Conjunctival impression cytology (CIC) to detect subclinical vitamin A deficiency: comparison of CIC with biochemical assessments

Conjunctival impression cytology vitamin A deficiency: comparison Anne M Gadomski, Anamaria R Rosas,

Chris L Kjolhede, John and Michele R Forman

Wittpenn,

(CIC) to detect subclinical of GIG with biochemical Jesus

Bulux,

=

KEY

Vitamin

WORDS

tein, relative

dose

response,

A deficiency, sensitivity, specificity, conjunctival impression cytology

Introduction Mild xerophthalmia has been associated with increased risk of morbidity and mortality (1-3). Animal studies of experimental vitamin A deficiency demonstrated that changes in immune function, growth, and ocular cytology occurred well before the development of clinical signs (4, 5). Clinical signs of xerophthalmia can occur late, are at times rapidly progressive and irreversible, and are relatively rare, requiring large sample sizes to determine prevalence (6). Given the above, there is a need for ways of assessing marginal or subclinical vitamm A deficiency. Marginal vitamin A deficiency, defined as that portion ofthe spectrum ofvitamin A status that is less than adequate, ie, < 0.70 imo1/g liver, and greater than the critical level, ie, > 0. 17 tmo1/g liver (7), has been assessed via hepatic biopsy and relative dose response. Because > 90% of the total body vitamin A is stored in the liver, hepatic biopsies are the closest approximation to a gold standard for assessment of total body vitamin A status (8). Obviously, this technique cannot be used in field settings. The relative-dose-response (RDR) technique was developed as an indirect method to estimate hepatic reserves. RDR is based on the accumulation of aporetinolbinding protein (apo-RBP) in the liver when vitamin A Am J Clin Nuir

I 989;49:495-500.

Printed

in USA.

© 1989 American

Society

retinol,

retinol-binding

pro-

status is deficient or marginal (9). Small doses of retinol yield a rapid release ofholo-RBP and increases in serum retinol levels among deficient and fasting individuals. This test measures the rise in retinol level 5 h after a test dose of retinyl palmitate is given and RDR > 20% was correlated with liver concentrations of retinol < 0.070 imol/g among hospital patients (10, 1 1). RDR was also used under field conditions to assess vitamin A deficiency (12, 13). Its limitations however may be its maccuracy in sthtes of protein malnutrition or liver disease where RBP levels may be too low to produce a response to the test dose given as part ofthe RDR. Prior studies of alcoholic patients encountered this limitation (14). RDR the Department of International Health, Johns Hopkins and Public Health, Baltimore, MD; the Department ofOphthalmology, Los Angeles County/University of Southern Callfornia Medical Center and the Centei for Studies ofSensory Impairment, Aging and Metabolism, Dr Rodolfo Robles V Hospital, GuateI

From

School

mala

ofHygiene

City, Guatemala.

2 Supported by cooperative agreement #DPE-595 1-A-OO-505 1 between the Office ofHealth, the United States Agency for International Development, and the Institute for International Programs, Johns Hopkins School ofHygiene and Public Health. 3 Reprints not available.

ReceivedDecember7, 1987. Accepted for publication March 29, 1988. for Clinical

Nutrition

495

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ABSTRACT By use of sensitivity and specificity analysis, conjunctival impression cytology (CIC) was compared with fasting serum vitamin A levels and relative dose response (RDR) of Guatemalan children. One impression was taken from the temporal bulbar aspect of each eye, fasting serum vitamin A levels were then drawn, 480 RE ofoil-based retinyl palmitate was given, and a 5-h postdosing vitamin A level was drawn (RDR procedure). For a 20% RDR cutoff, the sensitivity ofCIC was 23% with a specificity of 80% and a positive predictive value of 9% (n 213 children). Compared with fasting vitamin A levels alone (with 0.70 mo1/L as abnormal), the sensitivity ofCIC was 26%, specificity was 81%, and positive predictive value was 22% (n = 221 children). There was no significant difference in the mean serum retinol level between those with abnormal and normal CIC. In this study population CIC does not identify the same group of children with marginal vitamin A as identified biochemically. Am J Clin Nutr 1989;49:495-500.

496

GADOMSKI

To evaluate

the validity

ofCIC,

the technique

must be

applied in a general population whose vitamin A status can be defined by other established means of assessment. Therefore, we undertook this field trial to see how CIC compares with relative dose response, fasting serum retino!, and RBP levels in individuals suspected to have subclinical vitamin A deficiency but with little evidence of clinical xerophthalmia. and methods

Subjects Study

were

the child’s

ferred as necessary. The mothers were instructed to bring their children in a fasting state to a predetermined site the morning

ofthe study. Any children vitamin

infection infection

corded

reported

to have

fever

or serious

infection

or at the time of the study were excluded because A levels are depressed in the presence of fever and/or

overnight

(21, 22). The observation of mild upper respiratory or other obvious conditions without fever was rebefore the study procedures were done.

Procedures

The procedures for conjunctival impression cytology were described in the first part of this study (23). After the impressions were obtained, 3 mL venous blood was drawn from an antecubital site, allowed to clot in glass tubes shielded from light, kept cool, and centrifuged within 2 h at 600-1000 X g for 10-15 mm. Immediately after the first blood was drawn, the child received by mouth 0.480 mg retinyl palmitate (HoffmanLaRoche, Nutley, NJ) in corn oil delivered via a precision mlcropipette. The children and mothers were then fed breakfast composed of4O g white bread with a spread offried black beans (-35 g), a small 24-g sweet roll, 240 mL hot chocolate, and 100 g ofwatermelon. On the basis ofthe Latin American Food Composition Table (24), this breakfast supplied 24 RE. A second

blood

specimen

was drawn

5 h after the first blood

level.

After the second blood was drawn, mothers and children were fed a lunch rich in vitamin A. During the 5 h between blood draws, the study pediatrician led a discussion of relevant public health issues with mothers while the heights, weights, and head and arm circumferences of the study children were measured. Children with a history ofnight blindness or clinical signs ofvitamin A deficiency were treated with 200 000 IU of vitamin A (60 mg RE vitamin A plus 36 mg vitamin E in oil) at the conclusion ofthe study procedures.

areas

Recent

surveys

and school-age

of various

select

populations

of preschool

in Guatemala revealed a range of 1624% serum < 0.70 mo1/L (N Solomons, personal communication, 1986). The present study was conducted in several areas ofGuatemala: an orphanage in Guatemala city, three low-socioeconomic barrios ofGuatemala city, an isolated village 30 km from the capital, and a village along the Rio Dulce in the eastern Guatemalan lowlands. The latter population had been assessed in November 1986 by use of serum retinol levels. The children in this group had had levels < 0.70 mol/L, were being located for follow-up, and were included in this study before treatment with vitamin A. children retinol levels

Enrollment

Laboratory

procedures

After the blood specimens were centrifuged and separated in the field, the specimens were stored at -20 #{176}C until transported to Boston on dry ice. There the specimens were stored at -20 oc until they were analyzed for retinol content by the Bieri method of reverse-phase high-pressure liquid chromatography (25). The coefficient of variation for retinol determination in this lab is 3% within

A levels,

component

children

of this study required

were enrolled

1-3 d before

runs and 6% over the course

by an immunoturbidimetric Centrifugal Analyzer (Roche yule, NJ)(26)and antiserum was supplied

was

analyzed Cobas Fara

tics, La Jolla,

CA. Prealbumin

an immunoturbidimetric

Because the RDR vitamin

performed. Informed consent was obtained from mother or guardian after the study’s objectives and procedures were explained. The protocol and procedures for this study were approved by the Committee oflluman Vo1un. teers at Johns Hopkins University and by the corresponding committee at the Center for Studies of Sensory Impairment, Aging and Metabolism in Guatemala. A 7-d history offever or ifiness was recorded for each child as well as a 6-mo history f daily nondietary vitamin A intake. Those children with fever, history of supplemented nondietary vitamin A intake for > 2 wk, or serious infection were excluded from the study and re dures

fasting

the reagent

the proce-

ME) (27).

supplier,

of 1 d. RBP

procedure with a Diagnostics, Bdllerby Behnng Diagnos-

determinations were made with method of analysis, described by Atlantic Antibodies, inc (Scarborough,

Downloaded from www.ajcn.org by guest on July 10, 2011

is also limited by the need for two venipunctures and by effects oftransport, storage, and handling on serum retino! levels. Serum retinol levels are not accurate indicators of vitamm A status except at extremes of excess or deficiency (8), and for this reason retinol levels > 0.35 zmol/L do not closely correlate on an individual basis with RDR or hepatic biopsy results (1 1). However, retinol levels were used in aggregate form to estimate the prevalence of vitamm A deficiency in a population (15). For example, the effect of vitamin A fortification of sugar in Guatemala was evaluated by comparing the percentage of preschool children having serum retinol < 0.70 Mmol/L prefortification (2 1.7%) with the percentage postfortification (9.1%) (16). Conjunctival impression cytology (CIC) was proposed as a method ofdetecting early xerophthalmia (5, 17) and subclinical vitamin A deficiency (18). Prior studies of dc in India and Indonesia involved application of CIC to subjects with clinical signs of xerophthalmia, stages XN and/or XIB, compared with control subjects without clinical signs (17, 19). Indonesian subjects and control subjects had decreasing mean serum retinol levels with increasingly abnormal CIC stages. After vitamin A treatment 50 of 53 children with abnormal cytology reverted to normal cytology at some point during the 6mo follow up. CIC was felt to be closely correlated with vitamin A status and more sensitive than a clinical ocular exam (19). However, calculation ofsensitivity and specificity from these studies would be misleading because the dc was tested where the number of cases equal the number ofcontrols; this could lead to an artificially high sensitivity and positive predictive value (20).

ET AL

IMPRESSION TABLE

CYTOLOGY: TABLE

1

Populations

studied:

aggregate

univariate

3

Sensitivity,

descriptors

and positive

specificity, with RDR*

compared Number

497

SENSITIVITY

predictive

(PPV) of CIC

value

Percent

RDR Age (y)

5

2 50 45 50 64

6 >6

11 13

20% and abnormal CIC. Perhaps the sensitivity of CIC relative to the other methods used to assess vitamin A status may be better in populations that have more severe vitamin A deficiency. For this reason it would be valuable to replicate this study in such populations. Additionally, it would be valuable to replicate this study in settings similar to Guatemala to evaluate the effect ofcomorbidities ofthe eye on CIC results. U retinol,

r

CYTOLOGY:

GADOMSI(1

500 17. Wittpenn JR. xerophthalmia

Tseng S. Sommer A. by impression cytology.

Detection 1#{243}f #{232}a’TI)’ Arch Ophthalmol

1986; 104:237-9. 18. Amedee-Manesme 0, Luzeau R, Wittpenn JR, Hanck A, Sommer A. Impression cytology detects subclinical Vitamin A deficiency. Am 3 Clin Nutr 1988;47:875-8. 19. Natadisastra G, Wittpenn JR. West KP, Muhilal, Sommer A. Impression cytology for detection of vitamin A deficiency. Arch Ophthalmol 1987; 105:1224-8. 20. Ransohoff DF, Feinstein AR. Problems of spectrum and bias in evaluating the efficacy ofdiagnostic tests. N Engi J Med 1978;299:

926-30.

DC: US Government Pribting Office, 1961. Bieri JG, Tolliver TJ, Catignani GL Simuftaneous determination of aipha-tocopherol and retinol in plasma or red cells by high pressure liquid chromatography. Am J Clin Nutr 1979;32: 2 143-9. 26. Bankson DD, Rifai N, Silverman LM. Immunoturbidimetric

measurement

ofserum retinol binding protein in renal and hepatic Ann Clin Chem (in press). 27. Atlantic Antibodies, Inc. Quantification of prealbumin using the Cobas Bio Centrifugal Analyzer in conjunction with Atlantic Antibodies reagents. Scarborough, ME: Atlantic Antibodies, mc, 1988. (Ad-0054.) 28. Center for Disease Control. Standard deviation-derived growth reference curves from NCHS/CDC reference population. NCHS growth curves for children, birth-18 years. Atlanta, GA: CDC, 1978. (Series 1 1 number 165, DHEW publication [PHSI 78-1650.) 29. Mobarhan S, Russell R, Underwood BA, Wallingfordi, Mathieson IW, Al-Midani H. Evaluation of relative dose response test for vitamin A nutriture in cirrhotics. Am J Clin Nutr 198 1;34:2264disease.

70. 30.

Statistical Analysis System Institute. 5th ed. Cary, NC: SAS, mc, 1985.

SAS user’s guide:

statistics.

31. Snedecor OW, Cochran WG. Statistical methods. 6th ed. Ames, IA: Iowa State University Press, 1967. 32. Vahiquist A, Rask L, Peterson PA, Berg T. The concentrations of retinol-binding protein, prealbumin, and transferrin in the sera of newly delivered mothers and children ofvarious ages. Scand J Clin Lab Invest 1975;35:569-75. 33. Driskell WJ, Lackey AD, Hewett JS, Bashor MM. Stability of vitamin Am frozensera. ClinChem 1985;31:871-2. 34.

25.

Smith FR, Underwood Depressed

35.

plasma

BA, DenningCR, VarmaA, Goodman DS. retinol-binding protein in cystic fibrosis. J Lab

Clin Med 1972;80:423-33. Ingenbieck Y, Van Den Schrieck H, De Nayer P, Dc Visscher M. The role ofretinol binding protein in protein-calorie malnutrition. Metabolism

1975;24:633-41.

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21. Arroyave G, Calcano C. Descenso de los niveles sericos de retinol y de su proteina de enlace (RBP) durante las infecciones. Arch Latinoam Nutr 1979;29.233-60. 22. Kozlowski B, Taylor M, Baer M, Blyer E, Trahms C. Anticonvulsant medication use and circulating levels of total thyroxine, RBP, and vitamin A in children with delayed cognitive development. Am J Clin Nutr 1987;46:360-.8. 23. Kjolhede CL, Gadomski AM, Wittpenn J, et al. Conjunctival impression cytology; feasibility ofa field trial to detect subclinical vitamin A deficiency. Am J Clin Nutr 1989;49:490-4. 24. Interdepartmental Committee on Nutrition for National Defense. Food composition table for use in Latin America. Washington,

ET AL