Diagnosis of Poisoning: Selection and Submission of Samples for Laboratory Investigations

An experienced and alert veterinarian can make a tentative diagnosis of poisoning based on case history, clinical findings and environmental evidence. However, like any other disease, a complete diagnosis of poisoning should include identification of the clinical manifestation produced by the causative agent, abnormality of structure or function produced by it and finally the identification of specific cause (poison) of the illness. As such confirmatory laboratory assistance is essential for making complete diagnosis. Laboratory investigations are crucial for patient management as well as to address a number of socio-economic and legal issues involved with the cases of poisoning, especially those related to industrial toxicities and mass mortality. Laboratory tests also help to assess the extent of toxic effects, and also to understand pathogenesis and underlying organ dysfunction specific to identified poison.

Laboratory investigations, especially involving chemical analyses for poisons are quite laborious and expensive. It is therefore necessary to submit only rightly selected and preserved specimens, transported in appropriate conditions to the laboratory for optimal efficiency for confirmation of the diagnosis.  Following broad guidelines may be useful for collection and submission of samples to laboratory for diagnosis of poisoning:

 Veterinary diagnostic laboratory

• Contact a diagnostic laboratory beforehand to know what tests are offered and preferred protocol if any followed for collection and submission of samples for diagnosis of poisoning.
• Most veterinary colleges and animal sciences institutes of the Indian Council for Agricultural Research (ICAR), including Centre for Animal Research and Diagnosis (CADRAD) at ICAR-Indian Veterinary Research Institute, Izatnagar have well established veterinary diagnostic laboratories in the country. There are also a number of private veterinary diagnostic labs in major cities of India, which provide a range of veterinary diagnostic services including molecular diagnostic and toxicological testing. The standard medical diagnostic laboratory can also be approached for testing of veterinary samples.

Samples for investigation of poisoning cases

• In addition to samples from live/dead animals, specimens for laboratory analysis should invariably include material suspected as the source of poisoning to animals e.g. feed and feedstuffs, forage, plants, water, soil, baits, discarded materials, etc.
• Whole blood, urine, milk, rumen aspirate, hair, feces are easily available samples ater, soil, baits, discarded materials, etc from live animals for laboratory investigation. Liver biopsy (100-200 mg) may be collected from cattle suspected for metal poisoning.
• Invariably collect samples of alimentary tract together with ingesta including contents from rumen and intestinal segment, and liver when poisoning is suspected via oral route.
• Skin or lung specimens should also be collected when exposure is suspected via cutaneous and respiratory routes.
• Additional specimens required for laboratory testing from live or dead animals may vary with type of poisoning suspected and some of which are listed here:

Types of samples for laboratory investigations in poisoning

Collection and storage of samples

• Select right samples and collect them appropriately both for histological and toxicological testing.
• Submit specimens in adequate quantities for chemical analysis for example at least 10-20 ml each of blood or serum, 20 ml each of urine and milk, 20 g hair,  100-250 ml rumen content and ingesta, and   100 g each of liver,  kidneys, fat, brain, and other tissue,  250 g  each of feed,  forage, vegetation, and soil,  and 250 ml water.
• Plant samples should be collected for identification as well as for chemical analysis for plant toxins. For identification, submit the whole plant along with roots and leaves. Place whole plant between two clean papers (don’t use newspaper) or in a big paper envelope.  Wrap it carefully in waterproof plastic cover. A good picture of plant can also be sent to facilitate identification.
• Vacutainer tubes (preferably high density polyethylene-HDPE) with anticoagulants are very handy for the collection of blood samples. Urine should be collected in 50 ml commercially available screw cap plastic vials. Ocular fluid can be collected in small HDPE tubes and frozen. High-quality aluminum foil zip lock pouch (available commercially for packing food items) can be used for collecting ingesta and rumen content, faeces, hair as well as soil and feed and fodder samples. Milk should be collected in clean HDPE jar or tubes and refrigerated.
• Collect rumen contents from multiple locations in the rumen and mixed thoroughly.  Similarly, samples form large organs like liver and those of feed, fodder, forage, water and soil should be collected from multiple locations.
• Each sample should be stored in thoroughly cleaned separate containers with proper labeling and identification. Do not use plastic ware in suspected cases of organic chemical poisonings.
• Do not add anything to the samples for chemical analysis i.e. No preservative, No antiseptics and No fixatives unless otherwise recommended as in case of whole blood and ingesta for nitrate/nitrite poisoning. In exceptional conditions, ethyl alcohol (1ml/ g of tissue) can be used as preservative.  Separately send sample of the preservative along with the specimen consignment.
• Samples for histopathological examination should not be more than 0.5 cm width and preserved in 10% neutral-buffered formalin in the ratio of 1:10 (tissue: formalin). Samples for histopathology may be pooled within the same container with proper marking.

Shipping of diagnostic specimens

• Get specific guidelines (if any) and instructions from the regulatory authorities and identified laboratory for proper transport of samples for diagnosis of poisoning.
• The samples should be accompanied with a submission report consisting of complete clinical history, signalment of the animal, postmortem findings, treatment details and feeding, management and environmental information. A note on legal implications, if any, should also be furnished.
• Pace samples in properly sealed airtight, waterproof containers using double or triple layer packaging. Each container should be clearly labelled by using a waterproof marker to indicate type of tissue/ fluid collected, the animal sampled, date and place of sampling and name of the sender.
• Arrange samples in transport box in such a manner that the formalin container does not come in contact with frozen material during shipment. Commercially available insulated icebox can be used for short-distance transportation of laboratory material.
• Keep a track of transport of material and its delivery. If it is possible, try to send specimens to the laboratory by a personal courier. Always maintain record of entire correspondence and copy of submission report in your records

It is important to note that the above broad guidelines can be modified as per the field situation by the attending veterinarian. However, ensure that right samples are collected and submitted timely to reach the laboratory in the proper condition.