Sample collection

 
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The most important aspect of diagnosis of hemostatic disorders is the collection and submission of an optimal sample for testing. Unlike many other clinical pathologic tests, coagulation assays are unforgiving with respect to poor sample collection and handling. Many causes of prolonged clotting times or decreased factor concentrations are artifactual, due to poor sample collection and handling. View other sample collection pages specific for hematology, chemistry, urinalysis and cytology.

Technique

  • Clean venipuncture is essential: If venipuncture is difficult or blood flow ceases in the middle of collection, a new blood draw will be required. Poor venipuncture will result in activation of coagulation due to contamination with tissue factor. This means that large peripheral veins are optimal sites for obtaining coagulation samples (to ensure rapid blood flow) and if the vein is not entered on the first attempt, a fresh needle should be used. See detailed instructions and video.
  • Anticoagulant: Collect blood into the following anticoagulants:
    • EDTA: Platelet count
    • Citrate (3.2 or 3.8%): All other hemostatic tests. The remaining guidelines apply to citrate tubes.
      • For citrate, collect the appropriate volume of blood: This is essential!!
      • This is nine parts blood to one part citrate (i.e. 5 ml citrate vacutainers contain 0.5 ml of citrate and draw 4.5 ml of blood).
      • This maintains the correct blood:citrate ratio which is essential for testing. Under- or overfilling of the citrate tube will affect coagulation test results (particularly underfilling, which prolongs times of clotting assays). This means that if the needle comes out of the vein during sample collection, you need to start from scratch (new needle, new collection tube)
      • Vacutainers can be used but these sometimes underfill. The best method for blood collection is to draw up the required volume of citrate for the desired amount of blood directly into a syringe. There is a video of this procedure available on the
      • The sample should be mixed with anticoagulant rapidly to prevent clotting occurring within the tube. This is achieved with the above blood method. Long butterfly catheters are used for sample collection and slow sample draws can be problematic. Only short butterfly catheters (3") should be used for blood draws.
      • There is controversy on the need to discard the first few ml of blood. If clean venipuncture is achieved and there is anticoagulant in the syringe, this is not necessary.
      • The hematocrit of the patient should be considered. Severe hemoconcentration can result in overcitration of the plasma, which will falsely prolong clotting times (especially the APTT). The converse is true in anemic animals, in which a sample may clot due to undercitration. In very severely anemic (PCV < 15%) or erythrocythemic (PCV > 65%) animals, the amount of citrate should be adjusted using the following formula (however, in reality, most people do not compensate for hematocrit when taking blood):

Volume of citrate = 0.00185 x blood volume to be collected x (100 - Hct [%])

View the technique for collection of whole blood in citrate anticoagulant recommended by the Comparative Coagulation Laboratory of the Animal Health Diagnostic Center of Cornell University.

Handling and shipping

Coagulation factors are notoriously unstable. Samples should be submitted as soon as possible after collection. As most of us do not have access to a clinical laboratory laboratory on site, this usually means shipping the sample. It is far better to ship a sample to a veterinary diagnostic laboratory, then to run the sample up to the local hospital, because not all coagulation testing is created equal. Studies have shown that clotting times are stable for 6-24 hours in whole blood, with stability being more assured if plasma is separated from cells.

Citrate-anticoagulated blood samples for hemostasis testing:

  • Centrifuge the sample as soon as possible after collection and remove plasma from the cells (keep the sample refrigerated until centrifugation).
  • Store plasma refrigerated and shipped (with a cool pack) to reach the laboratory within 24 hours of collection.
    • If the delay before submission is longer than that (the usual situation), the sample should be frozen in a dedicated freezer (coagulation factors degrade in frost-free freezers) and kept frozen until it reaches the laboratory (shipped on dry ice).

EDTA-anticoagulated blood samples for platelet counts:

  • Transport EDTA (and citrate-anticoagulated plasma) on ice packs.
  • The EDTA blood should not in direct contact with the ice (wrap with a paper towel); ice may freeze cells in the EDTA tube and cause platelet clumping.

Test ASAP!!

  • Platelets become activated with storage (particularly refrigerated) and clump, which decreases (and can invalidate) platelet counts.
  • Coagulation factors are unstable in whole citrated blood and are more stable in citrate-anticoagulated plasma (centrifuged whole blood) when stored refrigerated (see above).

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