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General Principles and Communication in animal sample collections:
• Protocols and diagnostic tests are subject to frequent changes.
• Critical Communication: Essential between the practitioner and diagnostic laboratory staff for efficiency and optimal service.
• Practitioners must be specific and clear in their test requests.
• The laboratory staff provides guidance on sample collection, handling protocols, and assistance in interpreting results.
• Most laboratories publish user guidelines with preferred protocols.
Detailed Case History (Submission Form)
• A detailed case history is critical, especially for interpreting cytologic and histologic evaluations.
• The submission form must be protected in a waterproof bag to prevent damage from fluids.
• Required Elements:
◦ Owner's first and last name.
◦ Species, breed or size/weight (if breed unknown), sex, age, and animal identification.
◦ Relevant clinical signs.
◦ Gross appearance (including size and anatomic location) of lesions.
◦ Previous treatment, including response to treatment.
◦ Results of relevant diagnostic testing (e.g., CBC, serum biochemical analysis, imaging studies).
◦ For population health studies, morbidity and mortality in the group.
🚨If a zoonotic disease is suspected, it must be clearly indicated on the form to alert laboratory personnel.
Packaging of Samples for Shipment:
• Shipment must comply with courier protocols, possibly including IATA regulations for hazardous materials if air transport is used.
• Formalin Volume Restrictions (IATA): No more than 30 mL of free liquid solution in each inner container, and no more than 1 L in the entire outer package.
◦ Alternative: Tissue samples can be fixed in-house for ≥24 hours and then transferred to a smaller volume of fresh formalin for shipment.
• Labeling for Fresh Tissue Samples (Infectious Substances): Shipments must be clearly labeled:
◦ UN2814 (affecting humans)
◦ UN2900 (affecting animals)
◦ UN3373 (Biological substances, Category B)—Most veterinary samples fall into Category B.
• Contact state and federal veterinary agencies if high-risk or reportable diseases are suspected.
• 3-Layer Barrier Approach: A fundamental approach to packaging:
1. Primary Container: Holds the sample (sealed jar, bag, or tube).
2. Secondary Container: Encloses the primary container and includes absorbent material.
3. Tertiary Container (Shipping Box): Houses the secondary container; often includes refrigerant/cold packs and cushioning materials (e.g., polystyrene foam). A sturdy polystyrene refrigerator box or a cardboard box with a fitted polystyrene lining is ideal.
• Unacceptable Items for Shipment: Syringes, obstetrical gloves, containers without sealable orifices, needles, scalpel blades, or broken microscope slides.
• Liquid Samples: Must ship in a sealable jar, not plastic bags.
• Labeling: Use waterproof markers; critical information includes patient identification and specimen type (e.g., urine, serum).
• Coolants: Must be sealed in separate plastic bags to prevent condensation damage.
• Refrigerant or cold packs should not be placed in direct contact with samples (e.g., tubes of whole blood) that could be adversely affected by freezing.
• Dry Ice Usage: Must be noted on the cardboard box label, and the lid should not be sealed with tape to prevent pressure buildup from CO2 release.
Preparation of Samples for Histologic Evaluation
• Autolysis Avoidance: Autolyzed tissues are generally useless; prompt necropsy and organ sampling are critical.
• Tissue should not be frozen prior to fixation.
• Tissue Size and Fixation:
◦ Samples (other than CNS tissues) should never be >1 cm thick, preferably 5–7 mm.
◦ Must be immediately placed in ≥10 times their volume of phosphate-buffered 10% formalin.
• Lesion Representation: Biopsies should be representative of lesions. Cutaneous punch and endoscopic biopsies should be centered directly on visible lesions.
◦ Wedge biopsies or necropsy samples should include the interface between normal and abnormal tissue.
• Tumor Processing: Small tumors (<1.5 cm) may be cut in half; larger tumors may be sliced like bread or several representative samples (7 mm wide, including interface) may be collected.
• Fixation Time: Prolonged formalin fixation (storage for >7 weeks) can adversely affect advanced testing, such as immunohistochemical testing.
• Containers: Use laboratory-approved unbreakable containers with screw-top lids.
• Specific Necropsy Tissues:
◦ GI Mucosa: Short sections of gut must be opened lengthwise for adequate fixation due to rapid decomposition.
◦ Spinal Cord: The dura mater should be carefully incised lengthwise to permit rapid penetration.
◦ Brain Fixation: Ideally, requires a whole, intact, fixed brain. Requires immersion in a large volume of formalin for many days.
▪ Shipping Brains: A chilled, carefully packaged, unfixed brain may be acceptable if shipped overnight. Often, the brain is halved longitudinally (one half sent unfixed/refrigerated for microbiology; other half partially fixes in transit).
▪ Slicing the brain introduces fixation artifact and should be avoided; fixing the intact (or halved) brain in a large volume of formalin for >24 hours is preferred.
Preparation of Samples for Microbiological Testing
• Suspected infectious agents should be noted on the submission form due to specific growth requirements (e.g., anaerobic culture).
• Tests rely on intact viable organisms or nucleic acid/protein detection.
• Handling: Unfixed specimens (tissue, fluid) must be collected aseptically and shipped promptly.
• Culture of Fluid: A separate aliquot must be submitted in a sterile, additive-free container. Tubes with EDTA or other preservatives are generally unacceptable for culturing.
• PCR Assays: Cross contamination must be strictly avoided.
◦ Swabs for PCR: Avoid agar/charcoal-based transport media and calcium alginate swabs. Use sterile cotton- or polyester-tipped applicators, shipped either dry or with a few drops of sterile saline solution or viral transport media (consult lab).
• Pooling: Generally, each tissue should be collected into separate, clearly labeled sterile bags or tubes. Gut samples must never be pooled with other tissues.
• Freezing: Tissues/fluids may be frozen, but chilling and delivery within 24 hours is generally preferred.
◦ Exception: Prompt freezing is critical for toxin analysis (e.g., Clostridium perfringens and C botulinum) to prevent degradation.
• Fecal Samples (Parasitology): Submit chilled in sealed containers. Freezing prevents Baermann analysis.
• Ectoparasites/Nematodes: Submit in vials containing 70% alcohol.
Preparation of Samples for Toxicological Testing
• A specific analysis must always be requested; laboratories cannot simply "check for poisoning".
• A complete description of clinical and epidemiological findings helps differentiate poisoning from infectious diseases.
• Most Critical Samples: Gastric contents, liver, kidney, whole blood, plasma or serum, and urine.
• Freezing Requirement: Freezing is critical to prevent degradation of certain analytes like cholinesterase and zinc; otherwise, chilled shipping is sufficient.
• Containers: Plastic containers are ideal; containers must be free of chemicals, and jars with metal tops should be avoided. Samples must be packed individually and labeled.
• Legal Action: Containers must be sealed for tamper detection or hand-carried; chain of custody must be accurately documented.
• Feed/Water Samples (if suspected source): Submit chilled samples along with the feed tag. A representative composite sample from the suspect lot (aliquots from top, middle, and bottom) should be submitted.
Preparation of Samples for Hematological Testing
• Requires anticoagulated whole blood filled to the appropriate volume; incorrect blood-additive ratio causes unreliable results.
• Blood Smears: Should be prepared immediately after collection to minimize artifacts.
• Storage: Anticoagulated blood must be refrigerated; blood smears stored at room temperature.
• Anticoagulant Choice:
◦ Mammals: EDTA (anticoagulant of choice for CBCs).
◦ Nonmammals: Heparin is commonly used for species where EDTA causes hemolysis (e.g., stingrays, some fish, reptiles, and avian species).
• Coagulation Testing: Requires a citrate (blue-top) tube filled to the appropriate volume; underfilling causes falsely prolonged clotting times.
• Plasma Separation (Coagulation): Citrated plasma must be separated from whole blood within 6 hours (for PT/aPTT) by centrifugation (1,500g for 15 minutes).
• Avoid Freezing Whole Blood: Causes cell lysis and gross hemolysis, interfering with testing.
Preparation of Samples for Serum Biochemical Analysis
• Serum is preferred because anticoagulants in plasma can cause artifactually increased or decreased analyte values (e.g., potassium-EDTA raises potassium and decreases calcium).
◦ Consultation is recommended for nonroutine tests where heparinized plasma might be required.
• Fasting: For dogs and cats, withholding food for 12 hours is advisable to avoid lipemia interference.
• Collection: Use a serum (red-top) or serum separator (tiger-top) tube.
• Clotting: Hold at room temperature for 20–30 minutes for complete clot formation.
◦ Incomplete clotting may lead to latent fibrin formation/gelling.
• Separation: Loosen the clot by gently rimming the tube walls, then centrifuge (~450 rpm for 10 minutes).
◦ If a serum separator tube is used, inspect the polymer gel layer for cracks and re-centrifuge if necessary.
◦ If a red-top tube is used, serum should be removed and transferred to a clean tube to minimize artifacts (e.g., glycolysis reducing glucose).
• Storage: Serum should be refrigerated or frozen until analyzed.
• Hemolysis: Avoid rough handling or incomplete separation of erythrocytes from serum.
• Volume: Typically 0.5 mL is preferred.
Preparation of Samples for Urinalysis
• Labeling: Must be clearly labeled with specimen type (urine).
• Collection Method: Note the method (free catch, catheterization, cystocentesis) as it is helpful for interpretation (e.g., contaminants in free catch).
• Protection: If evaluation is delayed beyond 30 minutes, protect the sample from ultraviolet light, which degrades constituents like bilirubin.
• Containers: Use clear containers; lids must be securely screwed on to prevent leakage or volatilization of ketones. Containers must not be reused.
• Storage/Shipping: Urine should be stored and shipped chilled (note that crystal precipitation may occur).
• Cell Degradation: Urine is caustic and rapidly degrades formed elements.
• Sediment Smears: If urinary tract disease is suspected, submit air-dried cytologic smears prepared from fresh urine sediment along with the sample.
• Volume: Generally 5–6 mL preferred.
Preparation of Samples for Serologic Testing
• Generally requires serum, though plasma is often satisfactory.
• Collection procedures follow those for serum biochemical analysis.
• Samples must be free of hemolysis.
• Paired Samples: May be required for diagnosis; the acute sample is followed by a second sample collected 10–14 days later, and both are submitted simultaneously.
Preparation of Samples for Cytologic Evaluation
• Sampling Method: Fine needle aspiration or coring is typically preferred for superficial masses over surface impression smears, which are prone to contamination.
• Slide Preparation: Aspirated material must be spread into a monolayer (cells roughly touching but not overlapping).
◦ Preferred Technique: Gentle horizontal smearing using a second slide. This is superior to the vertical pull-apart technique.
◦ Avoid excessive specimen density and cytolysis. Cytolysis introduces artifacts that can mimic malignancy.
• Fluidic Samples: Highly cellular fluids can be smeared directly; low cellularity fluids should be centrifuged to concentrate cells.
• Storage: Slides should be stored at room temperature, protected from dust, excessive humidity, and prolonged light exposure.
• Labeling: Slides must be physically labeled with patient identification and the source of the sample.
• Contaminants: Excessive ultrasound or lubricant gel makes the sample microscopically opaque and hinders cellular stain uptake.
• Practical Guidelines: Use fresh slides, avoid coverslips and acetate tape, and deposit cellular material toward the center of a single side of the slide.
• Formalin Hazard: Exposure to formalin or formalin fumes interferes with staining. Never submit blood or cytologic smears in the same package as formalin-fixed tissues.
• Immunocytochemical (ICC) Staining: Usually requires air-dried, unfixed smears; consult the laboratory if special stains are anticipated.
Preparation of Samples for Fluid Analysis
• Analysis of cavitary effusions (peritoneal, pleural, pericardial) and other fluids (synovial, CSF) includes protein content, cell counts, and cytology.
• Sample Tubes:
◦ Routine Analysis: EDTA (purple-top) tube.
◦ Microbial Culture/Biochemical Analysis: Serum (red-top) tube.
• Shipping: Samples should be shipped chilled but not frozen.
• Cytologic Smears: Must be prepared immediately after collection to minimize cell deterioration. Slides should be labeled with patient ID, source, fluid, and type of preparation.
• CSF Samples: Should be collected into small EDTA tubes and shipped immediately with high priority due to rapid cytologic degradation.
Preparation of Samples for Flow Cytometry
• Can be performed on naturally fluidic samples (whole blood, effusions) or organ aspirates (lymph node, bone marrow) collected into fluid medium.
• Time Sensitivity: Intact, well-preserved cells are required; samples 48 hours old or less are usually necessary to ensure cell viability.
• Consult the lab regarding specimen volumes.
• Practical Tip: It may be advisable to submit samples early in the week to avoid weekend transit delays.
Preparation of Samples for Genetic Analysis in Animals
• Tests range from karyotype analysis to identification of specific genes.
• Consult Lab: The laboratory offering the test should be contacted to determine specific collection/handling requirements, as samples range from hair to skin or blood.
• Blood Samples: Many analyses require collection into acid-citrate-dextrose (yellow-top) tubes and overnight shipment of chilled tubes.
• Tissue Samples: Must be unfixed and shipped immediately after collection.
• Aseptic collection and prevention of cross-contamination are critical.
References: Merck Veterinary Manual
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