Hemolysis Shortens PTTs When Heparin is Present

I received this cheerful note from Nichola Marcus, a graduate of our UAB Clinical Laboratory Science program, now on the laboratory staff of Huntsville Hospital, Huntsville, Alabama:

Hey George!

Check out the AACC abstract poster D-54 from your former UAB student–I never would have guessed that my first abstract would be coag related.

I have a question for you: Do you think that platelet factor 4 is responsible for shortening partial thromboplastin times (PTTs) when specimens are hemolyzed and the patient is on heparin? If that is the case, it seems that we are indirectly measuring platelet disruption via a hemolysis index – since, of course, our method is mechanical clot detection, not spectrophotometric.

Hi, Nichola, it is good to hear from you, and congratulations on having your project accepted for a poster presentation by AACC. For those who would like to see it, Ms. Marcus’s poster will be presented Wednesday, July 28, 2010, 2:00 to 4:30 PM in the Anaheim Convention Center at the AACC/ASCLS annual meeting.

For help with your question I contacted my friend and collaborator, Dave McGlasson at Wilford Hall USAF Hospital in San Antonio. We agree with your position. Platelet factor 4 is a heparin-neutralizing protein that is secreted from α-granules of damaged or activated platelets, it is likely that it is shortening the PTT. This is the same reason you must centrifuge and separate plasma specimens for heparin assay by PTT within one hour of collection. Upon standing, platelets release platelet factor 4, neutralizing and shortening the PTT result. The result becomes significantly shortened after one hour.

One additional step you may want to try if you have specimens left over is to run anti-Xa heparin assays on your heparin specimens; pre- and post-hemolysis. You may find the heparin concentrations in the post-specimens are reduced compared to the pre-specimens. This suggestion also comes from Dave.

I hope to see you and review your poster at the meeting. Thanks for contacting me, and I wish you continued success. Geo.

Additional information from Nichola:

Here’s a little background on the abstract. Our Roche MPA system processes >90% of our blood specimens. It is directly connected to three of our chemistry analytic lines and in the near future, our refrigerated specimen archiving system. Last year, we connected our Stago STAR Evolution to the pre-analytical line. We decided that if we are going to treat our coagulation specimens like chemistry specimens, we should perform specimen integrity checks. The MPA can take an aliquot of specimens and perform a ‘serum index‘–this gives a quantitative result for hemolysis, icterus, and lipemia. The big question was how to establish serum index cut-off values for coagulation specimens. I found no published information and there is a lot of conflicting information about the effects of hemolysis on coag specimens. I really thought doing our own studies would be very easy since our routine coags are tested using mechanical clot detection. The data really surprised me – my dreams of nice linear data were crushed.  After a lot of head scratching,  we established our hemolysis index cut-offs using a 95% confidence interval for % difference between paired non-hemolyzed & hemolyzed specimens (with acceptable biological variation as the reference interval). We are also applying a quantitative lipemia result to D-dimers since it is a turbidometric assay.