Mixing Studies 4:1? (November 4)
“Mirnaibarra” in El Paso asks, “Do you recommend the 4:1 mix to be used routinely? Also, what is the most common and preferred way of interpreting mixing studies correction; percent correction, a percent of PNP or the Rosner Index?
George answers that most labs use the 1:1 mix of patient plasma and NP for initial mixing studies, occasionally using a 3:1 or 4:1 mix when a thrombotic picture suggests LA but the 1:1 mix corrects. Dr. Larry Brace also advocates for the 1:1 mix on the grounds that a lupus anticoagulant-sensitive PTT reagent is at least as likely to detect lupus anticoagulant as is a 4:1 mix.
George posted a Quick Question in June polling our participants on their criteria for mixing study correction. The majority of people use correction to within 5 seconds of the normal control plasma. George advocates for correction within 10%, using the Rosner index, whereas Dr. Dorothy Adcock of Esoterix Coagulation requires correction to within the upper limit of the normal range.
Heparin Recall (November 16)
Steve Duff of Precision BioLogic alerts us to a new recall of unfractionated heparin posted by the US FDA on October 27, 2010. The supplier, Scientific Protein Laboratories, in testing retained crude heparin lots revealed traces of oversulfated chondroitin sulfate. This is the same problem that created a public health risk in 2008.
Anti-Xa and Lipemia (November 18)
“McDowekm” performs anti-Xa heparin testing on a hybrid curve using Stago’s Rotachrom reagent. If a specimen is lipemic, it can falsely raise the anti-Xa result. Can lipemic specimens be ultra-centrifuged to obtain a more accurate Anti-Xa result?
George responds that it would be helpful to review the data that indicate lipemia is raising the anti-Xa results. When performing the Rotachrom assay according to published protocols using the hybrid curve, the original specimen reaches a final dilution, counting diluents and reagents, of 1:22. Given this dilution, and according to the manufacturer’s package insert, interference will occur only when the original triglyceride concentration rises to above 360 mg/dL. Also, lipemia would be expected to reduce, not raise the anti-Xa result. This post generated three comments.
Mixing Studies Interpretation (November 18)
Stacy Askvig at the University of North Dakota Medical Center asks, “I do only PTs and PTTs, and my references seem to give me conflicting interpretations. Our patient has a prolonged PTT and DRVV, both are shortened in mixing studies:
PTT-LA 107 seconds, mix 79.5 (reference interval 0-50)
DRVV 73 seconds, mix 56.7 (reference interval 0-44)
George responds the PTT and DRVVT are both prolonged and do not adequately correct in the mixing studies. This is presumptive evidence for the presence of a lupus anticoagulant (LA). To confirm an LA Stacy would need to next perform a phospholipid neutralization step using a high phospholipid reagent.
Mixing Studies and The Thrombin Time (November 20)
“Skierktc” wonders if anyone screens with the TT plus protamine sulfate to rule out heparin in the specimen. If yes, what is their policy for continuation of the study?
George responds that when you encounter a prolonged PTT that is not corrected by NP, perform a TT to determine if the patient is receiving heparin. Heparin will prolong the TT from its normal mean to 40 seconds or longer. Once you have concluded heparin is present, treat the specimen with Hepsorb or Hepzyme, then repeat the mixing study.
Apixiban Phase 3 Trial Halted (November 22)
On 11/19/10, Pfizer and Bristol Myers Squibb discontinued the APPRAISE-2 phase 3 trial of Apixiban in high-risk patients with acute coronary syndrome. Apixiban bleeding risks were not offset by its antithrombotic benefit in the high-risk patient population. At least two trials appear to show that Apixiban, which is a direct anti-Xa oral antithrombotic, is effective at stroke prevention in atrial fibrillation. The oral direct anti-thrombin drug Dabigatran (Pradaxa, Boehringer-Ingelheim) was released by the US FDA for stroke prevention in October.
Short APTT Sensitivity Survey (November 22)
We recently posted a brief survey asking laboratory scientists to characterize the sensitivity of their “routine” APTT assay for LA. This is a new three-question follow-up survey for physicians. The APTT is used to monitor anticoagulant therapy, screen for coagulation factor deficiencies as a means to predict risk of bleeding, and to screen for LA, which may be associated with thrombosis and or recurrent miscarriage. A number of APTT reagents are available that are highly, moderately, or minimally sensitive to lupus anticoagulants. We propose to learn which of these three applications of the APTT are of most importance to clinicians, and to determine clinician understanding of APTT reagent LAC sensitivity. Investigators are Ellinor Peerschke, PhD, Elizabeth Van Cott, MD, Dorothy (Dot) Adcock-Funk, MD, and Marisa B. Marques, MD. Ankush Randhawa and George Fritsma assisted with survey development.
More on Mixing Studies and the Thrombin Time (November 23)
Herb Crown (St. Louis Coagulation Consultants) provides a comment on mixing studies.
We use the following testing scheme: 1) perform a PT, PTT and TT. If the TT is significantly prolonged, treat the plasma with Hepzyme; repeat the PTT and the TT. If heparin is the offending agent, the PTT may or may not correct, but the TT will correct. We will use the treated plasma to perform the mixing study.
We also have a “home-grown” control consisting of factor VIII deficient plasma and NP mixed 1:1 and tested immediately and after 60 minutes’ incubation at 37°C. We have established normal reference ranges for zero time and 60 minutes so we can evaluate patients’ results to a reference range. One could build an inhibitor positive control using lupus anticoagulant positive plasma mixed with NP.
Also mentioned is physician reluctance to canceling a mixing study. Even though heparin is removed from plasma, a mixing study may still be of value whether it is positive or negative. Also, just because a plasma tests negative for inhibitor studies doesn’t mean an inhibitor is not present.
Herb’s discussion also reminds George of the saline mixing study. Mix one aliquot of patient plasma 1:1 with NP, and a second aliquot 1:1 with saline. If the result obtained with the NP mixture corrects and the saline mixture prolongs dramatically, suspect a factor deficiency or a specific factor inhibitor. Conversely, if the result obtained with the NP mixture shows no correction and the saline mix shows correction, suspect a lupus anticoagulant.
Effect of Dabigatran on PT and PTT (November 24)
Judy Miller asks, can you provide a guideline for how dabigatran therapy will affect our PT, PTT and fibrinogen test results?
George answers that dabigatran, like argatroban, prolongs the PT and PTT. It also prolongs the TT, which means it generates a false decrease in fibrinogen assay results. Dabigatran’s concentration peaks at two hours, and its half-life is 12 to 17 hours, so the PT, PTT, and fibrinogen assays should be made at least 24 hours after discontinuing dabigatran.
Another question is how do we monitor dabigatran (and rivaroxaban). We will need to monitor in the case of renal disease (dabigatran), liver disease (rivaroxaban), or when there is drug interference. We’ll also need a way to determine on a stat basis when one of these new drugs is responsible for bleeding episodes.
Dr. J. Amiral, president of Biophen recommends Biophen’s Hemoclot Thrombin Inhibitor assay for dabigatran and Biophen’s direct anti-Xa assay (which differs from the heparin anti-Xa assay) for rivaroxaban.
Pre-op Plavix Assay (November 24)
Dan Hood asks, “What testing do you recommend for Plavix effect for patients preoperatively?”
George indicates that Plavix suppresses platelets by blocking ADP receptors. The reference method for Plavix effect is platelet aggregometry using ADP as the agonist. However, if your laboratory is not performing aggregometry, you may want to use the Accumetrics VerifyNow P2Y12 or the Helena PlateletWorks ADP Kit. Further, DiaPharma distributes the Multiplate 5.0 Analyzer, which is currently under FDA review. Joe Lamb and Donna Lawler add comments recommending the Thromboelastograph.
False Positive Phospholipid Neutralization (November 29)
G Pihan from Beth Israel Deaconess asks, “What is the incidence of false positive hexagonal phospholipid neutralization tests in the presence of a strong (400-800 BU) factor VIII inhibitor?”
George answers the hex-phase phospholipid is the most specific platelet membrane surrogate for identifying lupus anticoagulants when testing in a PTT system. There are no data published on the incidence of false positives, although it is possible that a high-titer factor VIII inhibitor could cross-react in the Staclot-LA system.
The ISTH requires that we use at least two parallel testing systems to confirm LA, and most of us use a PTT-based kit and a dilute Russell viper venom (DRVVT)-based kit. In instances when the DRVVT result is negative but the PTT-based hex-phase phospholipid result is positive for LA, the lab should follow up with a factor VIII assay to rule out factor VIII deficiency or a factor VIII inhibitor.
Incubated Mixing Studies (November 29)
Joe Lamb, St Francis Hospital, Columbus, GA suggests testing patient and NP after incubation to ensure the labile factors haven’t deteriorated and agrees with doing the saline mixture. He asks, “Do you report these “extra” results?”
George responds that you should always incubate an aliquot of the NP alongside the patient-NP mixture so that you can compare the incubated mixture PTT to the incubated NP PTT. The incubated PTT is likely to be a little longer, reflecting minor deterioration of the labile factors. We should publish mixing study results with an interpretive comment that clearly explains the meaning of the data for the ordering clinician.