The Fritsma Factor

Cheat Sheet for October, 2011

Proficiency Testing for Mixing Studies (October 1, 2011)

Janet Smick is looking for proficiency testing for PT/PTT mixing studies other than CAP. George recommends NASCOLA, which provides a series of proficiency testing modules, including lupus anticoagulant, factor deficiency, and factor VIII inhibitor modules. Internal control plasmas are available from Precision BioLogic Inc; George King Bio-Medical, Inc; and Aniara.

Rivaroxaban Update (October 1, 2011)

The European Medicines Agency Committee for Medicinal Products for Human Use now recommends rivaroxaban for stroke prevention in atrial fibrillation and for venous thromboembolism treatment.

Case: A 5-YO with Ecchymoses (October 4, 2011)

From the ASCLS Consumer Forum, a 5-YO female experiences chronic ecchymoses with minimal trauma. CBC results are normal; platelet count 343,000/uL with normal morphology, bleeding time is 4 minutes. PT, PTT, fibrinogen, and TT are normal. There is no platelet response to 2 uM arachidonic acid, 2 mg collagen, and none to ADP at 1 and 10 uM. Platelets also do not respond to 0.8 and 1.2 mg/mL ristocetin. What condition is likely, and what would you do next? George suggests Glanzmann thrombasthenia, but is curious about the ristocetin results. (No comments as of 11-11-11).

Hepzyme Source? (October 5, 2011)

Donna Robert needs a source for Hepzyme, which she used to get from Dade. George checked with Laura Taylor at UAB, they order Hepzyme from Siemens, which bought out Dade a few years ago. Hepzyme does not appear on the Siemens web site.

Clottable Protein C and S (October 6, 2011)

Pam Owens cites a specimen from a pregnant patient with a low clottable protein C. Her INR is normal, but she is a heterozygote for both factor V Leiden mutation and the prothrombin 20120 mutation. Could the prothrombin gene mutation could be interfering with the assay?

Dean Willett, Director of Marketing at Precision BioLogic, responds that Clot C is unaffected by FVIII levels up to 600% so we can probably rule out the possibility that high FVIII levels commonly observed during pregnancy are the culprit. In Precision’s Clot C validation studies we did not observe any influence from FV Leiden. We haven’t done any studies related to the effect of the prothrombin gene mutation, so there’s no way to be definitive, but, in theory, the excess thrombin in circulation could potentially be causing a procoagulant effect in-vitro that might be shortening clotting times, resulting in lower PC activity results.

George questions whether the mild prothrombin activity elevation associated with the prothrombin gene mutation is enough to account for a decreased Clot C. Given the physiology, he would first look to the factor V Leiden mutation gene. The pregnancy alone could account for the reduced protein C, independent of factor VIII activity levels.

Dabigatran and Lupus Anticoagulant Testing (October 7, 2011)

Pam Owens has a patient on dabigatran because he had multiple DVTs while on warfarin. Pam got a positive result on the DRVVT (ratio 1.27 with a cutoff of 1.18). Can dabigatran cause a false positive DRVVT? Dean Willett responds that we haven’t done any studies on dabigatran patient specimens (or any other DTIs) but assumes it interferes with many clot-based assays. George remarks that here is another (perhaps off-label) application of dabigatran: a substitute when warfarin is ineffective. Dean and George agree that any clot-based assay is likely to be prolonged by a DTI.

Is Ecarin Available? (October 7, 2011)

Gretchen Schaef Johns, M.D., Mayo Clinic, Jacksonville, FL asks, is ecarin available in the US and is the ecarin clotting time or ecarin chromogenic assay RUO? George responds that Diagnostica Stago has developed both the ecarin clotting time and the ecarin chromogenic assay, however the assays are RUO. Jeff Light of Centerchem reminds us that ecarin remains available from Centerchem as a raw reagent for developing in-house ecarin clotting times. Also, Centerchem will soon have FDA clearance for the Pefakit PiCT UC for monitoring all anticoagulant therapy.

Case: DRVVT Positive While on Warfarin (October 7, 2011)

From the ASCLS Forum: “I had a cerebral DVT in March 2009 at the age of 22. I have been tested 7 times for LAC while on warfarin over the past 18 months and 4 of 7 have come back positive. This has resulted in my hematologist prescribing warfarin for the ‘foreseeable future.’” George indicates from lab results the questioner was consistently negative for LAC using hexagonal phase phospholipid neutralization of the PTT, but generated ratios of 1.3 or 1.4 for LAC using the DRVVT with neutralization 4 out of 7 times over 12 months. The patient was tested once for ACL, the results were negative.

“My hematologist is uneasy with taking me off anticoagulants because of the location of my previous clot. How do I ease her mind? How will Lovenox injections affect the test results? I know the objective is to get me completely off the anticoagulants for a “clean” test, but if my doctor suggests this, is it an option?”

George issues disclaimers about avoiding diagnosis and treatment discussions online and suggested the DRVVT results seem to indicate LAC, but the best possibility for diagnosing or ruling out antiphospholipid syndrome while on warfarin was to test for ACL and beta-2-glycoprotein 1 antibody (IgG and IgM). While switching to Lovenox is a medical decision to be made by the physician, LAC testing by DRVVT while on Lovenox is reliable. George asked: while it is clear warfarin prolongs the DRVVT, it seems the ratio of the neutralized to non-neutralized clotting times could still be conclusive, especially when the test was repeatedly positive. Do you agree? Does she have LAC? Also, do you agree the patient could be tested while on Lovenox? No comments as of 11/11/11.

Post-partum Hemorrhage and Component Therapy (October 12, 2011)

Crystal Azevedo asks, “Why do healthy women who experience post-partum hemorrhage seem to ‘autocorrect’ their coagulopathy with very little help from component therapy? Once the bleeding site has been tended to, it seems the patient requires relatively little support with factor/fibrinogen replacement.”

George didn’t have a ready answer, so he posted this question for our expert participants and directed Crystal to Callaghan WM, Kuklina EV, Berg CJ: Trends in postpartum hemorrhage: United States, 1994-2006. Am J Obstet Gyn 2008;202:353. No comments as of 11/11/11.

Factor X Assay in APL (October 19, 2011)

Shaun Seth: I read Dave McGlasson’s commentary on factor X assay and its role in antiphospholipid antibody syndrome. I was curious to know when or why a lab would use the factor X assay vs. the factor II assay. George replies that the factor X chromogenic assay is an excellent means for monitoring warfarin (oral anticoagulant therapy) in place of the PT/INR whenever you suspect interference, such as prolongation by lupus anticoagulant. In theory, a chromogenic factor II (prothrombin) should work just as well, but Mr. McGlasson responds there just aren’t many reliable chromogenic factor II assays available, in contrast to the chromogenic X, which has excellent precision characteristics.

D-dimer Lot-to-lot QC (October 20, 2011)

From Toni Okada, Bellevue Medical Center, WA: “I attended your session on coagulation problems at the NW Medical Lab Symposium. Thank you for a great presentation and for putting the symposium information on your website. I would like to know what protocol you use for checking out new lots of D-dimer reagent? Siemens used to provide product insert information about target QC ranges for each lot of reagent but they’ve stopped.

George contacted a Siemens representative for follow-up on the question, but received no answer as of 11/11/11. He also checked with Cari Reed at UAB, where they use Stago kit controls, which auto-calibrate when placed on the instrument. Meanwhile, Crystal Azevedo, Affiliated Laboratory, Inc., Bangor, Maine commented: “My laboratory is using the Liatest D-Dimer by Diagnostica Stago on a STAR Evolution. When we switch lots of reagent, I run 15-20 normal plasmas. I can then validate my cut-off value using a local population. As far as QC, we do run at least 30 points of each of 2 levels to be included in a peer group with Stago.

Is PTT Monitoring of UFH a Thing of the Past? (October 21, 2011)

George received this note from Stephen Duff, Co-CEO of Precision BioLogic Inc about his October 19 “Top Ten” post: “I noted with interest your Top Ten summary over the years and was puzzled to see ‘PTT Heparin Therapeutic Range’ 2006 with a check mark beside it for ‘Resolved’. I think many labs still struggle with getting a sufficient number of samples to perform the Brill-Edwards exercise and many do not have an anti-Xa up and running. Any insights?”

George responds: “I may have had a brain cramp when I typed that check mark in the ‘Resolved’ column, but my thinking at the moment was that when you add up those who have changed to LMWH and fondaparinux, plus those who have gone from the PTT to the anti-Xa heparin assay, and throw in the new oral anticoagulants rivaroxaban, apixiban, and dabigatran, surely the need for the Brill-Edwards curve has gone on the endangered, if not the extinct list.” In follow-up, George posted a reprise of a 2008 Quick Question to learn how far we’ve come in monitoring heparin, and whether we can really claim the “ex vivo” calibration curve has been resolved.

Quick Question Summary: PFT Blood Collection (October 25, 2011)

In a summary of our October, 2011 Quick Question, “What specimen do you require for platelet function testing,” George comments that a few of us “die-hards” continue with older collection methods to avoid platelet activation. Whether the butterfly set activates platelets or reduces platelet activation in comparison to standard collection remains an open question.

Specimen Freezing (October 25, 2011)

From Kim Kinney “Hi George, Even though I am not in coag much any more I still get a ton of questions. One of our TC’s was questioning our storage of PT/INR specimens. We follow the current recommendations of room temperature for 24 hours. We do not accept refrigerated whole blood or spun plasma refrigerated. We do accept frozen plasma. He wanted to know why you can freeze plasma without activation of VII but refrigeration may activate VII and therefore, shorten your PT. I was not sure of an answer other than frozen is a quick freeze; can you help?”

George responds, as documented in CLSI H21-A5, specimen chilling activates factor VII and also causes large von Willebrand factor multimers to precipitate upon centrifugation. These effects are avoided if the specimen is frozen rapidly, for example, by placing platelet free plasma in a -70°C freezer, and thawed rapidly, typically in a 37°C water bath. Prolonged chilling over several minutes, such as transporting on ice or storing in the refrigerator, or repeated freeze-thaw cycles may unpredictably shorten or prolong PTs or PTTs.

Study Sites Needed (October 25, 2011)

From Lauri Ketch, Manager of New Product Development at Precision BioLogic Inc: “We are gearing up to begin external studies for a new product, the results of which will be used in a 510(k) submission next year. We are searching for labs to partner with for these studies, which would compare our product to an existing, in-market predicate device. Should anyone be interested in learning more they can contact me at 902.468.6422 x 265, lketch@precisionbiologic.com

Does Beta2-GP1 Affect the SRA? (October 31, 2011)

From Dr. Jeanine Walenga at Loyola University Medical Center in Chicago, “Are there data on whether β2GP1 interferes in the SRA for HIT testing? Dr. Jon Geske, senior R&D scientist at Precision BioLogic Inc, responds: “I am not aware of any studies that suggest that β2GP1 interferes with the SRA test. I found one paper (Nimpf et al. Atherosclerosis 1987;103: 109-14) that suggested that β2GP1 inhibited serotonin release induced by ADP, but platelet activation in HIT is through the Fc gamma receptor.” George found one additional reference: Shi W, Chong BH, Chesterman CN. Beta 2-glycoprotein I is a requirement for anticardiolipin antibodies binding to activated platelets: differences with lupus anticoagulants. Blood 1993; 81:1255-62. The authors state: “In neither case does LA nor ACA have an effect on thrombin-induced release of serotonin or beta-thromboglobulin nor do they affect platelet aggregation induced by a number of agonists. This antibody binding may play an etiological role in thrombocytopenia associated with aPL, but does not explain thrombosis on the basis of hyperaggregability or increased platelet release.”

PTT Specimen Storage Time (October 31, 2011)

Joe Lamb, St. Francis Hospital, Columbus, GA notes that recent updated test directories for the two big reference labs state the specimen stability for PTT is 24 hours at room temperature. This causes confusion when we reject a specimen more than four hours old for PTT testing. Did these guys do their own studies? Who’s right, the Clinical and Laboratory Standards Institute (CLSI) or the Big Guys? Hi, Joe, and thanks for alerting me to this apparently new requirement. I checked the “Big Guys’” web sites and confirmed your statement, which seems to contradict standard H21-A5 that limits the PTT to four hours’ room temperature storage. I’ve forwarded your question to several colleagues to see if they have some new data.