Comparing the protein concentration of unstimulated and stimulated tears, the data displayed an equal variance with a T test P-value of 0.224. The P-value signifies there was no significant difference in protein concentration between stimulated and unstimulated tears. Fullard and Snyder (1990) concluded eight out the twelve proteins of tear concentration were found to be significantly higher in unstimulated tears compared with that of stimulated tears. These proteins found to be higher were IgA, IgA-SC, IgA1, IgA2, IgM, IgG, Transferrin, Serum Albumin. Lactoferrin, Prealbumin and Lysozyme levels were observed to be similar in both samples. The insignificance found in the experiment measured the total protein concentration of tears. Conversely, the study conducted by Fuller and Snyder investigated the significant difference of individual proteins hence they concluded a difference in significance. The method of collecting tears by Fullard and Snyder (1990) was similar to the method in this experiment whereby a flame polished micropipette was used. It should be considered, the method for collecting tears may influence data. Collection of tears using the Schirmer’s tear test indicated an increase in tear protein concentration (Farias et al. 2013; Posa et al. 2013; Stuchell et al. 1984) as opposed to using a microcapillary tube, as used in the study by Fullard and Snyder and this experiment. The increase in protein concentration seen in the Schirmer strip test could be attributed to contamination from the conjunctiva or epithelial cells (Dor et al. 2019)
Contact lenses are worn as an alternative to spectacles to correct a patient’s refractive error (Kavanagh et al. 2002). The difference in protein concentration between individuals wearing contact lenses and those not, was concluded to be insignificant with the data showing an equal variance between the two samples, p=0.173. This shows a disparity in the results compared with a study conducted by Sariri and Khamedi (2007). They determined the total tear protein concentration of contact lenses wearers to be greater compared to non-wearers. Similarly, Kramann et al. (2011) found a significant difference in tear protein concentration of individuals wearing soft and rigid gas permeable lenses as opposed to non-contact lens wearers. Specifically, Sariri and Khamedi also looked at the tear lysozyme concentration and found these to be greater in contact lens wearers as well, although the biological activity of lysozyme was decreased which they out down to contact lens use. Lysozyme is an antibacterial enzyme which aids in protecting the eye from infections. In the same study conducted by Sariri and Khamedi, it was also noted most immunoglobulins were found in subjects wearing contact lenses. Immunoglobulins are an antibody located in the body which support the immune system by defending against pathogens. An increase of total tear protein, along with lysozyme and immunoglobulins would suggest frequent contact lens wear due to their presence.
Despite the statically insignificance of contact lens wearers unstimulated and contact lens wearers stimulated results, p=0.653. It can be deduced from the above findings, whereby an increase in protein concentration was reported in unstimulated tears and separately in contact lens wearers; that a significant difference in protein concentration, showing an increase in protein would be found in contact lens wearers with unstimulated tears.
Schirmer’s tear test and tear film BUT (break-up time) test is utilised to show the tear production, and stability and composition of tears. Although a decline was observed with age in both sexes in tear production, no statistically significant difference between genders was found (Ozdemir and Temizdemir, 2010). However, Sullivan et al. (1990) reported there was “little or no impact” of the protein level in tears between gender. Though, little research appears to be available when comparing the implications of gender on tear protein concentration. Nonetheless, these results reflect the findings in the experiment when comparing the results of male and female protein concentration, p=0.914, showing the data to be insignificant.
Comparing non-contact lens wearers to contact lens wearers where both subjects were unstimulated, the results demonstrate p=0.263, signifying the difference between subjects is statistically insignificant. Comparing this result to other research conducted, similarly, no significant difference was found on analysis of total tear protein when comparing asymptomatic contact lens wearers to spectacle wearers (Subbaraman et al. 2015).
There were several limitations to the experiment conducted. The experiment required donors to collect their own tears in order to calculate the total protein concentration of stimulated and unstimulated tears. The results from this data showed there was statically no significant difference between the two samples, however an analysis of individual proteins on the tears would give a better representation, as when analysing individual proteins, significant difference was found between the two samples (Fullard and Snyder, 1990). Additionally, when calculating statistical significance, it must be remembered when using a smaller sample size, as used in this experiment, its likely insignificance would be found as there is not enough data to exploit.
A limitation due contact lens wear could be understanding more from the individuals about their wear of lenses. Subjects were asked to confirm if they wore lenses or not? With the results showing there was no significant difference between the two samples. However, further information from subjects could’ve been obtained in order to determine their significance. Additional information such as type contact lenses worn, as studies indicate soft lenses compared with rigid gas permeable lenses exhibit an increase in protein concentration of soft lenses as opposed to RGP lenses (Kramann et al. 2011). Additionally, no specificity was made as to whether the contact lenses the subjects were wearing were daily disposable lenses, bi-weekly, monthly or extended wear contact lenses. Although Carnet et al. (1999) reported major tear proteins doesn’t appear to be affected, their study was based solely on extended wear of hydrogel lenses.
Dry eye disease affects millions of people worldwide. It is a disease affecting the ocular surface of the eye whereby discomfort and dryness is caused by abnormalities of the tear film due to a reduction in aqueous tear production or increased tear evaporation (Lee et al. 2102). Subjects with dry eye disease produce a reduction in tear protein (Ohashi et al. 2003) therefore, in this experiment, donors with dry eye disease were excluded from the experiment. A limitation to this could be subjects not being aware they have dry eye disease and so altering the accuracy of the results.
In addition to the limitations explained above, other limitations can be caused through systematic error. Donors were required to collect 10µl of tears and carefully hold the microcapillary tube in a way after collection so to prevent loss of tears. In not done correctly, loss of tears could potentially affect the results. Furthermore, due to tear production rate and evaporation of tears, to collect 10 µl of unstimulated tears this would take at least ten minutes. If collection of tears took substantially quicker than with, even without a source of stimulation, these tears would be classes as stimulated tears rather than unstimulated tears. When logging the information on the spreadsheet for data collection, if donors note their tears to be unstimulated whereby, they were in fact stimulated this may skew the results.
To conclude, the results of this experiment displayed all comparable samples were statistically insignificant. Further information would be needed from donors so to make additional comparisons and observe they this would influence the results. Additionally, testing for individual proteins rather than total protein concentration in the tears would likely give further information on protein concentration of the donors.