Thesis (Ph. D.)--Ohio State University, 2004. / Title from first page of PDF file. Document formatted into pages; contains xiii, 122 p.; also includes graphics Includes bibliographical references (p. 80-94). Available online via OhioLINK's ETD Center

Assessment and diagnosis of dry eye disease (DED) is a challenging task. The conventional ways of diagnosing DED are problematic due to their invasiveness, poor test reliability and significant test duration. Previously, ocular surface thermography has been shown to be able to detect early inflammation and dry eye. However, its diagnostic ability and ocular temperature metrics that can best diagnose DED are not clear. The objectives of this thesis were manyfold. First, the prevalence of dry eye in Singapore population was investigated as a helpful basis for the rest of the project. A cross-sectional dry eye survey was carried out using McMonnies dry eye questionnaire. Members of the public were interviewed at 46 (out of 62) selected mass rapid transit stations in Singapore and its vicinity. 1004 questionnaires were collected from participants aged 15 - 83 years and various ethnicity. Prevalence of symptomatic dry eye (SDE) was found to be 12.3% (about 0.5 million Singaporeans). Risk factors associated with SDE were found to be age, gender, ethnicity, hypertension and contact lens wear. Smoking was not associated with SDE.The main part of this thesis sought to evaluate the efficacy of ocular thermography in diagnosing DED. A new infrared detector (NEC Thermo Tracer TH 9260) with relatively high resolution was used. Inter-image, inter-occasion and inter-examiner repeatability was first studied on 21 healthy and 15 DED subjects. Ocular surface marking and ocular surface temperature (OST) acquisition was performed with a novel 'diamond' method using a custom-designed OST analysis V2 software. Ten out of the twelve tested OST indices were shown to be highly repeatable for three studied time points: 0 s, 5 s and 10 s. They were temperatures of the geometric center of the cornea (GCC), mean temperature (MOST) of the region of interest (ROI), maximum (MaxT) and minimum (MinT) temperatures of the ROI, extreme temporal (T1) and nasal conjunctiva (T4), mid temporal (CT) and nasal conjunctiva (CN) and temporal (LT) and nasal limbal (LN). Another 62 DED and 63 age- and sex-matched controls were then recruited and the ten static and dynamic OST indices were evaluated. Static measures were study of absolute OST at t = 0 s, 5 s and 10 s after eye opening. Dynamic measures were study of mean change and net change in OST over 10 s of sustained eye opening. Static measures on eight OST indices (GCC, MOST, MinT, MaxT, T4, CT, LT and LN) at t = 0 s, 5 s and 10 s and dynamic measures on two OST indices (T4 at 3 s onward and MaxT at 5 s onward) were found to be valuable in detecting DED. The temperature metrics (static and dynamic) were identified for further investigation. Thereafter, the diagnostic ability of the temperature metrics were evaluated singly and as combinations in terms of their area under the curve (AUC), Youden index and discrimination power. Receiver operating characteristic curves were plotted for each metric. Best detectors for DED were found to be the T4 temperature metrics: particularly T4-5 and T4-10 (i.e. absolute temperature of the extreme nasal conjunctiva at 5 s and 10 s). Values of T4-5 of < 34.8 °C were found to give sensitivity and specificity of 87.1% and 50.8% respectively and values of T4-10 of < 34.6 °C were found to give sensitivity and specificity of 77.6% and 61.9% respectively. The two temperature metrics had highest Youden index as compared to other metrics and were shown to be useful in view of AUC > 70% but of limited performance in view of their discrimination power. Nevertheless, measuring T4-5 and T4-10 was found to be comparable to other conventional methods for DED. T4-10 was better than T4-5 in view of higher AUC and Youden index. None of the tested dynamic metrics was good detector for DED and combining metrics were not able to increase the diagnostic ability. The last part of this thesis was to validate the effectiveness of some common conventional dry eye tests, to study their correlation with T4 temperature metrics and derive the best composite/combined tests for DED. Sixty two DED patients and 82 controls were studied. The conventional clinical tests examined were: symptom evaluation using McMonnies dry eye questionnaire (Mscore) and symptom count (Scount), fluorescein break-up time (FBUT) and corneal epithelial staining (CES), non-invasive break-up time (NIBUT) and tear meniscus height (TMH). Mscore and Scount was the best detectors for DED, followed by FBUT and CES. Discordance between signs and symptoms for DED was further confirmed. Combining CES with T4-10 (series) can be future objective tests for DED. Further research is warranted, particularly to (1) validate the ability of T4-10 as a stand-alone test for DED and (2) work out an algorithm and validate the diagnostic ability of the recommended combined test (CES and T4-10) using newly recruited subjects.

Introduction Menopause which is defined as a permanent physiological, or natural, cessation of menstrual cycle, plays an important role in the development of ocular surface dryness symptoms and there is an increased prevalence of dry eye in women, especially those aged over 50. Despite the high prevalence of dry eye in post-menopausal women (PMW), very few studies have been undertaken to understand dry eye disease in a group of PMW who are not on Hormone Replacement Therapy (HRT). Studies in the past on PMW have primarily focused on the relationship between HRT and dry eye. Hence, a series of studies were undertaken to understand the clinical aspects of dry eye and their relationship to a variety of tear film components, in a group of PMW with and without symptoms of dry eye. The specific aims of each chapter were as follows: • Chapter 4: To characterize symptoms of dry eye using questionnaires, namely Ocular Surface Disease Index Questionnaire© (OSDI) and the Indiana Dry Eye Questionnaire (DEQ). • Chapter 5: To characterize clinical signs and symptoms in participants who present with and without symptoms of dry eye. • Chapter 6: To compare tear osmolality and ferning patterns in participants with and without dry eye symptoms. • Chapter 7: To investigate the potential relationship between subjective symptoms and clinical signs with tear film lipocalin and lysozyme concentrations in participants with and without dry eye symptoms. • Chapter 8: To optimize a technique for the isolation of total RNA (ribo nucleic acid) and total protein derived from conjunctival epithelial cells collected via conjunctival impression cytology (CIC). • Chapter 9: To quantify the expression of MUC1 (mucin1) and MUC16 (mucin16) mRNA and protein and to investigate the potential relationship between mucin expression and tear film breakup time in a group of participants with and without dry eye symptoms. Methods • Chapter 4: Participants were categorized as being symptomatic or asymptomatic of dry eye based on their response to the OSDI questionnaire. These results were then compared to the DEQ, which has questions related to the frequency of ocular surface symptoms and their diurnal intensity. • Chapter 5: Non invasive tear breakup time (NITBUT) was evaluated using the ALCON Eyemap®. Tear volume was assessed using the Phenol Red Thread (PRT) test and bulbar conjunctival hyperemia was measured using objective (SpectraScan PR650© Spectrophotometer) and subjective (slit lamp) methods. • Chapter 6: Tears were collected via capillary tube. A freezing point depression osmometer was used to measure the osmolality of the tear film. The tear ferning test was performed and evaluated for the quality of ferning, based on the Rolando grading system. • Chapter 7: Tears were collected via capillary tube and an eye wash method. Tear lysozyme and lipocalin concentrations were determined via Western blotting. • Chapter 8: CIC was collected using either Millipore (MP) or Poly Ether Sulfone (PES) membranes. RNA and protein isolation was performed using two different RNA isolation techniques. Two methods of protein isolation from CIC discs were evaluated. RT-PCR of mRNA for MUC1 and western blotting of lipoxygenase type 2 protein (LOX2) was performed to confirm the collection of intact RNA and total protein respectively. • Chapter 9: Tears were collected via capillary tube and an eye wash method. CIC was collected using MP membrane. Expression of MUC1 and MUC16 mRNA was assessed via real time PCR. Expression of both membrane-bound and soluble MUC1 and MUC16 were quantified via Western blotting. Results • Chapter 4: The OSDI total score and sub scores for the Non Dry Eye (NDE) and Dry Eye (DE) groups were significantly different (NDE =7.43 ± 7.71 vs DE = 24.87 ± 13.89; p<0.001). The DEQ scores showed that the DE group exhibited a higher frequency and intensity of symptoms than the NDE group, which worsened as the day progressed (p<0.001). • Chapter 5: The DE group exhibited a significantly shorter NITBUT (5.3 ± 1.7 vs 7.0 ± 2.7 secs; p=0.0012). Tear volume was significantly lower for the DE group (19.3 ± 5.1mm vs. 16.3 ± 5.6mm; p=0.031). Bulbar hyperemia was significantly higher in the DE group for both objective (u’ = 0.285 ± 0.006 vs. 0.282 ± 0.006; p=0.005) and subjective techniques (48.4 ± 10.0 vs 40.6 ± 10.4; p=0.0011). • Chapter 6: Osmolality values in DE individuals were significantly higher than the NDE (328.1 ± 20.8 vs. 315.1 ± 11.3 mOsm/kg; p = 0.02). There was a significant difference between the DE and NDE participants for the ferning patterns (p = 0.019). No significant correlation between tear osmolality and tear ferning was noted (DE: r = 0.12; p > 0.05, NDE: r = -0.17; p > 0.05). • Chapter 7: No difference in tear lysozyme or lipocalin concentration was found between DE and NDE groups, irrespective of tear collection method. Method of collection significantly influenced absolute concentrations (p<0.008). • Chapter 8: There was no significant difference between the two procedures used to isolate RNA and protein from CIC membranes (p>0.05). Total RNA yield was greater with the MP membrane. The mean yield of protein extracted from MP membrane using the two protein isolation techniques also did not show a significant difference. • Chapter 9: No difference was found in the expression of either MUC1 or MUC16 protein or mRNA expression between symptomatic DE and NDE (p>0.05). Weak correlations were found between the NITBUT values compared with either soluble or membrane bound MUC1 and MUC16 expression. Conclusions • Chapter 4: Questionnaires are useful tools to symptomatically divide participants into dry eyed and non dry eyed candidates. However, the questionnaire used to categorise patients can impact on the outcome variables determined. • Chapter 5: Post-menopausal women with dry eye symptoms demonstrate shorter NITBUT, lower tear volume and increased bulbar conjunctival hyperemia than those who have no symptoms. • Chapter 6: Tear osmolality in DE is higher than in NDE. There is a tendency towards less ferning in persons over 50 years of age, regardless of their symptoms. • Chapter 7: Comparison of clinical data with lipocalin and lysozyme concentrations failed to reveal statistically significant correlations. The concentration of either protein was not associated with tear stability or secretion. • Chapter 8: The total RNA yield was greater with the MP membrane. RNeasy Mini (RN) (Qiagen) method is recommended due to enhanced speed as well as on-column isolation and DNase digestion capabilities. CIC with MP membranes followed by immediate freezing and then extraction and processing facilitates the collection of total protein from human conjunctival cells. • Chapter 9: No difference was found in the expression of either MUC1 or MUC16 protein or mRNA expression between symptomatic PMW and asymptomatic controls.

Sjogren’s syndrome (SS) is a systemic autoimmune disease that presents to eye care practitioners with the hallmark symptom of “dry eye.” Stratifying dry eye patients as Sjogren’s positive or negative is a critical differential diagnosis, as SS patients have numerous systemic complications and a forty times greater risk of developing lymphoma. As such, management of this relatively common dry eye sub-population requires specialized care. Since a firm diagnosis requires testing that is both invasive and expensive, patients should be protected from these tests if they are not warranted. In this thesis, studies were therefore undertaken to determine if SS dry eye could be differentiated from other forms of dry eye using two methods: 1) standard clinical tests used in a multi-disciplinary Sjogren’s syndrome clinic and 2) subsequent biological evaluation of collected tear samples and cells from the ocular surface. The former would allow eye care practitioners to conduct appropriate tests and pose suitable questions to ifferentiate these subgroups, and the latter might serve in the future as a relatively non-invasive quantitative means of differentiating such groups through biomarkers.

Introduction: Tear film osmolality is a product of the varying concentrations of dissolved solutes (proteins, lipids and mucins) in the tear fluid. Research suggests that a hyperosmotic tear film is a trait common to all forms of dry eye, and it may be the driving force causing the discomfort, ocular surface damage and inflammation found in both evaporative and tear deficient forms of dry eye disease. Tear film osmolality has been proposed to be the “gold standard” diagnostic test for the evaluation of dry eye disease, as a distinct separation between tear film osmolalities in normal and dry-eyed (aqueous deficient or evaporative) populations has become evident. Historically, tear film osmolality could only be measured in a laboratory setting and required a highly skilled technician to use the instrumentation. The recent development of easy-to-use, small volume osmometers has made it possible for tear film osmolality to be measured clinically. As these instruments are quite new, there has been very little research completed with them. Therefore, a series of studies was conducted to investigate the utility of one of these new osmometers – the Advanced Instruments Model 3100 Nanolitre Osmometer. The specific aims of each chapter were: - Chapter 3: To determine if the Advanced Instruments Model 3100 Nanolitre Osmometer was capable of quantitatively measuring tear film osmolality in a normal population, using 0.5μL tear samples. - Chapter 4: Previous studies have shown the Advanced Instruments Model 3100 Nanolitre Osmometer not significantly different from another commercially available osmometer (Wescor Vapor Pressure Osmometer) for the measurement of human tears. This chapter examined the repeatability of the new instrument over multiple measurements on the same sample and over multiple days. - Chapter 5: To determine if tear film osmolality values varied significantly over the course of a normal working day in a population that was primarily free from symptoms of dry eye. - Chapter 6: To investigate the relationships between tear film osmolality and other commonly used clinical tests for dry eye disease. The clinical tests examined included various questionnaires designed to assess patient symptoms (Single Item Dry Eye Questionnaire (SIDEQ), the Ocular Surface Disease Index (OSDI), and the McMonnies Dry Eye Questionnaire (MMDEQ) and a linear analogue comfort scale (LACS)), a non-invasive tear break-up time test (NIBUT), and examination of ocular surface redness and tear ferning (TF). Secondarily to determine if the other clinical tests demonstrated significant diurnal variations over the course of a normal working day. - Chapter 7: To measure tear film osmolality in a population with mild to moderate symptoms of dry eye disease, and to compare this value with the osmolality of a population of age-matched controls without the disease. Secondarily, to investigate the relationship between tear film osmolality and patient comfort in a population with mild to moderate symptoms of dry eye disease. Methods: - Chapter 3: Tears were collected from 40 volunteer participants with a capillary tube. Some participants were non-contact lens wearers (Non-CL), while others wore either soft or rigid contact lenses (CL). Tear film osmolality was measured with the Advanced Instruments Model 3100 Nanolitre Osmometer. - Chapter 4: Tears were collected from 10 volunteer participants using two different collection techniques. Collections were repeated on three separate days (6 study visits total); three osmolality measurements per collection were taken using the Advanced Instruments Model 3100 Nanolitre osmometer. - Chapter 5: Tears were collected from 40 volunteer participants in two separate studies (n=80 in total). Tears were collected with a capillary tube three times a day (morning, mid-day and afternoon), on two separate days (6 study visits total). Tear film osmolality was measured with the Advanced Instruments Model 3100 Nanolitre Osmometer. - Chapter 6: Clinical tests were administered and tear samples were collected using a capillary tube from 40 volunteer participants. Measurements were taken three times a day (morning, mid-day and afternoon), on two separate days (6 study visits total). Tear film osmolality was measured with the Advanced Instruments Model 3100 Nanolitre Osmometer. - Chapter 7: Participants were classified as either having dry eye disease (DE) or not having dry eye disease (NDE) based on a clinical examination that included a case history, phenol red thread test and biomicroscopy (white light and sodium fluorescein assessment). Tear samples were then collected from all participants using a capillary tube and tear film osmolality was measured with the Advanced Instruments Model 3100 Nanolitre Osmometer. Participants also completed the SIDEQ, the OSDI, and the MMDEQ. Results: - Chapter 3: The mean tear film osmolality of the population was 298.7±11.4mOsm/Kg. CL wear (soft or rigid) did not appear to have a significant effect on tear film osmolality (CL: 298.5±11.2mOsm/Kg vs. Non-CL: 298.9±11.5mOsm/Kg), although this study was not designed to specifically look at the effects of contact lens wear on tear film osmolality. - Chapter 4: There was reasonably good concordance between measurements of tear film osmolality taken with the Advanced Instruments Model 3100 Nanolitre Osmometer (intraclass correlations range from 0.6497 (F= 0.0582) to 0.9550 (F = 0.5893)). Repeatability appeared to be affected by significant changes in ambient humidity (>10% per day). Concordance was similar with both sampling techniques. - Chapter 5: In the first study, no significant diurnal change in tear film osmolality was found (p>0.05), although a significant difference in measurements taken on Day 1 compared to Day 2 was found (p=0.040). When the first and last 10 participants enrolled were compared, the difference between days was present in the first 10 participants, but not in the last 10; it is likely that the investigator underwent a learning process during the period of the study, and that reflex tearing occurred more often in the early portion of the study compared with the latter portion. In the second study, no significant diurnal change in tear film osmolality was found (p>0.05) and no significant difference in measurements taken on Day 1 compared to Day 2 was found (p>0.05). When tear film osmolality was compared with the number of hours participants were awake, no significant correlation was found (r = 0.07044). - Chapter 6: Significant correlations were not found between tear film osmolality and SIDEQ (r = 0.1347), OSDI (r = 0.0331), MMDEQ (r = 0.2727), LACS (r = -0.1622), NIBUT (r = -0.2280), subjectively graded redness (r=-0.2280), or objectively measured redness (r = 0.1233). A weakly significant correlation was found between TF and tear film osmolality (r = 0.3978). None of the clinical measures (LACS, NIBUT, subjective or objective redness or TF) varied significantly over the course of the day. - Chapter 7: Tear film osmolality was higher in both the right (DE = 311.1±12.4mOsm/Kg, NDE = 306.2±11.2mOsm/Kg) and left eyes (DE = 313.2±11.9mOsm/Kg, NDE = 304.0±7.5mOsm/Kg) of participants, but the difference was only statistically significant in the left eye. Tear film osmolality did not correlate significantly with DE patient symptoms using any of the questionnaires (SIDEQ, OSDI, MMDEQ). Conclusions: - Chapter 3: The Advanced Instruments Model 3100 Nanolitre Osmometer appeared to be capable of measuring tear film osmolality in a normal population. Our population mean was slightly lower than what is reported to be normal (305mOsm/Kg), but it still fell within the range of values reported as normal (297 – 318mOsm/Kg). - Chapter 4: The Advanced Instruments Model 3100 Nanolitre Osmometer demonstrated reasonably good repeatability for the measurement of human tear samples. Unfortunately, the instrumentation appeared to be affected by dramatic weather changes. Maintaining the instrument in a humidity controlled environment may resolve this problem. - Chapter 5: Tear film osmolality did not appear to vary significantly over a normal working day. Inducing reflex tearing, perhaps with an unskilled investigator collecting the tears, can be a significant source of error (as demonstrated in the first study). - Chapter 6: Tear film osmolality did not correlate well with other clinical instruments designed to assess either patient symptoms or signs of dry eye disease in a normal population. Tear film osmolality and tear ferning did demonstrate a weakly significant positive correlation. None of the clinical measures assessed demonstrated a significant diurnal variation over the course of a normal working day. - Chapter 7: Tear film osmolality appeared to be higher in participants with mild to moderate symptoms of dry eye when compared with age matched, asymptomatic controls. Tear film osmolality did not correlate well with patient symptoms in a population of mild to moderate severe dry eyed individuals.

Introduction Menopause which is defined as a permanent physiological, or natural, cessation of menstrual cycle, plays an important role in the development of ocular surface dryness symptoms and there is an increased prevalence of dry eye in women, especially those aged over 50. Despite the high prevalence of dry eye in post-menopausal women (PMW), very few studies have been undertaken to understand dry eye disease in a group of PMW who are not on Hormone Replacement Therapy (HRT). Studies in the past on PMW have primarily focused on the relationship between HRT and dry eye. Hence, a series of studies were undertaken to understand the clinical aspects of dry eye and their relationship to a variety of tear film components, in a group of PMW with and without symptoms of dry eye. The specific aims of each chapter were as follows: • Chapter 4: To characterize symptoms of dry eye using questionnaires, namely Ocular Surface Disease Index Questionnaire© (OSDI) and the Indiana Dry Eye Questionnaire (DEQ). • Chapter 5: To characterize clinical signs and symptoms in participants who present with and without symptoms of dry eye. • Chapter 6: To compare tear osmolality and ferning patterns in participants with and without dry eye symptoms. • Chapter 7: To investigate the potential relationship between subjective symptoms and clinical signs with tear film lipocalin and lysozyme concentrations in participants with and without dry eye symptoms. • Chapter 8: To optimize a technique for the isolation of total RNA (ribo nucleic acid) and total protein derived from conjunctival epithelial cells collected via conjunctival impression cytology (CIC). • Chapter 9: To quantify the expression of MUC1 (mucin1) and MUC16 (mucin16) mRNA and protein and to investigate the potential relationship between mucin expression and tear film breakup time in a group of participants with and without dry eye symptoms. Methods • Chapter 4: Participants were categorized as being symptomatic or asymptomatic of dry eye based on their response to the OSDI questionnaire. These results were then compared to the DEQ, which has questions related to the frequency of ocular surface symptoms and their diurnal intensity. • Chapter 5: Non invasive tear breakup time (NITBUT) was evaluated using the ALCON Eyemap®. Tear volume was assessed using the Phenol Red Thread (PRT) test and bulbar conjunctival hyperemia was measured using objective (SpectraScan PR650© Spectrophotometer) and subjective (slit lamp) methods. • Chapter 6: Tears were collected via capillary tube. A freezing point depression osmometer was used to measure the osmolality of the tear film. The tear ferning test was performed and evaluated for the quality of ferning, based on the Rolando grading system. • Chapter 7: Tears were collected via capillary tube and an eye wash method. Tear lysozyme and lipocalin concentrations were determined via Western blotting. • Chapter 8: CIC was collected using either Millipore (MP) or Poly Ether Sulfone (PES) membranes. RNA and protein isolation was performed using two different RNA isolation techniques. Two methods of protein isolation from CIC discs were evaluated. RT-PCR of mRNA for MUC1 and western blotting of lipoxygenase type 2 protein (LOX2) was performed to confirm the collection of intact RNA and total protein respectively. • Chapter 9: Tears were collected via capillary tube and an eye wash method. CIC was collected using MP membrane. Expression of MUC1 and MUC16 mRNA was assessed via real time PCR. Expression of both membrane-bound and soluble MUC1 and MUC16 were quantified via Western blotting. Results • Chapter 4: The OSDI total score and sub scores for the Non Dry Eye (NDE) and Dry Eye (DE) groups were significantly different (NDE =7.43 ± 7.71 vs DE = 24.87 ± 13.89; p<0.001). The DEQ scores showed that the DE group exhibited a higher frequency and intensity of symptoms than the NDE group, which worsened as the day progressed (p<0.001). • Chapter 5: The DE group exhibited a significantly shorter NITBUT (5.3 ± 1.7 vs 7.0 ± 2.7 secs; p=0.0012). Tear volume was significantly lower for the DE group (19.3 ± 5.1mm vs. 16.3 ± 5.6mm; p=0.031). Bulbar hyperemia was significantly higher in the DE group for both objective (u’ = 0.285 ± 0.006 vs. 0.282 ± 0.006; p=0.005) and subjective techniques (48.4 ± 10.0 vs 40.6 ± 10.4; p=0.0011). • Chapter 6: Osmolality values in DE individuals were significantly higher than the NDE (328.1 ± 20.8 vs. 315.1 ± 11.3 mOsm/kg; p = 0.02). There was a significant difference between the DE and NDE participants for the ferning patterns (p = 0.019). No significant correlation between tear osmolality and tear ferning was noted (DE: r = 0.12; p > 0.05, NDE: r = -0.17; p > 0.05). • Chapter 7: No difference in tear lysozyme or lipocalin concentration was found between DE and NDE groups, irrespective of tear collection method. Method of collection significantly influenced absolute concentrations (p<0.008). • Chapter 8: There was no significant difference between the two procedures used to isolate RNA and protein from CIC membranes (p>0.05). Total RNA yield was greater with the MP membrane. The mean yield of protein extracted from MP membrane using the two protein isolation techniques also did not show a significant difference. • Chapter 9: No difference was found in the expression of either MUC1 or MUC16 protein or mRNA expression between symptomatic DE and NDE (p>0.05). Weak correlations were found between the NITBUT values compared with either soluble or membrane bound MUC1 and MUC16 expression. Conclusions • Chapter 4: Questionnaires are useful tools to symptomatically divide participants into dry eyed and non dry eyed candidates. However, the questionnaire used to categorise patients can impact on the outcome variables determined. • Chapter 5: Post-menopausal women with dry eye symptoms demonstrate shorter NITBUT, lower tear volume and increased bulbar conjunctival hyperemia than those who have no symptoms. • Chapter 6: Tear osmolality in DE is higher than in NDE. There is a tendency towards less ferning in persons over 50 years of age, regardless of their symptoms. • Chapter 7: Comparison of clinical data with lipocalin and lysozyme concentrations failed to reveal statistically significant correlations. The concentration of either protein was not associated with tear stability or secretion. • Chapter 8: The total RNA yield was greater with the MP membrane. RNeasy Mini (RN) (Qiagen) method is recommended due to enhanced speed as well as on-column isolation and DNase digestion capabilities. CIC with MP membranes followed by immediate freezing and then extraction and processing facilitates the collection of total protein from human conjunctival cells. • Chapter 9: No difference was found in the expression of either MUC1 or MUC16 protein or mRNA expression between symptomatic PMW and asymptomatic controls.

Sjogren’s syndrome (SS) is a systemic autoimmune disease that presents to eye care practitioners with the hallmark symptom of “dry eye.” Stratifying dry eye patients as Sjogren’s positive or negative is a critical differential diagnosis, as SS patients have numerous systemic complications and a forty times greater risk of developing lymphoma. As such, management of this relatively common dry eye sub-population requires specialized care. Since a firm diagnosis requires testing that is both invasive and expensive, patients should be protected from these tests if they are not warranted. In this thesis, studies were therefore undertaken to determine if SS dry eye could be differentiated from other forms of dry eye using two methods: 1) standard clinical tests used in a multi-disciplinary Sjogren’s syndrome clinic and 2) subsequent biological evaluation of collected tear samples and cells from the ocular surface. The former would allow eye care practitioners to conduct appropriate tests and pose suitable questions to ifferentiate these subgroups, and the latter might serve in the future as a relatively non-invasive quantitative means of differentiating such groups through biomarkers.

Introduction: Tear film osmolality is a product of the varying concentrations of dissolved solutes (proteins, lipids and mucins) in the tear fluid. Research suggests that a hyperosmotic tear film is a trait common to all forms of dry eye, and it may be the driving force causing the discomfort, ocular surface damage and inflammation found in both evaporative and tear deficient forms of dry eye disease. Tear film osmolality has been proposed to be the “gold standard” diagnostic test for the evaluation of dry eye disease, as a distinct separation between tear film osmolalities in normal and dry-eyed (aqueous deficient or evaporative) populations has become evident. Historically, tear film osmolality could only be measured in a laboratory setting and required a highly skilled technician to use the instrumentation. The recent development of easy-to-use, small volume osmometers has made it possible for tear film osmolality to be measured clinically. As these instruments are quite new, there has been very little research completed with them. Therefore, a series of studies was conducted to investigate the utility of one of these new osmometers – the Advanced Instruments Model 3100 Nanolitre Osmometer. The specific aims of each chapter were: - Chapter 3: To determine if the Advanced Instruments Model 3100 Nanolitre Osmometer was capable of quantitatively measuring tear film osmolality in a normal population, using 0.5μL tear samples. - Chapter 4: Previous studies have shown the Advanced Instruments Model 3100 Nanolitre Osmometer not significantly different from another commercially available osmometer (Wescor Vapor Pressure Osmometer) for the measurement of human tears. This chapter examined the repeatability of the new instrument over multiple measurements on the same sample and over multiple days. - Chapter 5: To determine if tear film osmolality values varied significantly over the course of a normal working day in a population that was primarily free from symptoms of dry eye. - Chapter 6: To investigate the relationships between tear film osmolality and other commonly used clinical tests for dry eye disease. The clinical tests examined included various questionnaires designed to assess patient symptoms (Single Item Dry Eye Questionnaire (SIDEQ), the Ocular Surface Disease Index (OSDI), and the McMonnies Dry Eye Questionnaire (MMDEQ) and a linear analogue comfort scale (LACS)), a non-invasive tear break-up time test (NIBUT), and examination of ocular surface redness and tear ferning (TF). Secondarily to determine if the other clinical tests demonstrated significant diurnal variations over the course of a normal working day. - Chapter 7: To measure tear film osmolality in a population with mild to moderate symptoms of dry eye disease, and to compare this value with the osmolality of a population of age-matched controls without the disease. Secondarily, to investigate the relationship between tear film osmolality and patient comfort in a population with mild to moderate symptoms of dry eye disease. Methods: - Chapter 3: Tears were collected from 40 volunteer participants with a capillary tube. Some participants were non-contact lens wearers (Non-CL), while others wore either soft or rigid contact lenses (CL). Tear film osmolality was measured with the Advanced Instruments Model 3100 Nanolitre Osmometer. - Chapter 4: Tears were collected from 10 volunteer participants using two different collection techniques. Collections were repeated on three separate days (6 study visits total); three osmolality measurements per collection were taken using the Advanced Instruments Model 3100 Nanolitre osmometer. - Chapter 5: Tears were collected from 40 volunteer participants in two separate studies (n=80 in total). Tears were collected with a capillary tube three times a day (morning, mid-day and afternoon), on two separate days (6 study visits total). Tear film osmolality was measured with the Advanced Instruments Model 3100 Nanolitre Osmometer. - Chapter 6: Clinical tests were administered and tear samples were collected using a capillary tube from 40 volunteer participants. Measurements were taken three times a day (morning, mid-day and afternoon), on two separate days (6 study visits total). Tear film osmolality was measured with the Advanced Instruments Model 3100 Nanolitre Osmometer. - Chapter 7: Participants were classified as either having dry eye disease (DE) or not having dry eye disease (NDE) based on a clinical examination that included a case history, phenol red thread test and biomicroscopy (white light and sodium fluorescein assessment). Tear samples were then collected from all participants using a capillary tube and tear film osmolality was measured with the Advanced Instruments Model 3100 Nanolitre Osmometer. Participants also completed the SIDEQ, the OSDI, and the MMDEQ. Results: - Chapter 3: The mean tear film osmolality of the population was 298.7±11.4mOsm/Kg. CL wear (soft or rigid) did not appear to have a significant effect on tear film osmolality (CL: 298.5±11.2mOsm/Kg vs. Non-CL: 298.9±11.5mOsm/Kg), although this study was not designed to specifically look at the effects of contact lens wear on tear film osmolality. - Chapter 4: There was reasonably good concordance between measurements of tear film osmolality taken with the Advanced Instruments Model 3100 Nanolitre Osmometer (intraclass correlations range from 0.6497 (F= 0.0582) to 0.9550 (F = 0.5893)). Repeatability appeared to be affected by significant changes in ambient humidity (>10% per day). Concordance was similar with both sampling techniques. - Chapter 5: In the first study, no significant diurnal change in tear film osmolality was found (p>0.05), although a significant difference in measurements taken on Day 1 compared to Day 2 was found (p=0.040). When the first and last 10 participants enrolled were compared, the difference between days was present in the first 10 participants, but not in the last 10; it is likely that the investigator underwent a learning process during the period of the study, and that reflex tearing occurred more often in the early portion of the study compared with the latter portion. In the second study, no significant diurnal change in tear film osmolality was found (p>0.05) and no significant difference in measurements taken on Day 1 compared to Day 2 was found (p>0.05). When tear film osmolality was compared with the number of hours participants were awake, no significant correlation was found (r = 0.07044). - Chapter 6: Significant correlations were not found between tear film osmolality and SIDEQ (r = 0.1347), OSDI (r = 0.0331), MMDEQ (r = 0.2727), LACS (r = -0.1622), NIBUT (r = -0.2280), subjectively graded redness (r=-0.2280), or objectively measured redness (r = 0.1233). A weakly significant correlation was found between TF and tear film osmolality (r = 0.3978). None of the clinical measures (LACS, NIBUT, subjective or objective redness or TF) varied significantly over the course of the day. - Chapter 7: Tear film osmolality was higher in both the right (DE = 311.1±12.4mOsm/Kg, NDE = 306.2±11.2mOsm/Kg) and left eyes (DE = 313.2±11.9mOsm/Kg, NDE = 304.0±7.5mOsm/Kg) of participants, but the difference was only statistically significant in the left eye. Tear film osmolality did not correlate significantly with DE patient symptoms using any of the questionnaires (SIDEQ, OSDI, MMDEQ). Conclusions: - Chapter 3: The Advanced Instruments Model 3100 Nanolitre Osmometer appeared to be capable of measuring tear film osmolality in a normal population. Our population mean was slightly lower than what is reported to be normal (305mOsm/Kg), but it still fell within the range of values reported as normal (297 – 318mOsm/Kg). - Chapter 4: The Advanced Instruments Model 3100 Nanolitre Osmometer demonstrated reasonably good repeatability for the measurement of human tear samples. Unfortunately, the instrumentation appeared to be affected by dramatic weather changes. Maintaining the instrument in a humidity controlled environment may resolve this problem. - Chapter 5: Tear film osmolality did not appear to vary significantly over a normal working day. Inducing reflex tearing, perhaps with an unskilled investigator collecting the tears, can be a significant source of error (as demonstrated in the first study). - Chapter 6: Tear film osmolality did not correlate well with other clinical instruments designed to assess either patient symptoms or signs of dry eye disease in a normal population. Tear film osmolality and tear ferning did demonstrate a weakly significant positive correlation. None of the clinical measures assessed demonstrated a significant diurnal variation over the course of a normal working day. - Chapter 7: Tear film osmolality appeared to be higher in participants with mild to moderate symptoms of dry eye when compared with age matched, asymptomatic controls. Tear film osmolality did not correlate well with patient symptoms in a population of mild to moderate severe dry eyed individuals.

Aim: The aim of this study was to establish normal values for Schirmer tear test (version 2) and tear break up time (TBUT) in a South African young adult Black population. Method: Following ethical approval by the biomedical research and ethics committee, KwaZulu-Natal, participants were recruited from the city of Durban in South Africa via personal invitations, poster advertisements and University of KwaZulu-Natal optometry clinic clients. McMonnies questionnaire for dry eye diagnosis was administered and those who failed were excluded from the main study. Two hundred (100 males and 100 females) participants who met the inclusion criteria were included in the study. Following a slit lamp examination of the eye, the Schirmer test was administered and the following day, the TBUT was measured. A re-test version of the two procedures were conducted one week after, at about the same time of the day for each subject. Results: The participants were aged 18-30 years, mean = 20.77 ± 2.29 years. The mean Schirmer test values for all participants (N = 200; 400 eyes) was 15.96 ± 6.86mm. The values for the males and females (200 eyes each) were 16.34 ± 6.93mm and 15.58 ± 6.81mm respectively. The mean TBUT (400 eyes) was 7.18 ± 1.89 secs. The mean values for the males and females (200 eyes each) were 6.90 ± 1.88 secs and 7.32 ± 1.67 secs respectively. A strong positive correlation (r = 0.895) and (r = 0.914) respectively was found between the right and left eyes in the two tests. Conclusion: Generally, the mean values found in this study for the Schirmer test are similar to those that have been reported in the literature. However, values for TBUT differ from the values that have been previously reported, being higher in some instances and lower in others. These findings have implications for dry eye diagnosis and also contact lens practice in South Africa. / Thesis (M.Optom.)-University of KwaZulu-Natal, Westville, 2012.

M.Sc. (Biochemistry) / Dry eye disease (DED) and keratoconus (KC) continue to affect the quality of life of many South Africans (and elsewhere) and in the case of KC often leads to blindness. It is estimated that DED affects 14% to 33% of the population worldwide, while 1 in 2000 of the worlds population is affected by KC. However, details of the etiology of these diseases and their biochemical ‘fingerprint’ remain uncertain. In this study, emphasis was placed on the investigation of immunological proteins in the precorneal tear film of DED and KC subjects and meibomian lipids in these individuals. Tear fluid and meibum were collected from control, DED and KC volunteers. Control subjects were non-contact lens wearers and free from ocular diseases, whereas DED subjects were diagnosed by means of an ocular surface disease index (OSDI) questionnaire. DED subjects were divided into two groups: ‘moderate DED’ and ‘severe DED’ based on OSDI. KC subjects were diagnosed by the use of a slit-lamp biomicroscopy exam. Enzymelinked immunosorbent assays were performed to quantitate secretory immunoglobulin A (sIgA), tumour necrosis factor-alpha (TNF-á) and matrix metalloproteinase-1 (MMP-1) in the collected tear fluid. Meibum was analysed with proton nuclear magnetic resonance (1H-NMR) spectroscopy and Fourier transform infrared spectroscopy (FTIR). Multivariate data analyses (PCA) were used to extract interpretable information from the multidimensional data generated from the aforementioned techniques and used to build a broad picture of the general lipidomic differences between DED, KC and healthy subjects. Tear levels of sIgA and MMP-1 were significantly decreased in patients with KC compared to control. In contrast, the tears of severe DED subjects were characterised by higher levels of TNF-á and lower levels of sIgA. In subjects with moderate DED, TNF-á levels were significantly elevated. The results of this study re-emphasize that KC and DED individuals are associated with differential expression of specific tear proteins and support the view that the severity of DED is reflected in the levels of immunological proteins present in basal tears. Differences in the chemical composition of meibum from subjects with severe DED and KC compared to control were observed, more specifically in the aliphatic region of 1H-NMR spectra and C-C rocking region of FTIR spectra. The results therefore point towards the saturated components of fatty acids (and their chemical environments) as key targets for future investigations to elucidate compositional differences between DED, KC and healthy meibum.