HMB45) on UK NEQAS-ICC slide.
Optimal staining.
Plate 52. Sub-optimal staining of melanoma cells in UK NEQAS-ICC slide.
Melanoma markers
Introduction
There is a role for immunocytochemistry in the identification
of metastatic melanoma (Nasiell et al, Nasuti
et al, Filie et al). This role is particularly useful in FNA
preparations of metastatic samples with epithelioid
differentiation (Filie et al) and patients in clinical trials
undergoing immunotherapy (Fetsch et al).
Features of Optimal Staining (Plates 50 and 51)
• Up to 75% of the cells should be positive for HMB45
(Plate 50).
• Almost 100% of the cells should be positive for S100.
Polarisation of S100 was shown by some participants
(Plate 51).
• The expected cellular location for all melanoma
markers is cytoplasmic
• The intensity of the counterstain should be such as to
show nuclei.
164
Plate 51. Melanoma marker (S100) on UK NEQAS-ICC slide. Optimal
staining, note the polarization of S100 positive reaction.
Plate 53. Pan-cytokeratin on UK NEQAS-ICC slide. Optimal staining.
Features of Sub-optimal staining (Plate 52)
• Loss of morphology. This was most often seen when
digestion methods were employed using cell conditioning
fluid on automated staining systems,
enzymes or heat mediated antigen retrieval. Any
form of antigen retrieval of alcohol-fixed cytospins
is not usually necessary. If employed, retrieval
should be minimal.
• Poor counterstain quality. Quite often, participants
didn’t seem to employ any counterstain, or if
used, this was too weak to be of use. In cytological
preparations, nuclear morphology can help to
identify cell type.
Note on primary antibodies and pre-treatment methods:
The majority of participants used antibodies to S100 or the
HMB45 clone. One participant used an antibody to
MelanA. Both of the best methods highlighted did not use
any form of pretreatment.

Note on In House controls: Of the 84 participants who submitted
In House controls, only 15 used cytology controls,
including cell blocks. All others used histological sections
of melanoma/skin.
REFERENCES
1. Fetsch PA, Steinberg SM, Riker AI, Marincola FM, Abati A. Melanoma
antigen expression in serial fine-needle aspiration samples in patients
with metastatic malignant melanoma participating in immunotherapy
clinical trials: a preliminary look. Cancer. 2001; 93: 409-14.
2. Filie AC, Simsir A, Fetsch P, Abati A. Melanoma metastatic to the
breast: utility of fine needle aspiration and immunohistochemistry.
Acta Cytol. 2002; 46: 13-8.
3. Nasuti JF, Gupta PK, Baloch ZW. Clinical implications and value of
immunohistochemical staining in the evaluation of lymph node
infarction after fine-needle aspiration. Diagn Cytopathol. 2001; 25: 104-7.
4. Nasiell K, Tani E, Skoog L. Fine needle aspiration cytology and
immunocytochemistry of metastatic melanoma. Cytopathology.
1991; 2: 137-47.
Pan-cytokeratin
Introduction
The use of fine needle aspiration cytology is now
considered a valid and standard method in the
diagnosis of various primary and metastatic tumours.
The use of a pan-cytokeratin marker can be useful in
highlighting epithelial cells in such samples.
Features of Optimal Staining (Plate 53)
• The samples circulated for staining were cytospin
preparations from a clinical sample (pleural aspirate)
of adenocarcinoma of the lung. Cytoplasmic staining
of cytokeratin should be seen.
BEST METHODS
Melanoma markers
Participant Scored 19/20 (UK NEQAS-ICC slide) using this
method.
Method: DakoCytomation ChemMate EnVision
kit K5007
Automation: DakoCytomation TechMate 500 plus
Buffer and pH: DakoCytomation ChemMate EnVision
K5007 buffer
Blockade Type: DakoCytomation ChemMate EnVision
K5007 solution
Antigen Retrieval: None
Primary Antibody: DakoCytomation S100 polyclonal Z0311
dilution 1:3000, for 60 minutes at room
temperature
Chromogen: DakoCytomation ChemMate EnVision
K5007 DAB for 7.5 minutes
165
Immunocytochemistry — Cytology
• As this is a clinical sample, there is a population of nonepithelial
cells, which should remain negative.
• In keeping with all cytological preparations, nuclear
morphology can help to identify cell type. The
intensity of the counterstain should be such as to
show nuclei.
Features of Sub-optimal staining
• The major feature of sub-optimal staining was one of
non-specificity. In this clinical sample, background
reactive cells such as polymorphs should be negative.
A poor score was usually attributable to positive
cytoplasmic staining of these reactive cells.
• Often, in those participating laboratories which scored
below an acceptable level, there was also a loss of cell
morphology, particularly of nuclear detail. Loss of
morphology was often seen when the material was
pretreated either by enzyme, cell conditioning fluid or
microwaving. One participant went so far as to both
microwave followed by enzyme digestion.
Pretreatment regimes are not recommended on this
alcohol-fixed material.
• In cytological preparations, the appearance of the
nucleus is important and needs to be seen.
Notes on primary antibodies and automation: The two
clones, most often used were the MNF116 (DakoCytomation)
and the mix of AE1/AE3 (various sources). One laboratory
used CAM5.2, which although not a pan-cytokeratin marker,
was positive in the sample distributed. Twenty-three of the
participating laboratories used some form of automation.
Note on controls: Twenty-five laboratories used cytology
controls, including cell-blocks. All others used histological
sections of appendix, normal colon or normal
colon/colonic adenocarcinoma.
Pan-cytokeratin
Participant Scored 18/20 (UK NEQAS-ICC slide) using this
method.
Method: Ventana iView DAB kit
Automation: Ventana NexES
Buffer and pH: Ventana APK wash buffer
Blockade Type: Ventana blocker solution
Antigen Retrieval: None
Primary Antibody: Biogenex MU-071-UC clones AE1/AE3
dilution 1:200 for 30 minutes
Chromogen: Ventana iView DAB for 8 minutes

Immunocytochemistry — Cytology
Melanoma markers
Participant Scored 18/20 (UK NEQAS-ICC slide) using this
method.
Method: DakoCytomation ChemMate K5001 kit
Automation: DakoCytomation TechMate 500 plus
Buffer and pH: DakoCytomation ChemMate K001 buffer
Blockade Type: DakoCytomation ChemMate K001 solution
Antigen Retrieval: None
Primary Antibody: DakoCytomation HMB45 M0634 dilution
1:5, for 60 minutes at room temperature
Chromogen: DakoCytomation ChemMate K5001
DAB for 15 minutes
Table 28. Primary Antibody (Melanoma markers)
Antibody Details N %
Biogenex MU 058 UC S100 1 100
DakoCytomation Z0311 S100 40 70
DakoCytomation M0634 (Clone HMB45 ) 34 74
ENZO C34930 HMB45 1 100
Incomplete data 3 100
Other 1 100
Novocastra NCL-HMB45 2 50
Novocastra NCL-Melan A 1 100
Novocastra NCL-S100p 2 0
Ventana 760 2523 S100p 1 0
166
Pan-cytokeratin
Participant Scored 20/20 (UK NEQAS-ICC slide) using this
method.
Method: DakoCytomation ChemMate K5001 kit
Automation: None
Buffer and pH: DakoCytomation ChemMate K001 buffer
Blockade Type: DakoCytomation ChemMate K001
solution
Antigen Retrieval: None
Primary Antibody: DakoCytomation MNF116 M082 dilution
1:300, for 40 minutes at room temperature
Chromogen: DakoCytomation ChemMate K5001
DAB for 15 minutes
MAIN TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE CYTOLOGY MODULE
The following tables record the number of participants (N) using each system. The percentage (%) refers to the
proportion of these participants achieving acceptable staining (a score >12/20). For example in the first table,
34 participants used DakoCytomation HMB45 code M0634, of whom 74% achieved acceptable staining.
Table 29. Primary Antibody (pan-cytokeratin)
Antibody Details N %
Biogenex MU071-UC (Clone AE1/AE3) 6 100
Becton Dickinson (CAM 5.2)* 7 100
BMA T1302 1 0
Dakocytomation M0821(Clone MNF116) 35 94
Dakocytomation M 3515 (Clone AE1/AE3) 18 100
Home Made cocktails 2 100
Novocastra NCL-AE1/AE3 3 100
Novocastra NCL-PAN CK 1 100
Incomplete data 3 100
Immunotech 1918 and 1077 (KL1) 3 100
Sigma C2562 (?) 1 0
Ventana 760 2135 (AE1/AE3) 1 100
Zymed 18-0132 (AE1/AE3) 4 100
*Although CAM5.2 is not a pan-cytokeratin antibody participants who used it
were not penalized at this assessment.

Table 30. Pre-treatment
Melanoma PAN CK
Pre-treatment N % N %
DakoCytomation Pronase S2013 1 100 - -
Incomplete data 3 67 1 100
Microwave Oven 8 63 6 100
None 69 75 72 97
Other - - 1 100
Pressure cooker 3 33 2 50
Sigma Chymotrypsin C-4129 - - 1 100
Sigma Protease P8038 - - 1 100
Ventana benchmark CC1 1 0 - -
Ventana Protease 1 - - 2 100
Water bath at 98 o C for 40 minutes 1 0 - -
Table 32. Chromogen
Melanoma PAN CK
Chromogen & Supplier N % N %
Biogenex HK 1535K 4 50 4 100
DakoCytomation Envision 5 80 5 100
DakoCytomation K3466 DAB 2 0 2 100
DakoCytomation K5007 10 90 10 70
DakoCytomation K3464 AEC 1 100 1 100
DakoCytomation K3461 AEC 1 0 1 100
DakoCytomation K5001 23 83 23 100
DakoCytomation K5005 4 75 4 100
Incomplete data 2 50 2 100
Lab Vision DAB 3 67 3 67
Other 7 71 7 100
Sigma D5637 DAB 2 50 2 100
Sigma K3468 DAB 6 83 6 100
Ventana Fast red 1 100 1 100
Ventana iView DAB 15 53 15 100
167
Immunocytochemistry — Cytology
Table 31. Detection System
Melanoma PAN CK
Type, Supplier & Product Code N % N %
Biogenex Super Sensitive Multi link/
HRP LP000-UL
2 50 2 100
Chemicon HP 1000 1 0 1 100
DakoCytomation ChemMate L.St.Av/
HRP K5001
24 83 24 100
DakoCytomation ChemMate
Envision K5007
11 82 11 73
DakoCytomation ChemMate L.St.Av/
ALP K5005
4 50 4 100
DakoCytomation Duet St.ABC K0492 5 80 5 100
DakoCytomation Envision 7 71 7 100
DakoCytomation LSAB Kit/HRP K0675 3 67 3 100
DakoCytomation K0690 1 0 1 100
Incomplete data 1 100 1 100
LabVision TS 125 HR 2 50 2 100
Other 1 100 1 100
Vector Elite Universal ABC PK6200 4 100 4 100
Vector PK 7200 RTU 2 50 2 100
Vector PK 6100 1 0 1 100
Ventana Basic system 4 0 4 100
Ventana Alk Phos system 3 67 3 67
Ventana iView system 10 70 10 100
Table 33. Automation
Melanoma PAN CK
Instrument & Supplier N % N %
Biogenex Optimax 7 86 7 100
Biogenex Genon MX i6000 2 50 2 100
DakoCytomation Horizon 1 100 1 100
DakoCytomation TechMate 500 13 92 13 92
DakoCytomation Autostainer 17 65 17 94
Incomplete data 1 0 1 100
LabVision Autostainer 5 80 5 100
None 23 78 23 96
Shandon Sequenza 1 0 1 100
Ventana ES 2 0 2 100
Ventana NexES 7 71 7 100
Ventana Benchmark 6 50 6 83
Ventana Gen 11 1 0 1 100

Immunocytochemistry — Alimentary Tract
The Alimentary Tract (Pilot) Module
Merdol Ibrahim
Antigens assessed: CD117 (c-KIT)
Material circulated: A composite block comprising of a
Gastro Intestinal Stromal Tumour (GIST) and appendix.
Number of participating laboratories: 50
Guidelines used in the assessment of slides:
SCORE STAINING PATTERN
0 No returns
1 Little or no staining of the GIST or cells of Cajal
2 Very weak demonstration of the GIST or cells
of Cajal
3 Weak demonstration of the GIST or cells of Cajal
4 Good demonstration of the GIST or cells of Cajal
5 Excellent demonstration of the GIST or cells of
Cajal
NB. These are only very general guidelines and marks were deducted for such
things as poor localisation of staining or diffuse staining, inappropriate staining
of certain cell types, excessive background staining, excessive counter-stain,
uneven staining or other factors which made interpretation difficult.
Distribution of scores
frequency of returns
Run 65V Cd117 without retrieval on UK NEQAS Sections
Summary
Scores >12/20 25 (81%)
Scores 10-12/20 4 (13%)
Scores 12/20 34 (78%)
Scores 10-12/20 5 (11%)
Scores 12/20 22 (67%)
Scores 10-12/20 3 (9%)
Scores 12/20 36 (80%)
Scores 10-12/20 7 (16 %)
Scores

Plate 54. Strong staining of mast cells in section of appendix from
UK NEQAS-ICC section of appendix, however there is also some
background staining present which is particularly noticeable within
crypt epithelial cells.
Plate 56. GIST from the UK NEQAS-ICC section showing intense and
even staining.
Plate 58. Good demonstration of a GIST from a participant’s In
House section (HIER had been used).
169
Immunocytochemistry — Alimentary Tract
Plate 55. Interstitial cell of Cajal, from UK NEQAS-ICC section,
demonstrating intense staining.
Plate 57. Poor demonstration of mast cells on UK NEQAS-ICC section,
showing excessive background and non-specific staining.
Plate 59. Poor demonstration of same GIST as that shown in Plate
58. In this case HIER had not been used.

Immunocytochemistry — Alimentary Tract
BEST METHODS
CD117
Participant Scored 20/20 (UK NEQAS-ICC slide), and
20/20 (In House slide) using this method.
Method: DakoCytomation ChemMate K5001
Automation: DakoCytomation TechMate 500 plus
Buffer and pH: DakoCytomation ChemMate K5001
buffer
Blockade Type: DakoCytomation ChemMate K5001
blocking solution
Antigen Retrieval: Pressure cooker to heat 2L citrate
buffer pH6.0 for 3 minutes at full
pressure
Primary Antibody: DakoCytomation A4502 polyclonal
c-KIT (dilution and incubation time
were not stated)
Chromogen: DakoCytomation ChemMate K5001
DAB for 10 minutes
170
CD117
Participant Scored 20/20 (UK NEQAS-ICC slide), and
20/20 (In House slide) using this method.
Method: Ventana Benchmark DAB kit
Automation: Ventana Benchmark system
Buffer and pH: Ventana buffer
Blockade Type: Ventana blockade system
Antigen Retrieval: Ventana Cell Conditioning I solution
Primary Antibody: DakoCytomation A4502 polyclonal
Dilution 1:100 for 32 minutes at 37 o C
Chromogen: Ventana DAB for 8 minutes
MAIN TECHNICAL PARAMETERS EMPLOYED BY PARTICIPANTS IN THE ALIMENTARY TRACT
(PILOT) MODULE
The following tables record the number of participants (N) using each system. The percentage (%) refers to the
proportion of these participants achieving acceptable staining (a score >12/20). For example in the first table,
30 participants used DakoCytomation Polyclonal antibody A4502 of whom 80% achieved acceptable staining using
an antigen retrieval system.
Table 34. Primary Antibody (CD117)
With retrieval Without retrieval
Antibody details N % N %
DakoCytomation A4502 polyclonal 30 80 38 92
Incomplete data 0 0 2 100
NeoMarker RB 1518 0 0 1 0
NeoMarker RB 9038 0 0 1 0
Novocastra NCLCD117 0 0 1 100
Ventana 790 2939 0 0 1 100
Table 35. Pre-treatment
With retrieval Without retrieval
Pre-treatment N % N %
Incomplete data 2 50 - -
Microwave Oven 13 77 n.a. n.a.
Pressure Cooker in Microwave Oven 2 100 n.a. n.a.
Pressure Cooker 10 90 n.a. n.a.
Ventana Benchmark 2 100 n.a. n.a.
Water bath 95-98 o C 1 0 n.a. n.a.
None n.a. n.a. 44 89
n.a. = not applicable

Table 36. Detection System With retrieval Without retrieval
Type, supplier and product code N % N %
DakoCytomation ChemMate K5005 1 100 1 100
DakoCytomation ChemMate K5001 8 88 13 85
DakoCytomation ChemMate – 6 83 8 88
Envision K5007 DAB
Dakocytomation Duet St.ABC K0492 1 0 1 100
DakoCytomation Envision Plus systems 3 67 6 100
DakoCytomation St.ABC/HRP K0377 1 0 1 100
Incomplete data 2 100 2 100
Power Vision DPVB 999 HRP 1 0 0 0
Vision Biosystem D59404 1 100 1 100
Vector Elite ABC PK 6100 1 100 1 100
Vector Elite Universal ABC PK6200 1 100 1 100
Ventana iVIEW system 4 100 7 88
171
Table 37. Chromogen
With retrieval Without retrieval
Chromogen and Supplier N % N %
DakoCytomation Envision Plus kits 3 33 5 100
DakoCytomation K3468 DAB 3 67 3 100
DakoCytomation ChemMate - 8 88 13 85
K5001 DAB
DakoCytomation ChemMate - 1 100 1 100
K5005 Alk phos
DakoCytomation S3000 DAB 1 100 1 100
DakoCytomation ChemMate - 5 80 7 86
Envision K5007 DAB
Table 38. Automation
With retrieval Without retrieval
Instrument and Supplier N % N %
Biogenex Optimax 1 100 2 50
Biogenex Genon MX 6000i 1 100 2 100
DakoCytomation Autostainer 9 67 11 82
DakoCytomation TechMate 500 9 78 15 73
LabVision Autostainer 2 50 1 0
None 4 100 6 100
Ventana Benchmark 3 100 4 75
Vision Biosystem 1 100 1 100
Immunocytochemistry — Alimentary Tract
Incomplete data 2 100 2 100
Other 3 67 3 100
Vision Biosystem Bond-x DAB 1 100 1 100
Ventana iVIEW DAB 3 100 6 83

Immunocytochemistry
Instructions for Authors
AIMS
The journal will concentrate on the use of modern
immunocytochemical techniques in prognostic and
diagnostic clinical settings, and clinical research.
Methodological and application based papers will be
equally welcomed. Articles may be:
• Research Reports
• Technical Articles
• Technical Reviews
• Applications Reviews
• Quality Assurance Articles
The main objective of the journal is the timely dissemination
of information relevant and valuable to the practitioners
and users of immunocytochemistry.
SUBMISSION OF MANUSCRIPTS
All material submitted for publication should be sent to
the Editor-in-Chief:
Mr. Andrew R Dodson
Editor-in Chief
Immunocytochemistry
Royal Liverpool University Hospital
Liverpool L69 3GA, UK
Include a covering letter that gives contact details:
name, address, telephone, and e-mail.
Manuscripts should be submitted in English, in duplicate
on A4 paper. All copy must be typed double-spaced
on one side of the paper only. Photographs should be
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drawings other illustrations, and tables should be
submitted separately in duplicate as one item per
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Acceptable formats for text are MSWord for Windows,
Rich Text Format, and Adobe Acrobat PDF. Photographs
should be submitted as high-resolution JPEG or TIFF files.
In exceptional circumstance high-quality transparencies
will be accepted in place of digital copies. The journal
recognises the utility and importance of colour photomicrographs
to illustrate staining results, and will
make no page charges to authors for their inclusion.
Immunocytochemistry
172
FORMAT
Title: Full title
Authors: Full names of all authors, given in the order
they should appear in the published article; indicate
which is the contact author and insure that full contact
details are given for this person.
Summary: A synopsis of the papers contents, brief and
factual, to indicate the full scope of the paper. Avoid
phrases such as ‘…will be discussed’. Length should be
50 to 150 words.
Main body: Full word count (including title, summary,
main body and references) should not exceed 2,000
words. Structure methodological papers in a standard
format: Introduction, Materials and Methods, Results,
Discussion, Conclusions (Conclusions section is optional).
References: Numbered consecutively as they appear
in the text. Use the following style:
1. Shi SR, Key ME, Kalra KL. Antigen retrieval in formalinfixed
paraffin embedded tissues: an enhancement
method for immunohistochemical staining based on
microwave oven heating of sections. J Histochem
Cytochem 1991; 39: 741–748
2. Ellis IO. Immunocytochemistry in breast pathology. In
Theory and Practice of Histological Techniques (5 th
edn). Bancroft JD, Gamble M (eds). Churchill
Livingstone, London 2002; 499–516
3. Shepherd P, Dean C (eds). Monoclonal antibodies: a
practical approach. Oxford University Press, Oxford;
2000
4. http://www.histopath.ucl.ac.uk/neqas/frame.html
For multiple author references, cite only the first three
authors, followed by et al.
Journal names should be abbreviated using the Index
Medicus standard format.
Copyright
UK NEQAS-ICC for Immunocytochemistry (UK NEQAS-
ICC) holds the Copyright on all material it publishes in
Immunocytochemistry. Author(s) will be required to sign
a Copyright Transfer Agreement prior to publication.
Honorarium
UK NEQAS-ICC for Immunocytochemistry will pay an
honorarium to the value of £100 (less tax deducted at
source for UK residents). This will be made payable to
the lead (first named) author, unless specific written
instructions to the contrary are received at the time of
submission of the paper.