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10 Toxicologic Testing and Using Diagnostic Laboratories

SOURCES

Toxicologic testing may be available at many levels: in the veterinary clinic (for example, ethylene glycol testing), at a local or regional commercial veterinary diagnostic labo-ratory, or at a veterinary diagnostic laboratory associated with a veterinary school or state department of agriculture. When selecting a diagnostic laboratory the following cri-teria should be considered: cost, turnaround time, experi-ence with the sample type, available detection limits for the toxicant of concern, quality assurance and quality control programs, and assistance in the interpretation of results (Galey 2004 ).

The use of specialized instruments and highly trained personnel will often make toxicologic testing quite expen-sive compared to other diagnostic tests routinely per-formed for small animal patients. Many laboratories also charge additional fees for samples that are sent from another state, so be sure to ask about any additional fees that may apply.

Turnaround time refers to how long it will generally take to receive test results once samples arrive at the labo-ratory. Since some toxicologic tests may take several days or sometimes more than a week to complete, it will still be necessary to stabilize, decontaminate, and begin to treat the patient while waiting for results. Since some analyses can be completed more quickly, especially if the labora-tory receives prior notice, it is important to contact the laboratory directly before submitting samples for critically ill patients.

Common sample types submitted for toxicologic testing in veterinary medicine include blood, urine, stomach con-

tents, tissues, food, baits, water, and plant material. Each distinct sample type may require its own specifi c type of processing and analysis to detect the same toxic substance. Not all sample types may be appropriate for obtaining reliable results for all toxicologic tests. Diagnostic labora-tories should be able to recommend which sample types are best or preferred for specifi c toxicologic tests.

Detection limits refer to the minimum amount of a toxic substance that must be present to reliably produce a posi-tive test result. Detection limits will often be listed on a diagnostic report as the MDL, the minimum detection limit or method detection limit. When a toxic substance is either not found in a sample or appears to be present at a very low level below the MDL that is not confi rmable, the result may be reported as either “ none detected ” or less than the MDL. If a specifi c level of detection of a toxicant is needed for either diagnostic or treatment purposes it is probably worthwhile to contact the veterinary toxicology laboratory ahead of time to be sure that its method is able to adequately provide the desired information.

Quality assurance and quality control are the practices laboratories use to ensure that the results they report are consistent and accurate. This reliability is typically accom-plished by the use of standard reference materials, stan-dard data curves, spiked samples, regular use of check samples, and participation in multilaboratory profi ciency tests. When using an unfamiliar laboratory for the fi rst time, it may be prudent to inquire about its quality assur-ance and quality control program.

Once a test has been completed, interpretation of the results by a trained professional such as a veterinary

Small Animal Toxicology Essentials, First Edition. Edited by Robert H. Poppenga, Sharon Gwaltney-Brant.© 2011 John Wiley and Sons, Inc. Published 2011 by John Wiley and Sons, Inc.

Lisa Murphy

78 Section 1 / Fundamentals of Veterinary Clinical Toxicology

Environmental Samples

Many toxicants cannot be successfully detected in animal tissues or bodily fl uids, so the collection and submission of environmental samples may be critical to identifying an animal ’ s access to a potentially toxic substance. Common environmental samples that could represent sources of intoxication to small animals include foods, baits, plants, mushrooms, water, and other unidentifi ed substances or medications. For a description of typical sample amounts and how they should be collected, stored, and submitted for toxicologic testing, see Table 10.1 .

Antemortem

Common sample types that can be obtained from a live patient for toxicology testing include whole blood, serum, urine, vomitus, stomach contents, feces, biopsy speci-mens, and hair. Samples should be obtained preferably at the earliest feasible opportunity after the animal arrives at the clinic. If specimens cannot be collected before exten-sive medical treatments are instituted, any drugs adminis-tered or treatments given should be noted as part of the case history when submitting toxicology samples for testing. Suggested sample amounts and how they should be stored and transported are described in Table 10.2 . The sample size that can be safely collected from tiny patients

toxicologist, can be critical in determining its signifi cance to the case as a whole and may be used to direct additional patient care or diagnostic testing. To determine whether a patient ’ s result is abnormal or diagnostically signifi cant, something should be known about what “ normal ” results are expected to be for an animal of a particular species, age, or gender. As an example anticoagulant rodenticides would not be expected to be present in the blood of an unexposed animal, while a low level of lead in the same animal ’ s blood could be considered normal and not expected to be associated with signs of lead toxicity.

An experienced veterinary toxicology laboratory will be able to provide reliable, accurate, and useful results in a timely matter.

DIAGNOSTICS

The appropriate selection of diagnostic tests in cases of suspected intoxications will rely heavily on obtaining a good case history. Based on the historical information provided, a veterinary toxicology laboratory ’ s professional staff should be able to assist veterinary clinics with ques-tions about which tests should be selected for a given case, the best and appropriate sample types to submit, and the desirable sample quantities needed to run the requested analyses.

Table 10.1. Environmental sample types and amounts for toxicologic testing

Sample Type Amount Collection/Storage Method

Human or animal foods 500 grams (approximately 2 cups) Refrigerate or freeze in a well - sealed plastic bag or container.

Baits Entire amount present up to 500 grams (approximately 2 cups)

Refrigerate or freeze in a well - sealed plastic bag or container.

Plants Whole green plant including the roots; a fresh tree, or shrub branch with leaves (and fl owers if present); entire nut, fruit, berry, or other characteristic part of the plant

Press or wrap in newspaper, place in a sealed plastic bag, and then send to the diagnostic laboratory either fresh or frozen.

Mushrooms Whole mushroom or as much as possible if the only remaining source is from the animal ’ s vomitus or stomach contents

Keep relatively cold and dry in a paper bag to reduce moisture accumulation and subsequent spoilage.

Water 0.5 – 1 L (approximately 2 – 4 cups) Ideally, collect into a new, unused, clean, and dry glass container that is free of any detergent, chemical, food, or beverage residues.

Other unidentifi ed substances or medications

Representative amount of what is present up to 500 grams (approximately 2 cups)

Refrigerate or freeze in a well - sealed plastic bag or container.

Chapter 10 / Toxicologic Testing and Diagnostic Laboratories 79

poisoning. Common postmortem sample types are described in Table 10.3 . Rarely, collection of other post-mortem sample types may be necessary for the detection of some compounds, such as lung tissue for paraquat. This kind of special testing information can be obtained by consulting with the toxicology laboratory.

MANAGEMENT OF EXPOSURES

Diagnostic testing should not be substituted for general patient care and case management. All patients should be evaluated as individuals and life - threatening signs such as respiratory distress, cardiovascular compromise, and central nervous system abnormalities such as seizures should be addressed fi rst. Once the patient has been stabi-lized, diagnostic tests can be performed and specifi c care can be provided. Even in the event that diagnostic toxico-logic testing is not readily available, most intoxicated animals can recover if close monitoring, symptomatic treatment, and good nursing care are provided.

such as neonates and exotic species may be limited. Some form of toxicologic testing can still be performed in most cases, though the number and types of tests along with the ability to verify any positive results may be severely limited.

Postmortem

When patients have died as a result of a suspected toxico-sis it may be benefi cial to refrigerate or freeze samples for potential toxicologic testing while awaiting results of other diagnostic tests such as complete blood counts, serum chemistries, gross necropsies, and histopathology. These results may provide valuable information about possible cause(s) of death and subsequently what specifi c toxico-logic analyses should be pursued. One exception may be if other animals in the same household or neighborhood are sick or at risk. In these cases toxicologic testing may be pursued more quickly when the history and clinical signs observed are strongly supportive of or suspicious for

Table 10.2. Suggested antemortem samples for toxicologic testing

Sample Type Amount Collection/Storage Method

Whole blood 2 – 4 ml Refrigerate in calcium EDTA or heparin tube. Serum 2 – 4 ml Spin and refrigerate or freeze the serum in a plain glass or plastic

blood tube to separate from the red blood cells. Use a special all - plastic royal blue top for accurate zinc levels.

Urine At least 10 – 20 ml Refrigerate or freeze in a clean, tightly sealed plastic container. Vomitus or stomach

contents At least 10 – 20 ml Refrigerate or freeze in a clean, tightly sealed plastic bag or container.

Feces At least 100 grams Refrigerate or freeze in a clean, tightly sealed plastic bag or container. Biopsy specimens At least 2 – 5 grams Refrigerate or freeze in a clean, tightly sealed plastic bag or container. Hair 5 – 10 grams Place in a clean, tightly sealed plastic bag or container.

Table 10.3. Postmortem samples for toxicologic testing

Sample Type Amount Storage Method

Stomach contents At least 10 – 20 ml Refrigerate or freeze in a clean, tightly sealed plastic bag or container. Liver At least 5 – 10 grams Refrigerate or freeze in a clean, tightly sealed plastic bag or container. Kidney At least 5 – 10 grams Refrigerate or freeze in a clean, tightly sealed plastic bag or container. Brain Half of the brain Cut in half sagittally, leaving the other half and the midline intact for

histopathology. Refrigerate or freeze in a clean, tightly sealed plastic bag or container.

Body fat At least 5 – 10 grams Refrigerate or freeze in a clean, tightly sealed plastic bag or container. Eye One globe Refrigerate or freeze in a clean, tightly sealed plastic bag or container;

allows testing of either ocular fl uid or retina Injection site Surrounding tissue Refrigerate or freeze in a clean, tightly sealed plastic bag or container.

80 Section 1 / Fundamentals of Veterinary Clinical Toxicology

1. Manny, a 6 - year - old, intact male, 65 - pound German shepherd dog, presents to the veterinary hospital with a history of vomiting and muscle tremors that began about 2 hours ago. Prior to development of signs, Manny had been roaming free on the owner ’ s farm. The owners are concerned that Manny may have gotten into the shed that houses the farm pesticides. They also noted that Manny was seen digging in the compost pile in the pasture. Besides blood or serum, list two other samples from Manny that might be helpful to aid in diagnosis.

2. For each of the two samples selected in Question 1, indicate the amount to collect and the best method of storage/handling of the sample for submission to a diagnostic laboratory.

3. Which of the following is not correct regarding diag-nostic toxicology samples? a. Freezing or refrigeration is an acceptable means of

preserving samples prior to submission to the diag-nostic laboratory.

b. Veterinary diagnostic laboratory personnel can assist the veterinary technician in determining the best test and sample for a particular suspected toxi-cology case.

c. When determining whether a laboratory result is clinically signifi cant, the diagnostic toxicologist will compare the result with a range of “ normal ” values.

d. Toxicology testing should always be the fi rst step in attempting to diagnose the cause of death in a suspected poisoning case.

e. Environmental samples that may be submitted in a suspected toxicology case include animal foods, plants, mushrooms, and water.

4. Which of the following is true regarding the MDL (minimum detection limit)? a. Refers to the minimum amount of toxicant in an

animal that will cause a toxicosis b. Refers to the highest amount of toxicant that the

diagnostic test can detect in a sample

c. Refers to the minimum amount of a toxic sub-stance that must be present to reliably produce a positive test result

d. Refers to the lowest level of toxicant that will be toxic to a particular animal

e. Refers to the smallest amount of a nontoxicant (i.e., contaminant) that will give a false positive result

5. The appropriate selection of diagnostic tests in cases of suspected intoxications will rely heavily on the diagnostic laboratory obtaining _____________. a. An adequate blood sample b. A good case history c. A freshly dead animal d. Frequent calls from the veterinary clinic asking for

results e. Payment in advance

CHAPTER 10 STUDY QUESTIONS

ANSWERS

1. Urine, vomitus (stomach content), feces. 2. Urine, collect at least 10 – 20 ml and submit

refrigerated or frozen in a clean, tightly sealed plastic container. Vomitus (stomach contents), collect at least 10 – 20 ml or g and submit refrig-erated or frozen in a clean, tightly sealed plastic container. Feces, submit at least 100 g and submit refrigerated or frozen in a clean, tightly sealed plastic container.

3. d. In most cases toxicology testing should be per-formed after other testing (necropsy, histopa-thology, clinical pathology) results are obtained in order to help narrow down the potential toxi-cants to analyze for.

4. c. 5. b. A good case history is essential in knowing

what kind of testing would be most appropriate for a particular case.

REFERENCE

Galey , Frank D. 2004 . Diagnostic Toxicology . In Clinical Veterinary Toxicology , edited by Konnie H. Plumlee , pp. 22 – 27 . St. Louis : Mosby .