Clinically Relevant Immune-Cellular Metrics of Inflammation in Meibomian Gland Dysfunction

Abstract

Purpose: To determine the reliability and clinical relevance of in vivo confocal microscopy (IVCM)-based immune-cellular metrics of palpebral conjunctival inflammation in meibomian gland dysfunction (MGD).

Methods: Sixteen MGD patients and 13 reference controls included in this cross-sectional, retrospective study, had an ocular surface exam, symptom assessment (Ocular Surface Disease Index questionnaire [OSDI]), and palpebral conjunctival IVCM imaging. Bland-Altman analyses, intraclass correlation coefficient (ICCa), Lin's concordance correlation coefficient (ρc), receiver operating characteristic (ROC) analyses, and correlations were performed. Clinical outcome measures were symptom severity (OSDI scores), tear break-up time (TBUT), and corneal fluorescein staining (CFS grade).

Results: Compared to controls, patients with MGD had variable symptom severity (average OSDI score: 48.3 ± 7.6, P = 0.0008, range: 8.3-85.42), shorter TBUT (6.8 ± 0.9 seconds, P = 0.002), comparable corneal staining (0.31 ± 0.19, P = 0.20), and greater conjunctival inflammation (epithelial immune cells [EIC]: 477.8 ± 54.2 vs. 123.3 ± 17.2 cells/mm2, P < 0.0001; intraglandular immune cells [IGIC]: 41.9 ± 3.3% vs. 20.33 ± 7.3%, P < 0.01). Immune-cellular metrics had high inter- and intraobserver agreement (ρc: 0.86-0.94; ICCa and Cronbach's α: 0.85-0.97, P < 0.0001). EIC correlated positively with OSDI (rs: 0.49, P = 0.03), while both EIC and IGIC correlated inversely with TBUT (rs: -0.47, -0.45, P < 0.05), and had high accuracy in detecting inflammation (ROC area under the curve [AUC]: 0.97 and 0.89, P ≤ 0.001).

Conclusions: EIC and IGIC are increased in highly symptomatic patients with MGD that have minimal corneal staining, and correlate with symptoms and clinical signs. EIC and IGIC may provide reliable and clinically relevant metrics of inflammation.

Figure 1

Image analysis of palpebral conjunctival in vivo confocal micrographs using ImageJ. En face in vivo confocal micrographs were analyzed using ImageJ. Immune cells were identified as hyperreflective, polymorphous structures ranging in size from 5 to 20 μm (red dots, arrowheads; A, B). EIC (A) and SIC (B) were expressed as number of cells per mm²; PGIC (C) were identified as the number of cells within a 20-μm radius around the glands; IGIC (D) were quantified as the percentage intraluminal area occupied by immune cells; IGIC⁺ (E) and IGIC⁻ (F) were assessed for lumen width (W), lumen internal length (L), glandular external length (EXT-L), and thickness of luminal hyperreflective ring presumed to be ductular epithelium (r) (arrow; r = [EXT-L − L]/2). Scale bars: 100 μm.

Figure 2

IVCM of the palpebral conjunctiva in controls and patients with MGD-associated palpebral conjunctival inflammation. Representative en face in vivo confocal micrographs of healthy controls (A, C, E, G) and patients with MGD (B, D, F, H), illustrating increased immune cells in the palpebral conjunctival epithelium (B) and stroma with periacinar fibrosis (D), periglandular cells around the acinar epithelium (F), and increased occlusion of presumed meibomian gland ductules leading to altered ductular dimensions in MGD (H). Scale bars:100 μm.

Figure 3

Bland-Altman plots assessing interobserver agreement for in vivo immune-cellular metrics of palpebral conjunctival inflammation. Each plot was generated using the interobserver difference in measurements (y-axis) against the mean of measurements for both observers (x-axis). The mean difference between interobserver measurements (bias) is represented by the middle solid line, accompanied by its 95% CI (broken lines) and 95% LoA (mean ± 1.96 SD; top and bottom solid lines). All metrics assessing immune cells, both in the palpebral conjunctival tissue (EIC, SIC, PGIC; A–C) and meibomian glands (IGIC; D), demonstrated very good interobserver agreement without proportional bias.

Figure 4

Correlation of immune-cellular metrics with symptom severity and clinical signs in MGD-associated palpebral conjunctival inflammation. r_s was used to determine the strength of correlation between in vivo confocal immune cell parameters that were significantly altered in MGD, and both clinical symptom severity (OSDI scores) and TBUT. Both palpebral conjunctival EIC (A, C) and percentage occlusion of presumed meibomian IGIC (Figs. 3B, 3D) showed direct correlation with OSDI (A, B), and inverse correlation with TBUT (C, D). P < 0.05 was considered statistically significant. N represents the number of subjects available with both measurements.

Figure 5

ROC curves and analyses illustrating the diagnostic utility of EIC and IGIC in the assessment of palpebral conjunctival inflammation. AUC and AR for both EIC and IGIC indicate that these immune-cellular metrics (EIC, IGIC) are very good to excellent diagnostic aids for palpebral conjunctival inflammation. ROC analysis: sensitivity (sens), specificity (spec), PPV, NPV, LR+, and LR−. EIC ≥ 195.8 cells/mm² and IGIC ≥ 21.1% may prove to be precise and accurate thresholds in screening for and confirming eyelid inflammation. Diagnostic accuracy grades based on AUC: excellent (AUC 0.9–1.0), very good (AUC 0.8–0.9), good (0.7–0.8), sufficient (AUC 0.6–0.7), bad (AUC 0.5–0.6), test not useful (AUC < 0.5).