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Coagulation Specimens

Introduction

To produce valid results for hemostasis/thrombosis test and factor assays, specimen integrity is crucial and must be maintained. All specimens sent for testing must be collected and transported in the following manner.

Coagulation Specimen Collection Guidelines

  • Obtain venous blood by clean venipuncture. Avoid slow flowing draws and/or traumatic venipuncture, as either of these may result in an activated or clotted specimen. Do not use needles smaller than 23gauge. Do not leave the tourniquet on for an extended length of time before drawing the sample.
  • Specimens containing heparin should not be used for coagulation studies. If possible, stop heparin therapy for up to two minutes before the draw to avoid contamination. Heparin interferes with most clotting assays.
  • Collect coagulation specimens in light blue-top vacuum tubes (3.2% buffered sodium citrate).
  • Draw a discard tube (non-additive or light blue-top tube) if other than PT or APTT are being drawn or they are being drawn with other tests. Dispose of the discard tube or use for another, non-coagulation assay.
  • Non-additive tubes are collection tubes that do not contain anticoagulant, gel or other additives.
  • Fill the light blue-top tubes to 90-100% and mix by gentle inversion. An exact ratio of nine parts blood to one part anticoagulant must be maintained. Inadequate filling of the sample tube will alter this ratio and may lead to inaccurate results.

Hematocrit Above 55%

  • It is important to adjust the sodium citrate volume when a patient’s hematocrit is greater than 55%. When a patient has a hematocrit greater than 55%, the plasma volume will be too low for the amount of sodium citrate solution in the tube. This may lead to false increase of the Protime INR or aPTT results because of excess anticoagulant. The anticoagulant must be reduced in the tube before filling with whole blood. Contact Laboratory if more information is needed.
  • Use the following formula to determine the amount of anticoagulant to use:

(1.85 X 10-3) (100-HCT) (V Blood) = C

C = adjusted volume of anticoagulant remaining in tube

HCT = Patient Hematocrit

V Blood = Volume of blood added (if a 3 mL collection tube is used, volume would be 2.7 mL)

Coagulation Specimen Processing and Transport

  • Deliver whole blood sodium citrate tube to the Laboratory within the stability time limits, transport at ambient room temperature (15-25°C). Refrigeration (2-8°C) or freezing of whole blood sodium citrate tube is unacceptable.
  • If unable to deliver whole blood sodium citrate tube to the laboratory within stability limits, follow the steps listed below:

1. Centrifuge the specimen at 1,500 x g for 15 minutes, or at a speed and time required to consistently produce platelet-poor plasma, platelet count less than 10,000 µL.

2. Immediately remove the platelet-poor plasma from the specimen using a plastic transfer pipet, taking care not to disturb the buffy coat layer or touch the sides of the tube, as this may recirculate platelets back into the plasma. Place platelet-poor plasma into properly labeled transport tubes. Centrifuge a second time. Then remove the platelet poor plasma without disturbing the cell button on the tube bottom and place 1 mL aliquots of the plasma in properly labeled polypropylene transport tubes, clearly marking the vial contents as CITRATED PLASMA. This second spin will ensure that no platelets are accidently pipetted into the final frozen aliquot tubes. Note: Use of glass transfer pipets will result in activation and/or clotting of the plasma.

3. Immediately freeze the plasma in a non-frost-free freezer. Samples may be stored frozen at (- 20°C) for two weeks or preferably at (-70°C).

NOTE: Freeze aliquot tubes in upright position.

NOTE: Specimens must remain frozen during storage and shipment.

Coagulation Specimen Processing Stability


Coagulation Specimen Processing Stability Table




TEST

NOTE: If unable to deliver whole blood to testing laboratory within stability time, it is best practice to submit frozen platelet poor plasma aliquots.

Whole Blood

Processed Platelet Poor Plasma

Room Temp (15-25°C)

Refrigerated (2-8°C) or Frozen

Frozen (-20°C)

Frozen (-70°C)

Protime INR

24 hours

Not Acceptable

2 weeks

6 months

APTT

4 hours

Not Acceptable

2 weeks

6 months

APTT - Drawn to monitor heparin therapy

1 hour

Not Acceptable

2 weeks

6 months

Heparin Assay

1 hour

Not Acceptable

1 month

6 months

Fibrinogen

4 hours

Not Acceptable

2 weeks

6 months

D-Dimer (DDHS)

4 hours

Not Acceptable

2 weeks

6 months

Special Coagulation

4 hours

Not Acceptable

2 weeks

6 months

Causes for Specimen Rejection

  • Hemolyzed samples
  • Clotted samples
  • Samples collected in incorrect anticoagulant
  • Underfilled or Overfilled whole blood (light blue top tube)
  • Refrigerated whole blood (light blue top tube)
  • Frozen whole blood (light blue top tube)