Conjunctival impression cytology--a study of normal conjunctiva.SS Gadkari, SD Adrianwala, AS Prayag, P Khilnani, NJ Mehta, NA Shaha
Department of Ophthalmology, Seth G.S. Medical College, Parel, Bombay.
Conjunctival impression cytology was used to study conjunctival ocular surface in 30 normal individuals in the age group of 20 to 60 years. The cytology was graded according to the classification of Nelson. Out of 30 subjects, 26 showed the features of Grade O (average age 28.33 years) and 4 showed grade 1 (average age 56.66 years). The method was very easy to perform, cheap and fast. The relationship of various cells to each other was maintained. This method can be used to study the conditions like trachoma, avitaminosis A and other disorders of conjunctival surface. It can be undertaken by a paramedical worker in an out patient clinic.
Keywords: Adult, Biopsy, methods,Conjunctiva, pathology,Conjunctival Diseases, pathology,Female, Human, Keratoconjunctivitis Sicca, pathology,Male, Middle Age, Trachoma, pathology,
Conjunctival impression cytology using cellulose acetate filter paper is a recent technique for the study of the conjunctiva. It is fast, non invasive, easy to perform, and economical. The technique involves the use of a millipore filter paper to pick up a layer of cells from the conjunctival surface.
In this study normals were studied. The slides were stained with periodic-acid Schiff, haematoxylin-eosin, Giemsa stains.
In view of the fact that conjunctival disease such as trachoma and conditions leading to dry eye are major causes of blindness, any study which helps us to know more about the conjunctiva assumes significance.
Thirty normal patients who attended the out patients department were selected. The patients were evaluated to make sure that there is no clinically obvious conjunctival disease.
This involved taking a detailed history to rule out redness of the eyes, itching, discharge, sticking of the eyes in the mornings etc. All patients prior to selection for this study were subject to slit lamp biomicroscopy. This was to look for and rule out the presence of papillae, follicles, congestion, cicatrization, etc. Examination of the conjunctiva was also performed with supravital stain Rose Bengal 1% to detect any early changes of kerato-conjunctivitis sicca. Schirmer test was performed to ensure wetting of greater than 15mm at the end of 5 minute in all the selected cases. Tear film break up time was evaluated. Normals were found to vary between 15 and 35 sec. The patients were explained the procedure and their consent were taken.
The patient was asked to lie down in the supine position. Lignocaine 4% was instilled into the conjunctival sac. The patient was asked to open the eyes when the watering ceased. The excess pool in the conjunctival sac was gently swabbed. The impression was taken from the temporal bulbar conjunctiva. The upper lid was everted and an impression was taken from the palpebral conjunctiva. Cellulose acetate filter paper (Millipore, HAWP 34) was prepared into 3mm x 3mm pieces. A blunt, smooth ended forcep grasped one corner of the filter paper, while a smooth glass rod held in the other hand was used to gently press the paper onto the conjunctiva. The filter paper was kept on the surface for approximately 3 to 5 seconds. The filter paper was than removed with a peeling motion. This was then applied to a clean glass slide, at room temperature and the impression was transferred by uniform, gentle pressure. Fixing was done using 95% ethanol and 1 % formalin. The slides were stained with Per- iodic acid Schiff, Wright, hematoxylin-eosin, and Giemsa stains.
The slides were studied under low and high power with a light microscope.
The cytology was graded according to the scheme suggested by Nelson (2) the details of which are given in [Table - 1].
Thirty normal subjects were studied. Their age sex distribLition is given in [Table - 2] and grading of conjunctival compression cytology is given in [Table - 3]. Grade 0 was found more commonly in young persons and Grade I was found in the older age group.
Conjunctival impression cytology was found to be a suitable technique to obtain information of the conjunctival surface. To study the conjunctiva in patients previous investigators had used excised pieces of tissue or scrapings of the conjunctiva. Needless to say, both these methods were traumatic to the patient.
Conjunctival impression cytology was used for the first time in 1974 by Egbert et al (1), which they called a simple conjunctival biopsy. After experimenting with a variety of methods, including cellophane tape, photographic film, and various synthetic filters (Duralon, Polyvic, Mitex) they found that the original Millipore (mixed esters of cellulose) filter seemed effective for the purpose. After taking the impression, and fixing the impression, the filter paper was cleared by immersion in immersion oil for microscopic observation.
The method of staining the filter paper has been modified by Guazzi et al. She transferred the material obtained by the cellulose acetate paper on to a slide at 4 degree celsius.
We modified the method further by transferring the impressions form the filter paper to the slide, which was at room temperature, with very good results.
This study was undertaken in a age group from 20 to 60 years. This age distribution of the series is mentioned in [Table - 1]. It was found that out of 30 patients, 26 patients had features of grade 0 cytological appearance, while the remaining four had grade I cytological changes. The average age of patients with grade 0 cytological appearance was 28, 33 yr. While that of patients with grade I cytological appearance was 56.66 yr. While our series was too small to comment conclusively, it is possible that the ageing process itself along with the exposure to environmental conditions, results in gradual change from grade 0 to grade I cytological features, with age.
Lignocaine 4% eye drops were used as a local anesthetic. These are known to free corneal epithelial cells. Precautions such as preventing rubbing of eyes after instillation, swabbing the tear pool prior to the procedure, and gentle manipulation were taken. An impression taken from the corneal surface in a fresh cadaver eye showed presence of a sheet of epithelial cells with complete absence of goblet cells unlike the conjunctiva. In adults this procedure may be performed with minimal discomfort in absence of topical anaesthesia.
Amongst the various strains used by us, we found per iodic acid schiff to be more suitable for studying the conjunctival morphology, especially the goblet cells. The conjunctival impressions showed that the entire layer of superficial conjunctival cells maintained their normal relationship to each other.
The cytological features of epithelial as well as goblet cells were studied. The goblet cells are identified conclusively by the PAS positive cytoplasm or by their accentrically placed nuclei and plump shape and large size. The epithelial cells are small and round with easinophilic cytoplasm. The nuclei are large basophilic, with a nuclei cytoplasmic ration of 12. A morphological change to grade I is accompanied by epithelial cells that are slightly larger and more polygonal. The nuclei are smaller with a nucleocytoplasmic, ration of 1:3. The goblet cells are abundant and plump, oval and have an intensely PAS positive cytoplasm. With grade I change, their numbers reduce. Still the morphology is unchanged. (See [Figure:1], [Figure:2] & [Figure:3])
Nelson et al,, have successfully counted the goblet cells using a calibrated grid at 200 or 400 over an area of 0,03 or 0.008 sq.mm. respectively. The mean total of each such 10 areas was recorded for each specimen. Unfortunately such grid was not available to us at the time of this study.
This method enables to study and standardize this useful procedure, which is extremely useful to visualize the state of the superficial conjunctival cells. It is easy to perform, the impressions can even be taken by the paramedical workers. It is cheap the cost of the filter paper, required in small quantities is also very affordable. The results of the techniques are very fast and the time taken for the entire procedure from taking impression to seeing the slide does not take more than 8-10 minutes. The relationship of the various cells to each other is maintained, unlike that in a scraping, where only individual cells can be studied. This procedure can be performed easily in the outpatients department, and is atraumatic to the patient. It can be incorporated as one of the route investigation for patients with conjunctival diseases.
The baseline information obtained from this study of normal conjunctiva by this method can be used in trachoma. Various dry eye syndromes, avitaminosis A, giant papiflary conjuctivities, and other disorders of the conjunctival ocular surface
[Table - 1], [Table - 2], [Table - 3]