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.
“Kaarin” wonders what labs are using for a deficient FXIII assay control when using clot-lysis methodology. George found that Hyphen BioMed, (Aniara), provides a FXIII deficient plasma. Crystal Azevedo responded hat she performs the monochloroacetic acid solubility testing, using George King’s Factor XIII deficient plasma. On August 18, George noted that Precision BioLogic Inc’s CryoChek controls provide a FXIII level.
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.
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.”
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.
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.”
Summary of our July, 2012 Quick Question:
How do you assay for von Willebrand factor (VWF) activity?
a. VWF ristocetin cofactor (VWF:RCo) by aggregometry: 21 (34%)
b. Automated VWF ristocetin cofactor (VWFRCo): 21 (34%)
c. VWF activity immunoassay (VWF:Act): 15 (25%)
d. Collagen binding assay (CBA): 4 (7%)
It seems the CBA, despite slightly superior reproducibility compared to the VWF:Act (Favaloro reference given), has not caught on. It would be interesting to learn whether anyone is currently validating or planning to adopt the CBA.
From Terezinha Paz Munhoz, Laboratório de Patologia Clínica, Porto Alegre, Brazil: We started to use recombinant thromboplastin, RecombiPlasTin 2G (ISI 1.0) on our IL ACL TOP 500. The PT results are quite different from rabbit brain reagent with a range of 8–220 seconds. The linearity in this equipment is 8,0–320 seconds, and the % activity (that we still report) is “crazy” with values from 5%–140%. INRs are very high, too. What is the importance in reporting these results? Should we limit as <10% and >130% or less? If we decide to stop reporting % activity we still have seconds and INR in a very wide range. We can describe prolonged results as incoagulable, but there is a clot.
When we tested donors samples (n = 20) the results range (Med ± 2 SD) were 9,28–12,3 sec; activity 83,5%–127,3%; and INR 0,8–1,09. With our patients we’ve been having results like I told above. Including more normal patients from the lab we achieved a bit larger range that we are using: 12,1–16,5 sec; 75,6–130,4% and INR 0,8–1,14. How can we manage this situation?
George responds, the results you are seeing are typical of synthetic low ISI thromboplastins. I discussed your question with reagent expert Dave McGlasson, and we agree that you should probably set your instrument to cut off at about 200 seconds, as INR values that are generated from PTs longer than 200 seconds have little clinical significance. You are also wise to continue to educate your clinicians and anticoagulation clinic experts on the value of the INR over the use of PT results expressed as % activity.
Also, to establish your RI, your healthy laboratory professional subjects probably more closely match your patient population than do donor samples and provide you with a more workable interval, thus 12.1–16.5 seconds may be realistic.
From Janice Stringfield, Jennie Stuart Medical Center, Hopkinsville, KY: We have an extremely lipemic specimen that we have a PT and PTT ordered from the ED. How do you get a result? We can’t find anyone that does a mechanical method anymore. Do you have any suggestions?
George suggests that if you have access to someone with a “high-end” optical instruments that assays at the short-wavelength end of the spectrum, like the ACL TOP 700, you may be able to achieve valid results from a moderately lipemic specimen, though not one that is extremely lipemic. Alternatively, does anyone in town have a Stago STA-R? All levels of Stago equipment, including the Compact or Satellite use an electro-mechanical principle.
Most of us have long ago donated the last of our Fibrometers to the schools in our neighborhoods, but one of those may still be around. As a final alternative, you may have a “seasoned” tech in your lab who can accurately do a tilt-tube PT and PTT. In these latter two instances, you’d be reporting a result from an un-validated assay, so you’d want to run controls or calibrators, and indicate to the attending physician how you generated your result.
I’d be curious to learn whether the patient’s lipemia is the result of a recent fatty meal, or a congenital condition. If it is the former, you will be able to collect a measurable specimen within a few hours. Often, however, extreme lipemia is inherited, in which case you will have to continue to search for a way to perform the PT and PTT.
From “Balasubm” at Einstein Healthcare in Philadelphia: I have a 74 YO female renal dialysis patient who is asymptomatic with prolonged PT of 38 seconds and PTT of 116 seconds, with a normal TT. Mixing performed immediately reveals PT of 30.3 seconds (N 9.5–13.5) and APTT of 84.3 seconds (N 22–38). After incubation the PT went to 31.6 seconds and APTT to 86.6 seconds. The specimen was drawn from the dialysis catheter; however, these lines do not use heparin. Could you please tell us what you think these results mean?
George suggests that, given that there was no correction upon immediate mixing, you have presumptive lab evidence for LA. The normal TT supports the absence of heparin, and additionally, the absence of dabigatran. George recommends you perform an LA profile on the specimen, typically one that uses a DRVVT kit and Sta-Clot LA.
George knows of no therapeutic used in renal dialysis that could account for the prolonged PT and PTT and the lack of correction, however the patient’s physician will want to order a confirmatory LA profile 12 weeks or more from this positive sample, and it may be possible to collect the new specimen at a time between dialysis treatments to eliminate the possibility of interference.