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Defining an Optimal Sample Size for Corneal Epithelial Immune Cell Analysis Using in vivo Confocal Microscopy Images
Xin Yuan Zhang1, Mengliang Wu1, Holly R Chinnery1
1Department of Optometry and Vision Sciences, University of Melbourne, Parkville, VIC, Australia.
Insights
Determining the optimal number of in vivo confocal microscopy (IVCM) images for quantifying corneal immune cell (IC) density is crucial. This study found 12 images for central cornea and 7 for peripheral cornea ensure accuracy, while subjective selection methods lack consistency.
Area of Science:
- Ophthalmology
- Cell Biology
- Medical Imaging
Background:
- In vivo confocal microscopy (IVCM) is vital for quantifying corneal immune cell (IC) density in clinical research.
- Current practices often use arbitrary sample sizes for IVCM images, lacking evidence-based guidance for reliable IC density estimation.
- Establishing a standardized image sample size is essential for consistent and accurate clinical study outcomes.
Purpose of the Study:
- To determine the optimal number of randomly selected IVCM images for accurate quantification of corneal epithelial IC density in central and peripheral cornea.
- To evaluate the consistency and precision of subjective image selection methods (three representative images) compared to random selection.
Main Methods:
- Utilized all combinations of 2-15 non-overlapping IVCM images from 20 healthy participants to derive IC density estimates.
- Defined the 'true mean' IC density by averaging quantifications from 16 images.
- Assessed accuracy using mean ratios against the true mean and evaluated observer consistency with intraclass correlation coefficients (ICCs).
Main Results:
- 12 randomly selected IVCM images yielded an IC density estimate within 30% of the true mean 95% of the time for the central cornea.
- Seven randomly selected IVCM images provided equivalent precision for the peripheral cornea.
- Subjective selection of 'three representative images' showed poor inter-observer consistency (ICC <0.5) and similar precision to random selection.
Conclusions:
- The study provides evidence-based sample size recommendations for IVCM image selection in corneal IC density quantification.
- 12 images for central and 7 for peripheral cornea are recommended for achieving reliable IC density estimates.
- Subjective image selection is less reliable than random selection for consistent and precise IC density quantification.
Purpose:
In vivo confocal microscopy (IVCM) images are frequently used to quantify corneal epithelial immune cell (IC) density in clinical studies. There is currently limited evidence to inform the selection of a representative image sample size to yield a reliable IC density estimate, and arbitrary numbers of images are often used. The primary aim of this study was to determine the number of randomly selected, unique IVCM images required to achieve an acceptable level of accuracy when quantifying epithelial IC density, in both the central and peripheral cornea. The secondary aim was to evaluate the consistency and precision of an image selection approach where corneal epithelial IC density was quantified from "three representative images" selected independently by three experienced observers.
Methods:
All combinations of two to 15 non-overlapping IVCM images were used for deriving IC density estimates, for both the central and peripheral cornea, in 20 healthy participants; the density value from averaging quantifications in the 16 images was defined as the "true mean". IC density estimates were compared with the true mean in each corneal region using a mean ratio. Intraclass correlation coefficients (ICCs) were used to evaluate the consistency of the mean ratios of IC density estimates derived from the method involving the manual selection of "three representative images" by the observers. The precision of the IC density estimates was compared to a scenario involving three randomly selected images.
Results:
A total of 12 randomly selected, non-overlapping IVCM images were found to be required to produce a corneal epithelial IC density estimate that was within 30% of the true mean, 95% of the time, for the central cornea; seven such images produced an equivalent level of precision in the peripheral cornea. Mean ratios of corneal IC density estimates derived from "three representative images" methods had poor consistency between observers (ICC estimates <0.5) and similar levels of precision when compared with using three randomly selected images (p > 0.05 for all comparisons), in both the central and peripheral cornea.
Conclusions:
Data presented in this study can inform image selection methods, and the sample size required for a preferred level of accuracy, when quantifying IC densities in the central and peripheral corneal epithelium using IVCM images.
