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Preparation of Platelet Poor Plasma for Special Coagulation Testing

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Published: October 2008

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Preanalytic variability affects all laboratory tests but can be especially problematic for assessment of coagulation disorders. Joel Cayou discusses the preparation of platelet-poor plasma for coagulation testing.

Presenter: Mr. Joel Cayou of Division of Hematopathology at Mayo Clinic

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Introduction

Welcome to Mayo Medical Laboratories' Hot Topics. These presentations provide short discussions of current topics and may be helpful to you in your practice.

Preanalytic variability affects all laboratory tests but can be especially problematic for assessment of coagulation disorders. Mr. Joel Cayou discusses the preparation of platelet-poor plasma for coagulation testing.

Evaluate Specimen

A coagulation specimen arrives for processing before certification; take a moment to evaluate your specimen. Sodium citrate tubes must be filled to at least 90%  full and it is easy to create a sample tube for comparison like calculating 90% of the blood volume, for example; if you use 4.5mL tubes, fill a sodium citrate tube with 4.05mL of colored liquid; 4.05 is 90% of 4.5mL. Under-filled tubes should be rejected because the proportion of the anticoagulant plasma will be incorrect and could lead to misleading test results.

Check for clots by inverting the specimen gently; reject any specimens with observed clots.

Centrifuge Specimen

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 centrifugation speed too slow or too short for a spin time may all lead to an increase number of cells in the plasma which could contribute to a clotted specimen. Recommended centrifuge 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.

Evaluate Specimen

Evaluate your specimen for an acceptable hematocrit and the presents of hemolysis.

Hematocrit

An acceptable hematocrit will be between 25-55%; for hematocrits less than 25% the plasma calcium is in an excess proportion to sodium citrate anticoagulant. The plasma will be prone to clots and plotting time results will be falsely low.

For hematocrits greater than 55%, the sodium citrate anticoagulant is in excess proportion to plasma calcium. The plasma will produce falsely-prolonged clotting times.

Hematocrit Test Tubes

Here is an illustration to help us gage the hematocrit and 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 50% hematocrit with a 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) will not be acceptable.

Hopefully you can verify questionable hematocrit with CBC results; if too high or too low, hematocrit should be rejected. The patient needs to be redrawn using tubes that have had the level of anticoagulant in them adjusted to the proper proportion.

Hemolysis

If the plasma appears red tinged instead of amber, your specimen is hemolyzed. A common cause of hemolysis is a traumatic venipuncture. Hemolyzed specimens are prone because of the live cells and excess tissue thromboplastin present in the specimen. If possible, redraw the patient.

Remove Plasma

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, gently draw specimen into 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.

This 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.

Here 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

Pour residual plasma and cells onto the gauze. Observe for clots. Clots will appear as red globular clumps that sit on top of the gauze, the buffy coat will be white and disc like and can be pushed into the gauze. Reject any specimens that contain any clots found in this manner.

Second Spin

Plasma separated from cells goes back into the centrifuge for a second spin.

Balance and centrifuge the tubes. Carefully remove and rack tubes after certification. 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

Pipette plasma from second spin tube into labeled aliquot tubes without disturbing the bottom 0.5mL as indicated by the dash line.

Specimen Storage

Promptly freeze specimens after processing.

Conclusion

I have presented the procedure for preparing platelets 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, a clot activator, in it there is a hypothetical possibility for contamination and subsequent clotting.
  • 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 mixing may activate the platelets.
  • For hematocrits less than 25%, the plasma calcium is in excess proportion to 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, which is DICICF, will 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 then 10,000 per mcL.
  • Higher cell counts in licenses of those cells after a subsequent freeze cycle may cause the sample to clot.
  • Recommended speed is 1500 x G; recommended time of spin is 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 and the potential will be caused for pouring off plasma, rough handling, and pipetting into the cellular layer.
  • Specimens found with 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.

Not performing the second spin will also leave too many cells remaining in the specimen and therefore will also be prone to clotting.


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