Preanalytic variability affects all laboratory tests but can be especially problematic for assessment of coagulation disorders. Christopher Desens discusses the preparation of platelet-poor plasma for coagulation testing.
Christopher Desens, MLS(ASCP) is a Technical Specialist in the Hematopathology Division within the Department of Laboratory Medicine and Pathology in Rochester, Minnesota.
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Preanalytic variability affects all laboratory tests but can be especially problematic for assessment of coagulation disorders. Speaking today is Mr. Chris Desens. Thank you, Chris.
Thank you for that introduction. My name is Christopher Desens from the special coagulation laboratory at the Mayo Clinic.
I have no disclosures at this time.
Before processing a coagulation specimen, take a moment to evaluate the specimen. Check that all sodium citrate tubes are filled to at least 90% full. Improperly filled tubes can lead to inaccurate coagulation results.
Check for clots by inverting the specimen gently or inserting and removing two wooden sticks. Reject any specimens with observed clots.
To start processing, centrifuge the tubes. Be careful that the tubes are evenly balanced when placing them into the centrifuge. When the centrifuge stops, carefully remove and rack tubes. Be aware that an unbalanced centrifuge, speeds that are too slow, or spin times that are too short may all lead to an increase number of cells in the plasma which could contribute to a clotted specimen. The recommended centrifugation speed is 1500 x G for at least 15 minutes.
Sodium Citrate Specimen
Here is an example of a sodium citrate specimen after centrifugation. Review the differences in relative positions of the plasma, buffy coat and cell layer.
After centrifugation, evaluate your specimen for an acceptable hematocrit and the presence of hemolysis.
An acceptable hematocrit will be between 25-55%. For hematocrits less than 25%, the plasma calcium will be in an excess proportion to the sodium citrate anticoagulant. The plasma will be prone to clots and clotting time results will be falsely low.
For hematocrits greater than 55%, the sodium citrate anticoagulant will be in excess proportion to plasma calcium. In this scenario, the plasma will produce falsely-prolonged clotting times.
Hematocrit Test Tubes
Here is an illustration to help us gage the hematocrit in the draw tube. Please note that the brown layer is just a graphical representation of the citrate anticoagulant. The anticoagulant will not appear this way in the draw tube, but you do need to be aware of the volume it displaces when estimating the hematocrit.
The tube on your left depicts approximately a 50% hematocrit with the cell volume and the plasma volume being roughly equal. The tube on our right depicts greater than 55% and is not acceptable. Any cell volume less than 25% (noted here with the dotted line) has a greater chance of clotting.
Hopefully you can verify questionable hematocrits with CBC results. If the hematocrit is outside of the normal ranges, consider re-drawing using tubes that have had the sodium citrate adjusted to the proper proportion to avoid inaccurate test results or clotted samples.
Also evaluate the specimen for hemolysis after centrifugation. If the plasma appears pink or red, it is likely hemolyzed. A common cause of hemolysis is a traumatic venipuncture, and hemolyzed specimens are prone to clotting because of the lysed cells and excess tissue thromboplastin present in the specimen. If possible, redraw the patient.
When removing the plasma layer, be careful not to disturb the cell layers. Transfer the top three-quarters of plasma into labeled plastic tubes using a transfer pipette.
Never pour off plasma; pouring off plasma directly from the draw tube will introduce excess cells to the specimen.
To remove plasma, start from the top of the liquid, gently drawing specimen into the pipette as you go further down tube.
Leaving approximately 0.5mL of plasma (shown here with a dash line) will insure that you do not disturb the buffy coat and cell layer. Note how the buffy coat is still well defined in this picture.
The picture on the left is an example of pipetting too far into the buffy coat and cell layer. You can actually see evidence of the buffy coat and cell layer disruption; the buffy coat is less defined and you can see red cells in the plasma. Using this portion of the specimen will put to many cells into the plasma and potentially cause the specimen to clot later on.
The picture on the right is an example of going much too far into the specimen by actually pulling the cell layer into the pipette. Please note that it is not acceptable to purge the cells from the pipette and then use the plasma that remains.
Check for Clots
After harvesting the plasma, check for clots by pouring the residual plasma and cells onto the gauze.
Clots will appear as red globular clumps that sit on top of the gauze as seen on the left. The buffy coat will be white and disc like and can be pushed into the gauze as seen on the right. Reject any specimens that contain any blood clots found in this manner.
Plasma separated from cells goes back into the centrifuge for a second spin.
Balance and centrifuge the tubes. Carefully remove and rack tubes after centrifugation. Failure to perform a second spin will result in plasma containing too many platelets which interfere with some coagulation testing and is a contributing factor for a clotted specimen.
Transfer Plasma to Aliquot Tubes
After the second spin, transfer plasma to aliquot tubes. Use a plastic pipette and start from the top of the liquid and gently draw specimen into the pipette.
When pipetting the plasma, be careful not to disturb the bottom 0.5mL of the tube you are pipetting from.
Finally, after processing the specimen, promptly freeze them in a freezer that does not self-defrost.
I have presented the procedure for preparing platelet poor plasma for special coagulation testing. If carefully followed we will have the best specimen possible from which your patient will get accurate, high-quality results.
In conclusion, the preprocessing risk factors for clotted specimens are as follows:
- Traumatic venipuncture and the resulting tissue thromboplastin and hemolysis may activate the clotting cascade.
- If a coagulation tube is drawn after a tube with a procoagulant or a clot activator, there is a hypothetical possibility for contamination and subsequent clotting of the sodium citrate tube.
- If the specimen is not mixed, the anticoagulant is not evenly mixed into the blood potentially allowing clots to form in the area absent of anticoagulant.
- Too vigorous of mixing may activate the platelets.
- For hematocrits less than 25%, the plasma calcium will be in excess proportion to the sodium citrate anticoagulant. The plasma will be prone to clots and clotting time results will be falsely low.
- A patient in a hypercoagulable state such as, Disseminated Intravascular Coagulation or Intravascular Coagulation Fibrinolysis will also be at risk for producing a clotted specimen.
The processing risk factors for clotted specimens are as follows:
- Centrifugation recommendations for coagulation specimens must be followed to insure that the plasma platelet levels are kept to a minimum, less than 10,000 per mcL.
- Platelet counts greater than this number may cause the sample to clot after a subsequent freeze-thaw cycle.
- Recommended centrifugation speed is 1500 x G for the recommended time of 15 minutes. If the speed is too low, timed to short or the centrifuge is unbalanced there may be cellar contamination and the potential for clots.
- Always use a plastic pipette for removing plasma.
- Never pour off plasma directly from tube.
- Excessive cellar contamination will be caused by pouring off plasma, rough handling, and pipetting into the cellular layer.
- Specimens found with a clot either before or after centrifugation must be rejected; the clotting cascade has started. The specimen will continue to form clots and test results would be potentially misleading.
Lastly, not performing the second spin will also leave too many cells remaining in the specimen and therefore will also be prone to clotting.