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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

RESULTS

Table 1

Breast Aspirate Samples Prepared By the Double Embedding

Technique

Results removed from this portfolio sample. If you would like

to see the complete project, then please contact Bridget on

bridget@eclipsecopywriting.com

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

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

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

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

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

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

APPENDICES

Appendices removed from this portfolio sample. If you would

like to see the complete project, then please contact Bridget

on bridget@eclipsecopywriting.com

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