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Year : 1997  |  Volume : 45  |  Issue : 1  |  Page : 25-29

Conjunctival impression cytology in xerophthalmia among rural children

Dept. of Pathology, M. L. N. Medical College, Allahabad

Correspondence Address:
M Singh
Dept. of Pathology, M. L. N. Medical College, Allahabad

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Source of Support: None, Conflict of Interest: None

PMID: 9475008

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The advent of Conjunctival Impression Cytology (CIC) has opened a new chapter in the detection of Vitamin A deficiency as it is a simplified field technique. The need for such a technique was of paramount importance in India where as estimated 5 to 7 percent children suffer from eye signs of Vitamin A deficiency. In the present study the results of clinical evaluation of apparently healthy children and those having clinical sings and symptoms of Vitamin A deficiency have been compared with CIC results. Serum Vitamin A levels have also been estimated wherever possible. The findings indicate that CIC results are superior to the clinical assessment and it is suggested that CIC can be used as screening tool for children of school going age (and earlier) for early detection and correction of Vitamin A deficiency.

Keywords: Conjunctival Impression Cytology (CIC), Vitamin A deficiency, Serum Vitamin A

How to cite this article:
Singh M, Singh G, Dwevedi S, Singh K, Kumar D, Tiwari A, Aggarwal M. Conjunctival impression cytology in xerophthalmia among rural children. Indian J Ophthalmol 1997;45:25-9

How to cite this URL:
Singh M, Singh G, Dwevedi S, Singh K, Kumar D, Tiwari A, Aggarwal M. Conjunctival impression cytology in xerophthalmia among rural children. Indian J Ophthalmol [serial online] 1997 [cited 2019 Nov 14];45:25-9. Available from:

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Vitamin A deficiency and resultant ocular diseases are among the most common debilitating nutritional disorders that afflict the rural children in India. An estimated 5 to 7% children in India suffer from eye signs of Vitamin A deficiency. Recent evidence suggests that even mild Vitamin A deficiency probably increases morbidity and mortality in children, emphasising the public health importance of this disorder.[1] A simplified field technique for early diagnosis and identification of populations at risk would be a useful tool. Such a technique can be useful in monitoring the intervention programmes for Vitamin A as well.

In the recent past the advent of Conjunctival Impression Cytology (CIC) has opened a new chapter in this field and has proved to be a reliable and valid measure of physiologically significant Vitamin A deficiency, specially in view of serious shortcomings associated with all other approaches available for assessing Vitamin A deficiency associated conditions in the field. Clinical surveys will require larger sample size to detect relatively rare transient conditions like Bitot' spots and night blindness, dietary assessment is neither very accurate nor precise, and measurement of Vitamin A in serum poses significant logistic problems as well as non cooperation from the people and requires sophisticated equipment and trained personnel for analysis. Serum Vit. A levels also suffer from poor correlation with body stores except under condition of severe depletion. The relative dose resonse (RDR) may resolve the last mentioned problem but it compounds logistic difficulties.

The technique of CIC was described by Egbert[2] in 1977 who first used cellulose acetate filter paper to take conjunctival surface smears. Sommer[3] in 1982 reported that conjunctival biopsy specimens from children with mild vitamin A deficiency revealed generalised metaplasia throughout the bulbar conjunctiva. Hatchell and Sommer[4] in 1984 reported detection of early conjunctival changes in vitamin A deficient rabbits using impression cytology; they showed progressive disppearance of goblet cells and appearance of enlarged epithelial cells in those rabbits. Since then several other reports[5][6][7][8], have added ample evidence that CIC has come to be the main stay in assessing the vitamin A deficiency problem in the community being easy, accurate, low cost and precise with high acceptability.

  Materials and methods Top

The study was conducted in the rural areas of Jasara block of Allahabad district. A random sample of 530 children from the primary schools were screened. Each child was thoroughly examined and the personal, social and medical histories were recorded on a pre-tested schedule. A qualified ophthalmologist assessed the ocular condition of the children for the presence of clincial signs of xerophthalmia and wherever necessary slit lamp examination was done. Xerophthalmia was clinically classified according to the WHO classification based on ocular signs[9] [Table - 1]. Cases where any pathology other than xerophthalmia was seen were excluded. Conjunctival smears were collected from all the 530 children.

Serum Vitamin A levels could be estimated only in 120 cases, others having refused the invasive technique. The estimation was done by spectrophotometric method in ultra-violet range as described by Wooton.[10] The calorie intake of the children was determined by questionnaire method. Socio-economic status was graded by the Kuppuswamy scale.[11]

  The Technique Top

The conjunctival cells samples were collected on Millipore filter material (HAWP 304; Millipore Corp, Bedford) with help of disk applicator. The applicator eliminates any hand contact with the filter paper specimen, applies a disk of paper of fixed area to the conjunctiva, reduces variations in applied pressure, improves cells adhesion and permits more precise targetting of sample sites. It was used for one inferotemporal and one inferonasal location. Local anaesthetic was not needed. After taking the specimen topical antibiotic (Ophthomycetin) was instilled in each eye.

The CIC specimens were stained with periodic acid Schiff and Hematoxylin. All CIC specimen were examined and staged according to the degree of squamous metaplasia as described by Wittpenn[12][Table - 2].

All CIC specimens were examined in a masked faslion i.e. the child's identity, clinical and serun Vitamin A status as well as the status of the other specimens from that case were not known to the examiner at the time of interpreatation. They were graded according to the degree of squamous metaplasia as described above.

Each child was assigned to the lowest stage (i.e. the most normal) found among all the specimens obtained from that child. Impression cytology from a normal child showing transition from a abundant normal epithelium to abnormal epithelium specimen, was graded as normal.

  Results Top

The study comprised of 530 primary school children studying in I-V classes. Of these 310 children were clinically diagnosed to have xerophthalmia with or without night blindness and were designated as the study group while the rest (220) without any apparent sign of xerophthalmia were designated as the control group.

There were 370 (69.8%) male and 160 (30.2%) female students in the age range between 5 and 12 years. Of these the majority (88.7%) belonged to the lower middle or upper lower socio-economic status while the rest (11.3%) belonged to the upper middle socio economic status. It was found that 401 children were consuming an average daily diet between 1200-1600 k calories and only 42 children were consuming average daily diets between 1600-1800 k calories.

History of diarrhoea during the last two weeks was given by 230 (43.40%) children, while 60 (11.32%) cases gave history of worm infestation and 80 (15.09%) children were found to be suffering from upper respiratory tract infection.

The CIC analysis of the 530 children revealed that not only all children clinically diagnosed as having xerophthalmia showed altered CIC pattern but amongst the apparently healthy group also only 70 (31.8%) had normal CIC status, the rest having deficiency to some extent.

In the normal goup the predominant cells were small epithelial cells found in sheets together with presence of goblet cells and mucin spots. The goblet cells showed a tendency to aggregate into groups, similar to the observation of Norn[13] [Figure - 1].

In those having abnormal cytololgy the predominant cells were large discrete epithelial cells with rare or no goblet cells and mucin spots [Figure - 2]. The borderline abnormal group showed cytology similar to abnormal except that few goblet cells could also be seen, whereas in the borderline normal the picture was similar to normal except that the epithelial cells were abnormal[Figure:3 & 4] Similar findings have been reported in previous studies conducted by various workers[4][5],[12],[14][15].

In this study of 310 cases of the study group none showed normal CIC. Abnormal CIC was seen in 41.9% cases, while borderline abnormal was seen in 32.3% cases. The rest 25.8% were borderline normal [Table - 2].

In the control group normal CIC pattern was seen only in 70 (31.8%) cases. The rest 68.2% showed varying degrees of abnormal CIC pattern in the form of loss of goblet cells and squamous metaplasia of normal conjunctival epithelium, indicating subclinical deficiency of Vitamin A.[6]

In [Table - 3] an attempt has been made to show the clinical as well as the CIC status of the children together.

Serum Vitamin A levels could be assessed only in 120 cases. Of these 70 children belonged to the sutdy group and 50 were from the control group. The mean serum levels and ranges were calculated for both separately and the difference was found to be highly significant (t = 4.734 p < 0.001) [Table - 4].

Similarly the comparison of serum vitamin A levels with CIC pattern [Table - 5] shows a significant difference. It was observed that the mean serum vitamin A levels in the normal CIC pattern group was significantly higher than the group showing any abnormal pattern (t = 5.78 p < 0.001).

  Discussion Top

The Jasra block of Allahabad district is socio-economically[13] backward area prone to droughts and famines. Hence calorie diets and resultant morbidity levels are an expected feature especially in children. Our observations confirm this state and are in conformity with the observations of Singh et al.[16]

In this study of the 310 cases, none of the children showed normal CIC pattern thus confirming the clinical diagnosis of xerophthalmia, indicating the specificity of CIC changes in vitamin A deficiency. In the control group normal CIC pattern was seen in 70 (31.8%) cases only. The rest 68.2% showed abnormal CIC pattern of varying degrees, indicating sub-clinical deficiency of vitamin A[6] [Table - 3]. This suggests that abnormal CIC pattern preceedes the ocular signs of xerophthalmia[5]and CIC is a more sensitive test. Thus this study also confirms the specificity and high sensitivity of CIC and is in conformity with other reports.[5],[6]

Mean serum vitamin A levels in the control group and the study group were 10 2.3/ug/100 ml (range 1 to 15/ug/100 ml) and 7.5 3.55/ug/100 ml (range 3 to 9/ug/100 ml) respectively. The difference was statistically significant [Table - 4]. These values are slighlty higher than the values reported in a previous study in the same distribution area.[16] Similarly when the serum vitamin A levels were compard in the group showing normal and abnormal CIC pattern, the group having normal pattern had significantly hgher vitamin A levels. [Table - 5], though the serum vitamin A levels were lower on the whole ranging from 3 to 15 ug/100 ml. This reflects poor Vitamin A nutrition even in the normal population. The relationship between the CIC pattern and serum vitamin A levels could have been highlighted further had more cases consented to giving blood samples. But as already mentioned this invasive technique has poor acceptability in the rural population.

In this study the over all accuracy of CIC prodecure as compared to serum vitamin A levels, was 83.3% while the overall accuracy of CIC compared to clinical assessment was 100%. Despite the relatively low accuracy of this procedure vis-a-vis serum vitamin A assessment it is the prodecure of choice because of the high acceptability in the community. The relatively small number of cases accepting the invasive technique required for serum vitamin A assessment is in itself an indicator of the poor acceptability of that procedure.

It is hereby suggested that CIC should be included as a screening test at the primary school level (or earlier) so that Vitamin A deficiency is detected and corrected at an early stage. This will help in prevention of ocular lesions which might subsequently lead to blindness especially in the presence of precipitating factors like acute respiratory tract infections and diarrhoea.

CIC can also serve as a useful technique for monitoring and evaluation of the vitamin A prophylaxis programme. Hence thought should be given to integration of this component in the primary health care system.

  References Top

Ministry of Health and Family Welfare Government of India, Policy of Management of Vitamin 'A' deficiancy.  Back to cited text no. 1
Egbert PR, Lauber S, Maurice D: A simple conjunctival biopsy. Am J Ophthalmol. 84:798-891, 1977.  Back to cited text no. 2
Sommer A, Green,WR: Goblet cell response to Vitamin 'A' treatment for corneal xerophthalmia. Am J Ophthalmol. 94: 213-215, 1982.  Back to cited text no. 3
Hatchell LD,Sommer A: Detection of oculation surface abnormalities in experimental Vit.A deficiency. Arch Ophthalmol. 102: 1389-1393, 1984.  Back to cited text no. 4
Wittpenn J: Tseng SCG, Sommer A: Detection of early xerophthalmia by impression cytology. Arch ophthalmol. 104:2237-2239, 1986.  Back to cited text no. 5
Natadisastra G,Wittpenn JR, West KP, Jr et al: Impression cytology for detection of Vitamin 'A' deficiency Arch Ophthalmol. 105: 1224-1228, 1987.  Back to cited text no. 6
Amedcc-Manesme O, Luzeau R, Wittpen R, et al: Impression cytology detects subclinical Vitamin 'A' deficiency. Am J Clin Nutr. 47: 875-878, 1988.  Back to cited text no. 7
Natadiasatra G, Willpenn R, Mihilal et al: Impression cytology: a practical index of Vitamin 'A' status. Am J Clin Nutr. 48:695-701, 1988  Back to cited text no. 8
W.H.O. Technical Report Series No. 590. Vitamin 'A' deficiency and Xerophthalmia. Report of a joint WHO/ USAID meeting, Geneva, 1976.  Back to cited text no. 9
Wooton DP: Mocro analysis in Medical Biochemistry, 4th ed J & A Churchill Ltd., London, 1964.  Back to cited text no. 10
Kuppuswamy B: Manual of Socio-economic status Scale. Manasayan, Delhi-6  Back to cited text no. 11
Wittpenn JR, Gadomski AM, K-johede et al. Conjunctival Impresion Cytology: Feasibility of field trial to detect subclinical vitamin 'A' deficiency. Am J Clin Nutr. 49:490-494, 1989.  Back to cited text no. 12
Norn MS. Cytology of the conjunctival fluid. Acta Ophthalmol. 59 (Suppl): 11, 1960.  Back to cited text no. 13
Tseng ACG: Staging of conjunctival squamous metaplasia log impression cytology. Ophthalmology 92:728-733, 1985.  Back to cited text no. 14
Reddy V. Rao V, Arun Jyothi, Reddy M: Conjunctival impresion cytology for asesment of vitamin A status. Am J Clin Nutr. 50:814-817, 1989.  Back to cited text no. 15
Singh G., Haque MA, Banerji SC, Singh M. Ocular health of rural children with special reference to Vitamin 'A' deficiency. Ind J Pediatrics. 50:387-390, 1983  Back to cited text no. 16


  [Figure - 1], [Figure - 2], [Figure - 3], [Figure - 4]

  [Table - 1], [Table - 2], [Table - 3], [Table - 4], [Table - 5]


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