Meibum

Clinical assessments were performed sequentially as follows: symptom questionnaires, tear meniscus height (TMH), tear break-up time (TBUT), corneal fluorescein staining (CFS), lid margin abnormality, MG expressibility, and meibography. All of the subjects completed the Ocular Surface Disease Index (OSDI) questionnaire, and they were asked whether they had any of the 14 MGD-related ocular symptoms.¹⁰ (link)
The Keratograph 5M (K5M; Oculus, Wetzlar, Germany) was used to measure TMH and to perform the meibography scans. TMH was measured 5 seconds after blinking, and we measured the central TMH of the lower eyelid. TBUT was measured and CFS was performed after the instillation of fluorescein. TBUT was measured three times, and the mean value was recorded. CFS was graded using the Baylor grading scheme from 0 to 4.¹¹ (link) Lid margin abnormalities were scored from 0 through 4 according to the following four parameters: anterior or posterior displacement of the mucocutaneous junction, vascular engorgement, plugged meibomian gland orifices, and irregularity of the lid margin. We assessed the meibum quality and quantity of the 15 glands on each lower eyelid. The MG expressibility score ranged from 0 to 45.¹² (link)^,13 (link)
Images of both the upper and lower MGs were captured by the K5M. To assess the degree of MG atrophy, we used the method described by Arita et al.¹⁴ (link) to calculate the meiboscore: 0, no atrophy; 1, atrophy of <1/3 of the total lid area; 2, atrophy of 1/3 to 2/3 of the total lid area; and 3, atrophy of >2/3 of the total lid area. The meiboscore results ranged from 0 to 6.
Parameters including MG density and MG tortuosity were calculated from the meibography images. We further analyzed the images using VIA 3 software (http://www.robots.ox.ac.uk/∼vgg/software/via/).¹⁵ First, we created a grid over the whole tarsus of the eyelid and then marked out the boundaries of each MG point by point. These points were then transformed into pixel coordinates for further analysis. MG density was defined as the ratio of the sum of the area of MGs to the total area of the tarsus:
$\mathrm{MG}\mathrm{density}=\frac{\mathrm{Sum}\mathrm{pixels}\mathrm{of}\mathrm{all}\mathrm{MGs}}{\mathrm{Total}\mathrm{pixels}\mathrm{of}\mathrm{the}\mathrm{tarsus}}$
This study defined MG tortuosity as the ratio of the imaginary straight length between the two nodes and the actual length of each MG. A similar method was used previously to measure the vessel tortuosity of the retina.¹⁶ (link) MG tortuosity was calculated as follows:
$\begin{array}{c}\hfill \begin{array}{c}{\displaystyle \mathrm{MG}\mathrm{tortuosity}=\frac{\mathrm{MG}\mathrm{perimeter}}{2\times \mathrm{height}\mathrm{of}\mathrm{the}\mathrm{minimum}\mathrm{external}\mathrm{rectangle}\mathrm{of}\mathrm{the}\mathrm{MG}}-1}\hfill \end{array}\end{array}$
The MG perimeter was the pixels at the edge of the MG. As the width of each MG was small, we used half of the MG perimeter to represent the actual length of the MG. Also, because the outlines of MGs are irregular and some MGs are tilted, the minimum external rectangle was used as the outline of the MG, and its height was calculated as the imaginary straight length (Fig. 1G). To start MG tortuosity at 0, the ratio minus 1 was recorded.