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T. Hillenaar, V. A. D. P. Sicam, B. Braaf, R. H. H. Cals, L. Remeijer, J. F. De Boer; Calibration of Corneal Light Scatter Analysis by in vivo Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5659.
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© ARVO (1962-2015); The Authors (2016-present)
To report a new calibration method for corneal light scatter (CLS) measurement by in vivo confocal microscopy (IVCM)
Three, 3 millimeter thick, polymethylmethacrylate (PMMA) slabs (Stout, Rotterdam, The Netherlands) with different light transmittance values (65, 49, and 26%) were examined by IVCM (Confoscan4, Nidek, Padova, Italy). First we examined the 3 PMMA slabs with 60, 72, and 80% light intensity (LI). Based on the 3 slabs, which covered the whole spectrum of gray values, we calculated two 6th degree polynomials to describe functions that convert scatter values measured with 60 and 80% LI to our arbitrary standard of 72% LI. Secondly we examined 1 normal cornea with the 3 before-mentioned LIs. To test the conversion functions, we converted CLS values measured with 60 and 80% LI to our standard of 72% LI. Thirdly we examined the 3 PMMA slabs using fixed LI (72%) with 2 similar confocal microscopes (both CS4). All data was analyzed with Mann-Whitney U-test.
The functions that convert CLS values measured with 60 and 80% LI to 72% LI values were respectively:y = -1.7E-11x6 + 1.3E-8x5 - 3.6E-6x4 + 4.3E-4x3 - 2.3E-2x2 + 2.0x + 2.3R2 > 0.999andy = 1.5E-11x6 - 9.8E-9x5 + 2.5E-6x4 - 3.1E-4x3 + 1.8E-2x2 + 0.30x + 1.8R2 > 0.999Mean CLS was 19.6, 34.5, and 48.7 for 60, 72, and 80% LI respectively. There was a strong statistical difference between CLS values measured with 60 and 80% LI compared to 72% LI values (p<0.0001 and p<0.0001). After application of the conversion functions mean CLS was 35.8 for 60 and 35.4 for 80% LI and did not statistically differ from 72% LI values (p=0.62 and p=0.43) (Fig 1). Slab measurements showed a statistically significant difference in mean scatter value of 17% (p<0.0001) between both microscopes.
CLS analyses show large differences between manufacturer calibrated confocal microscopes. To enhance intra- and interdevice comparison, an objective and repeatable calibration method is mandatory. Though lot-to-lot variation and stability over time need to be tested, PMMA slabs with standard opacifications are a promising calibration tool for CLS measurements.
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