Imaging assessment of conjunctival goblet cells in dry eye disease.

Dry eye disease (DED) is a widespread, multifactorial, and chronic disorder of the ocular surface with disruption of tear film homeostasis as its core trait. Conjunctival goblet cells (CGCs) are specialised secretory cells found in the conjunctival epithelium that participate in tear film formation by secreting mucin. Changes in both the structure and function of CGCs are hallmarks of DED, and imaging assessment of CGCs is important for the diagnosis, classification, and severity evaluation of DED. Existing imaging methods include conjunctival biopsy, conjunctival impression cytology and in vivo confocal microscopy, which can be used to assess the morphology, distribution, and density of the CGCs. Recently, moxifloxacin-based fluorescence microscopy has emerged as a novel technique that enables efficient, non-invasive and in vivo imaging of CGCs. This article presents a comprehensive overview of both the structure and function of CGCs and their alterations in the context of DED, as well as current methods of CGCs imaging assessment. Additionally, potential directions for the visual evaluation of CGCs are discussed.

Assessment of goblet cell size and density in relation to epithelial cell (multi)layering on conjunctival impression cytology samples.

PURPOSE: To assess goblet cell size and numbers in relation to the extent of multilayering of conjunctival impression cytology (CIC) samples as a basis for reducing variability in image selection for goblet cell density (GCD) estimates. METHODS: CIC was undertaken immediately postmortem off the superior bulbar conjunctiva of healthy young adult rabbits onto Millicell-CM Biopore filter units. After fixation with buffered glutaraldehyde and Giemsa staining, two × 200 images were selected from each sample representative of either slight multilayering or substantial multilayering, projected at × 1000, an overlay of the outlines of the goblet cells was made, and their dimensions and areas were measured. RESULTS: From measures of 4918 goblet cells, the average value (+/- SD) for the longest dimension was 17.7 ± 6.4 µm and 14.6 ± 5.3 µm for the shortest dimension. The GCD values ranged from 210 to 2069/mm2, with a mean of 1074 ± 601/mm2, but was lower for slightly multilayered images (at 537 ± 239 cells/mm ) compared with multilayered regions (at 1612 ± 601 cells/mm2; p < 0.001). The measured areas ranged from 72 to 491 µm2, with average values from any particular image ranging from 110 to 370 µm2, which were inversely correlated with the estimated GCD (Spearman's rho = - 0.722, p < 0.05). CONCLUSIONS: Larger goblet cells but in fewer numbers were predictably found across the filter surface where there were fewer layers of cells and vice versa. This difference could be considered in selection of images for counts of goblet cells from CIC specimens.

Assessment of conjunctival goblet cell density using laser scanning confocal microscopy versus impression cytology.

PURPOSE: To determine the association between conjunctival goblet cell density (GCD) assessed using in vivo laser scanning confocal microscopy and conjunctival impression cytology in a healthy population. METHODS: Ninety (90) healthy participants undertook a validated 5-item dry eye questionnaire, non-invasive tear film break-up time measurement, ocular surface fluorescein staining and phenol red thread test. These tests where undertaken to diagnose and exclude participants with dry eye. The nasal bulbar conjunctiva was imaged using laser scanning confocal microscopy (LSCM). Conjunctival impression cytology (CIC) was performed in the same region a few minutes later. Conjunctival goblet cell density was calculated as cells/mm(2). RESULTS: There was a strong positive correlation of conjunctival GCD between LSCM and CIC (ρ=0.66). Conjunctival goblet cell density was 475±41 cells/mm(2) and 466±51 cells/mm(2) measured by LSCM and CIC, respectively. CONCLUSIONS: The strong association between in vivo and in vitro cellular analysis for measuring conjunctival GCD suggests that the more invasive CIC can be replaced by the less invasive LSCM in research and clinical practice.

A systematic assessment of goblet cell sampling of the bulbar conjunctiva by impression cytology.

The purpose of this study was to assess the apparent goblet cell density (GCD) from conjunctival impression cytology (CIC) samples in relation to the number of conjunctival cells collected onto the filters. CIC specimens were collected from the superior-temporal bulbar conjunctiva of 16 pigmented rabbits onto Biopore (Millicell-CM) membranes, fixed with buffered glutaraldehyde and stained with Giemsa. Different numbers of microscope fields of view in each of the specimens were imaged by light microscopy using a 20× magnification objective lens (200× final magnification), and the goblet cells marked and counted. The GCD values/sq. mm were calculated. The same conjunctival region of 3 other rabbits was also prepared for transmission electron microscopy (TEM) by fixation, in situ, with the same buffered glutaraldehyde. Mean values for GCD estimates were found to vary from 399 to 1576 cells/sq. mm, depending on the image sampling and analysis strategy chosen, with the lowest inter-sample variance of around 10% being found if a maximum goblet cell count was taken on substantially multilayered regions of the CIC specimens. Counts of the number of goblet cells per 1000 visible conjunctival epithelial cells yielded a value of close to 90 (range 36-151), with modest inter-sample variability of around 30%. A three or ten 200× microscope field and random sampling strategy yielded mean GCD values between 542 and 670 cells/sq. mm, but with very high intra- and inter-sample variance of at least 60% and sometimes higher than 100%. TEM confirmed the multilayered organization of the conjunctiva and the deeper lying goblet cells. The general use of a goblet cell count as an objective marker for conjunctival normality or health is likely to be highly variable unless a more specific strategy is adopted. Beyond providing details of exactly the counting strategy used, it would be very useful to provide full details of the actual microscope field size used as well as information on the intra-sample variability in goblet cell counts.

Goblet cells of the normal human bulbar conjunctiva and their assessment by impression cytology sampling.

Goblet cells of the conjunctiva are the main source of mucus for the ocular surface. The objectives of this review are to consider the goblet cells as assessed by various histological, cytological and electron microscopy methods, and to assess the consistency of published reports (over more than 25 years) of goblet cell density (GCD) from impression cytology specimens from nominally healthy human subjects. Reported GCD values have been notably variable, with a range from 24 to 2226 cells/mm² for average values. Data analysis suggests that a high density of goblet cells should be expected for the healthy human conjunctiva, with a tendency toward higher values in samples taken from normally covered locations (inferior and superior bulbar conjunctiva) of the open eye (at 973 +/- 789 cells/ mm²) than in samples taken from exposed (interpalpebral) locations (at 427 +/- 376 cells/mm²). No obvious change in GCD was found with respect to age, perhaps because the variability of the data did not allow detection of any age-related decline in GCD. Analyses of published data from 33 other sources indicated a trend for GCD to be lower than normal across a spectrum of ocular surface diseases.

Sampling area selection for the assessment of goblet cell density from conjunctival impression cytology specimens.

PURPOSE: To assess the impact of using different sampling areas (fields of view) on the reliability of goblet cell density (GCD) estimates from conjunctival impression cytology (CIC) specimens from healthy individuals. METHODS: The CIC specimens were collected from the exposed nasal bulbar conjunctiva of 5 adult subjects (average age, 23 years) onto Biopore (Millicell) membranes and stained with Giemsa. A region from each of the specimens that contained abundant goblet cells was examined by light microscopy using a ×40 magnification objective lens, ×20 and ×10 lenses, the images were enlarged, and the goblet cells were marked and counted. The GCD values per square millimeter were calculated and then the impact of counting between 10 to many and 10 to few goblet cells assessed. RESULTS: The mean GCD estimates at ×400 magnification, ×200, and ×100 were 950 ± 226, 620 ± 154 and 471 ± 158 cells per square millimeter, respectively; these values were statistically different (P<0.05). The GCD estimates could change by as much as ±31.6%, ±12.2%, and ±4.2% for differences of ±10 cells counted per image. CONCLUSIONS: As a result of variability in goblet cell distribution across CIC specimens, the estimates of GCD can be expected to be different according to the sampling area used for goblet cell counts. Furthermore, the use of a small sampling area (high power field of view) is likely to result in an unacceptably large uncertainty (variability) in the GCD estimates.