Help us help you! Getting the most from a diagnostic toxicology laboratory

For most veterinary practitioners, a brief pathology rotation during their senior year of veterinary school may be the only time, if at all, they spend in a veterinary diagnostic laboratory. The lessons learned during this time often fade because of more pressing daily issues of private practice. In particular, the approach to diagnosing suspected toxicology cases may be suboptimal because of the infrequency with which these cases are encountered in routine practice compared with other medical cases.

Nevertheless, toxicoses do occur. The cases are often emotionally charged and can readily become legal cases. Successful outcomes in toxicology cases require thorough investigations. Specimens must be collected at the time of the animal's illness, and when necessary, necropsies and postmortem specimen collection should preferably be done by a board-certified pathologist at an accredited veterinary diagnostic laboratory with a toxicology section. Using a veterinary diagnostic laboratory allows for a complete diagnostic workup to determine whether an animal has become ill or died because of a toxicologic cause. This article provides practitioners with practical information about using the services of a diagnostic toxicology laboratory effectively to help ensure a satisfactory and rewarding outcome for all involved.

WHY USE A DIAGNOSTIC TOXICOLOGY LABORATORY?

Often, instead of submitting tissue specimens to a veterinary diagnostic laboratory, practitioners submit them to a contract laboratory with a request for a particular toxicologic test. This method provides a specific test result; however, practitioners are more likely to receive assistance with examination approach, differential diagnoses, appropriate sampling methods, test selection, and test interpretation from a diagnostician if an accredited veterinary diagnostic laboratory is used.1

Veterinary diagnostic laboratories are composed of a number of sections, such as pathology, microbiology, serology, and toxicology. Cases can easily be discussed and specimens easily passed among sections to most efficiently establish a diagnosis. Table 1 lists accredited veterinary diagnostic laboratories in the United States and Canada.

Table 1 Accredited* Veterinary Diagnostic Laboratories in the United States and Canada**

LABORATORY ERROR

It is important for veterinary practitioners, as users of toxicology laboratory services, to be familiar with factors that can contribute to variations in test results. Overall laboratory error includes all preanalytical, intra-analytical, and postanalytical phases.

Preanalytical errors occur at the time of patient assessment; test order entry; request completion and communication with the laboratory; and specimen collection, transport, or receipt in the laboratory. Intra-analytical errors occur during the analysis process itself. The intra-analytical phase begins when the specimen is prepared for testing and ends when the test result is interpreted in the laboratory and verified as ready to report.2 Postanalytical errors derive from inappropriate interpretation and use of laboratory results to make diagnostic and therapeutic decisions.

The limited body of literature examining the frequency of laboratory error in medical facilities indicates that most errors occur in the preanalytical phase followed by the postanalytical phase.3 My experience, from the diagnostic toxicology perspective, is that most errors occur in the preanalytical phase.

BEFORE SUBMITTING SPECIMENS

One of the most important things—if not the most important thing—practitioners can do to increase the likelihood of arriving at a diagnosis is to communicate with the diagnostic laboratory before collecting and submitting specimens. All too often, the diagnostic process stops short because of a lack of appropriate tissues or insufficient specimen quantity.

In addition, test results are most meaningful when all components of a case, including a thorough case history and properly collected and stored environmental, antemortem, and postmortem specimens, are presented to the toxicologist.

Obtain a complete case history

There is no such thing as a poison screen. Time, money, and specimens can be needlessly wasted searching for a toxin with a low probability of being present. A comprehensive history will help narrow and prioritize the list of possible toxic agents, thereby increasing the efficiency of the diagnostic approach.

Include on the submission form information on the recent circumstances surrounding the illness in question. Succinctly relate relevant information such as

• The patient's breed, sex, age, and weight

• The patient's recent medical history and current medications

• The total number of animals in the environment or at risk and the number affected

• The patient's clinical signs

• The substance ingested and the time at which it was ingested, if known

• The duration of the problem and the patient's response to treatment

• Diagnostic test results

• Differential diagnoses

• The date the specimens were collected

Other points to consider and information that may be valuable to the diagnostic process may include

• Environmental factors. What agents are in the animal's environment? What is the source of its food and water? Is the animal confined to a particular room in the house, garage, yard, or other area?

• The animal's behavior. Is the affected individual the alpha, or dominant, animal? Did this animal potentially eat more than others that had access to the same food or bait?

• Relationship of owners with others. How likely is a malicious poisoning in this case?

• Recent visitors to the home. Could the animal have had access to medications in luggage or dropped pills? This is especially a concern around holidays or other busy events involving high visitor traffic.

• Supervision of the animal outdoors. Is the animal confined to the yard, or does it roam the neighborhood? What potential hazards are in the area?

• The animal's vaccination status. Are the clinical signs truly a reaction to a toxicant, or does the animal have a preventable disease?

• Children in the home. Could a child have fed the animal food or medication believing it was a treat?

All paperwork accompanying the specimen should be placed in a plastic bag (Ziplock) in case specimen containers leak during transport. The submitting veterinarian's name, address, and contact information (office and cell phone numbers, fax number, and e-mail address) must be included. Enclosing copies of the animal's lifelong medical record is not encouraged and detracts from an efficient diagnostic process.

Collect appropriate samples

Most analytical quests run into problems because of inadequate sampling. Laboratory personnel can only work with the specimens submitted. Appropriate sampling is one of the most important steps in the analytical process. To help ensure a smooth process, make sure to acquire adequate amounts of specimen and to store the specimen correctly.

Toxicology specimens are often held until other components of the diagnostic process have been completed, such as histologic examinations. Because of this, it is recommended to collect a complete set of tissues, fresh and formalin-fixed, for examination by other diagnosticians in the laboratory. Information from these investigations, such as characteristic lesions, will help to direct the toxicologic investigation.

Which specimens? The toxicokinetics of the suspected agent must be considered since the tissue distribution and accumulation will vary depending on the toxicant. It is logical to analyze the tissue with the highest probability of containing the compound of interest. For example, the kidney is the best postmortem tissue specimen to analyze when ethylene glycol toxicosis is suspected. When in doubt, ask a veterinary diagnostic toxicology laboratory staff member what specimens to collect, if you have the opportunity to do so. If not, Table 2 lists antemortem, postmortem, and environmental specimens to collect when a toxicologic case is suspected. It is better to collect specimens that may not be used than to find out other specimens are needed after the remains are unavailable.

Table 2 Recommended Specimens to Collect for a Toxicologic Diagnostic Analysis

How much? For toxicologic analyses, more is better! Large amounts of specimen allow for concentration of compounds within the sample, increasing the likelihood of detection. Large specimen quantities also allow for repeat analyses if the results are questionable or for confirmation purposes. In addition, a plentiful supply of specimen permits further investigation for other compounds if a particular trail comes up cold.

Table 3 lists specimens to collect for a toxicologic investigation and the recommended minimum amounts. It should be noted that recommended amounts will vary depending on the purpose of the analysis. For example, loads of grain or large bags of feed may require larger specimen amounts than those listed in the table in order to provide a specimen representative of the entire batch. In some cases, it may not be possible to obtain the minimum suggested amount. In those instances, submission is still encouraged, but the possible limitations in the analytical process and in interpretation should be recognized.

Table 3 Recommended Specimen Amounts and Storage for Toxicologic Analyses*

How should I store the specimens? Most analyses cannot be performed on formalin-fixed tissue. Table 3 also lists recommended storage methods for specimens. Unless otherwise noted, specimens should be chilled as soon as possible after collection.

Package and store each tissue individually, and label each specimen. This will avoid any confusion about specimen identification and type, especially if the sample quality was questionable to begin with. For example, it is often difficult to distinguish autolyzed liver from kidney. Containers for specimen collection and storage should be clean to avoid contamination and potentially misleading results. In addition, containers should be able to withstand transport to the laboratory. Labels should include the date, specimen, clinician, and, in the case of multiple animals, animal identification.

SHIPPING SPECIMENS

In most cases, specimens should be triple-bagged and shipped as quickly as possible, preferably by overnight delivery, on ice or dry ice. In some cases, extra packaging is required to be in accordance with applicable regulations for shipment of hazardous materials (e.g. insecticides). Avoid shipping specimens on weekends or holidays because the parcel may sit for days in hot conditions. Senders should check with their couriers in order to meet current packaging and labeling requirements. Ultimately, the sender is responsible for ensuring that the package meets transportation requirements.

OTHER FACTORS TO KEEP IN MIND

Requests

Avoid requesting a specific instrument or method, such as an inductively coupled argon plasma emission spectroscopy (ICP). The laboratory will select the most appropriate analytical method based on the sensitivity needed and the sample matrix involved. Many laboratories have instruments dedicated to a particular analysis.

Discussing the diagnostic approach and appropriate analyses with a veterinary diagnostic toxicologist is always worthwhile. The diagnostician can refine the differential diagnoses list from the case history and clinical signs and prioritize testing to ensure a logical progression of analyses, while conserving valuable specimens and minimizing cost.

Analyses

Unrealistic expectations may exist regarding the scope, sensitivity, speed, and availability of toxicologic analyses. Complicated cases may require involved procedures such as repeated extractions, dilutions, and the use of multiple methods to arrive at a diagnosis. Such cases may take days and even weeks to complete. Discussion with the toxicologist will provide time frames and limitations of available tests and screens, as well as cost estimates to avoid surprises later.

Factors outside of the laboratory, such as specimen quality, directly affect analytical results and interpretation. Analytical equipment has become more sensitive and more precise. But regardless of the sophistication of the analytical procedure, the results will be suboptimal if the specimen quality is questionable.

Interpretation

The ability to detect smaller and smaller amounts of chemicals in a given matrix has posed challenges in interpreting the results. What is the clinical significance of these numbers? Reliable quantification of the amount of a compound in a tissue requires better tissue sampling and handling than does just determining its presence (a qualitative measurement). Quantification also poses challenges for toxicologists who must interpret these values in light of historical information, tissue selection, and specimen quality.4 For many species, information on toxic tissue concentrations is unavailable for many compounds.

Another factor affecting interpretation is the time from exposure to sampling. If the tissue specimen was obtained late in the course of the syndrome, a toxicant may have already been metabolized and excreted. Analyses may not detect any toxic substance at that point.4

CONCLUSION

Toxicologic testing can be a complex and intricate process. All toxicology laboratories vary in their analytical instrumentation, analyses offered, and preferred testing methods. An understanding of the analytical process, from collecting specimens to interpreting results, will help you better manage toxicology cases.

Petra A.Volmer, DVM, MS, DABVT, DABT

Veterinary Diagnostic Laboratory

College of Veterinary Medicine

University of Illinois

Urbana, IL 61802

REFERENCES

1. Meerdink GL, Hoffman WE. When is it worth sending it to the diagnostic laboratory? In Proceedings, Vol 12. North Am Vet Conf 1998;941-942.

2. Matlow A, Berte LM. Sources of error in laboratory medicine. Lab Med 2004;35:331-334.

3. Bonini P, Plebani M, Ceriotti F, et al. Errors in laboratory medicine. Clin Chem 2002;48:691-698.

4. Volmer PA, Meerdink GL. Diagnostic toxicology for the small animal practitioner. Vet Clin North Am Small Anim Pract 2002;32:357-365.