One of the technological advantages of the TearLab Osmolarity System is the simultaneous collection and analysis of the tear sample on a “lab-on-a-chip” platform eliminating or mitigating the risk of evaporation of the tear sample. In essence, the tear sample is collected and analyzed before evaporation can influence the sample, even in a sample as small as 50 nanoliters (nL). However, we are often asked how evaporation may impact research projects when the project requires the collection and storage of tear fluid samples for testing at a later date. This may occur in research requiring additional analysis of the sample for tests of biomarkers other than osmolarity.
To further investigate that question, we recently performed a study to determine the extent of evaporative error that would occur in osmolarity testing of contrived tear solutions stored in sealed 1.8 milliliter (mL) vials for a 24-hour period.
A bulk volume of contrived tear sample was formulated using Deionized (DI) Water,
BSS Plus Part I Intraocular Irrigation Solution (Alcon Laboratories, Ft. Worth, TX) and Sodium Chloride (NaCl) Certified ACS Crystaline (Fisher Chemical 235 M-11619, Fisher Scientific). From the bulk solution, five aliquots were dispensed in Wheaton 1.8 mL glass vials with screw top caps (Model 224700). The five vials were filled to capacity, with no air space remaining in the vial once the vials were tightly sealed with the screw caps.
At the same time and from the same bulk solution, another five aliquots were dispensed with 400 microliter (uL) of solution in the 1.8 mL glass vials, leaving approximately three-fourths of the vial filled with air space once the vials were tightly sealed with screw caps. The 400 uL samples were exposed to a sealed air space that would hypothetically allow evaporation from the solution to the air space within the sealed vials.
The solutions were left at room temperature for 24 hours at which time their osmolarity was tested with the reference TearLab Osmolarity System in a controlled laboratory environment. The same Test Card Lot/BHR# was used for all testing in order to mitigate any lot-to-lot variability that may exist within different Test Card lots.
The data is as follows:
| 1.8 mL Fill | Vials | ||||
| Tests | 1 | 2 | 3 | 4 | 5 |
| 1 | 317 | 314 | 322 | 320 | 311 |
| 2 | 318 | 320 | 315 | 319 | 315 |
| 3 | 310 | 313 | 317 | 320 | 310 |
| 4 | 313 | 305 | 314 | 312 | 317 |
| 5 | 312 | 308 | 319 | 314 | 315 |
| Mean | 314 | 312 | 317 | 317 | 314 |
| SD | 3.4 | 5.8 | 3.2 | 3.7 | 3.0 |
| 25 Test Mean | 314.8 | ||||
| 400 uL Fill | Vials | ||||
| Tests | 1 | 2 | 3 | 4 | 5 |
| 1 | 318 | 328 | 328 | 310 | 319 |
| 2 | 320 | 309 | 317 | 313 | 316 |
| 3 | 319 | 319 | 318 | 317 | 325 |
| 4 | 319 | 319 | 325 | 315 | 317 |
| 5 | 315 | 323 | 323 | 319 | 319 |
| Mean | 318 | 320 | 322 | 315 | 319 |
| SD | 1.9 | 7.0 | 4.7 | 3.5 | 3.5 |
| 25 Test Mean | 318.8 | ||||
After exposure to an air cushion in a sealed vial environment for only a 24-hour period, there was a statistically significant difference in the mean osmolarity between the 1.8 mL filled vials (314.8 mOsmol/L) and the 400 uL filled vials (318.8 mOsmol/L) (p=0.0027).
It can be surmised that the evaporation within the sealed vial over 24-hours contributed to the reduced osmolarity in the 400 uL partially filled vials. These results underscore the importance of treating vials with small tear sample volumes as individual samples, since effects from evaporation, chemical reaction or other vial-to-vial environmental influences may alter the actual value of the osmolarity of the solution within the vial.
As evaporation will concentrate the tear sample and thus impact quantitative analysis of all analytes or biomarkers of interest, this effect should be accounted for in study design and data analysis. Using osmolarity to normalize quantitative analysis of tear analytes may prove to be a solution to evaporative error or uncontrolled variation in tear concentration. There is precedence for the use of normalization ratios in other diagnostics tests in which the sample concentration is subject to uncontrolled variation such as the common “urine albumin/creatinine ratio” for diagnosis of kidney disease and the “amniotic fluid lecithin/sphingomyelin ratio” to assess fetal lung maturity testing.
Conclusion
This study has demonstrated that 400 uL of tear fluid stored in a 1.8 mL sealed vial (approximately 25% full) for 24 hours will experience a 1.3% shift (4.0 mOsmol/L) in osmolarity due to evaporation from the exposure of the fluid to the air contained within the sealed vial itself. For this reason, TearLab does not recommend the collection and storage of tear fluid samples for testing at a later date in normal clinical use. In addition, this evaporative effect should be taken into account when designing research studies requiring the collection and storage of tear samples.
