Fall 2012 Newsletter
Diagnostic Profiles: Use of Ocular Fluids and Retina in Portmortem Analysis
Including Use of Aqueous Humor for Diagnosis of Nitrate Poisoning
By Dr. Christina R. Wilson, Head Toxicology Section & Head of Analytical Chemistry and Dr. Stephen B. Hooser, Veterinary Toxicologist

Postmortem analysis for chemical constituents of the blood can be difficult unless the blood is collected very soon after death and prior to coagulation. However, ocular fluids (aqueous and vitreous humor) and the retina can be useful for the diagnosis of several pathologic conditions, or exposure to various chemicals, for some time after death has occurred. Vitreous (and in some cases aqueous) humor can be used to estimate the time of death and can be useful as an aid in the diagnosis of renal disease, nitrate poisoning, hypomagnesemic syndromes, calcium status and salt poisoning (Hanna, et. al., 1990, Lincoln and Lane, 1985, McLaughlin and McLaughlin, 1986, McLaughlin and McLaughlin, 1987). Retina can be used to diagnose organophosphate poisoning, or to prove livestock exposure to some chemicals such as clenbuterol.

Increased nitrate concentrations can also be detected in the postmortem vitreous humor for up to 24 hours at room temperature (if the concentrations are very high, they can be diagnostic of nitrate poisoning up to 60h following death). This has proven very useful in the diagnosis of cattle found dead from what was later diagnosed to be nitrate poisoning. The detection of high nitrate concentrations in forage combined with high nitrates in the vitreous humor can lead to a diagnosis of death due to nitrate poisoning (Boermans, 1990).

In all species that have been examined (cattle, dogs, swine, and rabbits), urea nitrogen and creatinine concentrations in ocular fluid correlate very closely with serum concentrations for up to 24 hours (8h in rabbits) after death at body temperature (37° C). At room temperature (20 to 24° C) or refrigerated (4°C), they can be stable for longer periods of time. Therefore, postmortem vitreous humor urea nitrogen concentrations can be useful to diagnose antemortem renal disease (Drolet, et. al., 1990, Henke and Demarais, 1992, Lane and Lincoln, 1985, Schoning and Strafuss, 1981).

The use of vitreous humor for the diagnosis of hypomagnesemia in cattle has received mixed reviews in various studies that have been performed. In some, vitreous and serum magnesium concentrations have been reported to correspond for 36 to 48h postmortem at 24°C (Lincoln and Lane, 1985). However, another study using over 250 cows concluded that serum and vitreous magnesium concentrations did not correlate closely enough to diagnose antemortem changes in serum magnesium (McCoy and Kennedy, 1994). Therefore, to diagnose hypomagnesemia in cattle, it would be necessary to combine clinical signs and an appropriate history with vitreous humor magnesium concentrations.

Also, the use of vitreous humor to estimate the antemortem calcium and sodium serum concentrations has had mixed reports. Some studies report that both are stable in the vitreous humor and substantially reflect the serum concentrations, while others dispute this. Therefore, the use of postmortem calcium and sodium concentrations in the vitreous must also be correlated to history and clinical signs.

Vitreous humor has been used to roughly estimate the time of death in several species. It can be useful up to 48 to 72h postmortem. This is done by measuring the increase of potassium or phosphorous into the vitreous over time. Since the amount of release varies with temperature, it is important to report the temperature of the animal during the postmortem time interval (Crowell and Duncan, 1974, Schoning and Strafuss, 1980).

Retina can be useful for postmortem diagnosis of organophosphate poisoning and for evaluation of antemortem exposure to certain illegal drugs such as clenbuterol. Since it is a neural tissue, retina contains a large amount of cholinesterase which is inhibited by organophosphate insecticides. This inhibition can be measured in the same manner as it is in brain or blood. Measurement of retina cholinesterase inhibition has been successfully used to diagnose organophosphate exposure up to 24h postmortem (Harlin et. al., 1989, Harlin and Dellinger, 1993). In addition, the retina can concentrate some drugs such as clenbuterol, and store them for long periods of time. Therefore, retina can be used to evaluate the exposure of livestock to this illegal drug (Biolatti, et. al., 1994).

Sample Collection:

Aqueous and vitreous humor can be collected in large animals with the use of a 16 to 21 ga needle attached to a 10ml syringe. In small animals, the smaller gauge needle and a smaller syringe may be used. The needle is inserted through the sclera 1 cm caudal to the limbus (corneoscleral junction). The needle is directed ventrad and slightly caudad for about 1 to 2 cm to avoid the lens before aspirating fluid. Usually, several millimeters can be recovered. Some studies indicate that it is best to centrifuge the vitreous following aspiration, therefore, if possible, centrifuge at approximately 833g for 10 minutes following collection. The vitreous should then be refrigerated for submission. If vitreous humor is not available, then aqueous humor from the anterior chamber can be collected. This is done by inserting a small gauge needle through the cornea into the anterior chamber and aspirating the aqueous into a 3 or 5 ml syringe.

For postmortem analysis of retina, the whole eyeball should be enucleated and submitted frozen. ADDL personnel will the remove the retina from the interior of the eye.

(The highlighted references should be of most practical value)

  • Boermans HJ. Diagnosis of nitrate toxicosis in cattle, using biological fluids and a rapid ion chromatographic method. Am J Vet Res, 51: 491-495, 1990.
  • Biolatti B, Bollo E, Re G, Appino S, Tartari E, Benatti G, Elliot CT, McCaughey WJ. Pahtology and residues in veal calves treated experimentally with clenbuterol. Res Vet Sci, 57: 365-371, 1994.
  • Crowell WA, Duncan JR. Potassium concentration in the vitreous humor as an indicator of the postmortem interval in dogs. Am J Vet Res, 35: 301-302, 1974.
  • Drolet R, D’Allaire S, Chagnon M. The evaluation of postmortem ocular fluid analysis as a diagnostic aid in sows. J Vet Diagn Invest, 2: 9-13, 1990.
  • Hanna PE, Bellamy JEC, Donald A. Postmortem eyefluid analysis in dogs, cats, cattle as an estimate of antemortem serum chemistry profiles. Can J Vet Res, 54: 487-494, 1990.
  • Harlin KS, Hamdy S, Beastly VR. Preliminary studies with bovine retina cholinesterase determinations in organophosphorous insecticide poisoning. J Vet Diagn Invest, 1: 356-358, 1989.
  • Harlin KS, Dellinger JA. Retina, brain, and blood cholinesterase levels in cats treated with oral dichlorvos. Vet Hum Toxicol, 35: 201-203, 1993.
  • Henke SE, Demarais S. Changes in vitreous humor associated with postmortem interval in rabbits. Am J Vet Res, 53: 73-77, 1992.
  • Lane VM, Lincoln SD. Changes in urea nitrogen and creatinine concentrations in the vitreous humor of cattle after death. Am J Vet Res, 46: 1550-1552, 1985.
  • Lincoln SD, Lane VM. Postmortem magnesium concentration in bovine vitreous humor: Comparison with antemortem serum magnesium concentration. Am J Vet Res, 46: 160-162, 1985.
  • Lincoln SD, Lane VM. Postmortem chemical analysis of vitreous humor as a diagnostic aid in cattle. Mod Vet Pract, 66: 883-886, 1985.
  • McCoy MA, Kennedy DG. Evaluation of postmortem magnesium concentration in bovine eye fluids as a diagnostic aid for hypomagnesemic tetany. Vet Rec, 135: 188-189, 1994.
  • McLaughlin PS, McLaughlin BG. A comparison of the chemical constituents of vitreous humor in four species. Am Assoc Vet Lab Diagn, 29: 183-190, 1986.
  • McLaughlin PS, McLaughlin BG. Chemical analysis of bovine and porcine vitreous humors: Correlation of normal values with serum chemical values and changes with time and temperature. Am J Vet Res, 48: 467-473, 1987.
  • Schoning P, Strafuss AC. Determining the time of death of a dog by analyzinf bloodm cerebrospinal fluid and vitreous humor collected at postmortem. Am J Vet Res, 41: 955-957, 1980.
  • Schoning P, Strafuss AC. Analysis of postmortem canine blood, cerebrospinal fluid and vitreous humor. Am J Vet Res, 42: 1447-1449, 1981.
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