*4.3. Limitations*

This study has several limitations. Serum samples were stored in a freezer ( −20 ◦C) at Kathmandu and were stored inside a freezer ( −80 ◦C) only after arriving at the University of Massachusetts, Amherst. No quality checks were conducted to assess sample degradation due to storage at higher temperature. The study was conducted from the subjects who have unusually high levels of daily occupational exposure, and therefore likely reflects results specifically to individuals who have extreme exposures. The study was also complicated by not having the same individuals followed during both seasons. There are several variables that were untested in this study and these variables may be very important in affecting the health of the subjects. Perspiration, stress, and exhaustion might be high during hot summers than in cooler spring conditions. Psychological stress may also elevate inflammatory biomarkers such as IL-6 [59], though stress induced by busy traffic was not likely much different between spring and summer in this cohort as traffic patterns were generally unchanged. There was also an issue of compliance on wearing masks in summer because comfort levels in wearing masks were lower during summer than in spring. Respiratory allergies and illnesses which were not included in this study may also be playing an important role. Additionally, average 24-hour PM2.5 used for daily exposure may not be the right indicator. Alternatively, a higher percentile closer to the peak occupational exposure may more accurately reflect the highly polluted environments of day-time traffic. There can be various other factors that were not considered in the current regression model such as age, lifestyles and years on the job. Though the subjects were living together at the dormitory at the sampling site during the study and their residential exposures were considered in this study, the prior residential history may still have played a role in their health. For example, female traffic police might have been using biomass for cooking activities in the past, and such history was not noted and considered in the current study.
