August 2012 Cheat Sheet

Crystal Azavedo asked if there exists any RCT data that compare bleeding and thrombosis risk in patients receiving UFH monitored with the PTT compared anti-Xa ?

George describes a lab that recently ran parallel PTTs and anti-Xa heparin assays on their UFH patients over several days. In comparing results, in 40% of cases, anti-Xa results that were in range paired with PTT results that were either above or below. Conversely, anti-Xa results that were high or low paired with PTT results that were within range. There were many instances in which patient dosages would have been inappropriately modified based on the PTT results. Dr. Paul Riley, Diagnostica Stago Inc, provided a bibliography of heparin monitoring studies that was linked in this post.

Sara Sneeden, Lee Memorial, said that their hematologists order mixing studies on patients who have been on Pradaxa®. These patients have VMax PT and PTT , and the mixing study is ordered on a subsequent draw. Is there anything to be learned from this study? Is there a better specimen with regard to the timing of this test?

George asserts that there is nothing to be learned from a mixing study while the patient is on dabigatran, and no physician would discontinue A/C therapy simply to get an accurate mixing study. If they were willing to discontinue, the half-life of Pradaxa is short, and they could probably get useful results after the patient is off the anticoagulant for three days. Perhaps they are attempting to identify the type of anticoagulant that is in use, or attempting to monitor its therapeutic effect. Also, if they are looking for a LA , you may be able to help them with the anti-cardiolipin antibody and anti-β-2 glycoprotein 1.

From Monica Truta: Because in Romania we don’t have books, guidance, or procedures about validation I want to know if for fibrinogen validation it is necessary to establish the LLD. If the mfr does not provide CV% for precision and accuracy what can I do?

George recommends that Monica establish LLD, and also precision and accuracy data. She may find everything she needs at the Westgard QC web site. Also, our audio modules 3 and 4, Method Validation 1 and 2, contain the information. Dave McGlasson and I have recently published Quick Guide to Laboratory Statistics and Quality Control, 2012, AACC Press, available from AACC , or Amazon.

Carol Gizzi at Baycare in Florida performs the FVIII assay with a silica-activated PTT reagent to monitor hemophilic patients. Recently she was asked to switch reagent to a PTT reagent using ellagic acid as the activator. We thought the silica-activated reagent was the most sensitive? Are we incorrect?

George knows of no reason to switch from a silica-based to an ellagic acid-based PTT reagent for the routine monitoring of coagulopathies, and in particular for factor VIII. He and participant Dave McGlasson checked with several reagent distributors, and none knew of any reason. However, tt is true that the ISTH recommends silica-based PTT reagents for the detection of lupus anticoagulant, as they are more sensitive than ellagic acid-based reagents, however both should work fine for monitoring factor deficiencies.

In an August 14 comment JLow reference our June, 2012 paper in AJCP (reference provided), where the recommendation was made that the screening PTT reagent should be lupus insensitive. Therefore, if Actin FS was recommended (rather then Actin FSL), this is the rationale for changing PTT reagent for screens and factor assays.

George responded on August 18, yes, an ellagic acid-based PTT reagent would be less sensitive to LA and therefore preferred for routine UFH monitoring and for factor deficiency screens.

On August 19, Dr. Emmanuel Favaloro commented that it is likely that the recommendation for Carol’s lab to switch to an ellagic acid-based PTT reagent is related to the perception that ellagic acid reagents are relatively ‘insensitive’ to LA. Such reagents are now considered to be preferred as a general screening test system to avoid unwanted detection of ‘asymptomatic’ LA positive individuals and the downstream problems this will cause in terms of investigative follow up testing, patient anxiety, and delays to surgery. Moreover, an LA-insensitive PTT reagent may be preferred for factor assay testing to avoid the influence of potential LA on factor assay results. However, it should be cautioned that not all ellagic acid PTT reagents are LA insensitive. In particular, both Actin FS and Actin FSL are ellagic acid based, but Actin FSL is not LA-insensitive. He provided a reference.

Jennifer Jacobsen, MLS (ASCP), Allina Health Laboratories, Minneapolis, asks about specimen management on patients being treated with TPA : She writes, “Both the prescribing information for Alteplase and Stago’s fibrinogen reagent package insert state that samples for coagulation testing from patients receiving TPA therapy should be collected in an anticoagulant containing an anti-plasmin agent such as aprotinin. Do you know of any lab that follows this recommendation? George does not know of any laboratories that use aprotinin to treat TPA patient specimens.

Here is the Activase package statement: “During Activase therapy, if coagulation tests and/or measures of fibrinolytic activity are performed, the results may be unreliable unless specific precautions are taken to prevent in vitro artifacts. Activase is an enzyme that when present in blood in pharmacological concentrations remains active under in vitro conditions. This can lead to degradation of the fibrinogen in blood samples removed for analysis. Collection of blood samples in the presence of aprotinin (150–200 units/mL) can to some extend mitigate this phenomenon.” Becton-Dickinson markets BD Pharmingen, a “Protease Inhibitor Cocktail.”

From “Kkehoe“: Recognizing that the preferred method for establishing a PTT therapeutic range for UFH is plotting the PTT against the anti-Xa , is there an alternative for labs that don’t perform anti-Xa testing? Are you aware of any commercial kits that can be purchased and used? We currently purchase a normal donor kit from Precision BioLogic Inc for validating our normal reference range when changing reagent lot numbers.

George responds that, regrettably, there is no alternative to plotting PTT against anti-Xa , often called the ex vivo “Brill-Edwards curve,” for establishing the PTT therapeutic range. This creates difficulty for labs that don’t perform the chromogenic anti-Xa heparin assay routinely and whose volume of UFH patient testing is low. Spiking normal plasma doesn’t work, as spiked curves don’t match up with ex vivo curves. George has had a couple of conversations with Stephen Duff, Co-CEO of Precision BioLogic about kits and he points out the practical limitations to providing a standardized commercial set of UFH plasmas. A distributor would need several large-volume sources for UFH plasma, implying the need for plasmapheresis of patients on heparin, an unlikely scenario. That’s why you won’t find commercial plasma sets. High-volume labs that collect and aliquot patient plasmas for internal validations are reluctant to share aliquots outside their health systems as the products must meet GLP standards to avoid liability.

Given these concerns, experts advise low-volume labs to identify, aliquot, and freeze candidate UFH specimens daily until at least 50 (plus 20 normal plasma aliquots) are available to meet the demands of the Brill-Edwards procedure and to continue the process to maintain an adequate inventory. Aliquots may be shipped to reference labs where the chromogenic anti-Xa heparin assay is available.

But why continue monitoring UFH with the PTT ? The anti-Xa assay is readily available on counter-top coagulometers and in manual chromogenic microtiter formats, and acute care labs now need the assay to test for LMWH , the synthetic pentasaccharide fondaparinux, and now the oral direct anti-Xa antithrombotics rivaroxaban and apixaban. Further, the PTT is notorious for producing spuriously prolonged results when a lupus anticoagulant or specific inhibitor is present or when there is a coagulopathy secondary to liver disease or vitamin K deficiency. Even more troublesome, the PTT is insensitive to UFH if the factor VIII activity is elevated or AT level is reduced, both are consequences of acute inflammation. A glimpse of the degree of scatter seen in a routine Brill-Edwards curve may be enough to convince you of the PTT ’s unreliability, as would a comparison of assay results on random UFH samples.

Rather than going to the effort of setting up the Brill-Edwards procedure, why not investigate a means for providing the more precise and accurate chromogenic anti-Xa heparin assay?

Herb Crown, St. Louis Hospital Coagulation Reference Lab (August 17) added that the alternative to performing the anti Xa assay in your lab is to store samples in a –70° freezer and send to a reference laboratory for testing. He continues, we were perplexed when we plotted the anti Xa values against the PTT seconds in a recent study. What we saw were elevated PTTs and minimal anti Xa values significantly beyond what we were expecting. When we assigned a therapeutic range of 0.3–0.7 to the clients PTTs , the therapeutic range of the PTT was much higher than what we thought we should be seeing. We found the samples were left sitting in a rack on the bench until the end of the shift and then were poured off and frozen. This allowed the platelets to release PF4 , which neutralized the heparin. The message is to carefully handle all heparin monitoring specimens and test them promptly. This is important whether a lab is using the anti Xa or PTT to monitor heparin therapy.

From “Doctor Mukesh:” a 3 month-old with an old intracranial bleed has a markedly prolonged PT and PTT and is bleeding from IV sites but looks pretty alright. What are the possibilities?

George responds that the PT and PTT RIs at three months vary slightly from adult ranges, at 10–14.2 and 29.0–50.1 seconds, respectively. Prolongation of both could imply vitamin K deficiency, immature or diseased liver, or a congenital deficiency of factors II (prothrombin), V, or X, or fibrinogen deficiency. For a 3-MO, the FV RI is 48–132% and the FX RI is 35–107%. If factors V and X are both decreased, suspect immature or diseased liver, confirm with a liver enzyme panel. If only FX is decreased, check also FII (prothrombin, RI 45–105%). If both are decreased, it is likely vitamin K deficiency, which may be treated with oral or IV vitamin K. A congenital single-factor deficiency of factors II, V, or X is also possible, and will be detected by factor assay. Include a fibrinogen assay, RI 150–379 mg/dL. Finally, although the PT and PTT results indicate a coagulopathy, be sure to also perform a platelet count.

Tonya Kiziah, Helena Laboratories, discussed Helena’s new hand-held Abrazo, currently RUO, which offers a POC DTI assay, DTM. DTM is a dry chemistry modification of the ecarin clotting time that employs paramagnetic iron oxide particle technology, the same technology that is used on Helena’s Cascade. DTM works with both fresh non-citrated and citrated whole blood, and is a sensitive measure of DTI therapy, including oral dabigatran. The DTM may be the first POC method for monitoring DTIs.

Crystal Azevedo is currently evaluating the ECA-T kit to monitor dabigatran therapy. Are there any labs/companies that have a set of validation plasmas?

George reports that no one is currently distributing dabigatran calibrators or controls, including the product developers at Diagnostica Stago, who have developed the RUO ECA for monitoring the DTIs bivalirudin, argatroban, and dabigatran. (Lepirudin was withdrawn in May 2012.) We’ll continue to watch the usual plasma distributors Precision BioLogic, Inc and George King Biologicals, among others, for developments.

It is no consolation to you, of course, but on July 16 Stago released information on its new rivaroxaban calibrators and controls, designed for use with their anti-Xa assay, according to Paul Riley, PhD, Stago’s Manager of Research Use Products. His press release is attached to the post.

Paul Riley, PhD, mentions Stago’s new RUO assay Asserachrom VIIa-AT that measures activated factor VIIa-antithrombin complex (VIIa-AT, similar to the familiar thrombin-antithrombin complex), an analyte elevated in hypercoagulable states. Dr. Riley suggests the assay may be a potential surrogate biomarker for activated coagulation states. The press release and a series of references are attached.