British Journal of Ophthalmology, 1980, 64, 700-704

Quantitative tear lysozyme assay: a new techniquefor transporting specimensD. V. SEAL,' I. A. MACKIE,2 R. L. COAKES,2 AND B. FAROOQI3From 2Moorfields Eye Hospital, London EC2 and WCJ, and the Public Health Laboratories,3St George's Hospital, Tooting, and 'Southampton General Hospital

SUMMARY We have developed a method for assaying the concentration of tear lysozyme usingeluates of tear fluid collected on filter paper discs. Specimens can be stored and transported toremote laboratories for assay. We have shown that the 'indirect' or eluate method gives statisticallycomparable results to the 'direct' method using fresh, neat tear fluid.

We have previously described a tear lysozymeassay using tear fluid collected on filter paper discs.'Discs must be placed as soon as possible directly onto an agar plate seeded with Micrococcus luteus(lysodeikticus). The results of the assay have givena reliable guide to lacrimal gland function.2 How-ever, the organisation of the test has virtuallyrequired that the patient attends the laboratory.This has been inconvenient and has often led topatients travelling long distances.Hypher3 has criticised the test suggesting that a

small amount of the tear fluid collected on to thedisc remains in the bottle. He has calculated acorrection factor for this.We have now developed a modified test that

allows the collection of tear fluid at a site distantfrom the laboratory and which minimises any lossof tear fluid from the disc.

Materials and methods

COLLECTION OF TEAR FLUIDTear fluid was collected on to Whatman No. 1filter paper discs, 5 mm in diameter, which wereplaced in the lower conjunctival sac. The volume oftear fluid collected was ascertained by weighing thedisc in its bottle before and after collection. For thispurpose a portable CAHN* automatic electro-balance was chosen (Fig. 1), as it could be set up

*Cahn Instruments, Ventron Corporation, 16207 SouthCarmenita Road, Cerritos, California 90701, USA. Avail-able in UK with Hewlett-Packard printing calculator andR51 balance from Oertling Instruments Ltd, Orpington,Kent.

Correspondence to Dr D. V. Seal, Public Health Laboratory,General Hospital, Southampton S09 4XY.

on a desk in an outpatient clinic. It was accurate to+/- 0-01 mg equivalent to +/- 0-01 ,ul tear fluid.A Hewlett-Packard printing calculator (Model

97Sl/0) was used, as it was compatible with theoutput of the balance and could be programmed tocalculate and print resu!ts of weight readings. Sinceno bottles were available commercially that weresufficiently small (maximum height 15 mm) or light(maximum weight 1500 mg), our Hospital PhysicsDepartment manufactured bottles by injectionmoulding from polypropylene beads.4 A tool-steelmould was made with brass inserts that producedthe bottle and lid as a one-piece mouldingt (Fig. 2).Each bottle, with lid closed, weighed approximately300 mg, measured 10 mm high by 11 mm wide andheld up to 200 ,ul fluid. The bottles were cleaned inan ultrasonic water bath and dried in a hot air oven;a filter paper disc was then placed in each bottle.They were sterilised in batches by 2-5 megarads ofirradiation.+

PREPARATION OF ELUATEAfter the bottle containing the tear fluid collectedon to the disc had been weighed approximately100 ,ul of phosphate-buffered saline was added froman Oxford pipette with a disposable tip and the lidwas fastened. The bottle was reweighed to ascertainthe exact volume of buffer added and the resultrecorded. After collection the bottles were trans-ported at atmospheric temperature and placedovernight in a +4°C refrigerator. Next day theywere transferred to a -20°C freezer in the laboratorypending assay.tManumold Mark 2 Injection Moulding Press, FlorinStreet, 457 Caledonian Road, London N7.

++Irradiated Products Ltd, Elgin Drive, Swindon, Wilts.700

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Quantitative tear lysozyme assay: a new technique for transporting specimens

Fig. 1 Portableelectrobalance with printingcalculator.

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Fig. 2 Injection mould andpolypropylene bottles.

PERFORMANCE OF ASSAYFor the assay, similar filter paper discs were pre-pared in 1OX 25 mm plastic tubes,* each tube beingweighed on an Oertling R51 laboratory balance,accurate to +/- 0-01 mg. With the use of forceps 2discs were dipped separately into the eluate of atear fluid sample, weighed, and the volume takenup by each disc calculated. Batches of two discswere similarly dipped into 5 standards of hen egglysozymet containing 005, 0-1, 0-2, 03, and 04mg/ml respectively, which were calibrated intounits of activity per gram by our original spectro-photometric method.1 All the discs were placed on*Metal Box Ltd, 30 Great Guildford Street, London SEI.

tKoch-Light Ltd, Colnbrook, Buckinghamshire.

an agar plate, previously flooded with Micrococcusluteus (lysodeikticus) ana incubated at 20°C for 4days. Zone diameters of bacterial lysis around thediscs were measured on an image intensifier andrecorded. After assay the eluates were frozen andstored at -20°C.

EVALUATION OF ASSAYThis new 'indirect' method of lysozyme assay wasevaluated by sampling tear fluid from the right andleft eyes of 42 'normal patients' (that is, patientswith glaucoma but with normal lacrimal functionclinically) aged from 20 to 79 years, with a mean of56 years. These results were compared with thoseobtained by our original 'direct' method from 86normal volunteers, aged 20 to 85 years, with a

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mean of 50 years. Eight patients with establisheddry eyes, aged 9 to 70 years, were also tested by the'indirect' method, and the results were comparedwith 23 patients with keratoconjunctivitis sicca,aged 42 to 68 years, tested by the 'direct' method.

TEST FOR ANTIBACTERIAL SUBSTANCES

Tear fluid was tested for the presence of antibac-terial proteins other than lysozyme; which couldtheoretically inhibit the growth of Micrococcusluteus. Agarose containing antihuman leukaemiclysozyme serum was seeded with the micrococcus.Two discs, each containing neat tear fluid fromnormal volunteers, were placed on the seededagarose and incubated at 37°C for 18 hours. Twodiscs, from the same volunteers, were also placed on

seeded agarose which did not contain antilysozymeserum and were similarly incubated.

Results

THE ASSAYThe hen egg lysozyme (HEL) standard had an

activity of 24 400 units per gram. This comparedwith an activity of 30 000 units per gram for ourHEL standard acquired in 1975. By expressing allresults in units of activity per ,ul, compensation wasmade for the 2 standards of different strengths, andresults of these methods could be directly compared.A standard graph was prepared for each plate

assay plotting the logarithms of the total quantities(in micrograms) of lysozyme on the discs against thesquares of the zone diameters of bacterial lysis (Fig.3). The concentration of lysozyme in the tear eluate,and hence that in the original tear fluid, was calcu-lated in units of activity per microlitre, and its

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relationship to the age of the patient is shown inFig. 4. The limit of sensitivity for the method was20 units of activity per .d tear fluid. The meannormal value decreased by 0 5 units of activity perul tear fluid per year. Values for +/- 2 standarddeviations (SD) of the means are shown togetherwith the critical lower limit of the 'direct' methodof assay which was 1-7 SDs below the mean at anyage by this method; values below this limit for anyage have only a 5% chance of normality, whilevalues below the 2SD limit have only a 2.5%chance.

In order to make direct comparison betweenresults of patients of different ages, adjustment wasmade by dividing each result, in units of activityper [L, by the value of the critical lower limit, given

Key x Normal Eye. Keratoconjunctivitis sicca Eye

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Fig. 4 Lysozyme concentrationby age in 42 normal patients and8 patients with sicca.

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Quantitative tear lysozyme assay: a new technique for transporting specimens

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Fig. 5 Distribution of tear lysozyme ratios.

in Fig. 4, for that patient's age (tear lysozymeratio (TLR)).

EVALUATION OF ASSAYThe distribution of the TLRs for the 'indirect' and'direct' methods of assay is shown in Fig. 5, wheremeans and SD limits for the normal range are shown,being different to those of Fig. 4 from which theyhave been derived; the TLRs are expressed on alogarithmic scale. There was no statistical differencebetween either method for the TLR values obtainedfrom normal patients. At any age the critical lowerlimit is equivalent to a TLR of 10, so that valuesless than this will have only a 5% chance of nor-mality. The lowest TLR value, and hence tearlysozyme concentration, that can be measured bythe 'indirect' method is 0 4, while the lowest valuefor the 'direct' method is 0 1, because neat tear fluidwas placed on to the agar plate resulting in largerzones of bacterial lysis.

For patients with keratoconjunctivitis sicca theTLR results by the 'indirect' method ranged from1 0 to <0*4 and for the 'direct' method from 1-3 to<0 1. A TLR value of <0 4 is a satisfactory lower

limit for this method, occurring in the patient withclinically established keratoconjunctivitis sicca andindicating that the lysozyme concentration is verylow. This has diagnostic and pathological implica-tions.

TEST FOR ANTIBACTERIAL SUBSTANCESThere was no lysis of the micrococcus around the2 discs on the seeded agarose containing anti-lysozyme antiserum, though immune complexprecipitates did occur under the disc, where lysiswas also present. There were large zones of bacteriallysis around the 2 discs on the seeded agarosewithout antiserum. This showed that the tearlysozyme had been prevented from diffusing awayfrom the disc and that there were no proteinspresent in the neat tear fluid which could inhibitgrowth of this micrococcus.

Discussion

There are various methods for lysozyme assay. Weconsider that the 'direct' method we previouslydescribed' added greatly to the accuracy of the test.Hypher5 has shown that the method of Bonavidaand Sapse,6 whereby a 5 mm piece of a wettedSchirmer strip is cut off and used for the assay, isprone to inaccuracy because of irregular diffusionof tears along the strip. The disadvantage of our'direct' method is the fact that discs have to becollected in the proximity of the laboratory andprocessed immediately. Processing can take up to 4hours and often continues after normal laboratoryhours. The 'indirect' method now described permitsprocessing of discs at any time after collection andtransportation of specimens to remote laboratories.

It has been pointed out7 that there is a diurnalrhythm of tear lysozyme secretion, and we suggestthat the discs are collected in the morning or after-noon to achieve maximum values.The use of live rather than killed Micrococcus

luteus has the advantage that a nutrient agar platecan be flooded when required and does not need tobe prepared in advance. It is a suitable techniquefor routine use in a hospital laboratory, and sophis-ticated equipment is not required. The zone sizesobtained are clear to read. We have found no errorfrom other antibacterial proteins known to bepresent in tear fluid, presumably because theirconcentration is too low or the M. luteus is notsensitive to them.Lysozyme concentration in transported tear fluid

has been studied before, when 1000 Schirmer teststrips were posted to a laboratory.8 The Schirmerstrips had been wrapped in plastic film to preservetheir moisture and eluates from them were tested by

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filter paper electrophoresis. A study was made ofthe relative proportions of tear lysozyme, globulin,and albumin with semiquantitation of the tearlysozyme concentrations. However, the paperelectrophoresis method is up to 300 times lesssensitive than an agarose-micrococcus plate assay,but standard values were not quoted.6 Rocketelectroimmunoassay is more sensitive than paperelectrophoresis but is not recommended for detect-ing low concentrations of lysozyme.9The normal range of lysozyme concentration by

the 'indirect' method has been found statisticallysimilar to that using fresh neat tear fluid (directmethod). This indicates that tear lysozyme remainsstable when held in solution as an eluate. Abnor-mally low values have been found in patients withkeratoconjunctivitis sicca, similar to those recordedby the 'direct' method, but a lower concentrationcan be detected by using neat tears. There is nopossible loss of tear fluid from the disc as a depositin the bottle with the 'indirect method' becausephosphate-buffered saline is pipetted over the discin the same bottle to form the eluate. In our earlierstudies we found filter paper discs containing neattear fluid, sealed in bottles, could not be held at+4°C, as they dried out by evaporation within thebottles. The lysozyme molecule is irreversiblydamaged at the liquid-air interface when this occurs,and assay values fell considerably.We envisage the development of enzyme linked

immunoassay or radioimmunoassay for measuringthe concentration of eluted lysozyme, using specificantiserum. It should be more sensitive, less time

consuming, and would allow the processing of alarger number of specimens.

We thank Merck, Sharpe and Dohme Ltd and the St.George's Hospital Slater Fund for financial assistance withthis study. We thank Professor G. R. F. Hilson, Dr D. G.Fleck, and Mr T. Evans for support and for providing uswith laboratory facilities. We thank Mr Ray Fautley,Hospital Physics Department, Soruthampton General Hos-pital, for manufacturing the mould and polypropylenebottles. We also thank Dr D. R. Gamble for his criticaladvice and Miss P. J. Stoffell for typing the manuscript.

References

1 Mackie IA, Seal DV. Quantitative tear lysozyme assay in;units of activity per microlitre. Br J Ophthalmol 1976;60: 70-4.

2 Mackie IA, Seal DV, Pescod JM. Beta-adrenergic receptorblocking drugs: tear lysozyme and immunological screen-ing for adverse reaction. Br J Ophthalmol 1977; 61: 354-9.

3 Hypher TJ. Uptake and loss of tears from filter paperdiscs employed in lysozyme testing. Br J Ophthalmol1979; 63: 251-5.

4 Brydson JA. Plastics Materials. 3rd ed. London: Newnes--Butterworth, 1975: 218-32.

5 Hypher TJ. Fluid uptake in Schirmer papers and itsrelevance to their employment in tear collection forlysozyme tests. Br J Ophthalmol 1980; 64: 696-9.

6 Bonavida B. Sapse AT. Human tear lysozyme. II. Quanti-tative determination with standard Schirmer strips. Am J-Ophthalmol 1968; 66: 70-6.

7 Horwitz BL, Christensen GR, Ritzmann SR. Diurnalprofiles of tear lysozyme and gamma A globulin. AnnOphthalmol 1978; 10: 75-80.

8 Erickson OF, Hatlen R, Berg M. Industrial tear study.Am J Ophthalmol 1959; 47: 499-508.

9 Osserman EF, Canfield RE, Beychok S. Lysozyme. NewYork: Academic Press, 1974: 347-53.

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