2012 Cheat Sheet

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.

George learned about an interesting case at the UAB Hospital special coagulation lab from specialty techs Patti Tichenor and Laura Taylor. A specimen arrived with an order for LA profile and anti-cardiolipin antibody. The PTT was prolonged and corrected on initial mix.

The UAB protocol recommends a two-hour incubation and repeat mixing study on the incubated sample to detect a specific inhibitor, which usually turns out to be an anti-FVIII antibody. Whether the result of the incubated mixing study is corrected, indicating a coagulation factor deficiency, or uncorrected, indicating a possible inhibitor, Patti and Laura typically go on to run a FVIII assay during the incubation, reasoning they will want to do so in either event. It turns out the FVIII activity was 38%, so they next ordered a VWF :Ag, which turns out to be 35%. The VWF :RCo results will follow, but a call to the ordering ophthalmologist reveals the patient was experiencing a retinal hemorrhage. Their appropriate and speedy application of the mixing study protocol provided an unexpected diagnosis and led to an appropriate treatment course.

On August 23, Herb Crown added, “And that my friend is what we do in the coagulation reference laboratory, why we do it, why we are so good at what we do and why we love our jobs. Congrats to the UAB staff. PS , make sure the doc gets written orders in the patient chart for your additional testing.”

“Japgi” has a 6 YO boy, well till a month back, when he had a post-traumatic gum bleed. The bleed was stopped after about half an hour, but a small ooze restarted and stayed for 3 days. His PT is 14s, PTT 46.4s. The prolonged PTT corrected with normal plasma 1:1 to 38.1 seconds. There was no correction with 1:1 mix of factor VIII-deficient plasma, 43.1 seconds; factor IX-deficient plasma, 43.1; or FXI-deficient plasma, 41.1. The entire exercise was performed on two separate samples a week apart. There is no history of bleeding in the family. After the first test the patient has been running a cold with fever. What is this and what can we do to confirm? Thankfully there are no further bleeds.

The results of your FVIII-deficient, FIX-deficient, and FXI-deficient plasma mixes led George to suggest there may be a LA present even though the prolonged PTT appears to correct with NP. It is unlikely the boy has deficiencies of all three factors. This is not unusual; there are examples of weak LAs that fail detection in immediate mixing studies. In childhood, as in adulthood, weak, transient LAs may be detected as a side-effect of acute viral infection. To follow up, George suggests a full LA profile, including DRVVT confirmation, at least 12 weeks subsequent to the last assay that was performed.

The possibility of an LA does not explain the bleeding. Assuming he has a chronic bleeding condition, George suggests a PLT count and perhaps PLT aggregometry, if available. The “small ooze’ description suggests the possibility of a mild PLT defect or thrombocytopenia.

From the ASCLS Consumer Web Forum: “How quickly does an elevated PT/INR decrease? I appreciate the answer may be ‘it depends,’ but for patients on warfarin and with an elevated INR , how quickly can we expect an INR to decrease per day when warfarin is stopped? The rule for a procedure encourages stopping warfarin five days before a procedure, suggesting a decrease in INR of ~0.5/day when warfarin is held.”

George found no published prospective RCT to determine an empiric timeline, however he predicts an interval based on the half-lives of the vitamin K-dependent coagulation factors II, VII, IX, and X. X and II are the longest at 52 and 60 hours, respectively, so the PT should drop to halfway between peak and normal in about 3.5 days.

A colleague contacted George about a relative who is an inpatient and is experiencing a pleural effusion subsequent to a MRSA infection. The MRSA has been treated and is resolved, but the effusion is mucinous and drains slowly. The surgeon was planning to use TPA to liquefy the fluid.

Approximately 350 mL of fluid had drained before the first infusion of TPA through the patient’s chest tube, which resulted in the further draining of 1000 mL. Physicians administered four more injections and over 2000 mL of effusion have drained. We would like to learn from participants if you are familiar with this procedure, how safe is it, and how long does the TPA last in pleural fluid? A September 6 follow-up: “They discharged my brother on Friday, August 31, after five successful treatments of TPA. His lung capacity is now just about normal.”