Symposium on Clinical Laboratory Medicine

Laboratory Diagnosis of Chemical Intoxications Frederick W. Oehme, D.V.M., Ph.D.*

While toxicologic problems are of general concern to all veterinarians, the small animal practitioner is recognizing the increasing importance of companion animal diseases produced by chemicals, and the future promises an increasing potential for chemical intoxications. The chemical and drug industries continue to produce and distribute everincreasing numbers and varieties of new and potent compounds. The widespread and often improper or overzealous use of such chemicals will increase the hazard for domestic animals. Growing public awareness of the potential danger from foreign chemicals has caused increasing client concern about proper use, possible biological and environmental adverse effects, and the probability of malicious poisoning. All too frequently, such inquiries result in medicolegal action, and the small animal practitioner is faced not only with the request for rendering appropriate prognostic and therapeutic measures, but also is challenged to provide confirmation for his working diagnosis. The role of the laboratory in assisting the clinician in arriving at his final diagnosis is a responsibility that is often unclear to all parties concerned and that is only beginning to be recognized in its own right.

ROLE OF TOXICOLOGY IN SMALL ANIMAL PRACTICE The initial recognition of potentially toxic disorders begins with the clinician. The small animal practitioner must continue to educate himself about the toxic hazards for his patients and should be capable of at least tentatively diagnosing the major clinical intoxications utilizing physical examination and office laboratory procedures. He should also be able to apply appropriate treatment to affected patients and render realistic prognoses. Finally, and perhaps most importantly, the clinician *Professor of Toxicology, Medicine, and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, Kansas

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should be able to utilize sound judgment in providing recommendations for the prevention of future poisonings and in giving counsel to emotionally involved clients intent on seeking legal recourse. Because of the wide spectrum of chemically-induced disorders, it is usual for each practitioner to recognize early those poisonings common to his particular practice situation. In addition, it is helpful to recognize the most commonly expected intoxications for each species of companion animal. In general, poisonings due to rodenticides appear to be most common, particularly those produced by strychnine, monofluoracetate, warfarin, and thallium. This incidence is followed in frequency by food intoxication· (enterotoxemias), insecticide poisonings (organophosphorus and carbamate compounds, chlorinated hydrocarbons), poisonings due to animals (snakes and toads), arsenic toxicity, metaldehyde poisoning, fungal intoxications, and lead and antifreeze toxicities. Strychnine and insecticide toxicities appear to be the most common poisonings observed in dogs. Cats often show toxic reactions to therapeutic agents and are especially sensitive to certain insecticides and to disinfectants and antifreeze. Birds frequently are presented with endogenous intoxications (auto-intoxications) due to inappropriate feedings, may be exposed to potentially toxic household chemicals, and are sensitive to pesticides and agricultural poisons. Exotic and zoo animals are often poisoned by rodenticides, by dietary contaminants, and by poisonous plants. The potential for adverse drug reactions or drug interactions when combination drug therapy is employed is always a potential hazard in any species of animal. Details of species toxicities have been reviewed individually, 4 and the reader is referred to a general review of diagnostic criteria for toxicologic disorders. 8 Additional suggested readings are provided at the end of this article. Regardless of the knowledge, experience, or degree of certainty provided by the examining clinician, there are numerous occasions where, because of clinical uncertainties, complicating factors, potential legal situations, or owner's request, the practitioner may wish to confirm his clinical diagnosis. It is at this stage that the toxicologic laboratory may offer a service, and indeed has a distinct responsibility.

ROLE OF THE TOXICOLOGY LABORATORY IN SMALL ANIMAL PRACTICE Purpose and Scope of a Toxicology Laboratory

Ideally, a laboratory is a support system for the practitioner and should be readily available to provide a source of information for the early diagnosis and treatment of patients. In this role it may be used to support a tentative diagnosis or to confirm a working diagnosis while the patient is still under treatment. The results of the laboratory procedures

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then offer guidance in continued therapy and provide valuable prognostic evaluations. The request for toxicologic services as a sequel to postmortem examination is less of an emergency situation, but may offer the most difficult challenges. Not only is confirmation of the clinical diagnosis desired, but the loss of animal life suggests that the owner may decide to recover his monetary loss. The results of the laboratory examinations are thus often crucial for potential future legal proceedings. The laboratory responding to requests for toxicologic analyses should preferably be readily accessible to the clinician and should offer rapid and reliable service. Although equipment necessary for the analytical procedure will vary with the individual request, a qualified laboratory will only accept responsibility for such assays that it can reliably and accurately perform. Minimal equipment for such procedures usually includes a spectrophotometer, chromatographic equipment, a variety of specialized glassware and measuring instruments, and a well qualified and competent professional staff. It is unusual for any one laboratory to offer qualified analyses for all potential toxicants, and the clinician will rapidly learn which procedures are most validly performed by the one or more facilities in his area. In some instances, specialized requests may have to be sent a considerable distance to a laboratory uniquely qualified to perform the desired assay. Once the clinician determines the appropriate laboratory for his particular purpose, it is important that he provide the laboratory with as much background information as possible. To this end, a telephone call is recommended, since it not only acquaints the laboratory with the practitioner's situation, but also offers the clinician a chance to determine if appropriate samples have been collected or if certain tissues are unnecessary for the requested procedure. Costs involved for the desired assays may also be clarified at that time and the client so informed. Embarrassment to all parties concerned may be avoided by a full understanding of the procedures, time, and expenses necessary. Frank discussion of the situation with laboratory personnel may often yield additional benefit by the toxicologist's suggesting alternative diagnoses that may have escaped the clinician's consideration. While some of the desired toxicologic laboratory procedures may be accomplished in a well equipped clinic or practitioner's laboratory, efficiency of performance and costs may require that the clinician have the toxicology tests referred to a local reference laboratory or to a more fully equipped, full-time commercial laboratory that offers toxicologic and other laboratory techniques. CLINIC OR PRACTITIONER's LABORATORY

The individual practitioner's clinic laboratory may be utilized to perform some preliminary and rudimentary toxicologic examinations. Minimal laboratory equipment and glassware are required, but a variety

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of fundamental items are necessary. These include a centrifuge, hot plate or Bunsen burner, separatory funnels, filtration equipment, assorted graduated cylinders, beakers and test tubes, and a variety of chemical reagents specific for the testing to be conducted. Distilled water and the ability to produce chemically-free glassware are also important. The tests should be performed by a laboratory technician who has the opportunity to become familiar with the individual procedures, and the practitioner should have some knowledge of the interpretation and application of the results. The clinic laboratory is best suited to perform screening tests that are relatively qualitative. Most practitioner's laboratories are appropriately utilized for these rapid procedures that give some indication for the confirmation or rejection of tentative diagnoses. The close proximity of the laboratory to the patient makes the utilization of samples from animals still undergoing therapy practical, but postmortem specimens may also be employed for these screening techniques. Vomitus, stomach washings, urine, and whole blood may be utilized from living patients. Digestive tract contents, urine, whole blood, and tissues from major organs (liver, kidney, lung) are available upon necropsy. Since the patient is readily available to provide sample material, an abundant sample should be taken of each specimen. A quantity may be preserved by freezing or refrigeration while the required amount is utilized for the test. At least I 0 ml of whole blood should be collected, 50 ml or more of urine are desirable, and at least 200 gm (and preferably all available) vomitus, stomach or digestive tract content should be collected. Entire organs may be preserved and portions utilized as appropriate. Suspected sources of the chemical agent may also be collected and assayed. Generous portions (preferably 1 pound) of feed, water, weeds, or suspected baits are desirable for determining the source of a toxicity. Screening Tests

Several chemical and biological procedures are of potential value to the clinician. Some are specific (such as the thallium procedure), whereas others provide nothing more than a possibility of the presence of the suspected chemical. Chemical procedures for thallium, arsenic, mercury and antimony, aflatoxins, drugs such as aspirin, phenothiazine and sulfonamides, and phenol may be considered, and biological tests are available to screen for herbicides-fungicides, insecticides, strychnine, warfarin, and the organophosphorus and carbamate chemicals. Thallium. Urine of suspected poisoned patients may be tested with the following procedure that is sufficiently sensitive to detect urine thallium up to 10 days following the onset of toxicity. Distilled water saturated with bromine (stored in a glass-stoppered amber bottle and allowed to stand 12 hours before use), 10 per cent sulfosalicylic acid in distilled water, concentrated hydrochloric acid, 0.05 gm Rodamine B in l 00 ml concentrated hydrochloric acid, a standard amount of thallium

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acetate or sulfate in distilled water, and benzene are the necessary reagents. Three test tubes are labeled, respectively, as blank, standard, and unknown (test). Four drops each of water, standard and test urine are placed in the respective tubes and to each is added bromine water until a slight yellow color persists. Sulfosalicylic acid is added by drops to each tube until the yellow color is barely removed. Then one drop of the concentrated hydrochloric acid and one or two drops of the Rodamine B solution are added to all the tubes. Each tube is shaken gently and briefly. Benzene (0.5 ml) is added to each tube, the tubes are shaken gently, and the two layers are allowed to separate. The presence or absence of color in the upper layer of each tube is important; a positive test is indicated by a reddish-purple color in the upper layer. This may be confirmed by observing the tubes under ultraviolet light, and a positive test will produce a yellow-green fluorescence in the upper layer. While the test rarely gives false positives, occasional false negatives may be observed. Repeated samples of urine should be analyzed from suspected cases since one negative urine test for thallium does not necessarily mean that a subsequent sample will not give a positive thallium response. Arsenic, Mercury, and Antimony. Practitioners may detect toxic quantities of several heavy metals in digestive tract content, suspected bait, or liver and kidney specimens by employing the Reinsch test. Although arsenic, mercury, antimony, and also bismuth and silver, and large concentrations of other less common metals will give a positive reaction to this procedure, laboratory technicians who are experienced in utilizing this technique will find it quite helpful in providing presumptive evidence of the presence or absence of a suspected toxin. Approximately 25 gm of finely chopped digestive tract contents, liver, kidney, or other tissue are placed in a chemically clean Pyrex container. Ten ml of concentrated hydrochloric acid mixed with 90 ml of distilled water are added to the container. A strip of bright, pure copper foil or wire, previously cleaned with dilute nitric acid or steel wool, is added to the mixture which is brought to a boil and maintained at a slow boil for 30 to 60 minutes. At the end of that time, the copper foil or wire is removed, rinsed in water, and permitted to dry. A discoloration or coating of the copper is presumptive evidence of the presence of one of the five most reactive metals and may suggest the presence of high concentrations of other less frequent contaminants. Arsenic and bismuth produce a gray to black deposit on the copper, whereas mercury and silver produce a shiny silver deposition. Antimony is revealed by a violet or blue-violet coating on the copper. To prove that the coating material is arsenic, the coated copper may be placed in a small test tube and the lower end of the tube heated gently. Upon heating, the arsenic forms a characteristic sublimate of glistening octahedral or tetrahedral crystals on the cooler (upper) portion of the tube. The characteristic shape of these crystals may be identified under low-power magnification.

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Aflatoxins. The rapid qualitative test for aftatoxins consists of placing 100 gm of feed material in a blender with 300 ml of a solvent consisting of 7 parts methanol and 3 parts water. The mixture is blended at high speed for approximately 5 minutes and then allowed to set until a layer of noncloudy liquid forms on the surface. Filtration through a muslin cloth with vacuum may be used to increase the rate of separation. Eighty to 150 ml of clear surface liquid are removed and placed in a 500 ml separatory funnel. Following the addition of 30 ml of benzene, the funnel is shaken for 30 seconds, and 200 ml of water added. Following separation, the lower layer is discarded. The upper layer is placed in a beaker, evaporated to dryness, and then resuspended to 0.5 ml of benzene. A small amount (50 microliters) is spotted on No.4 Whatman filter paper, allowed to dry, and then is placed under a long-wave ultraviolet light. If the spot on the filter paper gives a fluorescence, the sample is likely to contain aftatoxins. Aspirin. A presumptive test for salicylates may be performed on urine, whole blood, or serum samples. For urine, 2 ml of urine are added to 1 ml (20 drops) of 10 per cent ferric chloride solution. If salicylates are present, a purple color will result. This test is positive within 60 minutes after ingestion of the aspirin. Phenol derivatives, if present in the sample, will also give a positive result. If whole blood is to be tested, 5 ml of blood are acidified with 0.2 ml concentrated hydrochloric acid. This is extracted with 10 ml ethylene dichloride. After separation, the upper layer is discarded and 4 drops of 10 per cent ferric chloride and 2 ml distilled water are added to the remaining (ethylene dichloride) layer. The development of a purple color upon shaking is positive for the presence of salicylates. For serum samples, a white dish (of porcelain or similar material) is used. Two drops of serum are placed on the white dish, and one drop of 10 per cent ferric chloride is added. The development of a purple color is a positive reaction for the presence of salicylates. Phenothiazines. A presumptive test may be utilized on urine samples to confirm the presence of phenothiazines. Six drops of concentrated sulfuric acid are added to 2 ml of urine, followed by 2 drops of 10 per cent ferric chloride. Urine from animals with an overdose of phenothiazine or the phenothiazine derivatives (chlorpromazine, promazine, prochlorperazine) produces a light pink to purple color. Sulfonamides. Urine specimens from animals suspected of sulfanilamide nephrotoxicity may be used for this presumptive test. One drop of urine is placed on an unprinted portion of newspaper or cheap wood-pulp paper. On drying, one drop of concentrated hydrochloric acid is placed on top of the remaining stain. The production of an orange color indicates the presence of a sulfanilamide derivative. One drop of concentrated hydrochloric acid should be placed by itself on an unused portion of the paper as a control. The faint straw-yellow color that results should not be confused with the more orange color indica-

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tive of a positive test. This procedure may also be applied to tablets of medications suspected of containing sulfanilamide compounds. Phenolic Chemicals. Two simple presumptive screening tests for phenolic compounds may be employed on urine samples from animals suspected of phenolic poisoning. Positive reaction to both is presumptive evidence of poisoning. In the first procedure, 1 ml of a 20 per cent aqueous solution of ferric chloride is added to 10 ml of urine. If the resulting color is purple, the test is positive for phenol. In the second test, 10 ml of suspected urine is boiled with 1 to 2 ml of Millon's reagent. Millon's reagent is made by dissolving l 0 gm of mercury in 20 ml of nitric acid, diluting with an equal amount of distilled water, allowing the mixture to stand for two hours, and then decanting the excess water. A positive reaction of the reagent with the urine results in a red color. Biological Tests. A variety of biological procedures may be used to determine the specific or general toxicity associated with selected samples. Urine, vomitus, digestive tract content, dietary components, or baits may be assayed. A presumptive biological test for strychnine involves making urine, vomitus or stomach content, or a solution of the suspected bait material alkaline with 10 per cent sodium hydroxide. The mixture is extracted several times with ether, and the composite extracts are evaporated to dryness. The residue is redissolved in 1 ml of water, brought to neutrality, and injected into the abdominal sack of a live frog. Typical tetanic spasms will occur within 10 minutes if the sample is positive, and are especially induced when tapping the platform the frog is sitting on or directly stimulating the frog. Herbicides and fungicides may be detected in stomach contents or source material by utilizing the fact that this group of chemicals is selectively more toxic to fish than to other animals. A portion of digestive tract content or bait is made into a slurry with distilled water, and approximately l teaspoon of this slurry is added to a small to average sized aquarium containing several goldfish or similar small, hardy fish. These fish are then observed for unusual behavior or toxic reactions. If no death or obvious illness occurs within 30 minutes, approximately 15 ml (3 teaspoons or Y2 fluid ounce) more of the slurry is added and observation is continued for an additional hour. Death of one or more of the fish within that period of time is considered tentative evidence that the sample contained a herbicide or fungicide. Presence of insecticide may also be determined by a similar technique. Stomach content or suspected source is mixed with an approximate equal amount of water and placed in a sealed glass jar. Live insects (flies, mosquitoes, various garden insects) are placed into the container with the sample and the container is sealed. After 1 to 2 hours of observation, specimens containing an insecticide should produce an obvious kill of the introduced insects. For suspected nonspecific toxic substances in foods or stomach con-

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tents, the material may be fed to one or more test animals to determine if any toxic agent is present. For foods, three to five mice, rats, or guinea pigs may be used. For bird feed, one or more chicks may be employed. The test animals are weighed at the beginning and end of the procedure and the suspected material is fed free choice for 7 to 10 days. Daily consumption of the material is determined and the test animals are observed daily for evidence of toxicity. If the test animals remain normal throughout the test period and have consumed a reasonable amount of the test sample, it is assumed that the food does not contain a toxic substance. If stomach contents are to be tested, one or more laboratory animals may be force-fed a quantity of the suspected material. Evidence of toxicity should be observed within 24 hours. If this does not occur, the digestive tract contents must be assumed to be nontoxic, or deterioration or dilution of the sample may have occurred. This test must be interpreted with some caution since it is not always possible to extrapolate data from test animals to the original domestic species involved in the suspected poisoning. Biological responses to specific therapy may also be used as a screening test for certain intoxications. Animals suffering from organophosphorus or carbamate poisoning should respond promptly (within 1 to 5 minutes) to the intravenous administration of recommended amounts of atropine. Such a response is a valuable and immediate diagnostic tool. Animals not responding to such injections are probably not suffering from either organophosphorus or carbamate insecticide poisoning. Likewise, the treatment of suspected warfarin intoxications with whole blood and vitamin K1 should show a prompt clinical response in clotting time, prothrombin time, and general clinical condition. Patients that do not make a distinct improvement within 24 to 36 hours of initial treatment should undergo diagnostic reevaluation. Confirmation Needed. All positive reactions to screening procedures should be verified by duplicate sample submission to a competent analytical laboratory for quantitative analysis. This procedure confirms the tentative diagnosis and results and provides absolute evidence in the event of legal action. Negative reactions must be interpreted with common sense, since unfamiliarity with the procedure may result in faulty technique, or improper sample selection may produce false negatives. An experienced toxicologist should be consulted in problem cases or when any doubt of the test results is present. Advantages and Disadvantages The utilization of a practitioner's laboratory for toxicologic testing is a convenient and efficient diagnostic service. Samples may be secured and immediately processed for the desired analysis. If inadequate or inappropriate samples are taken, the patient is often still available for the securing of additional specimens. Further, as the patient's condition changes, additional tests may be performed as needed. The results from

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such assays are promptly available to the clinician and may be interpreted with full and direct knowledge of the patient's current condition. Unfortunately, most practitioners do not have the time, equipment, or personnel to adequately develop and maintain the appropriate testing procedures. The clinician must first have knowledge of the application of the various tests and then must provide laboratory help with sufficient time to develop reasonable expertise in conducting the desired procedures. Equipment and chemicals must be maintained in readiness, and personnel time and clinic space must be kept available for the performance of these procedures. Unless the clinic has sufficient demand for such assays, personnel and equipment may become outdated and inefficient in conducting the chemical determinations. In specific circumstances, however, the necessity for such on-thespot testing may prove of sufficient worth to result in a properly staffed and equipped facility. As more and repeated procedures are performed, increasing expertise and efficiency will develop. In such instances, the local practitioner's laboratory may begin attracting samples from surrounding practices and may serve as a reference laboratory for individual practitioners that do not have sufficient demand to warrant establishing their own facilities. The developing reference laboratory may then wish to grow and expand with the demands, in which case more specific analytical toxicologic procedures may be utilized. These are documented in the publications by Sunshine cited in the suggested references. 5 - 7 LOCAL REFERENCE LABORATORY

Practitioners' laboratories or specific chemically-oriented facilities within the local environment of the small animal practitioner may provide excellent service for conducting toxicologic analyses. Human hospitals, pharmaceutical or drug firms, research institutions, college and university laboratories, and full time commercial testing laboratories may exist sufficiently near the practitioner's clinic to warrant utilization as a toxicology reference laboratory. Such laboratories would be considerbly better staffed and equipped than the local veterinary hospital and would utilize more extensive chemical instrumentation and techniques in performing the tests. Assays would be quantitative, but each facility would usually have specific procedures which it would be capable of conducting, while it may be unable to perform other analyses. Such local laboratories are usually easy to become acquainted with, and practitioners considering utilizing these would be wise to visit the facilities and become personally acquainted with the personnel, their capability, and the general philosophy of their activities. If the visitation produces a favorable impression and contacts are made for the processing of toxicologic requests, it is wise to initially process duplicate samples through the new facility to confirm the reliability and precision of the

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results. Validity of the values of the procedures may be further confirmed by sending additional samples to known competent laboratories that have previously yielded satisfactory results. Such procedures should in no way be considered derogatory of the local laboratory, but rather should serve to provide practitioner confidence in the toxicologic results produced. Once such local laboratories have been demonstrated to produce efficient and reliable results, the small animal practitioner has a readily available and competent facility to handle his toxicologic laboratory demands. CoMMERCIAL LABoRATORY

Commercial laboratories are facilities that are in the business of conducting reliable and legally accepted chemical analyses. In most cases these are large institutions, often associated with research facilities or pharmaceutical and drug firms, that specialize in the conduct of chemical determinations. In certain instances, independent chemical or toxicologic laboratories have developed which specialize in industrial and biologic specimens. Such facilities are usually well staffed and equipped, but of necessity must demand fees appropriate for the costs involved in conducting the desired analyses. Such facilities are usually accredited by one or more professional, state, or federal agencies and are certified to be capable of producing accurate results. A variety of spectrophotometers, chromatographs, measuring equipment, digestion facilities, highly specialized glassware, numerous chemicals, and elaborate quality control procedures are almost always employed in such organizations. Under normal conditions, the cost of equipping such a laboratory would be prohibitive except for large and specialized group practices or hospitals. Commercial laboratories are usually able to perform a variety of toxicologic tests. In many cases, the laboratory will have specific ability to assay and quantitate for unique chemicals; an example is the Wisconsin Alumni Research Foundation Laboratory in Madison which is capable of specifically assaying for warfarin. These capabilities are usually well known and occasionally several different laboratories may be required so that the practitioner has the potential for requesting a range of toxicologic assays. Laboratories will usually accept a variety of samples, but the practitioner should be certain that proper samples are selected for the desired assays. Since most commercial facilities are chemically oriented, they are not accustomed to dealing with large amounts of biological specimens. Appropriate containers should be utilized for packaging these samples and liquids, such as blood, urine, stomach contents, or water, should be sealed in glass or heavy plastic containers that can be closed tightly. Specimens should always be packaged individually and wrapped so that the contents will not leak and contaminate other specimens. Identification of each specimen, together with history and

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the desired analysis, will ensure adequate communication between the practitioner and the laboratory. Packaging the entire set of specimens in a professional appearing format enables the laboratory to be enthusiastic about receiving, performing, and reporting the results of the requested testing. Specimens sent a considerable distance should be adequately preserved to prevent deterioration and the practitioner may wish to contact the laboratory to determine the most efficient way of transport. If one is in doubt about the appropriate selection of tissues or the procedure for submitting samples, considerable time and confusion can be avoided by telephoning the laboratory directly and determining the most desirable procedures. Since commercial laboratories are in the business of performing chemical analyses, the costs for their assays may be more expensive than those produced by a smaller laboratory or hospital. The practitioner should be well aware of the fees involved with any toxicologic requests so that the client and the veterinarian are not embarrassed upon receipt of the bill. On the average, a quantitative specific assay for toxin will cost $30.00 per sample. Qualitative or screening procedures may be somewhat less, whereas specific and highly specialized procedures for certain toxicants may prove considerably more costly. Because of this cost, and the many toxic chemicals for which analysis is feasible, the veterinarian should never request a laboratory to "check for all poisons," even if the animal died of unknown causes. Such a request not only indicates ignorance on the veterinarian's part, but may also produce an uncooperative attitude from the laboratory. Rather, the practitioner should supply as much information to the laboratory as possible, preferably should consult with a toxicologist at the laboratory by phone to determine one or more reasonable etiologic agents, and should then request assays for several specific and most likely chemicals. Such a philosophy will produce a good working relationship that will result in both parties being rewarded with the results. Advantages and Disadvantages The advantages of a commercial laboratory are its expertise and well equipped facilities. Most commercial institutions conduct numerous toxicologic analyses daily and are able to efficiently and validly offer the small animal practitioner quality service. Quality control procedures are usually well established and adequate to detect unexpected variations in instrumentation or technique. A quality and reliable result is the paramount benefit to be gained from a commercial facility. However, few commercial laboratories are located in the immediate vicinity of the practitioner's clinic. Hence, transportation of the sample to the laboratory and the associated packaging, shipment difficulties, and costs are hurdles that the busy practitioner may find cumbersome.

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Once these submission procedures are developed, the difficulties become less and the established pattern becomes relatively routine. Nevertheless, since certain specific tests require selected laboratories, the practitioner may be forced to utilize several different laboratories to secure a full array of toxicologic assays. The costs of such testings may also be relatively expensive and clients may be reluctant to have such procedures performed. A final major disadvantage of the commercial laboratory is the considerable lapse of time that may occur between the practitioner's submitting the sample and his receiving the results of the requested analyses. This is due not only to the length of time required for transport of the specimens to the laboratory, but to the laboratory's work load and schedule for performing the requested testing. In many cases two to three weeks or longer are necessary before the results are available, and in the case of an actively intoxicated patient, this is too long for the results to be of benefit in determining appropriate therapy. The ideal situation is to have the local practitioner located near a competent commercial laboratory with which he has a good working relationship. This would minimize the disadvantages associated with most commercial facilities, but would still provide the practitioner with reliable and legally sound toxicologic results.

COLLECTION AND SUBMISSION OF SAMPLES

The selection of tissues for analysis and their proper submission to the testing facility are important medical and legal procedures for securing a useful toxicologic analysis. Samples commonly collected for laboratory assays include liver, kidney, stomach or intestinal contents, urine, and whole blood. At least 10 ml of whole blood should be submitted, together with 50 ml or more of urine, and at least 100 gm each of liver and kidney. Digestive tract contents of at least 200 gm should be collected if available. In specific suspected intoxications, the collection of other selected organs is indicated. The entire brain, 100 gm or more of body fat and spleen, and generous samples of hair or nails and bone may be of special value in certain circumstances. Often overlooked is the submission of samples from suspected sources of the etiologic chemical agent. Feed, water, weeds in the area, and suspected sources or baits are excellent samples for determining the possible source of a poisoning. Generous portions (in excess of 200 gm and preferably 1 pound) of each sample should be provided to the laboratory. The possibility of laboratory analysis of samples from patients still ill should not be overlooked. Whole blood, urine, vomitus or stomach washings, and fecal samples in instances of chronic poisoning are all useful in securing rapid laboratory confirmation of suspected dog

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or cat poisonings. A listing of the suggested specimens and amounts of each specimen desired for the toxicologic tests commonly utilized in small animal practice is given in Table 1. For certain toxins, special attention must be paid to the method of handling and submitting the required samples. Specimens for submission should be taken free of chemical contamination and debris, and should not be washed because of the potential of contaminating the specimen or removing residues of the toxic material. Clean glass or plastic containers that can be tightly sealed are excellent for collecting specimens. Each sample should be preserved separately in an individual container labeled with the owner's and animal's identification and the type of tissue or specimen in the container. Preservatives, such as formalin, should never be added unless there is a specific reason for doing so. In those cases, such information should be included on the specimen label. Samples of the preservative, if used, should also be submitted separately for possible reference analysis. Serum or blood samples should be kept refrigerated, whereas tissue specimens are best frozen (and should be packaged so that they arrive at the laboratory while still frozen). The importance of supplying the laboratory with a complete account of history, signs, and lesions observed cannot be overemphasized. Such information is equally important when analyses originally requested prove negative and the need for intelligently selecting other analytical procedures becomes obvious. If adequate specimen material and a detailed history of circumstances, signs, and necropsy lesions are available, the laboratory is in an excellent position to provide optimal service and assistance. Such documentation also permits the firm establishment of responsibilities in the event that legal action occurs later. In certain toxicologic situations, a concurrent histopathologic examination is indicated. Samples submitted for histopathology should be preserved in I 0 per cent formalin and shipped in containers separate from those for which chemical analysis is requested. Since the tissues for chemical assay are usually frozen, this requires that separate specimens and separate packages be sent to assure that the formalinized tissues do not also undergo freezing and become worthless for histopathologic study. If in doubt, it is best to submit abundant samples for both procedures, since it is easier to discard excess specimen than to attempt to secure more after the carcass has been discarded. Plastic bags, cardboard, newspaper, and various forms of ice are good for packing specimens. Liquids should be shipped in leak-proof containers and individually wrapped in packing material to prevent leakage and contamination of other specimens or accompanying mail. Ideally, the best method of submitting samples to a laboratory is by personal messenger. Often the owner of an affected animal is sufficiently concerned to make the delivery himself.

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Table 1. Specimens Required for Selected Toxicologic Tests* AMOUNT OF ANALYSIS REQUESTED

Aflatoxin Ammonia ANTU Arsenic Calcium Carbon monoxide Chlorinated hydrocarbon insecticide Cholinesterase Copper Ethylene glycol

SPECIMEN REQUIRED

Food Whole blood, urine Stomach contents Stomach and intestinal contents, liver Liver, kidney Food, stomach contents Urine Serum Food Whole blood Whole blood Body fat, stomach contents Liver, kidney Whole blood Whole blood Liver, kidney Feces Serum, urine Kidney

SPECIMEN DESIRED

200 gm 5ml 100 gm 200 gm 50 gm 100 gm 50 ml 2m! 25 gm 15 ml 10 ml 100 gm 50 gm 10 ml 10 ml 50 gm 100 gm 10 ml Both kidneys

SPECIAL PRECAUTIONS

Keep dry and cool Maintain air-tight Freeze until tested Must be tested 12-24 hours after ingestion

Separate clot from serum; hemolysis must not be present Keep tissues separate and free of contamination; use only chemically clean glass jars to package Keep refrigerated

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Fix in formalin for histopathologic examination

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Fluoroacetate (I 080)

Lead Methemoglobin Nitrate, Nitrite Organophosphorus insecticide, Carbamate insecticide Oxalate Phenol, phenolic compound Phenothiazine or derivative Strychnine Thallium Warfarin Zinc

Stomach contents Kidney Urine Liver Bait, source Whole blood Liver, kidney Whole blood Water Source Body fat, stomach contents Whole blood Urine Food Kidney

All available One whole 50 ml 50 gm 100 gm 10 ml 50 gm 10 ml 50 ml 100 gm 50 gm 10 ml 50 ml 100 gm Both kidneys

Stomach contents Source Food or other source Stomach content, urine Liver Urine Liver, kidney Liver, food, source Liver, kidney Source

500 gm 200 gm 50 gm All available 50 gm 10 ml 50 gm 100 gm 50 gm 100 gm

Freeze until tested

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Use only heparin or citrate as anticoagulant

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Fix in formalin for histopathologic examination Pack in air-tight container

*Modified from Buck, W. B.: Use of laboratories for the chemical analysis of tissues. In Kirk, R. W. (ed.): Current Veterinary Therapy V. Philadelphia, W. B. Saunders Co., 1974, p. 166.

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EvALUATION OF RESULTS

The final phase in the utilization of the toxicologic analysis is the evaluation of the result. As with many laboratory tests, frequently too much reliance is placed upon an individual value, when many factors in the biology of the patient and the exposure circumstances are capable of producing considerable variation. In addition, the nbrmal laboratory variations inherent in any procedure add to the variability of results. Practitioners utilizing their own facilities or accustomed to results from a single commercial laboratory will be able to relate the significance of reported values to their previous observations. However, if a new procedure is attempted, or a different laboratory is used to conduct the analysis, some caution must be given to the specific interpretation of reported toxicologic values. The reporting laboratory may then be of assistance by providing the usual range of values that are included in their "normal" results and those that are considered significantly elevated. The significance of the individual laboratory data should be interpreted carefully by the practitioner, taking into consideration all available evidence. Because of species variations and the frequency of unusual circumstances of chemical exposure, interpretation of the results is often a difficult task. Although there appears to be slightly better consistency in instances of toxic concentrations in biological specimens than with routine biochemical determinations, the mere presence of the suspected toxicant is not always sufficient to confirm poisoning, nor is a negative finding conclusive evidence that a toxicosis did not occur. The persistence of certain compounds (such as the heavy metals and chlorinated hydrocarbon insecticides) and their common environmental occurrence ensure that these chemicals are usually detected in most animal tissues regardless of the cause of death. Other very rapidly metabolized toxins (such as the organophosphorus and carbamate insecticides) may not be detected on postmortem chemical analysis even though death was a direct result of exposure to one of those compounds. Proper evaluation of the laboratory results is greatly aided by familiarity and experience with animal toxicities. The practitioner's previous experience with similar toxicities should help him in evaluating the assay values, but it is important to recognize that toxic levels are relative to those concentrations normally found in the healthy animal; thus, it is important to determine that the chemical concentrations detected are meaningful and indeed reflect significance. To this end an experienced toxicologist is of potential value in helping to relate the data to the clinical case. The practitioner should not hesitate to contact such individuals for assistance at this crucial time. In general, given a proper history and the opportunity to consult with the practitioner, the analytic laboratory or a consulting toxicologist

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will usually be able to suggest proper interpretation of the test results. Often such consultation will bring into focus the history, clinical signs, and the results of histopathologic tissue examination together with the level of foreign chemical detected. Having available the complete data on the patient, including necropsy and histopathologic results, will greatly assist in relating the results of toxicologic assay to the potential etiology. However, the final responsibility for a diagnosis rests with the clinician after all possible circumstantial, clinical, postmortem and pathologic, and laboratory findings have been evaluated.

ROLE OF THE PRACTITIONER IN THE DIAGNOSIS OF SMALL ANIMAL TOXICITIES The diagnoses of small animal poisonings begin and end with the practitioner. He is the individual who initially receives and examines the patient, conducts preliminary evaluations, selects and submits specimens for analysis, and after accumulation of all available data and results, makes the final interpretation and diagnosis. It is thus important that the clinician be aware of all possible and probable toxic etiologies that may be involved in each case. A wide range of clinical syndromes must be considered and the differential diagnoses reduced to one or more of the most likely. Correlation of clinical signs to possible intoxications depends upon a knowledge of the variability in response and the observation that few intoxications are "textbook" examples. Support of his tentative diagnosis may then be provided by laboratory analysis, and the selection of appropriate specimens for the particular assay to be requested is vital. An awareness of which tests can or cannot be performed, and a realistic approach to the economics and practicality of the analytical situation, assures that the most beneficial utilization will be made of the available toxicologic procedures. If current trends persist, poisoning cases involving small animals are increasingly likely to be presented to the veterinarian. Implications, insinuations, tentative diagnoses, and general statements made by the veterinarian are often used by the client as a basis for legal action. Since such efforts are difficult to halt once direction is established, it is important that the veterinarian be factual and explicit in his statement to owners of poisoned animals. Many years may pass from the time of actual poisoning to the holding of a court trial, and it is of the utmost importance that records and strict documentation of circumstantial, clinical, pathologic, and laboratory findings be complete and accurate. A veterinarian dealing with any potentially legal situation must be absolutely certain of the facts involved and should see that detailed records are maintained and safely stored for possible future reference. Thus, the practitioner's role is an all important one. He is morally,

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ethically, and legally responsible to provide the best possible care for his patients and quality service for his clients. With the rapid development of toxicologic hazards and the increasing awareness of pet owners for potential chemical hazards in their animals, the practitioner is being asked for a greater understanding of the biological effect of toxins and the laboratory diagnosis of poisonings. The future only promises more of this concern and requires that the practitioner have continually new and updated information available. It is hoped that this will be continually provided through the close cooperation of practitioners and specialists such as toxicologists. REFERENCES l. Buck, W. B., Osweiler, G. D., Stahr, H. M., and Van Gelder, G. A.: Diagnostic proce-

dures in veterinary toxicology. Clin. Toxicol., 5:143, 1972. 2. Buck, W. B.: Use of laboratories for the chemical analysis of tissues. In Kirk, R. W. (ed.): Current Veterinary Therapy V. Philadelphia, W. B. Saunders Co., 1974, p. 166. 3. Oehme, F. W.: Toxicologic disorders. In Ettinger, S. J. (ed.): Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat, Volume l. Philadelphia, W. B. Saunders Co., 1975, p. 80. 4. Oehme, F. W. (ed.): Symposium on clinical toxicology for the small animal practitioner. VET. CuN. NoRTH AM., 5:587, 1975. 5. Sunshine, I. (ed.): Handbook of Analytical Toxicology. Cleveland, The Chemical Rubber Co., 1969. 6. Sunshine, I. (ed.): Manual of Analytical Toxicology. Cleveland, The Chemical Rubber Co., 1971. 7. Sunshine, I. (ed.): Methodology for Analytical Toxicology. Cleveland, The Chemical Rubber Co. Press, Inc .•. 1975. Kansas State University College of Veterinary Medicine Manhattan, Kansas 66506