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Veterinary Diagnostic Testing

Linda Mittel

Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, New York, USA

1.1 Introduction

Most veterinary diagnostic laboratories have websites or booklets describing requirements for diagnostic sampling. These resources have descriptions of the sample needed, volume, temperature requirements for shipping, and other valuable information to assist the referring veterinarian.

Obtaining diagnostic samples from animals may present zoonotic disease exposure to the veterinarian. The veterinarian should always be aware of zoonotic diseases, transboundary diseases and even potential bioterrorism acts when collecting diagnostic samples. One of the most recognized potential zoonotic exposures for veterinarians is rabies and this should be on the differential in any neurological case. Any neurological case should be carefully handled when obtaining brain or any samples from the horses.

Additionally, foreign animal diseases (FAD)/transboundary diseases should be on the differential when clinical signs suggest such. International movement of horses legally and illegally may introduce FADs into the United States and consultation with the USDA and state veterinarians should be done prior to any sampling should veterinarians have any concerns about these possibilities.

Veterinary diagnostic testing utilizes many of the rapidly developing testing platforms including PCR, sequencing, multi-array, and MALDI-TOF to assist in diagnosis. Testing procedures are changing frequently and veterinarians must familiarize themselves with their referral laboratories' website or contact the lab to stay abreast of new sampling requirements, and tests.

Many large state veterinary diagnostic laboratories are full-service laboratories and provide assistance to veterinarians in diagnostic plans, choosing tests and samples for suspected illnesses. State veterinary laboratories may be accredited by the American Association of Veterinary Laboratory Diagnosticians (AAVLD), which is an organization that promotes the improvement of veterinary diagnostics and standards for testing (see www.aavld.org/mission-vision-core-values). Veterinarians should work closely with their laboratory to be assured that they are familiar with the most current and correct sample collection and handling required by the laboratory.

Most laboratories have specialized sections for testing which include: clinical pathology, anatomical pathology, endocrinology, coagulation, bacteriology, virology, molecular diagnostics, and toxicology. Referral to other laboratories is routinely done by large laboratories due to the extensive testing requirements and recognized expertise of other laboratories.

1.2 Diagnostic Sampling

1.2.1 Whole Blood

One of the most frequently tested body fluids in the equine is blood.

• Most veterinary blood tests are done on whole blood, plasma or serum.
• A number of different blood tubes, transport vials, and so on, should be available to veterinarians at all times to obtain diagnostic samples such as CBCs and blood chemistries.
• Some blood tests require specialized collection tubes or containers that are not routinely stocked at veterinary practices and may be purchased from the laboratory.
• Consultation with your laboratory or review of their website should be done prior to blood sample collections to ensure quality and diagnostic samples.
• Special attention should be made to the specimen, the manner of collection, appropriate transport container, temperature requirements, correct test requests, and complete paperwork. Most laboratories welcome assisting veterinarians to help ensure the correct samples are collected.

1.2.2 Order of Draw

The order in which blood samples are drawn when multiple blood collection tubes are being collected from the animal is called "order of draw." Although this is not routinely practiced in veterinary medicine, it is suggested to follow the order of draw. Advanced techniques and the improved detection levels in diagnostic tests may cause inaccurate results from carry over between tubes with additives. It has been determined which additives affects test results and drawing the blood in the correct order is necessary, but some researchers feel the difference is minimum. The order of draw for most veterinary applications is: sterile tubes (blood cultures), light blue, red top, or SST, dark green, and purple (Box 1.1). If additional tubes are going to be drawn consultation with the lab should be done.

Box 1.1 Key points of blood sampling.

• Review the referral laboratory website or contact the lab to obtain information.
• Required sample type: plasma, serum, whole blood, etc.
• Animal preparation: fasting, at rest, after exercise, after medications, etc.
• Volume of required sample. The minimum volume allows one single analysis including instrument dead volume.
• Collection tube type and size: EDTA, heparin, citrate, glass, plastic tube, microtube, etc.
• Sample handling after collection: clotting time, centrifugation, temperature requirements.
• Shipping and handling requirements: receipt at the laboratory within stated time, chilled, frozen, room temperature, and so on.
• Do not freeze sera in glass tubes.
• Storage temperature is specified as room temperature (15-30?°C), refrigerated (2-10?°C), or frozen (-20?°C or colder).
• Samples after collection should immediately be placed in appropriate temperature holding areas until testing is begun or until prepared for shipping to referral lab.
• An air-dried blood smear should accompany EDTA samples for hemogram if testing not performed within 3-5?h post collection.
• Slides should be labeled with a pencil or diamond point pen.
• Cells in collection tubes with anticoagulants/additives may develop artifactual changes; therefore, air-dried slides should be made to prevent these changes.
• Slides should be placed in slide mailers away from moisture and formalized tissues/samples. Formalin fumes affect air dried slides and may render cytology smear nondiagnostic.

1.3 Collection, Preparation, and Handling

1.3.1 Blood Collection Tubes

Various types of evacuated blood-drawing supplies should be kept on hand in a clinic or in an ambulatory vehicle for equine diagnostic testing. Additional blood collecting supplies may include specialized blood-drawing needles, needle holders, and butterfly collection device needles.

There are numerous specialized blood collection tubes that are used in human medicine that can be used in veterinary diagnostic testing for special and routine tests (Figure 1.1). These tubes include: (1) trace element tube (royal blue cap), (2) thrombin based clot tube with activator gel for serum separation (orange cap), (3) glucose determinations (gray cap), (4) lead determination (tan caps), purple/lavender caps, and (5) blood culture collection tubes and DNA testing tubes (yellow capped with sodium polyanethol sulfonate (SPS) and others for specified tests.

Figure 1.1 Blood flow chart.

Source: Courtesy of Linda Mittel.

Important facts about evacuated blood collection tubes:

• Expiration date
  • Blood collection tubes expiration dates are stamped on the tubes.
  • Out of date tubes may lose vacuum because of dried out stoppers and cause incomplete seals, incomplete filling of tube, and additives may become inactive over time.
  • Plastic collection tubes may not maintain the same shelf life as glass.
• Tube size and complete fill
  • Evacuated tubes are designed to auto-fill to a designated amount and should be allowed to fill until blood stops flowing automatically.
  • Under-filling tubes with additives will adversely affect results.
  • If there is a likelihood that a tube will not be filled to the correct volume, smaller tube sizes should be used to ensure the correct dilution of blood to the additive. Blood collection tubes/containers come in various sizes.
  • Adhere to volume requested by laboratory because requested volume is used for verification of results, add-on tests, and parallel (acute and convalescent serology) testing.
  • Necessary volume should be calculated prior to collecting samples.
  • Mature normal sized horses should yield 4?ml of serum from each 10?cc blood drawn: 5?ml of plasma should be obtained from 10?ml of whole blood.
  • These volumes may vary with hydration, health status (anemia) and other conditions.
  • Foals or seriously anemic animals may require that smaller volumes of blood be taken.
  • Microtubes ranging from 200 to 600 microliters and other blood collection tubes are readily available ranging in sizes from 2 to 10?ml.
  • Butterfly collection lines/winged infusion sets may be used to obtain blood samples in the case of inaccessibility to the jugular veins, small vessel size, fractious animals, or difficult approaches.
  • Butterfly collection lines/winged infusion sets can be placed directly into the blood...