To test or not to test: Effective use of toxicology laboratories (Proceedings)

The establishment of a toxicologic diagnosis requires the same approach as that for any non-toxicologic diagnosis: obtaining a comprehensive history, establishing a minimum database, collection of appropriate samples for analysis and correct interpretation of laboratory results. In suspected intoxications, three scenarios are possible: an asymptomatic or symptomatic animal has been exposed to a known toxicant, an asymptomatic or symptomatic animal has been exposed to an unidentified toxicant or an animal is showing clinical signs due to an unknown cause for which toxicants should be included in a differential diagnosis. In many situations, a client calls asking whether an asymptomatic animal should be examined following a known ingestion of a chemical. In such situations, establishment of an exposure dose, if possible, and comparison of the exposure dose with specific toxicity information about a chemical is a critical first step in deciding subsequent advice (e.g., observing the animal at home or bringing the animal to the hospital for possible decontamination).

Case History

A proper history is an essential first ingredient for a successful toxicologic diagnosis. A good clinical history may provide specific clues or may only point the veterinarian and the diagnostician in a general direction. A good clinical history includes animal signalment, a thorough description of clinical signs, if observed, husbandry practices, environmental conditions and possible chemical exposures. Notation of pre-existing health problems is also important since underlying disease processes can contribute to increased sensitivity of an animal to a chemical. Important historical information includes the following: breed, sex, age, weight, current medical treatments, including any dietary supplements or herbs, number of animals in the group, number of animals affected, number potentially exposed, number exhibiting clinical signs, type, duration and severity of signs, possible time of exposure to identified chemicals, degree of exposure (dose) if known or, if worst case estimates can be made, and chemical formulations if possible [1, 2, 3, 4]. If an animal has been exposed or potentially exposed to an identified product, and a decision is made that the exposure is clinically significant, it is always recommended that the product be brought to the clinic for inspection if it can be handled in a safe manner. Examination of product labels can assist in the exposure assessment and provide a phone number if additional information about the product is needed.

It is important that the veterinarian not be misled by the perceptions of the owner. In many cases, owners are convinced that their animal has been "poisoned." This statement can lead many veterinarians to consider only a toxicologic etiology in lieu of other infectious or non-infectious causes of illness. Alternatively, the veterinarian should be careful in suggesting to a client that his or her animal appears to have been poisoned, when inadequate evidence exists to support such a conclusion.

Suspected cases of intoxication are often emotional and potentially involve litigation, especially when affected animals are rare and/or valuable. Because of the potential for litigation, it is incumbent upon the veterinarian to document all procedures and consider following a chain-of-custody process for all samples. Proper chain-of-custody involves creating a paper trail for each sample that documents in detail how a sample was stored, shipped, tested and discarded.

In many toxicoses, the only clinical sign is death. A thorough postmortem examination is essential in such circumstances. This may help eliminate non-toxicologic etiologies or perhaps narrow the list of possible toxicants. It should be kept in mind that many toxic agents may cause non-specific lesions or no lesions at all. Often, when a postmortem examination is done in the clinic, tissue samples are collected for either histologic or toxicologic examination, but not both. Two sets of tissue samples from animals with suspected toxicoses should be routinely saved. One set should be preserved in 10 percent buffered formalin for histologic evaluation and another set frozen for possible toxicologic analysis. A common and often unforgiving mistake is failure to submit brain, spinal cord, or peripheral nervous tissue when signs referable to the central or peripheral nervous system are present. A prudent and cost-effective procedure in unexplained deaths is to submit a full set of tissues for histologic examination following gross examination and to keep a second set frozen for later toxicologic analysis pending the histologic findings. It is always easier to dispose of unneeded frozen tissues than to collect tissues from an animal that has already been buried or otherwise discarded.

Ideally, a complete post-mortem examination should be conducted by a board-certified pathologist at an accredited veterinary diagnostic laboratory with toxicologic testing capabilities. A list of accredited veterinary diagnostic laboratories can be found on the American Association of Veterinary Laboratory Diagnostician's website at http://www.aavld.org/mc/page.do. Links to individual laboratories are provided. Some laboratories accept only certain animal species for evaluation and not all laboratories have toxicologic testing capabilities. Therefore, it is always useful to call the laboratory for specific information relating to the case at hand.

General sample collection protocols for investigation of possible toxicoses when live and/or dead animals are involved have been published (Table 1) Noteworthy are the references to urine and crop contents, which are samples that are often overlooked, but may sometimes be the most important to obtaining a diagnosis. Also, if an animal has vomited in the home environment, it can be critical that the owner bring a sample with the animal if a judgment is made that an animal should be examined.

Table 1. Comments on Selected Specimens for Submission to Veterinary Toxicology Laboratories

It is critical that samples are stored and shipped appropriately. Specimens should not be submitted in containers that have been used previously to store other materials, even if believed to be clean. When multiple samples are submitted, each container should be labeled with the name of the animal, the name of the owner, the date of collection and the identification of the specimen.

Depending on the toxicant of interest, samples should be either refrigerated or frozen. Refrigeration is generally sufficient if samples are to be shipped and tested soon after collection. If samples are to be retained pending other testing, freezing is more appropriate. When shipping samples, leaf-proof packaging and appropriate cooling is required. With a few possible exceptions, using ice packs provides sufficient cooling. Samples should be packaged in a way that avoids cross-contamination. Concentrated chemical sources such as baits or products can potentially leak through plastic bags; in such cases wrapping the sample in aluminum foil or placing in a separate metal container may be warranted. Use overnight delivery services, but avoid shipping samples to arrive on weekends or holidays, since the package can sit for a period of time prior to processing. Follow all applicable regulations regarding shipment of biological samples.

It is advisable to be familiar with the testing procedures of the diagnostic laboratory that you use. Although most laboratories offer a wide range of analytical tests, no one laboratory can run all possible analyses. If a particular test is not offered by an individual laboratory, it may be possible to forward the sample to a referral laboratory. Laboratory personnel are often knowledgeable about which laboratories have the capability and expertise to run an unusual test. The frequency of particular toxicoses varies for different regions of the country and thus the need for investing the resources and manpower to develop specific testing capabilities also varies. Generally, it is not cost-effective for diagnostic laboratories to develop analytical procedures for which they receive few requests, although some laboratories are willing to develop new chemical assays depending on their resources and circumstances of the case. Thus, lists of tests provided on websites or in users' guides may not always reflect what a laboratory is willing to or capable of providing.

Since many analytical procedures can detect the presence of certain compounds over a wide range of tissue concentrations, toxicology laboratories are not necessarily restricted to detection of high, potentially toxic concentrations of these agents. For example, some toxicology laboratories may offer therapeutic drug monitoring services or determinations of tissue mineral concentrations in order to rule out deficiency diseases.

The sophistication of analytical systems has enabled the diagnostic toxicologist to look for literally thousands of chemical compounds with a high degree of specificity and sensitivity. In cases in which there is no history of exposure to specific chemicals, powerful screening tools such as gas chromatography-mass spectrometry (GC/MS) and inductively coupled argon plasma emission spectroscopy (ICPAES) are available to detect a broad array of inorganic and organic compounds, respectively. For example, some diagnostic laboratories, using ICPAES or another metal screening technique called inductively coupled plasma mass spectrometry (ICP/MS), can test for several dozen metals with one analysis in tissue, fluid and environmental samples.

Sample size or type can be the limiting factors in the ability of a laboratory to test for a large number of compounds. Small sample sizes limit the number of tests that can be performed and can also decrease the sensitivity of a particular analytical technique. For example, if an analytical procedure typically requires 1 gram of tissue to maximize sensitivity, the availability of only 1/2 gram can decrease the sensitivity of the test by a factor of two.

It is important to note that, for certain toxicants, it is not necessary to quantify tissue concentrations; the presence of detectable toxicant in tissues along with compatible clinical signs is sufficient to yield a diagnosis. Alternatively, for agents that are ubiquitous in the environment, quantification of tissue concentrations may be critical for proper differentiation of a toxicosis from a background exposure.

References

Fitzgerald KT. Taking a toxicologic history. In: Peterson ME, Talcott PA, editors. Small Animal Toxicology, 2nd edition. St. Louis: Elsevier Saunders; 2006. p. 38-44.

Volmer PA, Meerdinck GL. Diagnostic toxicology for the small animal practitioner. Vet. Clin. Small Anim 2002,32:357-365.

Osweiler GD. Diagnostic toxicology. In: The national veterinary medical series. Toxicology. Philadephia: Williams and Wilkins; 1996. p. 37-46.

Poppenga RH, Braselton, Jr. WE. Effective use of analytical laboratories for the diagnosis of toxicologic problems in small animal practice. Vet Clin Small Anim 1990;20:293-306.

Fitzgerald KT. Establishing a minimum database in small animal poisoning. In: Peterson ME, Talcott PA, editors. Small Animal Toxicology, 2nd edition. St. Louis: Elsevier Saunders; 2006. p. 60-72.