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Sampling for histopathology analysis

General considerations

In order to correctly carry out the collection of samples for histopathological study, a number of considerations has to be taken into account:

  • Collecting samples from carcasses that have been dead for more than 12 hours should be avoided. The time should be shorter if the temperature of the cadaver is high (summer) or in case of clostridial diseases.
  • Samples for both histopathology and immunohistochemistry (IHC) should be fixed in 10% buffered formalin and stored at room temperature.
  • Frozen samples should not be sent as freezing forms crystals in the tissues and alters the original morphology.
  • Samples should be taken from all organs, regardless of whether gross alterations are observed or not, since histological modifications are not always seen grossly.

Tissue collection

  • El material que se use debe estar limpio y afilado (bisturís y cuchillos) para realizar cortes netos. No se han de usar tijeras ni pinzas de ratón para evitar dañar el tejido.
  • The thickness of the samples should be a maximum of 0.5 to 1 cm to allow the formalin to penetrate properly and thus avoid autolysis.
  • Samples should be taken from the edges of the lesions, always including one affected and one unaffected region.
  • The brain and eyeball should be fixed intact.
  • All tubular organs should be cut longitudinally and cleaned with water or saline before fixation in formalin (without damaging structures such as mucosa).
  • Parenchymal organs can be sampled regardless of the area and orientation of the cut.

Specimen fixation

  • The samples should be placed as soon as possible in the fixative of choice, placing the liquid first and then the organs or tissues, to prevent them from adhering to the walls of the container.
  • The specific container for formalin should have a wide mouth to allow the organs to be inserted and removed. In addition, the formalin volume: tissue ratio should always be respected (1:10), and all the samples of the animal under study can be placed in the same container.
  • Tissues that float (lung, lipomas) should be wrapped in wick paper to ensure that they are soaked by the fixative liquid.
  • The container has to be correctly identified, indicating the reference of the animal and with the pertinent pictograms and information on chemical risk.

Sampling for toxicological analysis

General considerations

Before taking samples for toxicological analysis, it is important to take into account a series of general considerations, including the following:

  • Preservation and transport in freezing immediately (-20ºC) of all samples after collection, with the exception of blood which should be refrigerated (4ºC) in order to be able to perform coagulation tests and the hemogram.
  • Avoid external chemical contamination from hair, dirt and dust in the environment.
  • Use a container for each organ (red lid duchesses or zip bags) correctly traced (individual and organ reference) and seal it hermetically. The set of samples from a case should be placed in a bag with a numbered flange-type seal.
  • It is advisable to include samples fixed for histopathology in order to confirm the diagnosis in case of doubt.

Type of samples

Since the variety of toxic compounds is very wide, whenever possible samples should be collected from: liver, kidneys, brain, whole blood, serum, urine and gastric or ruminal contents of dead animals. Likewise, samples of baits and suspicious substances or food should always be submitted.

In general, and in case a specific toxicant is not suspected, it is advisable to take the following samples to identify the possible toxic agent:

  • Esophagus and stomach contents: due to the fast action of most toxic agents these can be found in high concentrations in the esophagus and stomach, and not in the liver. Whenever a material likely to be toxic is found in the carcass, it should be collected in a container so that it does not mix with the food.
  • Liver: since it is the organ in charge of eliminating toxins absorbed through the digestive tract, it is important to sample it, especially if no significant findings are found in the digestive tract. Among the compounds that accumulate in the liver are anticoagulant rodenticides and heavy metals such as lead (which also accumulates in the kidney).
  • Brain (it is not necessary to remove it intact for toxicological studies): the poisons used are, in most cases, anticholinesterase pesticides. If this toxicant is present in the brain, brain acetylcholinesterase activity will be inhibited. In other cases, bioaccumulation of organochlorine compounds or mercury may be observed reaching lethal concentrations.
  • Bait and vomit: toxicants will be found in higher concentrations than in the cadaver, therefore, their detection will be more likely.
  • Blood: it is useful to determine lead poisoning (using tubes with lithium heparin); vitamin K antagonist anticoagulant poisoning (using tubes with sodium citrate); nitrate poisoning (brown blood) by determining the percentage of methemoglobin in the blood if it is analyzed in a few hours or preserved in liquid nitrogen. On the other hand, a routine hemogram can be performed.
  • Plasma: can be useful to determine the activity of plasma cholinesterases for the same reason given for brain sampling and can also be used for routine biochemical profiling.
  • Kidney: in case of dipyridyl herbicide (paraquat) poisoning by observing important lung lesions (congestion, edema and large re-epithelialization areas), as well as to detect cadmium accumulation.
  • Fat: it is a tissue where there is an accumulation of persistent lipophilic compounds such as organochlorines, polychlorinated biphenyls and polybrominated compounds.
  • Bone: lead accumulates in bone throughout the animal's life.
  • Hair, feathers and nails: some elements such as mercury and arsenic can accumulate in these attachments, indicating chronic or even acute lethal exposures.
  • Soil under the carcass: of special interest in cases of completely decomposed carcasses.

  Intestinal content Liver Encephalon Bait, Vomit Heart Skeletal muscle Kidney Fat Bone Fur, feathers, nails
Estrogenic agents   C                
Alkaloids   C                
Alphachloralose   C                
ANTU C C                
Arsenic   C         C      
Cadmium   C         C      
Carbamates (insecticides)     C              
Cyanide   C                
Copper   C         C      
Dicumarol   C                
Strychnine   C         C      
Ethylene glycol             C      
Drugs   C         C      
Fluoroacetate   C         C*      
Fluorides                 C  
Gossypol         F          
Dipyridyl herbicides   C         C      
Mercury   C C       C     C
Metaldehido   C                
Mycotoxins   C         C      
Monensin, salinomycin         C C        
Nitrates   F         F      
Organochlorines   C C C       C    
Organophosphates and carbamates   C C C            
Other metals and metalloids   C                
Others pesticides   C                
Lead   C         C     C
Anticoagulant rodenticides   C                

Table: Guidance as to which organs we are interested in analysing based on the lesions observed and the suspicion of toxic substances involved (C - frozen, F - formalin).

Sampling for microbiological analysis

General considerations

  • Each sample should be kept individually in sterile containers or zip bags to avoid contamination.
  • The conditions will depend on the type of pathogen suspected, therefore, it is important to consult with the laboratory about the conditions under which samples should be sent (medium, temperature, etc.).
  • They should be sent within 24 hours of collection.
  • Blood, urine, saliva, milk, cerebrospinal fluid or tissue samples should be collected as aseptically as possible for culture, virological analysis or PCR.
  • For parenchymal organ samples (lungs, liver, heart, kidneys or spleen), it is recommended to send a large portion of the organ, as it allows the laboratory to take aseptic samples. In the case of the brain, it should be sent whole.
  • Longitudinal organs (digestive tract) should be sent to the laboratory in unopened portions with the ends knotted.
  • In cases of necrosis or exudates, samples of the lesions can be taken with swabs.