Investigation of the accuracy of tear lysozyme determination by the Quantiplate method

The accuracy of lysozyme concentration determination by the method of measuring lysis diameter in agarose gel slabs containing Micrococcus lysodeikticus bacteria uniformly suspended throughout the gel was determined for various methods of tear sample collection. The effects of storage of lysozyme solution samples at subzero temperatures for several days was also examined. It was found that a power rather than the suggested exponential dependence between the lysozyme concentration and lysis diameter provides the most accurate fit and thus should be used for interpolation. Storing samples frozen in glass capillaries lowered the lysozyme concentration in a predictable manner. When Weck-Cel sponges were used to collect the samples the lysozyme concentration was greatly diminished in a nonlinear manner because of internal adsorption. The relative loss (cause by adsorption) depended on the actual lysozyme concentration as well as on the sample volume/sponge weight ratio. Storing samples absorbed by such sponges in a frozen state further altered the results in an unpredictable way. The observation that smaller tear samples for a given sponge size yielded lower apparent values for lysozyme concentration casts doubt on findings that have reported lower lysozyme concentration in the tears of keratoconjunctivitis sicca patients, where either cellulose sponges or filter paper discs were used for tear collection.