Ocular fluids | eClinpath

Some chemistry analytes can be measured on ocular fluids, both acqueous and vitreous. This is usually done in animals that have died and not as a premortem test and is used in an effort to identify biochemical changes that could explain the death of the animal. Numerous studies have been done to determine what tests can be done on ocular fluids post-mortem and the stability of the analytes (since we cannot always know when the animal has died). Results for many analytes will change markedly after death, affecting result interpretation, so this has to be taken into account when testing ocular fluids. For more on this, see common artifacts, although this is usually an uncommon artifact. Testing of ocular fluids has been used to help diagnose hypomagnesemia in ruminants (McCoy 2004), detect abnormalities in salt balance premortem, such as salt poisoning (Zilg et al 2016) and toxicologic testing and time of death estimates with forensics (Crichton et al 2015, Zilg et al 2015, Zilg et al 2016).

Vitreous fluid

Studies have been done comparing analyte concentration or activity in vitreous fluid compared to serum or plasma. In general, electrolytes and magnesium are found in similar concentrations in vitreous, whereas calcium and phosphate and markers of glomerular filtration rate (urea nitrogen and creatinine) are generally lower.

Vitreous versus Serum
Analyte	Bovine¹	Porcine¹	Canine²	Equine³
Sodium	Similar	Similar	Similar	Similar
Potassium	±30% lower	±30% lower		±9=20% lower
Chloride	Similar	Similar		±14% higher
Calcium	±50% lower	±50% lower		±40% lower
Magnesium	Similar	Similar		±10% higher
Phosphate	±85% lower	±90% lower		±75% lower
Urea nitrogen	±20-25% lower	±10% lower	±25% lower	±10% lower
Creatinine	±50-60% lower	±75% lower		±65% lower

The stability of vitreous fluid has been assessed in various species for various lengths of time and at various storage temperatures. Good studies generally keep the entire eye or the animal at these temperatures versus removing the fluid and maintaining at different temperatures. The former mimics (to the best of our ability) what would happen with death. In general, as for serum or plasma, analytes are more stable at colder temperatures for shorter times. Once collected, electrolytes and urea nitrogen are quite stable in human specimens frozen at-20ºC for 6-12 months, with only mild changes occurring in electrolytes (sodium, potassium, chloride) with refrigerated storage for a similar period of time (Gagajewski et al 2004).

Analyte	Bovine¹	Porcine¹	Canine²	Equine³
Sodium	Stable 4ºC, 24 hr 24ºC , 6 hr 36ºC , then ↓	Similar to bovine	Stable 4ºC, 12-24 hr 24-36ºC then ↓	Stable 24 hr 4-24ºC, 6 hr 36ºC then ↓
Potassium	Unstable: ↑ at all temps by 6 hours	Like bovine		Like bovine
Chloride	Stable 4 and 24ºC, 48 hr 24ºC, then ↓	Stable 4ºC, 24 hrs 24-36ºC		Stable all temp 48 hr
Calcium	↑ 6 hours, ↓ 24-48 hrs all temp	↑ 6 hours 4ºC, ↓ 12-24 hrs all temp		Stable 4-24ºC, 24 hours 36ºC, then ↓
Magnesium	Stable 24 hours all temps	Stable 4-24ºC, 6 hours 36ºC, then ↑		Stable 4ºC, 12-24 hours 24-36ºC, then ↑
Phosphate	Unstable, ↑ all temp by 6 hours	Like bovine		Like bovine
Urea nitrogen	Stable 48 hr 4-24ºC, 12 hr 36ºC then ↑	Stable 4ºC, 12-24 hours 24-36ºC then ↑	Variable at 4ºC, stable 12-24 hours 24-36ºC, then ↑	Stable 48 hr 4-24ºC, 12 hr 36ºC then ↑
Creatinine	Stable 48 hr 4-24ºC, 12 hr 36ºC then ↑	Stable 4ºC, 24 hrs 24ºC, 6 hr 36ºC, then ↑		Stable 48 hr 4-24ºC, 12 hr 36ºC then ↑

Ammonia: Ammonia concentrations have been measured post-mortem in cattle in an attempt to diagnose anhydrous ammonia toxicity (Fitzgerald et al 2006). The investigators collected vitreous samples from 8 reference cattle under similar storage conditions (collected after death and stored at 4ºC for 24 hours before analysis) and compared the 24 hour storage results with results from the same samples analyzed within 1 hour of collection. Ammonia concentrations in vitreous ranged from 24-365 umol/L, with a median of 78 umol/L in 1 hour samples but results had increased in samples to as high as 920 umol/L even if the median was unchanged (76 umol/L; range 21-920 umol/L) at 24 hours, indicating that ammonia concentrations will increase with storage. It is not clear how many of the 8 normal samples showed an increased ammonia concentration and if the samples with the highest increases were contaminated with blood or other cells which could produce ammonia. The samples in the affected cattle were higher than the median but within the range of the normal 8 cattle. Ammonia in vitreous humor increases linearly after death in human samples and is used to estimate post-mortem intervals (Henry and Smith 1980).

Aqueous fluid

There are some differences in electrolyte and mineral concentrations in aqueous versus vitreous fluid. One study in cattle showed that sodium, osmolality and phosphate concentrations were higher (particularly phosphate) whereas potassium, calcium and magnesium concentrations were slightly lower in aqueous fluid, respectively. Potassium and calcium concentrations increased in both fluids, with higher increases in aqueous fluid, after 24 hours of storage refrigerated. In contrast, higher increases in phosphate concentration were seen in vitreous fluid after similar storage⁴. Another study in cattle showed that magnesium is more stable in vitreous (24 hours in sheep and 48 hours in cattle) than aqueous humor (McCoy et al 2004).

Ammonia: Aqueous humor samples were also used to confirm a diagnosis of idiopathic hyperammonia in a horse (1108 umol/L versus 70-483 umol/L in the aqueous humor from 6 unaffected horses, collected similarly, i.e. after the carcasses had been maintained at 4ºC for 10 hours after storage, with storage on ice and analysis within 1 hour after collection) (Gilliam et al 2007).

References

McLaughlin & McLaughlin 1987: 6, 12, 24, 48 hours; 4, 24, 37ºC, n=10 per group
Schoning and Strafuss 1981: 3, 6, 12, 24, 48 hr, 4, 24, 37ºC, n=3-4 per group
McLaughlin & McLaughlin 1988: 6, 12, 24, 48 hours; 4, 24, 37ºC, n=8 per group
Hanna et al 1990: 24 hours, 24ºC, n=27-31, cattle only