COLLECTION AND TYPES OF CYTOLOGY SPECIMENS
Two types of smears are used for cytological examination depending on the preferred method of staining. Usually wet-fixed smears are preferred to air-dried smears. Wetfixed smears are prepared by immediately fixing the slide without allowing it to dry. The fixatives recommended are a mixture of equal parts of ether and 95% ethyl alcohol, formol alcohol or 95% ethyl alcohol alone. Not less than 15 min are required for adequate fixation though slides may remain in the fixative for 7-10 days without deterioration. Coplin jars made of glass or plastic are commonly used as containers for fixative. Papanicolaou and H&E stains are commonly used on wet-fixed smears. The airdried smears are simply prepared by allowing these to dry naturally. These smears are stained with Giemsa stain, Leishman stain, etc. Smears must be labelled regarding type of preparation besides patient identification and fixation so that they can be appropriately stained.
Request forms for cytological specimens must include identification data of the patient, type and site of specimen, clinical details and in case of slides, method of fixation used. In case of cervical smears date of last menstrual period, use of IUCD, hormone therapy, previous history, etc. must be mentioned on the request form.
2. Respiratory Tract:
A fresh early morning specimen produced by a deep cough should be collected and brought to the laboratory immediately without any fixation. If it is not possible to transport unfixed material to the laboratory, the sputum should be prefixed by asking the patient to expectorate into a widemouthed small jar half filled with 70% ethyl alcohol. In case of peripheral laboratories where cytology facilities are not available, fresh sputum should be examined grossly for tissue fragments and blood tinged areas. Smears from these areas and other randomly sampled areas should be prepared and fixed immediately (wet-fixed) in 95% ethyl alcohol. After fixation for 20 min these slides can be dried and transported to referral laboratory for reporting.
Bronchial Aspirates, Washings and Brushings:
Aspirates and washings collected during bronchoscopy may be centrifuged and smears prepared from the cell button. Direct smears can also be made and fixed in 95% ethyl alcohol. Direct smears should be prepared from bronchial brushings and wet-fixed in 95% alcohol. Alternatively they can be processed like aspirates and washings.
Bronchoalveolar Lavage (BAL):
BAL involves the infusion and re-aspiration of a sterile saline solution into the air passages. This fluid should be submitted as such immediately to the laboratory. If delay in transportation is expected, equal quantity of 95% ethyl alcohol should be added.
3. Urinary Tract:
Urine: Freshly voided urine is the specimen of choice in male patients. The first morning specimen should be avoided as the urine has been collecting in the bladder overnight and the cells have usually degenerated. In female patients catheterised urine is the preferred specimen. Approximately 50-100 ml of urine should be collected in an equal amount of 50% ethyl alcohol. If possible the patient should be sent to the laboratory for collection of a fresh sample.
Washings and Brushings of Ureter and Renal Pelvis, Bladder Washings:
All washings should be collected in an equal amount of alcohol for fixation. Brushings may also be added to alcohol for fixation. Alternatively direct smears may be prepared and wet fixed in an alcoholic fixative.
4. Pleural, Pericardial and Peritoneal Fluids:
Fluid should be collected in a clean, dry container, which need not be sterile, and should be sent to the laboratory as soon as possible. If it is not possible to send the fluid immediately, it should be stored in a refrigerator at 4°C and not allowed to freeze. The specimen can be preserved at refrigerator temperature for several days. In case of small peripheral laboratories without cytology facilities, smears should be prepared after centrifugation. Both wet-fixed and air-dried smears of the sediment, labelled as such should be submitted to the referral laboratory
Cerebrospinal fluid should be collected in a clean container and transported immediately to the laboratory for processing. If delay in transportation of more than a few hours is expected equal amount of 50:50 alcohol ether mixture, or 95% ethyl alcohol may be added.
6. Alimentary Tract:
Brush and wash samples may be collected from oesophagus, stomach, lower bowel and rectum. Brush smears should be prepared immediately by rolling on a clear glass slide. Five to six such smears may be wet fixed in alcohol for Papanicolaou staining. A few air-dried smears may also be prepared. These should be labelled as such and submitted for cytological examination. Wash samples from stomach must immediately be neutralised with N/10 sodium hydroxide (pH up to 6.0) and centrifuged rapidly in ice-cold siliconised tubes. Smears are prepared from the deposit in the same manner as mentioned above. Wash samples from lower bowel are also concentrated in the same way by centrifuging in ice-cold siliconised tubes and smears prepared.
7. Female Genital Tract:
Cervical Smear: The aim of collecting a cervical smear is to obtain a representative specimen from the squamocolumnar junction (transformation zone), using an Ayre’s spatula or similar device. Having exposed the cervix using a bivalve speculum a circumferential sample is obtained by rotating the spatula through 360°, thus obtaining cells from the entire junctional zone. If the smear does not show endocervical cells, an additional smear may later be obtained from the endocervical canal by using a cotton-tip applicator or endocervical cytobrush. Once the sample has been obtained it should be placed on a glass slide and spread smoothly down the length of the slide using a wooden spatula. The slide must be fixed immediately without allowing to, dry, by placing it in 95% alcohol for a minimum of 15 min or by using an aerosol spray fixative. After fixation the slide is allowed to dry and sent to the laboratory with a complete request form. Slide identification and labelling must be ensured at all times. In addition a sample may also be collected from the vaginal pool in the posterior fornix with the help of a pipette. Few drops of fluid are expressed onto a slide, spread and fixed in the same way as above. The advantage of this sample is that it contains cells from the entire female genital tract however the disadvantage is that the cells are dead and desquamated and may show degenerative changes, which are difficult to interpret.
Specimens for hormonal evaluation are taken from the lateral vaginal wall. If this is not possible, posterior fornix pool specimen may be used. The specimens should be taken by lightly dipping the applicator in the secretions avoiding forceful scraping. The smears are prepared, wet fixed immediately in the same manner as cervical smears, dried and submitted to the laboratory.
Nipple discharge: A few drops should be expressed by pressing the subareolar region. The drops are touched on to a clean glass slide and spread with the help of another slide. Smears should be wet fixed in alcoholic fixative and submitted.