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.

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.

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.

Peripheral nasal-temporal corneal asymmetry in relation to corneal thickness: a Scheimpflug imaging study.

PURPOSE: To investigate the asymmetry of the peripheral cornea up to 5 mm nasally and temporally from the centre and to assess correlations with regional peripheral corneal thickness. METHODS: Central and peripheral corneal thickness was measured by Scheimpflug imaging (Pentacam) in 113 eyes of 113 healthy, pre-presbyopic Caucasian subjects. Absolute and relative corneal thickness were analysed in 1 mm steps up to 5 mm to the nasal and temporal sides with the corneal apex as the central reference point. Nasal-temporal asymmetry was calculated as the thickness ratio between corresponding off-centre thickness measurements. RESULTS: The mean (±SD) central corneal thickness was 552 ± 36 µm. CT increased by 22% at 4 mm temporally to 672 ± 44 µm, and 32% at 4 mm nasally to 731 ± 45 µm. The nasal-temporal asymmetry became greater with increasing distance from the corneal centre, with a mean difference of 59 ± 22 µm at 4 mm from the apex. The nasal-temporal thickness ratio, based on this difference, was significantly related to the relative temporal (r = -0.41, p < 0.001, simple linear regression) and nasal corneal thickness (r = 0.61, p < 0.001). CONCLUSIONS: A substantial and progressively increasing nasal-temporal asymmetry in corneal thickness has been confirmed by Scheimpflug imaging, which is related to the magnitude of corneal thickness at peripheral locations. Pachymetry output data and models, including volume calculations, that assume symmetry to the corneal thickness profile may not provide optimum metrics for planning and predicting the outcome of corneal refractive surgery procedures.

Spontaneous eyeblink activity under different conditions of gaze (eye position) and visual glare.

PURPOSE: To further evaluate the spontaneous eyeblink rate (SEBR) of healthy adult human subjects according to direction of gaze, especially in the presence of bright light reflective glare. METHODS: On 32 subjects aged between 18 and 24 years, separate video recordings of 5 min duration were made with different conditions of gaze (horizontal, slightly upwards or slightly downwards) under normal lighting or a distant lighting glare source. RESULTS: The SEBR in primary eye gaze under normal lighting was 11.7 +/- 0.9 eyeblinks/min with a coefficient of variation (COV) of 20.5 %. A non-significant decrease in SEBR was noted with downward gaze, but a slight significant increase with upward gaze in both SEBR (to 13.0 +/- 1.1 eyeblinks/min) and COV (average 26.1 %). In the presence of glare, SEBR in primary eye gaze increased significantly (p < 0.001) to 14.4 +/- 1.3 eyeblinks/min, with an obvious time-related progressive increase (p < 0.001). On upward gaze in the presence of a glare stimulus, SEBR progressively increased even further (average 15.0 +/- 2.4 eyeblinks/min; p < 0.001), as did the COV (to 29.2 %). CONCLUSIONS: The results indicate that spontaneous eyeblink activity in silence can be affected by the presence of a glare light source, especially if the subjects are looking slightly upwards. This scenario should be avoided, if at all possible, in assessments of spontaneous eyeblink activity.

Effects of background lighting and retinal illuminance on spontaneous eyeblink activity of human subjects in primary eye gaze.

PURPOSE: To further evaluate possible effects of lighting conditions on the spontaneous eyeblink rate (SEBR) of normal young adult human subjects. METHODS: A baseline 5 min video recording was made followed by a second recording under test conditions in 7 different groups of 10 subjects. These test conditions were a simple repeat recording under reference conditions (subjects maintaining silence, with gaze directed towards a target on a 2 m distant whiteboard with the luminance of the reflected light of 35 cd/m, recording under lower (5 cd/m), higher (75 cd/m) and floodlit (150 cd/m) levels, after pupil dilation with phenylephrine 2.5 %, after sudden increase with floodlights (to 200 cd/m), as compared subjects engaged in conversation. RESULTS: The overall SEBR values ranged from 8.4 to 15.8 eyeblinks per minute (mean 12.3 ± 2.8 eyeblinks per minute). These did not significantly change with repeated assessments, no significant changes were seen comparing lower or higher illumination levels or after pupil dilation, but an increase to 20.0 ± 4.6 eyeblinks per minute was seen with a sudden increase in illumination. Conversation increased SEBR to 22.2 ± 6.4 eyeblinks per minute. CONCLUSIONS: Spontaneous eyeblink activity in silence may be slightly affected by background lighting conditions, but such effects are notably less than SEBR changes that can occur when there is a sudden increase in lighting levels (as a glare stimulus) or when assessments are made during conversation.

Assessment of the reliability of endothelial cell-density estimates in the presence of pseudoguttata.

PURPOSE: The purpose of this work is to assess the reliability of endothelial cell-density (ECD) estimates in corneas with different severity pseudoguttata. METHODS: Specular microscopy was undertaken on grade 1, 2, or 3 pseudoguttata patients and age-matched controls aged 52-83 years. On high magnification prints of central cornea, areas of complete cells (all sides visible) and partial 'cells' (one or more sides obscured) were measured manually. Sets of 45 complete cells were selected, as well as 75 cells that were a mixture of complete and partial cells on guttate endothelia. ECD was calculated by a progressive averaging technique. RESULTS: Each group comprised 12 patients with similar range of ECD values (1,230-4,587 cells/mm(2)). Based on 40 complete cells, ECD could be estimated to within ±3.1% for grade 3 pseudoguttata versus ±2.0% for controls. If a mixture of complete and partial cells were measured, ECD could be estimated to within ±2.8% for grade 3 pseudoguttata images (n = 70 cells) and ±1.1% for controls. The estimated variability increases to substantial levels of ±20% if only ten cells were measured. No statistical differences in ECD were noted between guttate and normal endothelia if only complete cells were measured, but could be different if partial 'cells' were included. CONCLUSIONS: Providing adequate numbers of complete cells are measured and in the absence of obvious polymegathism, ECD estimates can be made to within around ±3% in the presence of typical but significant pseudoguttata.

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.

Observations on the ultrastructure of equatorial scleral collagen fibrils in sheep eyes.

OBJECTIVE: To investigate collagen fibrils of the equatorial sclera in relation to the age-related changes in eye size in sheep. ANIMALS STUDIED: Lambs and outbred ewes. PROCEDURES: Sheep eyes (three lamb and three from adult outbred ewes), presumed disease-free, were processed for transmission electron microscopy (TEM) immediately postmortem. Tissue blocks from the equatorial region were sectioned across fibril bundles orientated along the equator. Micrographs including at least 500 fibrils were projected at 22,000× magnification for measures of fibril diameters (FDs). RESULTS: Lamb eyes were smaller than those of adult ewes but equatorial scleral thickness was only marginally less at 0.232±0.013 vs. 0.254±0.012 mm (P value not significant). Scleral tissue was composed of compacted bundles of collagen fibers that tended to be rounder in outer compared to being flatter in inner regions. In typical (normal) appearing regions, FDs were distinctly larger (68-410 nm) in outer sclera compared to inner sclera (63-281 nm). Outer sclera FDs were bimodal averaging 192±58 nm, compared to unimodal distributions at inner locations averaging 156±48 nm (P<0.001). Some atypical regions, especially at outer-mid sclera locations, were also noted where the FD distribution was bimodal but also included numerous microfibrils (<50 nm diameter), with similar appearances being found for both lamb and adult ewe eyes. CONCLUSIONS: The equatorial sclera is a mixture of rounder versus flatter collagen fiber bundles, the former being more likely to be made up of a mixture of both smaller and larger fibrils, as compared to slightly smaller fibrils.

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.

Suppressive effects of the obese tumor microenvironment on CD8 T cell infiltration and effector function.

Obesity is one of the leading preventable causes of cancer; however, little is known about the effects of obesity on anti-tumor immunity. Here, we investigated the effects of obesity on CD8 T cells in mouse models and patients with endometrial cancer. Our findings revealed that CD8 T cell infiltration is suppressed in obesity, which was associated with a decrease in chemokine production. Tumor-resident CD8 T cells were also functionally suppressed in obese mice, which was associated with a suppression of amino acid metabolism. Similarly, we found that a high BMI negatively correlated with CD8 infiltration in human endometrial cancer and that weight loss was associated with a complete pathological response in six of nine patients. Moreover, immunotherapy using anti-PD-1 led to tumor rejection in lean and obese mice and partially restored CD8 metabolism and anti-tumor immunity. These findings highlight the suppressive effects of obesity on CD8 T cell anti-tumor immunity, which can partially be reversed by weight loss and/or immunotherapy.

Contact lens wear and the development of squamous metaplasia of the surface cells of the conjunctiva.

PURPOSE: To review the reported effects of contact lens wear on the surface epithelial cells of the human conjunctiva as assessed by conjunctival impression cytology (CIC). METHODS: A literature search was undertaken to identify reports on the conjunctival health after contact lens wear, principally as assessed using CIC. RESULTS: Of 26 reports identified, 22 examined the bulbar conjunctiva, and 2 examined the tarsal conjunctiva. Just 16 reports provided data from which mean squamous metaplasia grades could be calculated, with the overall grade being just 0.7 on a 0 to 3 scale. Only 13 of these studies provided unambiguous data on the duration of contact lens wear, and only an apparent trend was evident in that grades of squamous metaplasia increased over early years of lens wear. Such a trend was not statistically significant either up to 6 years of average lens wear (P>0.05) or over all studies (P>0.5). The estimated variability in squamous metaplasia was substantially greater when low grades were reported, an observation that either reflects the heterogeneity in the cell response or highlights the difficulty in assigning low grades to cell samples. CONCLUSIONS: Based on subjective grading, CIC studies reveal no clearly definable relationship between the duration of contact lens wear and the extent of development of conjunctival squamous metaplasia. Logically, therefore, objective methods to assess squamous metaplasia are needed. Various options for quantitative CIC are discussed, including the use of in vivo confocal microscopy.

On the use of NIH image J for objective assessment of conjunctival cell and nucleus dimensions of impression cytology samples.

BACKGROUND: To assess the use of a public domain software Image J (NIH Image, Bethesda, MD) to make dimensional measures of human bulbar conjunctival cells. METHODS: Impression cytology samples were obtained from the nasal bulbar conjunctiva, and color images were taken at 200× magnification and projected and an overlay prepared or the image uploaded into Image J. The final image magnification for the overlays was approximately 2× that on the computer screen. For either overlays or screen images, linear measures were made from 30 or 25 cells of the cell longest dimension (LONG) and of the longest dimension of the nucleus (NUCLONG). The predicted variability in measures, from the calculated average values for any particular image, was systematically assessed, and the overall average results were compared. RESULTS: Image J measures were within ±2% agreement with overlays, with LONG and NUCLONG measures increasing with the grade of squamous metaplasia. The net difference in LONG measures was within -2.65 to + 2.93 µm, and NUCLONG measures were within -0.87 and +1.39 µm. There was a slight tendency for NUCLONG measures to be systematically overestimated on smaller nuclei when using Image J on the color images. CONCLUSIONS: The manual use of Image J on on-screen images can provide reasonably accurate objective measures of bulbar conjunctival cells, as compared with a higher magnification manual overlay technique. This should be suitable for comparisons between samples and to assess the effects of any disease-related changes or any interventions.

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.

pH dependent spectral properties of sodium fluorescein ophthalmic solutions revisited.

PURPOSE: To re-assess and further quantify the pH-related changes in the absorbance and fluorescence emission spectra of sodium fluorescein in buffered aqueous solutions. METHODS: Analytical grade sodium fluorescein (NaF) or commercial NaF ophthalmic products (Minims 2% sodium fluorescein or Fluorets sodium fluorescein ophthalmic strips) were prepared over a range of dilutions in 1% NaCl with the pH set with phosphate, borate or acetate buffer mixtures between pH 4.0 and 10.0 at room temperature. Absorbance and fluorescence spectra were recorded in 10 mm pathlength cuvettes. RESULTS: At pH 7.5-8.5, the main NaF absorbance in dilute solution (0.005% w/v, or c. 13 microM) is at 490 nm, but this peak shifts progressively to 460 nm as the pH is lowered below 7.5. The 490 nm absorption peak shows a 50% of maximum value at pH 6.75. For extremely diluted solutions of NaF (0.000002%, c. 50 nM), the fluorescence emission at 513 nm follows the same profile. At higher concentrations (>0.000005%), NaF solutions start to show marked fluorescence quenching at neutral pH with the emission wavelength progressively shifting to 530 nm and even to 560 nm at extremely high concentrations (0.125%) where the fluorescence is all but quenched. CONCLUSIONS: The present studies confirm and extend some earlier reports that pH (over the range of 4.0-8.5), as well as concentration, predictably determines the fluorescence emission of dilute aqueous solutions of NaF.

The orbscan acoustic (correction) factor for central corneal thickness measures of normal human corneas.

BACKGROUND: Orbscan scanning-slit optical pachymetry was introduced over a decade ago and yielded higher central corneal thickness (CCT) values to the "gold standard" of contact ultrasound pachymetry (U/S). An acoustic correction factor (AF) was introduced later to compensate for this difference. The goal of this review was to assess the magnitude and consistency of the difference, as well as to assess how useful the AF had been. METHODS: Using PubMed (Medline)-sourced citations, published articles were identified that included data on CCT from U/S and Orbscan, with the latter data checked to see whether an AF had been applied. Main comparisons were made between (1) Orbscan data without AF and U/S, and (2) Orbscan data with a 0.92 AF applied and the U/S data. RESULTS: From 46 studies involving a total of 6136 eyes (average number per study of 133, range 6-1214), the average CCT values by U/S ranged from 0.520 to 0.580, for a group mean of 0.545 mm. For Orbscan without AF, the average CCT values ranged from 0.557 to 0.624 mm, for a mean of 0.582 mm, a net difference of 0.037 mm from U/S, with all Orbscan data (average values from any particular study) being higher than U/S. With a 0.92 AF applied, the net difference was -0.009 mm. The calculated limits of agreement between the two methods ranged from 0.004 to 0.073 without AF, but from -0.041 to + 0.023 mm with the AF. The overall outcome was essentially the same if weighted for cohort size and sample variability, or if only studies reporting on one eye were considered. CONCLUSIONS: Orbscan pachymetry can be expected to yield CCT data that is approximately 7% higher than U/S. The global application of a 0.92 AF does not robustly align the Orbscan CCT data to that of U/S and, in fact, can easily result in the data being as much as 7% lower. Overall, the level of agreement between Orbscan and U/S is limited, and Orbscan data should simply be reported as measured without any adjustment.

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.