Cytology

Cytology

Cytology is the study of free cells from different organs of the body. These cells may have been shed by the body itself, aspirated by tube or needle; or scraped or washed from tissue surfaces. Therefore, effusions, secretions, aspirates and scrapings are all used in diagnostic cytology. Exfoliative cytology is the study of cells which are shed spontaneously from epithelial surfaces of the body.

This spontaneous shedding is a function of normal epithelium. The epithelial surfaces undergo constant growth and so they continue to shed worn out cells which are replaced by new ones. However, malignant tumour cells exfoliate more readily than those from healthy tissues. 

The detection of malignant cells in clinical specimens under microscopic examination is the most important role of diagnostic cytology. In addition, valuable information may be obtained about infections and infestations, reproductive defects and hormonal status. 

Clinical specimens are obtained with little or no discomfort to the patient.

All clinical specimens sent to the Cytology laboratory should be regarded as potentially pathogenic. Therefore, all safety precautions applicable to Microbiology laboratory should also be applied here. Safety cabinets are a must for processing sputum. Disinfectant jars should be placed at strategic points on the work bench and the staff should wear protective clothing.

Cytology has become a strong diagnostic tool for the early detection and diagnosis of malignancy in different organs of the body, in the assessment of hormonal activity which is valuable in some cases of infertility and in the evaluation of certain endocrine disorders.

In exfoliative cytology, the microscopic evaluation is based upon observation and characteristics of the basic cells. Since a majority of the cells are in the form of single cast off cells lying in a fluid medium, there is no distortion to their shapes. This is unlike histopathology where the arrangement of cell aggregates is the basis of diagnosis of malignancy. 

Most of the information from the study of discrete cells is based on the shape or size of the nuclei. Cytoplasm of the cells may only assist in identifying the cell type. Figure 16.1 shows some of the characteristic nuclear abnormalities associated with carcinomas. They are:

1. Nuclear enlargement with no increase in the overall cell size, showing a decreased cytoplasm/nucleus ratio.

2. Irregularity of nuclear outline with variation in size and shape.

3. Hyperchromasia. This is as a result of increased amounts of deoxyribonucleic acid (DNA); the nuclei of malignant cells often stain more intensely with basic dyes.

4. Multinucleate due to abnormal cell division.

5. Uneven distribution and variation in size of chromatin particles.

6. Increase in size and number of nucleoli.

To achieve a meaningful result and interpretation, smears should be prepared from fresh specimens and fixed while still wet. The nature of the specimen, the age of cell population, method of collection and fixation will all affect the morphology of the cells. Most importantly, a prompt and proper fixation is essential.

FIXATION

A good cytological fixative should penetrate rapidly with no distortion to the cell morphology.

The clinician, in most cases, prepares and fixes the smear in the clinic or at bed side. The laboratory should make available to the wards and outpatients' clinic, a container and carrier for smear fixatives in the form of a polythene screw-capped coplin jar grooved to take up to 8-10 slides.

If the smears are to be sent by post to a distant laboratory, aerosol fixative sprays give an effective form of fixation. These aerosol sprays consist usually of an alcohol and wax-the alcohol fixes

the smear and the wax sets to provide a water-soluble protective film. Some laboratories coat prefixed smears with glycerine or water soluble wax. Alternatively, the smears may be air-dried after complete fixation and then placed in suitable cardboard, plastic or wooden slide box-carrier designed and grooved to hold a few slides.

Fluids of larger volumes, e.g., pleural or peritoneal effusion, or sputum, should be sent to the laboratory immediately after collection and smears can then be made.

The most widely used fixatives are:

1. Alcohol-ether (equal parts of diethyl ether and 95% alcohol) mixture. 

2. 95% alcohol with or without 3% glacial acetic acid. 

3. A mixture of seven parts of tertiary butyl alcohol and three parts of 95% alcohol. 

4. Schaudinn's solution: 

Saturated aqueous mercuric chloride        66 ml 

Absolute ethyl alcohol                              33 ml 

Glacial acetic acid                                    0.3 ml 

Fixation time in any of these fixatives is at least 15 minutes and maximum 7 days.

SUBDIVISIONS OF CYTOLOGY

For practical purposes, cytology is subdivided as follows:

1. Gynaecological Cytology (Gynae-cytology) This deals with the cytology of the vagina, cervix and endometrium. 

2. Non-gynaecological cytology This is the cytology of cells suspended in body fluids. 

3. Fine needle aspiration cytology (FNAC) The 

study of cells aspirated from organs and structures of the body.

GYNAECOLOGICAL CYTOLOGY

There are three most important applications of gynae-cytology: 

(i) The detection of malignancy--mainly cancer of cervix. 

(ii) Assessment of hormonal activity 

(iii) Identification of vaginal infection andinflammatory conditions.

Specimen Collection and Preparation of Smears

1. Cervical smears Cervical smears are made from material collected with the help of a speculum (a metal or plastic device) which is inserted into the vagina and allows the uterine cervix to be readily visible. A specialised spatula known as the Ayre spatula or cervical spatula (Fig. 16.2.) is used for collection. The collection is made at the junction of the columnar epithelium by visualising

the cervix, the spatula is inserted via the speculum into the cervical os and rotated through 360 degrees. The material collected is quickly smeared over a pre-labelled microscope slide and fixed immediately

2. Aspiration from the posterior fornix With the aid of a speculum, cellular material is collected from the posterior fornix, using a disposable plastic pipette with a suction bulb (Fig. 16.3). Following aspiration, smears are prepared and fixed immediately.

3. Vaginal smears Vaginal smears are valuable for the assessment of hormonal function. Cellular material is collected by scraping the upper third of the lateral wall of the vagina with a wooden spatula. The cells are evenly and thinly smeared over a clean pre-labelled microscope slide and fixed. 

4. Endocervical smears This is used mainly for follow up cases where a surgical treatment has been used after a cone biopsy has been taken for assessment of dysplasia and malignancy or as a curative procedure. A cotton tip swab is inserted into the endocervix and rotated gently to cover a wide area of the endocervix. The material collected is smeared on a clean pre-labelled microscope slide and fixed.

5. Endometrial aspiration This procedure has to be performed under strict aseptic conditions so as not to introduce infection into the patient. A cannula is inserted into the uterine cavity and the cellular material is aspirated using a syringe. Thin smears are made on clean pre-labelled slides and fixed.

STAINING

The most commonly used stain in the gynae-cytology is the Papanicoloau stain. It is a stain specially suitable for cervical smears. It gives sharp nuclear staining and good differential colouring of acidophilic and basophilic cells. It makes the cytoplasm transparent. There are other stains that can be effectively used for diagnostic purposes in the gynae-cytology.

Papanicoloau Staining Method

This staining method can be performed either manually when there are only a few slides to deal with, or by using automatic staining machine when a large number of slides is to be stained.

Solutions

1. Harris haematoxylin (see Chapter 7) 

2. Orange G solution (OG 6) 0.5% Orange G (CI No.16230) in 95% alcohol 100 ml 

Phosphotungstic acid 0.015 g 

3. Eosin Azure 50 (E.A. 50 or E A 36): 

0.5% light green SF (Yellowish) (CI No. 42095) in 95% alcohol 45 ml

0.5% Bismark brown Y (CI No.21000) in 95% alcohol 10 ml 

0.5% Eosin (C1 No.45380) in  95% alcohol 45 ml 

Phosphotungstic acid 0.2g

Saturated aqueous lithium carbonat e1 drop 

Mix well and store in tightly capped, brown bottles.

Procedure for manual staining

1. Remove smears from fixative.

2. Rinse in descending grades of alcohol (80,70 and 50%) and water for 10 seconds each.

3. Stain in Harris haematoxylin for 2 minutes.

4. Rinse in water for 1-2 minutes.

5. Differentiate in 1% acid alcohol until only the nuclei retain the stain (a few seconds)

6. Wash and blue in tap water for 3-5 minutes.

7. Transfer to 70% alcohol for a few seconds.

8. Transfer to 95% alcohol for a few seconds.

9. Stain in OG 6 for 2 minutes.

10. Rinse in 2 changes of 95% alcohol.

11. Stain in EA 50 for 2-4 minutes.

12. Rinse in 2 changes of 95% alcohol.

13. Dehydrate in absolute alcohol, clear in xylene and mount in neutral synthetic medium.

Results

Nuclei:	Blue
Acidophilic (superficial) cells	reddish-pink
Basophilic (intermediate and parabasal cells)	:blue/green
Candida albicans	Red to pale pink
Trichomonas vaginalis	grey green

Note E A 50 or E A 36 and OG 6 are available commercially

Procedure for automatic staining Transfer fixed smears to:

Procedure for automatic staining Transfer fixed smears to:
Trough	Solutions	Time in minutes
1	70% alcohol	1
2	50% alcohol	1
3	Distilled water	1
4	Harris haematoxylin	3
5	Distilled water	1
6	Tap water 0.5% HCI in	1
7	70% ethanol  1/2	1/2
8	Tap water	1
9	Tap water	1
10	70% alcohol	1
11	70% alcohol	1
12	95% alcohol	1
13	95% alcohol	1
14	OG 6	2
15	95% alcohol	1/2
16	95% alcohol	1/2
17	E A 50 or EA 36	3
18	95% alcohol	1
19	95% alcohol	1
20	Absolute alcohol	2
21	Absolute alcohol	2
22	Xylene	1
23	Xylene	2
Remove slides from the machine into xylene dish and mount in a neutral synthetic resin medium.

Results Same as for manual technique.

HORMONE ASSESSMENT

As part of a general investigation into the cause of human reproductive disorder and sterility among women, hormone assessment is done.

Hormonal activity can be evaluated on the basis of microscopic examination of Papanicoloau stained vaginal smears. The karyopyknotic index (KPI) and the maturation index (MI) are the two methods commonly used to assess hormone activity.

Superficial cornified squamous epithelial cells show condensed, deeply stained, structureless (pyknotic) nuclei, with pink to red stained cytoplasm. The calculation of KPI is done by counting a total of 200 squamous cells in a Papanicoloau stained smear. The cornified squamous cells are expressed as a percentage of the total number of  assessment of the hormonal (oestrogenic) influence, smears are taken at about 3 or 4 days interval throughout the menstrual cycle

The maturation index (MI) is based on cell maturation which is determined by means of the morphology and staining reactions of the noncornified squamous epithelial cells. These are classified as superficial intermediate and parabasal cells. A total of at least 200 squamous cells are counted and each class of cells is expressed as a percentage. High oestrogenic activity is indicated by a preponderance of superficial intermediate cells while low activity is indicated by a predominance of parabasal cells. Another good staining

method used for the assessment of hormonal function is the Shorr's method.

Shorr's Staining Method

This method uses a single differential staining solution. It gives less cytoplasmic transparency and poorer nuclear definition than the Papanicoloau. It is not very useful in the diagnosis of malignant cells. 

Staining solution 

Staining solution
50% ethyl alcohol	100 ml
Biebrich scarlet (CI No.26905)  Water-soluble	0.5 g
Orange G (CI No.16230)	0.25 g
Fast green FCF (CI No.42053)	0.075 g
Phosphomolybdic acid	0.5 g
Glacial acetic acid	1 ml

Procedure

1. Fix smear in alcohol-ether for 1-2 minutes 

2. Stain in Shorr stain for 1-2 minutes. 

3. Rinse in 70% alcohol to remove excess stain. 

4. Transfer to 95% alcohol for 3 seconds. 

5. Transfer to absolute alcohol for 3 seconds. 6. Clear in xylene and mount in a neutral synthetic resin.

Results 

Nuclei	Red
Superficial cornified cells :	Bright orange-red
Non-cornified cells :	Green-blue

There are other stains which, though less effective, can be used in gynae-cytology. Some of these are:

(i) Haematoxylin and Eosin 

(ii) Acridine orange fluorescence technique 

(iii) Feulgen's reaction for DNA for researchwork.

Sex Chromatin (Barr bodies)

Sex chromatin is present in about 30% of cells from the female whereas cells from the male do not have the sex chromatin. The sex chromatin appears as a darkly staining dot in the nucleus. It is present

in the cells of the skin, cells of the buccal mucosa and in the cells of blood, notably the leucocytes. Specimens are conveniently obtained by scrapings of the buccal mucosa.

The Barr bodies are normally seen attached to the nuclear membrane of the epithelial cells. It is sometimes used to determine the sex of an individual.

Cresyl Fast Violet Acetate Method for Demonstrating Sex Chromatin

This is a rapid, simple and effective method for demonstrating sex chromatin. It requires no differentiation.

Staining solution

Cresyl fast violet acetate 1.0 g 

Distilled water100 ml

Procedure 

1. Fix smear while still wet in 95% alcohol for three minutes. 

2. Transfer to 50% alcohol for a few seconds and then to distilled water. 

3. Stain with cresyl fast violet acetate solution for five minutes. 

4. Rinse quickly in tap water. 

5. Dehydrate with 95% alcohol, then with absolute alcohol. 

6. Clear in two changes of xylene and mount in a neutral synthetic resin medium. 

Result

Sex chromatin deeply stained 

Cytoplasm faintly stained.

Note

This method is treated under Gynae-cytology for convenience. Specimens of bronchial washings or lavage should be centrifuged and smears made and fixed immediately.

NON-GYNAECOLOGICAL CYTOLOGY

This aspect of cytology involves the study of cells suspended in body fluids. The specimens are varied and are taken from various parts of the body.

Sputum

Sputum specimen is valuable for the study of respiratory tract disorders. It is used in the diagnosis of the following abnormal conditions.

(i) Malignant disease of the lower respiratorytract.

(ii) Pulmonary asbestosis

(iii) Pulmonary inflammatory conditions due tofungal infection, bacterial infection, viral infection or parasitic infection.

Sputum is normally collected as early morning deep cough specimens, and is preferably submitted on three consecutive days. It is not advisable to collect sputum specimen after a recent bronchoscopy has been done. 

The result will be invalidated by the presence of numerous inflammatory cells which could obscure some underlying pathology. As much as possible, sputum should be sent to the laboratory promptly and smears made as soon as possible. However, sputum specimens are well preserved when refrigerated at 4°C.

Preparation of smears 

Sputum must be processed in a biological safety cabinet. Purulent or blood stained particles are selected from the sputum with a microbiological wire loop and used to make thin smears, at least two smears from each specimen.

Bronchial washings are usually submitted in sterile containers. They are centrifuged without delay and smears made from the sediment. They can also be spun at 150 rpm for 10 minutes in a cytocentrifuge directly onto a clean pre-labelled microscope slides and fixed immediately, 

Fixation Fixation should be carried out while the smears are still wet. Many workers prefer to use 3% acetic acid in 95% alcohol.

Pleural Fluid and Ascetic Fluid

These are serous fluids that normally lubricate the walls of pleural and peritoneal cavities. They increase in volume and contain cells under certain pathological conditions. Cytological examination of these fluids may reveal malignant cells which may arise from tumours of the surrounding mesothelium or they may be metastatic deposits. 

Collection and preparation of smears 

By means of a needle or canula with an attached syringe, the specimens are aspirated from the pleural or peritoneal cavities. The aspirated material is transferred into a sterile container and sent to the laboratory. The specimens are centrifuged at 300 rpm for 10 minutes or cytospun at 1500 rpm for 10 minutes, and thin smears made, at least two smears from each specimen. Any clots that are formed are fixed and processed histologically, 

Fixation Fixation, as usual, should be carried out promptly and while still wet. However, if the choice of stain is Romanowsky, then the smear should be air dried and then fixed with methanol before commencing the staining procedure.

Gastric Brushing or Lavage

These types of specimens are very useful in the diagnosis of squamous cell carcinoma of the oesophagus or adenocarcinoma of the stomach. The collection of specimen is by the insertion of a small brush into the oesophagus or into the stomach under endoscopic guidance for by X-ray guidance. 

The brush is rotated at the particular spot of interest; the material is then smeared on a clean pre-labelled microscope slide and fixed immediately. If specimen of washing or lavage is obtained, then this is treated as the pleural or peritoneal fluids.

Urine

Urine cytology is of great value in the diagnosis of urethral tumours, urinary bladder carcinoma, car cinoma of the kidney and carcinoma of the prostate in males. Normal urine contains few or no cells; but under certain pathological conditions, the urine contains many abnormal cells. Early morning specimens of urine are preferred because they give larger concentration of cells due to relatively long residence in the bladder.

Fixation Urine tends to wash off slides during fixation and staining due to the low protein content. This difficulty may be overcome by 

(i) Centrifuging the sample and making smears with the sediment on albuminised slides; 

(ii) The urine sediment is mixed with some drops of egg albumin and then smeared or 

(iii) Celloidinising of the slides after fixation. An initial fixation or preservation can be done on urine sample that may not get to the laboratory in good time. This is by adding 50% alcohol to the specimen in equal proportion. 

Staining For non-Gynae-cytology, the Papanicoloau stain is equally as useful as in the Gynae-cytology but many workers prefer to use it as a confirmatory procedure. The common staining methods are: 

The Romanowsky staining method

Following fixation in ether-alcohol, smears may be stained by any of the Romanowsky's stains. Alternatively, a fluid specimen may be smeared and air dried and then fixed with methanol for five minutes before applying the Romanowsky stain. For technique of Romanowsky staining, please refer to the Haematology Section

The staining is satisfactory for cells in fluids and effusions. 

The most popular Romanowsky stains are the Giemsa and the Leishman stains. Methylene blue This single-stain, rapid and simple method is useful for the screening of fresh specimens, especially sputum, for malignant cells. The preparation is not permanent and should be examined immediately.

Staining solution

Methylene blue 1g

Distilled water100 ml

Procedure

1. Place a small amount of fresh purulent sputum, or two drops of centrifuged deposit of the body fluid on a clean microscope slide. 

2. Add one drop of the stain to the specimen on the slide and mix the two together thoroughly.

3. Cover the mixture with a clean cover slip and spread by gentle pressure 

4. Examine immediately.

Results Nuclei: shades of blue.

Any suspicious cell is confirmed using other more specific staining techniques.

Acridine Orange Fluorescence Method

This method is based on the principle that the fluorochrome dye, acridine orange, which has an af finity for nucleic acids, can emit visible light when excited by an ultra violet or blue light, usually of 350-400 nm Wavelength. At pH 6.0, this dye will demonstrate DNA green or greenish-yellow and RNA orange-red with fluorescence microscopy. 

Malignant cells have a large amount of RNA in their cytoplasm and so they are readily seen by their orange or red fluorescence under low power magnification. This method permits quick scanning of smear preparation. Positive or doubtful cases are usually confirmed by Papanicoloau stain. 

Solutions

 1. 0.067M Potassium dihydrogen orthophosphate: Dissolve 9.072g of potassium dihydrogen phosphate (KH,PO) in 1000 ml distilled water.

2. 0.067 M Disodium hydrogen orthophosphate: Dissolve 9.465g of disodium hydrogen ortho-phosphate, anhydrous (Na HPO) in 1000 ml distilled water. 

3. Phosphate buffer (pH 6.0): 87.8 ml of solution1 are mixed with 12.2ml of solution 2 

4. Acridine orange stock solution: Acridine orange (CI No.46005) 0.1 g Distilled water 100 ml Store in a dark bottle at 4 °C. 

5. Acridine orange staining solution: Acridine orange stock solution 10 ml Phosphate buffer (pH 6.0) 90 ml 

6. 0.1 M Calcium chloride differentiator: Calcium chloride (CaCl2) 11.099 g

Distilled water 1000 ml.

Procedure

1. Fix smear in ether-alcohol mixture for 15 minutes.

2. Pass through descending grades of alcohol (80, 70 and 50%) to distilled water.

3. Rinse briefly in 1% acetic acid and wash in two changes of distilled water for one minute each

4. Stain in acridine orange staining solution for three minutes.

5. Wash in phosphate buffer for one minute.

6. Differentiate with 0.1M calcium chloride until the nuclei are clearly outlined about one minute).

7. Wash thoroughly with phosphate buffer solution.

8. Mount with a cover slip using phosphate buffer as the mountant.

9. Examine by fluorescence microscopy.

Results

RNA fluorescence :	red
DNA fluorescence:	green

Note

Certain normal cells and micro-organisms also show varying degrees of orange-red fluorescence. Therefore experience is required to be able to identify cells and cellular morphology.

Haematoxylin and Eosin Method

Following fixation, it is possible to bring smears to water through descending grades of alcohol and then stain with haematoxylin and eosin as for sections. The H and E gives good result but lacks the transparency of cytoplasm that is seen in Papanicoloau technique.

FINE NEEDLE ASPIRATION CYTOLOGY (FNAC)

Before the advent of this procedure about two decades ago, the needle cone biopsy was the method employed to collect material from lesions in the organs that do not normally exfoliate cells. The fine needle aspiration technique has almost completely superseded the more traumaic method of cone biopsy.

For aspiration of specimens, the FNAC basically requires a sterile syringe and needle, the length of the needle depending on the location of the organ to be sampled. The needle thickness is usually in the range of 0.5-0.9 mm. A special handle can be attached to the syringe to allow single hand grip, freeing the other hand for palpation and fixation of the mass if it is mobile (Fig. 16.4)

To perform the aspiration, first of all, the skin is cleaned with a suitable antiseptic. The mass is then immobilised with one hand. Without the piston of the syringe being retracted, the needle is inserted into the mass. The cells are then aspirated into the needle. The needle can be inserted from different angles if the material from one site is too scanty. Release the piston before withdrawing the needle. This is to equilibrate the pressure in order to prevent the material being drawn into the barrel of the syringe.

The collected specimen is expressed on to a clean pre-labelled slide (or slides) and spread evenly. Fixation is done immediately and stained with Papanicoloau or H and E stains.Any tissue fragments seen in the aspirate are fixed, processed and stained histologically. Lesions that cannot be localised by touch (non-palpable) are visualised by means of ultrasonography or fluoroscopy. The ultrasonography is for abdominal aspiration while fluoroscopy is used for bone and thoracic lesions.

FNAC is very valuable for preliminary diagnosis of carcinomas as well as inflammatory conditions. It is rapid and fairly inexpensive. But experience is required to correctly interpret staining result. FNAC is particularly useful in dealing with suspected masses or lesions on the skin, lymph node, breast, thyroid, liver, kidney, lung and bone.