George thanked those who participated the November 1 Teleconference Network of Texas (TNT) presentation, “Monitoring the New Anticoagulants; Whatever Happened to the PT and PTT?” George referenced Jeff Light as his contact at CENTERCHEM, the company that markets the Pefakit® PICT, a means for monitoring the new oral anticoagulants.
Patti Richardson and Dr. Manjula Balasubramanian, St Christopher’s Hospital for Children, Philadelphia, PA asked, “Is it necessary to have a reference range for the INR? If so, should it be derived from the normal prothrombin time (PT) ranges?” George suggested that laboratory directors typically compute and publish an INR reference interval from the PT reference interval in addition to the target therapeutic range.
David Chance, Good Samaritan Medical Center, Corvallis OR, commented that overfilling tubes, unless clots are present, does not affect coagulation results. Overfilling may occur when a specimen is collected by syringe and pushed into a tube.
Herb Crown at St. Louis University Hospital Coagulation Reference lab suggested that transferring blood from a syringe exposes the blood collector to risk of needle-stick. Also, the specimen would start to clot when drawn in a syringe with no anticoagulant and could be hemolyzed because of the velocity in which the specimen is pushed. Also, results would be suspect because our reference ranges are set using tubes with a fixed vacuum that is standardized.
George agreed that hemolysis and clotting risks increase with the use of a syringe, however it is a standard method referenced in CLSI H21-A5. Replacing the needle with a 20-gauge or larger transfer needle and allowing the blood to flow down the side of the tube minimize hemolysis during whole blood transfer. Needle-stick injury is minimized by placing the evacuated tube in a rack to make the transfer; further, BD makes a shielded “safety transfer device” for this purpose.
On November 4 the FDA cleared oral anti-Xa Rivaroxaban for stroke prevention in atrial fibrillation.
Jose DeJesus, transfusion service manager for DCH Regional Medical Center in Tuscaloosa, Alabama has managed three warfarin overdose patients who were blood group AB. AB donor plasma is in chronic short supply and AB recipients should only receive blood group AB plasma to avoid acute intravascular hemolysis. Jose asked what he could do if another AB patient arrived and there was no AB plasma available.
Margaret G. Fritsma recommended you do not substitute A, B, or O plasma, as the possibility of hemolysis outweighs any possible hemostatic benefit. Chest 2008;133:160S-198S recommends, “In patients with serious bleeding and elevated INR, we recommend holding warfarin therapy and giving vitamin K (10 mg) by slow IV infusion supplemented with fresh frozen plasma, PCC, or recombinant factor VIIa, depending on the urgency of the situation. Although fresh-frozen plasma can be given in this situation, immediate and full correction can only be achieved by the use of factor concentrates because the amount of plasma required to fully correct the INR is considerable and may take hours to infuse.”
George suggested that in the absence of AB plasma, Proplex or NovoSeven are the answer. Both are managed carefully, as they may eventually lead to thrombosis, conversely, they would not lead to TACO.
Kerri Klemm asked if anyone has had problems with ristocetin-induced platelet aggregation? Kerri runs controls with ristocetin at 1.5 mg, which reacts and 0.75 mg, which shouldn’t react but does.
Herb Crown uses 4 concentrations of BioData ristocetin: 1.2, 1.0, 0.75 and 0.50. Based on the results, Herb may add 1.5 or 0.25. His reference range for 0.75 is lower than that of 1.2 and 1.0 and higher than the 0.50 concentration and it varies from time to time on the same control. Herb suggested you may consider the instrument as the source of the problem. BioData recommends sending instruments back to them periodically for “recertification.”
George contacted Kathy Jacobs, Sales Coordinator at Chronolog Corporation, Havertown, PA, who responded, “Your message comes at a very good time as we are expecting a new lot of ristocetin. I will be testing it against our current lot and will test some additional concentrations and let you know how it looks.”
Vilas Hiremath, United Labs, Bangalore, responded on November 22, “It is possible to have ristocetin no aggregation or no response in Glanzmann thrombasthenia. Please refer to Saunders Manual of Clinical Lab Science chapter 40, page 981.”
Jose DeJesus’ November 9 question about providing plasma to an AB patient generated further discussion.
First, if you routinely give incompatible platelet concentrate, why not incompatible plasma? The answer may relate to product volume. Platelet concentrate therapy typically involves infusion of up to 300 mL of product per unit, similar to plasma, however we usually infuse multiple plasma units, exposing the recipient to greater hemolysis risk, whereas platelet products may be administered more sparingly.
Second, why not give group A plasma? A current article, Immunohematology 2011, 27:61-5, suggests group A plasma with low-titer (< 1:16) anti-B antibody is safe to use as a universal donor plasma in acute blood loss. However, the article addresses acute blood loss, not hemorrhage secondary to warfarin overdose, where blood loss is probably not severe enough to warrant using a multiple transfusion protocol. NovoSeven or Proplex may restore coagulation without risking incompatibility.
Third, by anecdote, several transfusion service medical directors report they may theoretically choose to use low-titer group A plasma if AB is unavailable, though none has actually had to do so.
And finally, plasma may not fully restore hemostasis, as it provides only normal coagulation factor activity. There is the possibility of triggering TACO by using multiple plasma units, without restoring the INR.
Stephen Duff, Co-CEO of Precision BioLogic Inc reports the results of a phase 3 trial indicated that apixaban did not meet the primary efficacy outcome of superiority to enoxaparin for the endpoint of venous thromboembolism (VTE) and VTE-related death at day 30 in people with acute medical illness. The apixaban arm had a 13 percent lower rate of events than enoxaparin followed by placebo, which favored apixaban but was not statistically significant. The key safety outcome of major bleeding was low in both groups but occurred in more patients treated with apixaban than with enoxaparin, 0.47% versus 0.19% of patients in the enoxaparin group.
Adding low doses of rivaroxaban to standard antiplatelet therapy significantly prevented the occurrence of secondary cardiovascular events in patients with ACS, compared with standard therapy alone. Producers plan to seek US and European approval use in patients with ACS by the end of the year.
Dr Samuel Z Goldhaber (Brigham and Women’s Hospital, Boston, MA) at the annual AHA meeting indicated that there is a lack of randomized clinical trial evidence for bridging anticoagulation, though it is standard practice.
Michele Drejka, Isermann Special Coagulation Laboratory Frederick B. Cohen MD Comprehensive Cancer and Blood Disorders Center Newark NJ, participates in both CAP CGE and NASCOLA ECAT proficiency challenges. Michele is looking for acceptable criteria for the VWF:RCo activity by agglutination. She usually looks for ±2 SD, but cannot find criteria for VWF:RCo.
Dr. Emmanuel Favaloro, Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, New South Wales, Australia, responded: For EQA, every provider uses a different terminology and different acceptance criteria; not sure about CAP & NASCOLA ECAT, but NEQAS (UK), for example, uses the lowest and highest 10% of participant results to express ‘undesirable’ performance. In Australia, the RCPA uses ‘allowable limits of performance’ which is essentially an expert committee guideline which is test specific; for VWF:RCo, allowable limits are ±4 for values up to 10%, and ±40% for values >10%. These are fairly wide to take into consideration the test’s well-known variation.
In practice, as working examples; A group median result of 5% VWF:RCo will have an acceptable allowable limit range of 1-9% (i.e. 5 ±4), and any values reported below or above this will be identified as ‘undesirably low’ or ‘undesirably high’ respectively. A group median result of 50% VWF:RCo will have an acceptable allowable limit range of 50 ±(40% of 50%) = 50 ± 20% = 30-70%, and any values reported below or above this will be identified as ‘undesirably low’ or ‘undesirably high,’ respectively. Yes, very wide, but the RCPA will still get around 20% of participants falling outside the acceptable ranges in any given survey sample! As a comparator, allowable limits for VWF:Ag are ±3 for values up to 10%, and ±30% for values >10%, and allowable limits for FVIII are ±3 for values up to 10%, and ±25% for values >10%.
In terms of SD, however, this per-test variability is probably already taken into consideration, since SDs reflect a proportional variation; thus, a test result for FVIII including ±2SD will be narrower than a test result for VWF:RCo including ±2SD, and so ±2SD might be valid as an allowable range for both tests. ±2SDs basically covers about 95% of expected test results, and ±3SDs basically covers about 99% of expected test results. In quality control, we basically advise to follow established rules such as Westgard.
George also directed Michele to Chapter 5, Quality Assurance in Hematology and Hemostasis Testing, in Rodak BF, Fritsma GA, Keohane EM. Hematology Clinical Principles and Applications, 4th Edition, Elsevier, 2012.
Vilas Hiremath, United Labs, Bangalore, reported a one-day old baby citrate sample, unable to separate plasma after centrifugation. Repeated with fresh sample, same problem; what is the cause?
George asked, does this mean no plasma layer appears after centrifugation? If so, perhaps polycythemia has made the hematocrit exceptionally high, leaving very little plasma layer, or there is considerable hemolysis so the plasma layer is difficult to distinguish. If the plasma is clotted and therefore difficult to extract, it could mean there is chronic DIC causing excessive clotting.
From Robert Löfvenmark, MediRox AB, Nyköping, Sweden “I am curious about the international differences in the use of PT. Most parts of the world use PT Quick except Scandinavia, where we use the Owren method. Owren seems better in all aspects. It utilizes a buffer to pre-dilute the sample giving a final dilution of 21:1 and has a thromboplastin reagent with bovine factor V and fibrinogen. This gives full focus on vitamin K factors; is insensitive to factor V and fibrinogen levels so old samples are not a problem; lacks fibrinogen sensitivity; high INR in mechanical systems is not a problem; it is insensitive to optical disturbances owing to 1:21 dilution; it is suitable for capillary testing of whole blood after pre-dilution, can be used with plasma or whole blood, and has less variation among manufacturers. The only negative sides of PT Owren are that it adds a buffer and is a corner of a penny more expensive. Why do a majority prefer the Quick?”
George checked with Cindy Johns, who had written the following in a 1981 textbook: “The Thrombotest reagent contains an optimal concentration of calcium with adsorbed bovine plasma as a source of factor V and fibrinogen, and a tissue thromboplastin of bovine brain origin. This reagent gives longer coagulation times than those recorded with the standard one-stage Quick PT test and, according to Owren, reflects sensitivity to intrinsic-system factors, particularly to factor IX. This test system is also extremely sensitive to the concentration of factors VII and X. Since the method is designed primarily for the control of therapy with anticoagulants and not the diagnosis of hemorrhagic disorders, its accuracy is highest with activity levels below 50%. A therapeutic range for anticoagulant therapy is 5-15%.”
Cindy and George speculated that perhaps the Quick PT may have seemed more useful in 1981 because it functioned as a coagulopathy screen as well as a warfarin monitor. It is likely the PT has since become less often applied as a coagulopathy screen, and the Owren PT could see a comeback.
Rob responded the next day, “I was not around with the Thrombotest, but I have heard that it was the use of bovine thomboplastin that had some set backs. It was slower, more imprecise, but I think mad-cow disease was a topic of discussion as well? Owren brands used here in Scandinavia use rabbit thromboplastin. And beside MediRox which is a small local firm it is produced by global hemostasis companies like Stago and Axis Shield, so it should be available for the rest of the world if you wanted it.
So what can it be? Coagulopathy screening? Possibly, but I think teaching clinicians to order a PT Quick for those < 10% tests should easily balance the advantages for the remaining 90% OAT patients. Do you agree? Owren is slightly slower, that is true. Normal clotting time is 20-22 sec vs 10-14 for Quick. But again, there are many smart people out there that would figure out a faster Owren reagent if that was required. For example with a 1:15 dilution you would still avoid interference problems yet gain all the Owren pros. And as a parenthesis on speed; many automatic systems-like US market leader IL ACL TOP-have fixed analysis times on the tests, and are not at all optimized for speed, so the general lab can´t be in that hurry. it just the tradition? Opinions from opinion-leaders? Has a less good method won because of tradition and possibly marketing efforts? That would be kind of sad-I am really hoping that you could tell me different.”
George also received this reply from Dr. John D. Olson, Director of Pathology, University of Texas Medical Center: “I have had no experience with the Owren method. I suspect that there is little advantage of one over the other and your hypothesis makes sense to me. The Quick method is easy to automate and it is likely that once there was a marketing foothold, laboratories saw little reason to make a change. With INR gaining a very strong acceptance, I think the Quick method will remain dominant.”
And finally, George had a follow-up email from Flo Newlin, retired co-president (with Gordon Ens) of Colorado Coagulation Consultants. She, like Cindy Johns and Dr. Olson, considers the choice of the Quick over the Owren PT to be mostly driven by historical and perhaps economic forces. Likewise George spoke with Dr. Larry Brace of Edward Hospital, Naperville, IL reaching the same conclusions, and with Dave McGlasson, Wilford Hall USAF Medical Center in San Antonio, who advocates for the chromogenic X assay for its accuracy and reproducibility.
A November HeartWire describes problems reversing the bleeding associated with Dabigatran in trauma.
Ali Sadeghi-Khomami, PhD, senior research scientist at Precision BioLogic Inc sent a link to a new algorithm-driven Novo Nordisk app for iPhones, Coags Uncomplicated.
Mary Block, Cincinnati Children’s Hospital said she is fairly new to the special coagulation field and wants to know how labs set up contact factor assays. What do you use for calibrators and controls for HMWK and PK?
George responded, “You may have relatively few opportunities to assay HMWK or PK, as deficiencies are rare and have no clinical consequence. Factor XII deficiency is comparatively common, however there is no association with bleeding, though you may run a few factor XII assays in follow-up to an unexplained prolonged PTT. Obtain PK and factor XII-deficient plasmas from plasma distributors such as Precision BioLogic; HMWK-deficient plasma may be more difficult to locate. Precision also offers calibrators and controls for PK and factor XII assays. The assays are set up just as you would a factor VIII or IX assay.
Larry Brace, PhD, Edward Hospital in Naperville, IL, referenced the RE-LY study’s reported high bleeding rate for dabigatran, which may be linked to using the 150 mg/day dosage. Dr. Brace suggests on the basis of physiology that the anti-Xa anticoagulants such as rivaroxaban and apixaban may be safer.
“Ipaz” at Iline Microsystems asked, “For the dilute thrombin time, why is the patient plasma diluted with normal pool plasma and not with saline solution?”
George responded that the dilute thrombin time assay monitors the efficacy of DTIs dabigatran, hirudin, and argatroban. Patient plasma is first diluted (1:8 for dabigatran) using normal saline. Next, pooled normal plasma is added, then purified human α-thrombin, and the interval to clotting is recorded and compared to DTI calibrators. Diluting the patient plasma and adding normal plasma enhances assay reproducibility. The patient’s DTI acts upon a predictable level of thrombin provided in the normal plasma, and variations in patient procoagulant activity have little effect upon the clotting time.
Dr. Jean Amiral, President and Scientific Director of Hyphen BioMed and dilute thrombin time developer commented the plasma pool is used to render the assay insensitive to variable coagulation factors in tested specimen (mainly II and Fbg).
From Profa. Dr. Terezinha Paz Munhoz, Faculdade de Farmácia PUCRS, Chefe do Setor de Hematologia
Laboratório de Patologia Clínica Hospital São Lucas PUCRS:
“We are considering to exchange our coagulation equipment and the reagents as well. What is the best thromboplastin, recombinant or brain thromboplastin? What is the best application for each of them? We’ve been using brain thromboplastin for years and now that IL has both, we have the possibility to choose. Our population is composed of both inpatients and outpatients.
George has preferred recombinant thromboplastin for several years as its ISI is always near 1.0 and it is reproducible from lot to lot, however he hopes to generate some discussion relative to the best applications for each. (No responses as of 12.27.11).
Dorothy Zdanowicz, Chemistry/Coagulation Supervisor, Englewood Hospital & Medical Center, Englewood, NJ asked, “Has anyone been able to adapt a third-party reagent for ristocetin onto a Stago STAR-E or Compact? If so, would you be willing to share your experience?”
George knows of one facility that successfully adapted the Siemens automated ristocetin cofactor assay on a Stago STA several years ago, but knows another that was unable to use the reagent on the STAR-T. Dave McGlasson suggests using the Beckman/IL LIA VWF assay. He said it worked on the Evolution with CV’s less than 5% and had great sensitivity and specificity characteristics. Stago and Beckman/IL have test set-ups for the LIA method.
“Sunny” asked, what is the reason for not using PT to investigate LA, since it is also a phospholipid-dependent assay?
George confirmed that a particularly avid LA may on occasion prolong the PT, but PT reagent (thromboplastin) phospholipid is typically concentrated enough to neutralize LA so that it has no, or limited, influence on the assay. The tissue thromboplastin inhibition time (TTIT) test is a PT modification in which the reagent thromboplastin is diluted, and hence the phospholipid concentration is reduced. Although the LA panel used by most laboratories features the DRVVT and LA-sensitive (low phospholipid) PTT, the TTIT has been used successfully by many laboratories. In 2009, however, the TTIT, also called the dilute PT (DPT), failed to retain the recommendation of the International Society for Thrombosis and Haemostasis Subcommittee on Lupus Anticoagulant/Phospholipid-dependent Antibodies. The Subcommittee offered the opinion that the DPT suffers from variability in thromboplastin reagents. This recommendation is up for debate, and many laboratories continue to offer the TTIT as a means for detecting LA.
Steve Duff, co-CEO of Precision BioLogic received this question from a subscriber:
“Has Precision ever considered producing a set of plasmas containing varying levels of unfractionated heparin? I think there is a strong need for a commercial product that can be used to fulfill the CAP requirement for heparin therapeutic range development. It is a struggle for the smaller labs, such as those in community hospitals, to find 30-40 samples that meet the criteria. These are currently needed during initial installation of an analyzer, and every year when they change lot numbers of PTT reagent, so it would be an ongoing demand.”
Steve replied, “What you are describing is the ‘holy grail’ that we’ve been chasing for decades. We envisioned a set of 30 ex vivo samples on UFH that would have anti-Xa values assigned. The lab would then simply perform PTTs on the set, go online and input their PTT results and the lot number for the set. Software would immediately calculate and spit out their Brill-Edwards curve with the PTT therapeutic range in seconds. The challenge is getting a sufficient volume of plasma from individuals on UFH. Typically, we receive plasma via apheresis procedures on “healthy individuals.” It is virtually impossible to have someone on UFH cleared as a healthy individual by the medical director of an apheresis centre since UFH is nearly always tied to a previous event.
An article posted December 15 on TheHeart.org quotes Dr. Jawed Fareed, Loyola University, Chicago, who demonstrated using an in vitro experiment that rivaroxaban, apixaban, and dabigatran do not interact with the anti-platelet factor 4 antibodies responsible for HIT. The findings were reported at this year’s American Society for Hematology annual meeting.
Per Stephen Duff, co-CEO, Precision BioLogic, a December 19 “FirstWord” post announced EU clearance of rivaroxaban for stroke prevention and treatment of deep venous thrombosis.
An article being prepared by Dave McGlasson compares several DRVVT kits for their accuracy in detecting lupus anticoagulant. Dave advocates for DRVVT ratio normalization. The normalization formula is provided in the post.