1. Qualitative and Quantitative
Investigation of Cytology Material
from Carcinoma of the Breast with
Particular Reference to the
Demonstration of Estrogen Receptors
B. M. Brown A.I.M.L.S.
Department of Histopathology
St Thomas’ Hospital

2. Table of Contents
SUMMARY .......................................................... 3
Introduction ..................................................... 4
Aims of the project ............................................. 13
Materials and methods ........................................... 14
Summary of techniques ........................................... 16
Fine Needle Aspiration......................................... 16
Double Embedding Method........................................ 16
Periodic Acid Schiff (PAS)..................................... 18
Immunoperoxidase Indirect Method............................... 18
Results ......................................................... 20
Breast Aspirate Samples Prepared By the Double Embedding Technique
.............................................................. 24
Conclusion/Discussion ........................................... 25
Appendices ...................................................... 31
References ...................................................... 32

3. 3
SUMMARY
In total, 80 breast lumps were sampled by fine needle
aspiration for the demonstration of Estrogen Receptor Protein.
30 breast lump aspirates were used to prepare direct smears
and 50 breast lump aspirates were used to prepare pellets for
double embedding and histological sections.
Immunoperoxidase was carried out on each case by the Indirect
Immunoperoxide method, using Monoclonal Antibodies against
Carcinoembryonic Antigen (CEA), Epithelial Membrane Antigen
(EMA), Cytokeratin (CAM 4.2) (Cytok), DD9 (E7), and Estrogen
Receptor Protein (D5).
52/80 aspirates prepared were found to be positive for EMA.
16/80 aspirates were found to have Estrogen Receptor Protein
antigens. 20/80 were CEA positive. 56/80 Cytok positive and
22/50 were positive with DD9.
8/30 aspirates using a direct smear method were unreliable and
11/50 aspirates using the double embedding method were
uninterpretable.
The double embedding method gave unequivocal results proving
the aspirate was adequate. Whereas the smears gave equivocal
results due to cell adherence, smear techniques and background
staining.

4. 4
INTRODUCTION
One British woman in seventeen develops breast cancer during
her lifetime and nearly two thirds of these women eventually
die from the disease.1
In humans, breast malignancies probably arise largely from
basal cells of the ductal epithelium.
Ionizing radiation is the only known risk factor for carcinoma
of the breast and probably acts by damaging the D.N.A. of the
breast cells. Nevertheless, radiation exposure can only
account for a small proportion of tumours, and other factors,
causing risk, must occur. Many researchers believe that the
ovarian-pituitary axis may play an aetiological role.
By exerting hormonal effects, rates of cell proliferation may
be increased or decreased, and atrophy or differentiation of
stem or intermediate cells may occur.2
Women have approximately 100 times greater a chance of
developing breast cancer than men. From puberty to menopause,
the incidence of breast cancer increases rapidly but post-
menopausal, the incidence declines.

5. 5
Risk increases the longer a woman has normal ovarian function.
Premenopausal oophorectomy, without exogenous estrogen
supplements, is found to diminish the risk.2
It has also been found that child bearing alters the risk,
parous women being at greater risk than nulliparous women.
Parous women who had early pregnancies are at less risk than
parous women who have had late pregnancies. Women who suffered
interruption of their pregnancy during the first trimester,
are found to have an increased risk of breast cancer.3
Endogenous estrogens, progesterone and possibly androgens and
prolactin are thought to be involved in this risk factor and
are most likely to play a role in these pathogenic changes.
Estrogen causes proliferation of breast tissue and would
therefore be expected to increase the risk of breast carcinoma
by stimulating the growth stem and intermediate cells.
Progesterone causes alveolar cell growth in the estrogen-
primed breast, but also differentiation. It is unclear
therefore whether the net effect would be to increase or
decrease the risk.
Androgens depress mammary cell growth and would be expected to
be protective against breast carcinoma.

6. 6
Prolactin acts on the estrogen-primed breast to stimulate and
maintain lactation and since it is associated with the
function of differentiated cells rather than stimulation of
cell growth, it would be expected to decrease the incidence
risk.2
All estrogen secreting ovarian tumours have been related to an
increase in incidence of breast cancer.2
Hoover and associates4, observed an increase in risk with the
increase in numbers of prescribed estrogens. Ross and co-
workers5 observed the relative risk to increase with the total
cumulative amount of estrogens received.
High doses of estrogens can cause breast cancer. A report that
two transvestite men, receiving massive doses of estrogens to
induce breast development, subsequently developed breast
carcinoma.6 Oral contraceptives however, are a mixture of
Estrogen and Progesterone but produce a net progesteronal
effect and no alteration to risk is documented.3, 7, 8
Research is also looking at the relationship between diet and
breast cancer with special concern to dietary fat,9, 10
especially in the form of unsaturated fats, which is known to
produce mammary tumours in rodents. In the United States, an
increase in total fat consumption and an increase in

7. 7
polyunsaturated fats in daily diets has occurred. Little if
any change in the incident rate of breast cancer screening, in
white women, has been seen.11 However epidemiological studies
on inhibition or promotion of breast cancer in the hope of
finding a practical significance in the relationship between
diet and breast cancer will continue.
A lump in the breast is an important lesion for the patient.
In post-menopausal women, a single nodule in the breast is
almost certainly a carcinoma.
Screening for breast cancer is a valuable service and was
introduced in the attempt to detect the ‘early’ carcinoma and
so reduce the risk of spread and reduce the breast cancer
death rates. A bi-annual clinical examination is recommended.1
Education of women in self-examination procedures is important
especially in the detection of ‘interval cancers’ occurring
between the bi-annual check-ups.
Patients presenting with palpable breast lesions should have
samples taken for cytological examination. Samples are taken
by fine needle aspiration. This method is simple, inexpensive
and is most commonly used for preoperative assessment.12

8. 8
The advantages of the fine needle aspirate are; that it is
quicker to report than conventional surgical biopsy material;
less technically demanding; less traumatic to the patient and
viewed by the patient, as an extension of a doctor’s
examination.
Kun13 reported the first use of the Fine Needle in 1847 but
what use he made of his sample is unknown. In 1853, Sir James
Paget14 used fine needle aspiration to prepare a cell spread.
In 1930 Ellis and Martin15, a head and neck Surgeon, used a
need puncture and aspiration technique in preference to open
surgical biopsies. Dr F Stewart actually interpreted the
results and later went on to report a large series of
aspiration biopsies with repeated accurate diagnosis.16
There has been speculation as to spread of tumour cells when
using fine needle methods, but Berg and Robbens17 showed
identical 15-year survival rates in matched groups of fine
needle and surgical biopsy patients with breast cancer. In
some cases, local bruising is all that has been reported.
Breast cancer has been found in many cases to be hormone
responsive.
In 1896, an ovariectomy was found to produce regression of a
metastatic tumour.18 Since then, adrenalectomy and

9. 9
hypophysectomy have been used to achieve similar results. By
removing these organs, the source of circulating hormones,
which stimulate or support breast cancer, is removed.
This however is not considered the therapy of choice as only
20-40% of breast cancer patients are found to have tumour
regression and when chemotherapy is combined, 60% of patients
are found to have tumour regression.19
Normal target tissues for hormones, including mammary glands,
contain specific receptors. These receptor sites are
responsible for the initial interaction between the hormone
and the cell and function to trigger the biochemical chain of
events characteristic of that hormone.
It has been found that a proportion of breast tumours also
contain receptors while others do not. It is thought that when
malignant transformation occurs, the cell may retain all or
only part of the normal population of receptor sites. If the
cell retains the receptor sites, it has normal cell stimulus
by its hormonal dependence. If the cell does not retain the
receptor sites, the circulating hormones no longer recognise
it, as a target cell and endocrine control is lost. This
implies that the cells retaining receptor sites will benefit
from endocrine therapy, and those cells that do not have
receptor sites, will not.19 Therefore, steroid receptor assays

10. 10
assist the selection of patients with breast carcinomas who
may benefit from endocrine therapy. These biochemical assay
methods require fresh tissue, and elaborate equipment. By
utilising the aspiration samples and performing
immunoperoxidase methods directed against the estrogen
receptor protein, prognosis for endocrine therapy may be
aided.
Use of anti-estrogens are beneficial for patients who retain
estrogen receptors and do not undergo surgery. Drugs such as
Tamoxifen are thought to significantly reduce clonal growth,20
or may act as a competitive inhibitor to growth of the
cells.21, 22 For elderly patients, drugs such as Tamoxifen is a
save from trauma of surgery.
Immunocytochemical markers can be used to detect estrogen
receptors and with the use of other Immunocytochemical
markers, in the detection of other cell antigens, has been
found to be valuable in the diagnosis and prognosis of breast
proliferative disorders.
Estrogen receptor related protein (D5) [see note 1] is a
monoclonal antibody raised against affinity purified cytosol
estradiol receptor from human myometrium.23
Carcinoembryonic Antigen (CEA), [see note 1] is an
incompletely defined glycoprotein of 180,000 Daltons. It is an

11. 11
oncofetal antigen, which was originally found in colorectal
carcinomas but has been demonstrated in some human breast
cancers. Shousha et al, 24 suggested that there is a significant
relationship between the demonstration of CEA in breast
carcinomas and the presence of lymph node metastasis with five
and ten year survival rates.
There has also been suggestions that the presence of CEA
suggests a more aggressive tumour behaviour.25
Epithelial Membrane Antigen (EMA) [see note 2] is a large
glycoprotein >440,000 Daltons which is partially purified and
recognised by antisera raised against milk fat globule
membranes. It is found in all breast tissue and is therefore
used as a positive control for this study.26
Cytokeratin (CAM 5.2)27 (Cytok) [see note 3] is a murine
monoclonal antibody which recognises lower molecular weight
intracellular cytokeratin proteins within secretory
epithelium.
DD9 E728 is a murine monoclonal antibody, with some
discrimination for pancreatic adenocarcinomas, which binds to
a component of approximately 55.00 molecular weight. The
antigen detected has been found in breast carcinomas but its

12. 12
immunohistochemical pattern is different to that found with
antibodies against CEA and EMA28.
Notes
1. Amersham International PLC., Amersham, Bucks.
2. Dako Ltd., High Wycombe. Bucks.
3. Beckton Dickinson, Laboratory Impex, Middlesex.

13. 13
AIMS OF THE PROJECT
The aim of this project is to develop a method for
concentrating and processing the small number of cells
obtained by fine needle aspiration to paraffin wax allowing
histological sections to be cut.
The sections are then used to demonstrate and quantitate the
presence of the antibodies; CEA, EMA, Cytok, DD9, D5, and
compare with the results found with conventional smear
cytological preparations of fine needle aspiration samples.

14. 14
MATERIALS AND METHODS
In this project, 80 cases of patients with breast lumps were
investigated. 62 cases represent patients admitted into the
Outpatient Department for fine needle aspiration. Of these 62
cases, 30 had direct smears made from the sample, 32 cases
were sent to the Histopathology Department for double
embedding. The remaining 18 cases represents aspirations taken
from breast lumps received in the Histopathology Department
requiring frozen sectioning.
All the aspirates required for double embedding were fixed in
Methacarn within microcentrifuge tubes [see note 1]. All the
aspirates used for direct smear preparation were fixed in 95%
alcohol immediately following preparation.
Each case was examined Immunocytochemically by an Indirect
Immunoperoxidase method using Monoclonal Antibodies against
the antigens:
1. Carcinoembryonic Antigen (CEA)
2. Epithelial Membrane Antigen (EMA)
3. Cytokeratin (CAM 5.2) (Cytok)
4. DD9 E7 (DD9)
5. Estrogen Receptor Protein (D5)

15. 15
The following positive controls were used against each
antibody:
The positive control for CEA was a moderately differentiated
adenocarcinoma arising in the sigmoid colon.
For EMA a moderately differentiated ductal carcinoma of the
breast was used.
The control used for Cytok was a breast lump showing mild
fibrocystic disease with fibrosis and duct dilation.
A focally necrotic moderately differentiated adenocarcinoma of
the pancreas was used as a control for DD9.
Normal skin fixed using Methacarn, containing sebaceous glands
was used for D5 as a positive control.
In addition to Immunoperoxidase methods, each case was stained
by the following techniques:
1. Mayer’s alum Haematoxylin and Eosin
2. Periodic Acid Schiff (PAS)

16. 16
SUMMARY OF TECHNIQUES
Fine Needle Aspiration (Appendix 3)
In the Outpatient Department at the William Harvey Hospital,
fine needle aspiration was achieved by using a 21-gauge needle
and by a 10ml syringe pistol. [See note 1] (Fig. 1) By using
maximum suction and moving the needle tip back and forth
within the breast lump, cells were aspirated and fixed in
Methacarn fixative. (Appendix 1) or direct smears prepared and
fixed immediately following preparation in 95% alcohol. [Fig.
2a & b]
In the Histopathology Department at St. Thomas’ Hospital, fine
needle aspiration on frozen section breast lumps was achieved
by using a 21-gauge needle and a 20 ml syringe. Using a
standard technique, cells were aspirated and fixed in
Methacarn fixative
Double Embedding Method (Appendix 4)
The cells aspirated from breast lumps are received in
Methacarn fixative, within microcentrifuge tubes. By process
of centrifugation, and removal of supernatant, the cells are
transferred from Methacarn to absolute alcohol. This removes
the chloroform, which is one of the constituents of Methacarn
fixative. The absolute alcohol is removed and cells are re-

17. 17
suspended in distilled water and transferred to a ‘BEEM’
capsule, [see note 2] which allows a greater concentration of
cells. The distilled water is removed and the cells are
embedded in 5% agar. (See Appendix 4) Freezing the agar allows
better handling. They are placed onto a tissue processor, in
this form, commencing with 95% alcohol, processed and embedded
in paraffin wax. 4µ sections are cut and mounted onto slides
previously coated with a chrome gelatine solution. (See
Appendix 2)
Some of the aspirated samples contained fewer cells and these
proved difficult to see especially during the processing steps
and subsequent cutting stages. Dyes were used to aid these
stages so that the cell loss was minimised. The dyes used
were:
1% Eosin
Saturated Alcoholic Picric Acid
Lugols Iodine (1g Iodine, 2g Potassium Iodide, 100ml
D.H20)
1% Potassium Permanganate
Mayer’s/Celestine blue Iron Haematoxylin
Azure A
Notes
1. Syringe pistol (Cameco AB, Sweden)

18. 18
2. BEEM capsule 00. 1mm square tip pyramid shape. Supplied
by Agar Aids Ltd., Essex.
Periodic Acid Schiff (PAS) (Appendix 6)
This is a histochemical reaction, which demonstrates liberated
aldehydes in tissue/cells. These are found in glycogen and
neutral mucins. Glycogen and neutral mucins (and some acid
mucins) have a hexose carbohydrate fraction; it is the
adjacent 1:2 glycol groups of this that are oxidised by
periodic acid (HIO4) to give free aldehydes that re-colour
Schiff’s sulphate leucofuchsin reagent.29
Immunoperoxidase Indirect Method (Appendix 7)
In this method, the slides are incubated with two antibodies.
The first is unlabelled and the second, which is directed
against the immunoglobulin of the species in which the first
antibody is raised, is conjugated with horseradish
peroxidase.30
The slide is then treated with a substrate, usually
Diaminobenzidine (DAB), which in the presence of peroxidase
and hydrogen peroxide (H202) polymerizes to form an insoluble
brown material, which is deposited at the site of the initial
antigen/antibody reaction.

19. 19
Peroxidase and peroxidase-like enzymes are present in many
normal and neoplastic tissues including red blood cells. To
aid interpretation and to prevent false positive results,
these endogenous peroxidase substances require blocking prior
to incubation with the antibodies. This blocking procedure
includes the bleaching of haematin using hydrogen peroxidase
and periodic acid – borohydride method, which is shown in
appendix 6.
For D5, inhibition of endogenous peroxidase was inhibited
using 6% hydrogen peroxidase. The use of periodic acid and
potassium borohydride has been found to reduce the intensity
of the final staining reaction.
The use of the relevant controls is essential.31

20. 20
RESULTS
The results have been scored visually and no accurate means of
quantitation has been carried out.
The size of the pellet was estimated using an eyepiece
graticule calibrated by a stage micrometer using the following
calculation for each magnification:
number of stage micrometer divisions = µm
number of eye piece divisions
Mitosis were counted using x40 objective for five fields.
Table 1 represents the breast aspirate samples prepared by the
double embedding method.
The results were tabulated in detail and use the following
scoring system:
0 .... All cells negative
± .... Occasional positive cell
+ .... Small number of positive cells
++ .... Moderate number of positive cells
+++ .... Majority positive cells
++++ .... All cells positive
Freezing of the pellets caused no apparent distortion of the
cell morphology. Of the 50 samples prepared by the double

21. 21
embedding method. 11 samples were omitted from staining as the
nu8mber of cells present on the H&E, were found to be
insufficient for a reliable diagnosis.
14 cases were found to be CEA positive, 36 cases were EMA
positive, 32 cases Cytok positive, 22 cases DD9 positive and
11 cases were positive for D5. 30 cases were PAS positive.
If the pellets were not thoroughly washed in absolute alcohol
and distilled water to remove the Methacarn fixative, a black
precipitate was formed. It was later found that this
precipitate could be removed from sections prior to staining
by incubating the dewaxed section in saturated alcoholic
picric acid for at least five hours.
Six dyes were used on various pellets to help visualise the
cells during processing and cutting.
1% aqueous eosin was found to be valuable in the detection of
the cells during processing and cutting but could not be
removed by washing in water or 10% acid alcohol and was found
to some extent, mask the chromogen, diaminobenzidine reaction
product.
Iron haematoxylin helped to visualise the cells during
processing and cutting but this ‘dye’ also stained the nuclei

22. 22
of the cells which could cause difficulty with interpretation
of antigens located in the nucleus.
1% potassium permanganate appeared to be the best, staining
the cells clearly, allowing precise localisation of the cells
during processing and cutting. This stain required bleaching
with 1% oxalic acid prior to staining. During the
immunostaining, all the sections floated from the slides.
Saturated alcoholic picric acid demonstrated large pellets
well but small pellets were visualized during the initial
processing but when the pellets were processed to paraffin
wax, the colour was washed out and therefore did not aid
cutting.
Lugols iodine and Azure A did not stain the cells sufficiently
to be of any benefit during processing or cutting.
The pellets containing blood were the best. The blood could be
seen during processing of the pellets and cutting. When a
section was cut from the blood, cells were also found to be
present. The blood did not require special treatment prior to
staining especially with immunostaining as endogenous red cell
peroxidase-like activity was blocked prior to immunostaining.

23. 23
Table 2 represents the breast aspirate samples prepared by the
smear technique.
The results are not tabulated in detail and use the following
scoring system:
Pos .... Immunostaining present
Neg .... No immunostaining present
NT .... No tumour cells present
No cells No cells present
RBC .... Red blood cells only present
Of the 30 samples prepared by this method, 4 cases were CEA
positive, 15 cases were EMA positive, 22 cases were found to
by Cytok positive and 5 cases D5 positive.
DD9 and the PAS reaction were not used on these slides, a
colleague prepared the slides prior to this project, and
insufficient slides were found.

24. 24
RESULTS
Table 1
Breast Aspirate Samples Prepared By the Double Embedding
Technique
Results removed from this portfolio sample. If you would like
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bridget@eclipsecopywriting.com

25. 25
CONCLUSION/DISCUSSION
The quality of the pellets and smears rely mainly on the
expertise of the clinician sampling the patient’s breast lump.
If the clinician fails to sample the lump adequately, the
following procedures are of little value. Of the 18 breast
lumps sent for frozen section at St Thomas’ Hospital, which
were aspirated once a definite diagnosis had been made and
without prejudice to routine pathological investigation, 5
aspirates were found to be of no value due to insufficient
number of cells present. As the author was responsible for
aspirating these lumps, they can be regarded as taken from an
inexperienced hand. Of the 32 breast lumps received in the
histopathology department from the outpatients department at
the William Harvey Hospital, 5 aspirates were found inadequate
due to an insufficient number of cells present. It can be seen
therefore that an experienced clinician will yield a greater
number of adequate aspiration samples. 1 aspirate was
regrettably destroyed during the processing.
If an adequate breast lump aspirate is taken, the double
embedding method did seem to provide a more accurate result
section after section. At least 10 sections should be
available from even the smallest pellet and this is sufficient
to investigate the distribution of Immunocytochemical markers

26. 26
and histochemical stains with the use of adequate negative
controls. Sections can be reliably compared.
One of the advantages of the double embedding technique is the
small area within which the cells are concentrated. Little
time is spent screening the smear and valuable antibodies,
used to demonstrate markers Immunocytochemically, are not
wasted.
The smear technique proved to be unreliable as each smear was
unique. One slide from a patient may have malignant cells
present and another may not. Therefore with any immunostaining
performed, the results were likely to be equivocal.
The number of smears made from one breast lump aspirate are
minimal, usually a maximum of five slides. This leaves very
little material for other techniques.
The greatest disadvantage of the smear technique is the large
area over which the cells are spread. Time is wasted during
screening and valuable antibody is wasted as the larger area
needs to be covered.
The major disadvantage of the double embedding method is
seeing a small number of cells that may be present. The use of
dyes may not necessarily be of help as with 1% potassium

27. 27
permanganate, which caused the sections to fall off the slide
during immunostaining. Blood present in the pellet did however
aid processing and cutting and cause no problems with
subsequent staining. Perhaps the addition of one drop of blood
to each pellet is all the ‘dye’ required.
All pellets containing tumour cells were found to be EMA
positive, this would support the literature stating all breast
tumours are EMA positive.
16 pellets were found to be CEA positive but of these, not all
the malignant cells in each pellet were CEA positive.
Therefore CEA could not be used reliably to assess prognosis
of the patient as only a proportion of the malignant cells
were CEA positive. CEA was not detected in any pellets which
were benign or contained no tumour cells.
DD9 gave similar results to CEA but occasionally the result
was found to be stronger or a minimal positive reaction was
seen when CEA was negative.
34 pellets were Cytok positive.
11 pellets were found to have weak D5 positivity, which would
be regarded as patients who would react favourably to
endocrine therapy. Not all the malignant cells in a positive

28. 28
pellet were D5 positive which shows only a part of the normal
population of receptor sites were present.
The PAS reaction was found to be of no significant value and
in hindsight, a Diastase treated PAS reaction would be of more
value as this would aid in the diagnosis of adenocarcinomas of
the breast. EMA however was of value as this marker picked out
the acini of adenocarcinomas.
The number of mitotic figures was found to be of value as
their presence illustrated a more aggressive tumour. Methacarn
was a good fixative for the demonstration of mitosis, as it
arrests mitosis and then preserves the cells in this state.
No mitotic figures were seen in the breast aspirates prepared
by the smear technique. These samples were fixed in 95%
alcohol, and this may not have preserved mitosis if any were
present.
Methacarn was the fixative used in the double embedding
method, as D5 requires this fixative for optimum staining
reaction. In the smear technique, only 5/30 smears were weakly
D5 positive. This may be a false representation due to the
fixative used or this may be a true representation. As the
smears were fixed prior to this project, fixation was already
achieved using 95% alcohol and not Methacarn, which is the

29. 29
recommended fixative when demonstrating estrogen receptor
proteins with D5. Further research is required involving
various fixatives on the staining reaction of D5.
In the presence of tumour cells the immunostaining results of
the smear was similar to the pellets. However, as stated
above, each smear was unique and one slide per case could vary
from the next.
Methacarn was not the fixative of choice, but was required in
the demonstration of estrogen receptor protein. The EMA
positive cells showed intranuclear staining and Methacarn was
the suggested cause.
The aim of this project was to develop a method for
concentrating and processing fine needle aspiration samples to
paraffin wax. The use of plasma/thromboplastin as a method for
preparation of cell blocks has been documented33 but the
possibility of immunological cross-reactivity with foreign
human proteins and the fact that such reagents are not
immediately available in a histopathology laboratory, led the
author to use agar as an embedding medium.
The double embedding method used in this project is an ideal
method, which can be used by a routine laboratory requiring no

30. 30
expensive reagent or equipment. It can be used for any type of
aspiration sample or small friable histological specimens.
This method is more favourable to conventional cytological
preparations especially when a number of staining techniques
are required.
The demonstration of immunomarkers on these cases is a part of
a larger study correlating the distribution of these markers
in response to Tamoxifen and/or surgery in elderly women.
An additional required investigation is to establish whether
the role of progesterone is a significant factor in the
stimulation of carcinoma of the breast.

31. 31
APPENDICES
Appendices removed from this portfolio sample. If you would
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on bridget@eclipsecopywriting.com

32. 32
REFERENCES
1. Breast Screening project. From unknown source.
2. THOMAS, DAVID, B. (1984)
26. HYDERMAN, E., BROWN, B.M, RICHARDSON, T.C (1984)
Epithelial markers in prostatic, bladder and
colorectal cancer: An Immunoperoxidase study of
epithelial membrane antigen, carcinoembryonic
antigen and prostatic acid phosphatase
The full reference listing is not included in this portfolio
sample. If you would like to see the complete project, then
please contact Bridget on bridget@eclipsecopywriting.com