Assessment of the reliability (repeatability) of corneal thickness measurements in soft contact lens wearers using a non-contact specular microscope.

PURPOSE: To assess variability across 3 measures of central corneal thickness (CCT) obtained with a non-contact specular microscope and taken over a few minutes from habitual soft contact lens wearers. METHODS: One eye from 200 healthy adults (with an average age of 21 y, half of whom had a 3.5 ± 2.1 year history of successful daily wear of soft contact lenses while the control group had nominally normal eyes) were assessed using the auto-focus Topcon 2000P instrument to obtain an image of the endothelium and CCT. RESULTS: The individual CCT values encountered in the 200 subjects ranged from 0.449 mm to 0.591 mm, with the average of 3 measures ranging from 0.459 to 0.591 mm in the control group and between 0.449 and 0.585 mm for the SCL wearers. The group mean CCT values were the same for both groups (at 0.524 mm), but the group mean SD value was marginally higher (at 0.028 mm) for the SCL group as compared to controls (SD = 0.026 mm). The normalized intra-subject variability (as the group-mean coefficient of variation, COV value) was 0.843 ± 0.401 for the control group and higher at 1.08 ± 0.546 for the SCL group (p < 0.001). CONCLUSIONS: Repeat measures of central corneal thickness, using a non-contact specular microscope, is very similar to those taken on age-matched non-contact lens wearers. These results may not equally apply to similar pachymetry measures in patients wearing RGP lenses or for older patients wearing soft contact lenses.

Importance of standardizing the number of cells measured for coefficient of variation (COV) estimates of corneal endothelial cell area values as relevant to contact lens wear.

PURPOSE: To assess the impact of using different numbers of cells in calculations of the coefficient of variation (COV) value for normal and polymegethous endothelia METHODS: Four sets of 20 non-contact specular microscope images obtained from Caucasian individuals were assessed, and categorized according to the extent of polymegethism, i.e. grade 0 (none), grade 1 (mild), grade 2 (moderate) and grade 3 (substantial). Cell areas were measured manually and then values for between 2 and 100 cells were progressively added and averaged to generate COV estimates. These were then assessed in terms of their relative values (as percentages +/- SD) in relation to the value obtained on over 100 cells. RESULTS: For the 4 sets of endothelia with group-mean COV values of 25.8, 33.1, 45.1 and 56.8%, the reliability of the COV estimates realized asymptotic values of close to ±1.0, ±2.7, ±3.6 and ±5.0% with 90 cells, but with greater uncertainty with few numbers of cells, e.g. only to within ±3.4, ±5.3, ±6.1 and ±7.9% with 75 cells. CONCLUSIONS: COV estimates for the corneal endothelium are dependent on the number of cells used in the calculations. It is recommended that every effort should be made to not only assess 75-100 cells per endothelial image, but that this number should be the same or very similar for all endothelial images in a particular data set so that the uncertainly (or estimated proportional error) in the estimates is balanced.

Spontaneous eye blink activity during slitlamp-based assessments.

CLINICAL RELEVANCE: Slitlamp-type assessments of eye blink activity with head and chin support need to consider time-related differences that can occur. BACKGROUND: Previous studies have not assessed the predictability of changes in spontaneous eye blink rate occurring during slitlamp observations. METHODS: Video recordings were made of eye blink activity of 85 young adults who were either emmetropic or spectacle wearers for refractive errors between -8.25 D and +8.25 D. After an initial adjustment period of one to two minutes positioned at the slitlamp (including the time after removing spectacles), participants had a five minutes recording made in silence while seated with forehead and chin support and directing their gaze to a high-contrast target on a distant whiteboard under ambient luminance of 35 cd per square metre. RESULTS: The mean spontaneous eye blink rate values over five minutes were 13.4 ± 3.1 blinks/minute (± SD), ranging from 7.4 to 20.8 blinks/minute. Overall, incomplete eye blink events were noted 39 times in the total of 5,704 recorded (that is, 0.68 per cent of all eye blinks). There was a progressive decline in averaged spontaneous eye blink rate values (r = 0.897, p < 0.05), with 70.6 per cent of the participants exhibiting a higher spontaneous eye blink rate value in the first minute compared to the fifth minute. The inter-participant variability in spontaneous eye blink rate also progressively declined over time, but there was no detectable difference in either averaged values or the variability in spontaneous eye blink rate in relation to refractive error. CONCLUSIONS: In slitlamp-based assessments of eye blink activity, a small progressive time-related reduction appears likely but is not obviously related to visual blur in ametropic individuals.

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.

Short term effects of continuous lighting on the cornea of cage-reared laboratory rabbits.

This study was to assess the impact on the cornea and eye blink activity of adapting rabbits to continuous lighting (CL) compared to a 14:10 light:dark cycle. Female New Zealand White rabbits (2 to 2.5 kg) were maintained under a light: dark (L:D) cycle or switched to continuous fluorescent lighting (CL) for an average of 17 +/- 2 days. Animal behaviour in their cages was manually recorded using an event marker and in vivo slitlamp biomicroscopy at 40× undertaken in mid-afternoon. Animals were then euthanized and the corneas prepared for scanning electron microscopy (SEM). From images taken at 500× from the central region of the corneas, the number of exfoliating (desquamating) cells and the relative number of different cells with light, medium or dark reflexes were assessed for the corneal epithelial surface, while the number of cells/unit area were assessed for both corneal epithelium and endothelium. Exposure to continuous lighting was associated with higher number of eye blink events (15.7 vs 8.2/15 min) and mild corneal surface alterations evident by biomicroscopy with higher numbers of intra-epithelial 'granules' (32 +/- 14 vs. 4 +/- 3/sq. mm). SEM revealed low numbers of exfoliating cells on the corneal epithelial surface in all CL-adapted animals, but not in L:D controls. Trends were observed for there to be slightly higher numbers of epithelial cells/unit area, higher numbers of small light reflex cells and lower numbers of larger dark reflex cells in CL animals. The corneal endothelium showed no obvious adverse effects in CL-adapted animals but the percentage of 'hexagonal' cells was slightly higher compared to L:D controls. The results indicate that even a short period of exposure of laboratory-raised rabbits to constant lighting can be associated with mild adverse effects on the corneal epithelial surface.

On the in vivo assessment of goblet cells of the human bulbar conjunctiva by confocal microscopy - A review.

BACKGROUND: In vivo confocal microscopy (IVCM) has been used for over 10 years to assess the goblet cell density (GCD) within the human conjunctiva, but the reported values have been variable with no obvious indications as to why. METHODS: From publications between 2008 and 2019, representative GCD values were extracted, as well as on the image sampling strategy used. RESULTS: Average GCD values for any particular group of individuals ranged from 7 to 979 goblet cells / sq. mm, and with one notable outlier removed, an overall group-mean value for GCD (+/- SD) from single site locations was 207 +/- 143 goblet cells / sq. mm from 15 data sets for those usually designated as control subjects, with a value of 190 +/- 161 goblet cells / sq. mm calculated from 20 single site data sets from other (patient) groups. An overall analysis indicated that the reported average values for GCD from different groups of individuals increased according to the number of images assessed / individual (Spearman rho = 0.304), on the number of individuals evaluated to generate an averaged value for each group (rho = 0.367), and the total number of images assessed (rho = 0.346, multivariate analysis partial r = greater or = to 0.522). CONCLUSIONS: In the use of confocal microscopy to assess the number of goblet cells in the human bulbar conjunctiva, the substantial differences reported appear to be linked to the protocols used for image selection, and some type of standardization needs to be developed.

Multiple count sampling of goblet cells in microscope high-power fields using conjunctival impression cytology.

BACKGROUND: Published studies indicate that assessments of goblet cell density using conjunctival impression cytology has provided very variable results, but the reasons for this are unclear. Systematic analyses of the sources of variability are required. METHODS: From 20 healthy young adults, conjunctival impression cytology specimens were obtained using a supported filter unit applied to the superior bulbar conjunctiva. The filters were stained with Giemsa and 10 non-overlapping, randomly selected high-power field images were obtained from each specimen and the numbers of goblet cells per high-power field counted. RESULTS: From all 200 high-power fields assessed, the numbers of goblet cells ranged from zero to 74, with an overall mean value of 11.6 ± 14.8 per high-power field. From each successive set of 10 microscope field images from all individuals, the average number of goblet cells ranged from 23.2 in the first high-power field that obviously included numerous goblet cells down to 6.2 per high-power field. As the outcome from multiple counts/individual was systematically increased, these averages progressively decreased from 23.2 to 11.6 per high-power field, and while the standard deviation values also progressively declined (from 7.9 to 5.5 per high-power field), the relative variability (as the co-efficient of variation) did not, and increased to averaged values of over 100 per cent. CONCLUSIONS: These analyses indicate that there is a benefit of making multiple counts of goblet cells from different high-power fields, but that there is no obvious benefit of using more than five to seven high-power fields for any particular specimen.

Effect of distance vision and refractive error on the spontaneous eye blink activity in human subjects in primary eye gaze / Efecto de la visión a distancia y el error de refracción sobre la actividad de parpadeo espontáneo del ojo en sujetos humanos en la mirada primaria del ojo

Purpose: To evaluate whether visual target character and visibility affects spontaneous eye blink rate (SEBR) in primary eye gaze and silence. Methods: Video recordings were made of young healthy adults who were either emmetropic (n = 32) or who wore spectacles for refractive error (range -4.75 D and +4.50 D (n = 31). Emmetropes had 5 min recordings made whilst seated and looking towards a distant whiteboard. For spectacle wearers, recordings were made whilst looking towards the whiteboard with a 35 mm sized cross, and repeated after spectacle removal. The average number of eye blinks over 5 min was assessed, and its intra-subject variability as the coefficient of variation (COV). Results: Over 5min without a distance target, an average SEBR of 10.4 blinks/min was observed in emmetropes with a of COV = 38.1%, and a significant increase in SEBR over the 5th minute to 13.6 blinks/min. Hyperopes being asked to look towards a distant target showed the essentially same blinking rate of 11.1/min with or without spectacle wear with the intra-subject variability (COV) being 21.3%. Myopic subjects showed a slightly higher SEBR if looking towards a target without their spectacles (12.4 vs. 11.0blinks/min), with the COV being 18.8%. Conclusions: The studies indicate that some form of visual target could be useful to promote constancy of spontaneous eye blink activity over time, but that a distance visual target (when provided) does not need to be seen clearly

Objetivo: Evaluar si el tipo y visibilidad del objetivo visual afecta a la tasa de parpadeo espontáneo del ojo (SEBR) en posición primaria de la mirada y en silencio. Métodos: Se realizaron grabaciones de vídeo de jóvenes adultos sanos, emétropes (n=32), o que utilizaban gafas para el error refractivo (rango: -4,75 D y +4,5 D (n=31). Se realizaron grabaciones de 5min a los emétropes mientras permanecían sentados y miraban a una pizarra de lejos. Para los sujetos que utilizaban gafas, se realizaron grabaciones mientras miraban a la pizarra con una cruz de 35mm de tamaño, repitiéndose dichas grabaciones tras retirarles las gafas. Se evaluó el número medio de parpadeos durante 5 minutos y su variabilidad intra-sujeto como coeficiente de variación (COV). Resultados: Durante un periodo de 5 minutos sin objetivo visual, se observó una SEBR media de 10,4 parpadeos/min en emétropes con un COV=38,1%, así como un incremento significativo de SEBR a lo largo del quinto minuto a 13,6 parpadeos/min. En los hipermétropes a quienes se solicitó mirar a un objetivo de lejos se observó prácticamente la misma tasa de parpadeo de 11,1/min con o sin utilización de gafas, con una variabilidad intra-sujeto (COV) del 21,3%. Los sujetos miopes reflejaron una SEBR ligeramente superior cuando miraban al objetivo sin utilizar gafas (12,4 vs. 11 parpadeos/min), con un COV del 18,8%. Conclusiones: Los estudios indican que cierta forma de objetivo visual podría resultar útil para promover la constancia de la actividad de parpadeo espontáneo del ojo en el tiempo, pero que un objetivo visual lejano (de existir) no precisa ser visto con claridad

Effect of distance vision and refractive error on the spontaneous eye blink activity in human subjects in primary eye gaze.

PURPOSE: To evaluate whether visual target character and visibility affects spontaneous eye blink rate (SEBR) in primary eye gaze and silence. METHODS: Video recordings were made of young healthy adults who were either emmetropic (n=32) or who wore spectacles for refractive error (range -4.75D and +4.50D (n=31). Emmetropes had 5min recordings made whilst seated and looking towards a distant whiteboard. For spectacle wearers, recordings were made whilst looking towards the whiteboard with a 35mm sized cross, and repeated after spectacle removal. The average number of eye blinks over 5min was assessed, and its intra-subject variability as the coefficient of variation (COV). RESULTS: Over 5min without a distance target, an average SEBR of 10.4blinks/min was observed in emmetropes with a of COV=38.1%, and a significant increase in SEBR over the 5th minute to 13.6blinks/min. Hyperopes being asked to look towards a distant target showed the essentially same blinking rate of 11.1/min with or without spectacle wear with the intra-subject variability (COV) being 21.3%. Myopic subjects showed a slightly higher SEBR if looking towards a target without their spectacles (12.4 vs. 11.0blinks/min), with the COV being 18.8%. CONCLUSIONS: The studies indicate that some form of visual target could be useful to promote constancy of spontaneous eye blink activity over time, but that a distance visual target (when provided) does not need to be seen clearly.

Methods of Assessment of the Corneas of the Eyes Laboratory Rabbits Exposed to Solar Ultraviolet-B Radiation.

The goal of this review was to identify and discuss the specialized methods that have been used to assess the corneas of the eyes of living rabbits exposed to the damaging effects of ultraviolet-B (UV-B). From publications reviewed between 1916 and 2018, both albino and pigmented rabbits were used, usually being young adults weighing between 2 and 2.5 kg, and with many recent studies carried out under sedation. Older assessments were generally based on the use of in vivo slitlamp examinations, sometimes with the use of fluorescein or rose bengal to identify damaged cells, supported by light microscopy (histology) of excised corneas. In later years (after 1960), these structural studies have included in vivo and ex vivo specular microscopy, transmission and scanning electron microscopy as well as impression cytology. Early studies included measurements of the thickness of excised corneal specimens but in vivo pachymetry methods were widely used from the 1980s. Other assessments have included measurement of light transmission spectra of excised corneas, measures of metabolites in isolated corneas or staining corneal specimens for enzyme activities. While a wide range of specialized methods have been used, most of them have provided only descriptions of the effects of UV-B.

On the reliability (repeatability) of single pachymetry measures taken with specular microscope-based assessments of the human corneal endothelium.

PURPOSE: To assess agreement between 3 measures of central corneal thickness (CCT) taken over a few minutes from nominally normal eyes with a non-contact specular microscope. METHODS: 100 eyes from 100 healthy adults (with an average age of 22 y) were assessed using the Topcon 3000 P instrument to obtain a high quality image of the endothelium and pachymetry. RESULTS: The group mean CCT values from the 1st, 2nd and 3rd measures were 0.519, 0.520 and 0.520 mm, but the sets of values could differ by between - 0.020 mm and + 0.029 mm, i.e. between - 4.0 and + 5.6% of the average values. Paired comparisons (e.g. 2nd vs. 1st set) indicated limits of agreement (LoA) to be between - 0.020 and + 0.020 mm of the averaged value. Across the 3 measures, the averaged SD was 0.005 mm to give an estimate of the intra-subject variability (as the coefficient of variation, COV) of 0.91% (range 0 to 2.3%). The variability in the pachymetry measures was not predictably related to the averaged values of CCT (r = 0.022) or self-reported refractive error of the subjects (r = 0.012). Some repeat pachymetry assessments obviously included the same region of the endothelium. CONCLUSIONS: Single pachymetry measures with a non-contact specular microscope are only likely to be able to generate CCT estimates within +/- 4% of the expected average values. This repeatability is comparable to single image estimates of endothelial cell density and therefore acceptable in most cases.

Corneal structure, transparency, thickness and optical density (densitometry), especially as relevant to contact lens wear-a review.

Clinical instruments using Scheimpflug image-based methods to obtain optical sectional images of the cornea have been introduced in recent years along with proposals that it should be possible to routinely and reliably measure the optical density (referred to as the densitometry) of the human cornea in situ. Such a concept is reviewed from the perspective of what might be considered as the basic principles underlying the understanding of corneal transparency (from the 1950's) and the progressive changes in these ideas from subjective slitlamp-based clinical observations from the late 1960's, especially in contact lens wearers. Much more has been learned about the overall macrostructure (including corneal thickness) and the ultrastructure of the cornea from contemporary studies in the 1990's, and these aspects of the cornea will be reviewed alongside consideration of the methods of assessing the optical characteristics of the cornea in the living eye. From these perspectives, in this review systematic consideration will be given to what objective (quantitative) output one of these Scheimpflug-based systems provides and how this information might be actually related to corneal transparency characteristics that might be observed clinically, particularly after long-term contact lens wear.

Tear Film Stability and Tear Break Up Time (TBUT) in Laboratory Rabbits-A Systematic Review.

Several older studies on rabbits indicated that the pre-corneal tear film was unusually stable and this perspective was revisited recently. However, the methods used for these studies were very different from those generally used in human studies. A literature search was undertaken for the time period of 1965 through 2017, mainly using PubMed, to identify studies where values for the tear break up time (TBUT) were reported for the eyes of nominally normal (healthy) laboratory rabbits regardless of breed or age and where the methods were more similar to those routinely used in human clinical studies. For 20 reports identified where sodium fluorescein was used, the average TBUT values in any particular study ranged from 1.9 to 51 s, with a group mean of 21.8 ± 11.9 s (SD), with the inter-study variability in TBUT (as the coefficient of variation) being 19.4%. For four studies not using fluorescein, the mean break up time reported was 32.7 ± 16.2 s, while a separate study (also not using fluorescein) reported an average break up time of 1788 s. Most reports of the pre-corneal tear film stability in laboratory rabbits, especially as reported over the last 10 years, indicate break up times of less than 60 s have been observed, although has been little consistency in the methods used. Overall, this outcome is not consistent with a perspective that the rabbit (as routinely used in experimental studies) has an extraordinarily stable tear film.

Influence of mouth and jaw movements on dynamics of spontaneous eye blink activity assessed during slitlamp biomicroscopy.

BACKGROUND: To evaluate oculo-mandibular interactions during evaluation of spontaneous eye blink rate (SEBR) of normal young adult human subjects while seated at a slitlamp. METHODS: Repeat video recordings of five minutes duration were made on 76 young adult emmetropic subjects aged 18-25 years. The subjects were instructed to direct their gaze horizontally toward a distant target with the entire cornea of the left eye illuminated with a broad beam cobalt blue light. Repeat recordings were made: (a) on the following day in silence in group one; (b) immediately in silence for group two; and (c) immediately either while holding their mouth open slightly or while responding to casual conversation for groups three and four. RESULTS: For group one the averaged SEBR values for the first and second recordings were 13.5 and 14.6 blinks/minute, and similar results were obtained for group two if the subjects were relaxed (for example 13.9 and 12.0 blinks/minute). A slight, but statistically significant, time-related decline in SEBR was usually noted. For non-relaxed (restless) subjects exhibiting spontaneous mouth and jaw movements while being videographed, the averaged SEBR values were around 27 blinks/minute. For groups three and four, analyses of those individuals who managed to maintain a mouth open posture for the video recordings, the averaged SEBR was 7.2 blinks/minute, while those engaged in casual conversation had an averaged SEBR of 20.3 blinks/minute. CONCLUSIONS: Mouth and jaw movements and a non-relaxed state can substantially affect spontaneous eye blinking.

Goblet cell density estimate differences in impression cytology samples varies with different magnification of images.

PURPOSE: To assess the impact of using different microscope magnifications for the goblet cell density (GCD) estimates from conjunctival impression cytology (CIC) samples from healthy individuals METHODS: In a prospective study, CIC specimens were collected from the superior bulbar conjunctiva (12 o'clock, 5mm from limbus) of 20 adult subjects (average age 22 years) onto Millicell-CM membranes and Giemsa stained. A region from each CIC filter containing reasonably high numbers of goblet cells was imaged by light microscopy at a final magnification of 400X and then the same region assessed at 200X and then 100X. The images were enlarged, the goblet cells marked and counted and GCD values/sq mm calculated. RESULTS: The mean GCD estimates at 400X magnification, 200X and 100X were 644±180, 405±72 and 365±81 cells/sq mm respectively, and these values were statistically different (p<0.001). CONCLUSIONS: As a result of non-uniform distribution, a strategy to select a 400X high power microscope field (HPF) that appears to include a moderate number of goblet cells will have a probability (by at least 20:1) that the GCD estimates will likely be higher compared to those at 200X or 100X, and the probability for higher GCD values is at least 15:1 comparing assessments made at 200X to 100X. Investigators should use only one magnification, with that of a medium power field (200X final magnification) likely being the most useful.

Further Analysis of the Predictability of Corneal Endothelial Cell Density Estimates When Polymegethism Is Present.

PURPOSE: To assess variability in endothelial cell density (ECD) estimates when polymegethism (variance in cell areas) is present. METHODS: Using noncontact specular microscope images of the corneal endothelium, 4 sets of 20 cases were selected, which included 200 cells and had coefficient of variation values of less than 30% (group 1), 31%-40% (group 2), 41%-50% (group 3), and over 50% (group 4). A stepwise analysis was undertaken, 20 cells at a time, of the ECD estimates when using different numbers of cells for the calculations. RESULTS: The net differences in ECD estimates when comparing sets of 20 cells with 200 cells were 5.0% ± 3.9%, 8.1% ± 7.3%, 11.3% ± 9.4%, and 14.5% ± 12.4% for groups 1 to 4, respectively. For measures on 100 cells per image, the predicted variances in ECD values were 5.6%, 8.8%, 11.1%, and 13.7% for the 4 groups. CONCLUSIONS: Higher values of corneal endothelial polymegethism result in predictable increases in the variability (uncertainty) in ECD estimates, thus reducing the "accuracy" of ECD values. There is no obvious utility in assessing more than 100 cells in such endothelia.

A Grid-Based Nucleus Counting Method for Estimates of the Density of Superficial Conjunctival Cells from Impression Cytology Samples Taken from Normal Healthy Human Eyes.

PURPOSE: To develop and validate an easily procured objective estimate of the cell density of the superficial epithelial cells of the normal bulbar conjunctiva using conjunctival impression cytology (CIC). METHODS: From 20 healthy non-contact lens-wearing young adults, CIC was undertaken off the nasal aspect of the exposed bulbar conjunctiva, the filters stained with Giemsa, and images taken at 200× that only included an all-but-contiguous monolayer of epithelial cells. These images were projected at 1000× magnification and an overlay drawn of the location of the stained nuclei, from which two types of analysis were undertaken using either 100 micron circular or square regions of interest (ROI). From the former, the distances between all nuclei were measured and the number of nuclei/circle calculated to establish uniformity of cell nuclei distribution as a necessary prerequisite to using a grid-based counting method. From the latter, the number of nuclei in each square grid was manually counted and the grid-by-grid variability assessed. RESULTS: The average inter-nucleus distance was 16.1+/3.5 microns, with predictable positioning to successive nuclei in the circular ROI to give an estimated numerical density of 1944 ± 237/sq mm. Counting of nuclei in grid squares yielded numbers between 12 and 42 to yield an estimated cell density of 2390 +/ ̶330 /sq mm, with repeat counts off grid square ROIs being predictably within +/-15%. CONCLUSIONS: These studies indicate that it should be fairly easy to use carefully selected CIC samples to make estimates of the density of the superficial conjunctival cells. Such a method should be useful and offer advantage over the subjective assignment of morphological grades to CIC samples which have been shown to be highly variable.

An observational cross-sectional study on the corneal endothelium of medium-term rigid gas permeable contact lens wearers.

PURPOSE: To assess if polymegethism and pleomorphism were evident in corneal endothelium after medium-term rigid gas permeable (RGP) contact lens wear. METHODS: In a cross-sectional observational study over 12 years, single images of the central region of the corneal endothelium of one eye of 46 subjects were taken with a non-contact specular microscope, along with a measure of central corneal thickness (CCT). The images were printed onto A3-sized paper and 100 cells/image measured by planimetry. RESULTS: Subjects aged between 20 and 32 years, with an average cumulative RGP wear of 6.0+/- 1.6 years (range 3-9 years) were assessed; 26 of the subjects were Caucasian and 20 were Asian. The mean CCT was 0.515+/- 0.027mm. The group cell area value was 401+/- 42 sq micron to give an estimated endothelial cell density (ECD) of 2520+/- 273 cells/sq mm. As compared to a historical database, most endothelia (37/46) showed some changes with the mean coefficient of variation on cell area (COV) being 36.7+/- 8.0% and the percentage of 6-sided (HEX) being 51.8+/- 8.8%. There were modest correlations between years of RGP wear and both COV (p=0.009, r spearman=0.424) and HEX (p=0.025, r spearman=-0.291), but not for ECD or CCT. CONCLUSIONS: Corneal endothelial polymegethism appears to be a commonplace consequence of RGP lens wear with the magnitude of the change being related to the cumulative duration of the lens wear.

Relationship between Corneal Thickness and Radius to Body Height.

PURPOSE: To investigate the possible association between body stature (height) and corneal thickness and radius in younger-adult Caucasians, especially within the context of previously published literature. METHODS: Body height and weight were measured in 109 healthy subjects, with an average age of 24 ± 6 years (mean ± SD). Subjects underwent an ophthalmic assessment including anterior segment imaging by Scheimpflug topography and specular microscopy. Central and peripheral corneal thickness and corneal radius were analyzed. The relationship between body stature and corneal parameters was assessed using simple and multiple regression analysis. Effect size was determined by generating regression and correlation coefficients. RESULTS: Body height ranged from 1.54 to 1.86 m (mean ± SD 1.67 ± 0.08 m), central corneal thickness from 465 to 629 µm (554 ± 33 µm), whereas corneal radius measured between 7.16 and 8.49 mm (7.75 ± 0.24 mm). Body height was weakly associated with central corneal thickness and peripheral corneal thickness (r ≥ -0.180), and moderately with corneal radius (r = 0.351). Based on the regression equations, central corneal thickness decreases by 8 µm, whereas corneal radius increases by 0.11 mm for each 0.1-m difference in body height. No significant correlations were found for similar assessments using body weight or body mass index. CONCLUSIONS: Differences in corneal radius and corneal thickness can be linked to body stature. However, effect sizes were consistently small and no more than 13% of the variability in corneal curvature could be explained by variations in body stature.

Cell size and nucleo-cytoplasmic ratios of meibocytes in the anterior acini of the upper eyelid Meibomian glands in rabbits.

OBJECTIVE: To assess the morphological details of the acini of the normal meibomian gland. ANIMALS STUDIED: Six young adult pigmented rabbits. METHODS: The upper eyelid was prepared in extended configuration by glutaraldehyde fixation. Tissue block sections approximately 0.5-1 mm from the eyelid margin were assessed by light microscopy in sagittal sections and transmission electron microscopy (TEM) in coronal sections. TEM images at between 1000× and 2000× magnification were enlarged onto A3-sized prints and cell size and nuclei measured by planimetry. RESULTS: Light microscopy sagittal sections revealed clusters of variable sized acini, sometimes appearing to be slightly overlapping and without any obvious spatial organization of the internal cells (meibocytes). The estimated areas of the acini were close to 6500 sq micron. Coronal sections, as examined by TEM, allowed for visualization of small to large acini (average diameter 82 ± 17 microns, with an estimated area of 5500 sq. microns) containing variable numbers of immature (partly differentiated) or mature (fully differentiated) meibocytes with a distinct spatial organization. The average area of the meibocytes was 158 ± 81 square microns, and they usually appeared to have a single nucleus (with an average sectional area of 29 ± 12 square microns). Within individual acini, peripherally located immature meibocytes tended to be smaller and have higher nucleo-cytoplasmic area ratios, while more centrally mature located meibocytes tended to be slightly larger and had lower or much lower nucleocytoplasmic ratios. CONCLUSIONS: Comparative studies on meibomian glands can be undertaken with objective assessments to assess for normality or abnormality.