Reproducibility in measuring tear samples using a freezing point depression osmometer

Background Hyperosmolarity of tear fluid has been recognised as a common feature of all types of dry eye disease. This study was designed to assess the inter-session reproducibility of a freezing point depression osmometer (Fiske 110) as the most common and precise way of measuring osmolality, by using two different volumes of tear samples on healthy subjects, and to determine the possible applications of this device in tear film research and clinical practice. Methods Measurements were made by using the Fiske 110 osmometer under two different tear sample volumes (4 mu l and 2 mu l). In both cases, samples were diluted in purified water to obtain the 20 mu l required by the device to perform the measurement (1:4 and 1:9 dilutions, respectively). Inter-session reproducibility was determined in two groups of 40 healthy subjects, in two sessions, one week apart. In each group, one of the two different tear sample volumes was used to determine the reproducibility of each technique. Results No significant differences were detected between the measurements obtained in the two sessions using the 4 mu l (paired t-test, p = 0.772; mean difference +/- SD = -0.85 +/- 18.77 mOsm/L; 95 per cent limits of agreement [LoAs] = -37.64/+35.94) or the 2 mu l volume sample (paired t-test, p = 0.054; mean difference +/- SD = 9.27 +/- 29.44 mOsm/L; 95 per cent LoAs = -48.43/+66.97). Conclusions Whereas both techniques show an acceptable inter-session reproducibility, the bias range with the present protocol was higher using the 2 mu l tear sample volume than the 4 mu l one. Therefore, it seems that the diluted 4 mu l sample was the only dilution that could be acceptable for use in routine clinical practice for tear film analysis.