Kathy Jacobs at Chrono-Log, Inc asked, “When a patient’s platelets clump in sodium citrate, is there another anticoagulant that can be used to allow LTA platelet aggregation testing?” George replies, on two occasions I’ve used heparinized specimens for CBCs when EDTA and citrate specimens exhibited clumping, however, I doubt these would work for light transmittance (LTA) or for impedance aggregometry. I’ve heard of people mildly vortexing a sample with slight clumping, but, again, that probably would just activate or destroy the platelets.
Rrosales is purchasing a platelet aggregometer and is used to validating an instrument with an old instrument. How do you start the validation without an instrument to compare and does CAP allow this?
Kathy Jacobs of Chrono-log addressed this issue with Trudy Darden at CAP. Her response was: “The validation requirements can be found in the CAP General checklist. However, many of these requirements will not apply to platelet aggregometers as there are no commercial controls available. Manufacturers recommend that a normal range study be performed using 20-50 normal samples. The manufacturer provides broad guidelines regarding where this range should fall. The laboratory must define the normal population they will be using for this study. Since there are no commercial controls available an accuracy study cannot be performed. The laboratory can perform a precision study however, because the normal samples can be repeated for precision. The manufacturer provides guidance for interferences (low platelet counts) and recommends to discontinue the test after 6 minutes.”
Khaldee Lindsey Davenport-Landry, a graduate student in the University of Medicine and Dentistry of New Jersey’s online course, CLSC 5124, Advanced Hemostasis asks about mixing studies:
We do a rare PTT mixing study, and our procedure states that if the correction is within reference range, we should then do the incubated PTT mix. Just wondering if this procedure needs a face-lift.
George confirms Lindsey’s procedure is correct, if the PTT is prolonged and the prolongation is corrected in a 1:1 mix, the next step is to incubate the mix for two hours at 37ºC and repeat to detect any potential time- and temperature-dependent inhibitor. The specific inhibitors, in particular anti-factor VIII are usually IgG4, which is often detected only upon incubation. Though textbooks assert that the non-specific inhibitor, lupus anticoagulant (LA), is not time- and temperature-dependent, there are some LAs that in fact do require at least brief incubation. To create more confusion, some specific anti-FVIII antibodies may be detected in the immediate 1:1 mix. For this reason, some laboratory directors simply skip the immediate mix and do all mixes after a 1- or 2-hour incubation. The clinical picture differentiates: someone with LA may have experienced a thrombotic event or may be asymptomatic. Someone with anti-FVIII is nearly certain to be bleeding.
Thanks to Kathy Jacobs of Chrono-Log Corporation for her update on validation of aggregometry, added as a comment to Rrosales’ May 3 question. George had the opportunity to discuss the question with several experts colleagues at the Thrombosis and Hemostasis Summit of North America in Chicago, and all had the same conclusion, reflecting Kathy’s comment: Because aggregometry is a qualitative procedure, we don’t attempt to validate to a reference. To assure accuracy, we run reliable controls with each aggregation that we perform.
Back to the April 24 anticoagulation clinic manager’s question about when to validate a prolonged POC PT/INR. George spoke with representatives of Roche Diagnostics, distributor of Coag-U-Chek instruments, while attending the Thrombosis and Hemostasis Summit of North America meeting in Chicago. While Roche contends this is a local decision, their managers assert that most clinic operators choose 4.0 as the INR above which they send the patient for a follow-up venous PT/INR.
We’ve had three posts and several comments regarding LA mixing studies since March. In these posts several experts held that it is necessary to continue with LA detection studies even when the initial mixing study demonstrates correction. Their rationale is that a weak LA is neutralized in a 1:1 mix, and thus may go undetected. The LA may yet be detected and confirmed using DRVVT screen and confirm and the PTT-based hex-phase phospholipid neutralization method.
Thomas Ortel, MD, PhD, Duke University Medical Center spoke on LA profiling at the Thrombosis and Hemostasis Summit of North America in Chicago. His position parallels those of the experts who contributed to the previous three posts. He contends that it is necessary to perform the LA detection assays even when the mixing study corrects, given the potential for a weak LA. He further recommends that the PNP mixing study always be included as part of the profile. This is because the original order is most likely based upon some abnormal finding and the mixing study may detect and identify some additional, otherwise undetected abnormality. The addition of mixing study results to the DRVVT and PTT screen/confirm results provides a complete picture and helps develop the final findings. Dr. Ortel added in a private follow-up that in most of these types of cases, he sees mixing study results whose “correction” is borderline or equivocal.
Dr. Ortel’s comments attracted a detailed follow-up from Dr. Ali Sadeghi-Khomami, researcher at Precision BioLogic. Dr. Ali first clarified a few things in the case presented by Dr Cambereri. First, his patient was on Lovenox. Neutralization of anticoagulant activity of metabolized LMWH in the DRVVT assay is not reliable nor reproducible, partially because of a short incubation step (~3 min at 37°C) and poor performance of most heparin neutralizers used in reagent formulations. This could explain why after dilution with PNP and lowering the titer of LMWH a correction was observed. Second, the above case was an acute thromboembolic event, which is addressed by the ISTH, “Caution should be exercised in interpretation of the results of tests performed close to thromboembolic event as patients may be treated with UFH and/or VKA.” Third, application of platelet poor pooled normal plasma (PPPNP) is critical for LA-mixing study because any phospholipid released from platelet could correct clotting time. So LA-mixing is different from routine-mixing study in respect to platelet-debris content.
The mixing study helps overrule all factor deficiencies or possibility of VKA therapies as the cause of prolongation of LA-screen tests. Result of mixing should be significantly (+3SD) higher than normal plasma samples to be considered LA-positive. By the way, there are exceptional situations in which LA-cofactor proteins need to be added to patient plasma for maximum prolongation of clot time by LA-antibodies. I recall a report/question on this blog that even more prolongation was observed after mixing study in LA-detection. Obviously it makes sense to run PPPNP mixing prior to running more costly tests but the sequence of testing is not crucial for interpretation of result, for instance Staclot-LA is an integrated (screen/confirm) mixing test.
To answer some concerns raised by Herb Crown around dilution of LA-antibodies due to the mixing step with PPPNP, everything depends on the sensitivity of the method. If 2X dilution of patient plasma brings the titer of antibodies below limit of quantification of the test then we have a problem and the result of our PPPNP-mixing assay would be a false-negative. Therefore, if you are using a kit with a poor LA-sensitivity that is not optimized for mixing study or lacking a validation study with an adjusted cutoff to support its performance under mixing condition, this is a wrong choice. In practice, discovery of a weak LA has a very limited clinical value because most of the time it indicates transient situations. This is why repeating LA-test after 12 weeks is required to clarify presence or absence of real LA antibodies.
Dr. Marisa Marques University of Alabama at Birmingham Hospital, responded to a message posted April 27 on hemolysis and the heparin anti-Xa by “Danagah.” Dr. Marques provided a photo taken by Patti Tichenor of two specimens from the same patient, one hemolyzed, the other non-hemolyzed. Dr. Marques remarked that the heparin anti-Xa results from both specimens were identical!
On April 29, Crystal Azavedo asked about laboratories that monitor dabigatran with the ECT. At the Thrombosis and Hemostasis Summit of North America I became reacquainted with colleague, Dr. Leon Zuckerman, founder of Coagulation Consultants of Des Plaines, Illinois. Coagulation Consultants is a full-service hemostasis reference laboratory that also offers the ECT.
“SwimRose” had a case of a stroke patient on Pradaxa giving an INR of 2.0 with ISTAT, but the main lab instrument, an IL ACL, gave 1.1.
George responds that because the iSTAT prothrombin time/international normalized ratio (PT/INR) cartridge is cleared only for warfarin monitoring, there is little written about dabi. Dr Rajiv Pruthi, Mayo Hemophilia Center, asserts that the PT and PTT are prolonged by dabi, which is a DTI, but their degree of prolongation varies with reagent-instrument combinations and does not accurately reflect drug levels. One cannot compare dabi-affected INRs across platforms, nor can one use the INR to monitor dabi.
The thrombin clotting time (TCT, TT) is the current best way to check for dabi clearance, as small dabi levels significantly prolong the TT. Hyphen’s Hemoclot Thrombin Inhibitors assay, currently awaiting FDA clearance, may be used to monitor argatroban and bivalirudin as well as dabi. Likewise, Centerchem’s Pefakit PiCT, which can monitor all Xa inhibitors and DTIs looks promising, as does Stago’s ecarin clotting time. All these tests await the FDA.
Added by Dr. Pruthi: The degree of prolongation of INR with POC or venipuncture INRs vary with reagent/instrument combination. Since initial case reports demonstrating a discrepancy between venipuncture INR and POC INR, van Rynn confirmed such a discrepancy in a 4-subject study with POC INRs being disproportionately prolonged compared to venipuncture INR. Thus one needs to be cautious in interpreting POC INR in patients on dabigatran.
Kim Kinney at Indiana University Medical Center is being asked to make the TEG available in their special coag lab for clinical use. She asks to poll others: who is running the test and who is interpreting the results? TEGs seem to be mostly in operating suites where they are used to monitor heparin or thrombolytic therapy. Kim’s question generated three comments:
“Istiger415” runs the TEG for liver transplants and cardiac cases for pediatrics. Lab techs run the tests, and results are reviewed on monitors in the PR by the surgical teams as they are generated. The results are interpreted by Hematology. I know that UC is also running TEG in the adult setting for liver transplants as well as for massive trauma protocols, again by the techs in the lab.
Joe Lamb has had a TEG in hematology/blood bank for a little over two years. His technologists are performing the assays and the interpretations are done by a perfusionist.
“Dlawler” has been offering TEGs 24/7/365 by the hematology technologists. It is used mostly for liver transplant patients during surgery and is interpreted by the anesthesiologist during surgery. They are able to see the TEG tracings on monitors as they run in the lab. We also run TEGs on some ICU patients and pediatric traumas/ECMO patients. In the special coag lab we use the TEG analyzers for our platelet-mapping test for aspirin and Plavix.
From an inquirer to the ASCLS Consumer Forum (with permission): “I have a titanium aortic valve and sometimes experience afib. I take 5 mg daily of warfarin and my cardiologist wants my PT/INR to be 2.5-3.5. I also take a baby aspirin (81 mg) daily. Today my PT was 2.8 (perfect for me and it is usually pretty steady). My question relates to changes in altitude, as in a trip to Colorado, and the possible effects on PT/INR in staying at high altitude, 8-10,000, feet for a week. Will this raise or lower my PT/INR? What will happen to the value upon return home? I also consume beer and wine. The last time we went to Colorado, we were there for one week and consumed beer at the various breweries. When I came home, I was working in the yard and developed a hematoma on my wrist. When I sought treatment, my PT/INR was 5.8! Do you think the PT/INR elevation was attributable to alcohol consumption, going up in altitude for a week, or coming down, or a combination of all of the above?
George answered, short-term high altitude (several hours’ exposure) has no effect upon the PT/INR, as shown in several studies conducted using hypobaric chambers. These studies were mostly concerned with the possibility of thrombosis subsequent to prolonged air travel or the conditions that affect mountain climbers, high-altitude neurological or pulmonary edema (HANE, HAPE). There are no studies that employ several days’ exposure, though it is probably safe to generalize from the short-term hypobaric chamber studies.
Alcohol is a more likely culprit, although George does not conclude from your message that you are either a chronic or binge drinker. Chronic long-term alcohol consumption at a level that can damage the liver is a known cause of bleeding, as liver damage reduces the production of the coagulation factors. This can be verified by repeating the PT/INR and simultaneously testing for the liver enzymes. Binge drinking, by comparison, tends to suppress platelet function, which also leads to bleeding such as the hematoma you experienced, however platelet suppression is not reflected in the PT/INR.
Responses to our May, 2012 quick question: When using point of care testing, at what INR does your anticoagulation clinic require a confirmatory plasma-based (conventional) PT?
a. 4.0: 34 (65%); b. 5.0: 9 (17%); c. 6.0: 4 (8%); d. Never: 5 (10%)
Our responses closely parallel our April 24 and May 7 discussions; it looks like the majority of us prefer caution, knowing the risk of bleeding rises rapidly as the INR exceeds 4.0.
Most of us have maintained separate calibration curves for our chromogenic anti-Xa heparin assays, one each for UFH, LMWH, and fonda.
Dave McGlasson (Wilford Hall USAF Medical Center) has published two articles on the use of a hybrid curve that may be used for either UFH or LMWH, thus eliminating the need to inquire which formulation we are testing, and some have even extended the curve mathematically to use it for monitoring fonda.
At least two hemostasis assay distributors, Aniara and Stago, now make hybrid curve calibrators and advocate for this approach to heparin monitoring, however an informal poll at the recent Thrombosis and Hemostasis Summit of North America reveals that most of us are still using individual curves. Consequently, our current quick question asks us which approach we are taking.
Laura Paterek asks, are you sure the following statement is correct? “When a lupus anticoagulant is present, SCT Confirm becomes prolonged beyond the SCT Screen. Normalization renders the SCT insensitive to warfarin.” Doesn’t the confirm step decrease when LA is present?
George thanks Laura for locating this misstatement in his May 15, 2008 description of the Instrumentation Laboratory HemosIL Silica Clotting Time assay. The statement should say, “When a lupus anticoagulant is present, the SCT Confirm step produces a shorter clotting time than the SCT Screen.” George asks how many labs are using the SCT.
Dr. Kathryn Doig, Michigan State University writes: “Hi George. We have a grad student who is doing a project related to hemostasis. Along the way, she used the term “thrombokinase” which I had not heard in quite some time. Just thought I would verify that it is not used anymore.
George replies that factor X was first described in the 1950s as the Stuart factor and the Prower factor, named for the families in which the factor’s absence caused bleeding. Some time after, activated factor X was named “prothrombinase” because its substrate is prothrombin. Xa also somehow got assigned the term “thrombokinase,” coined by Morawitz in the 1920s, who postulated four related coagulation factors, a seminal event in coagulation understanding. We’ve almost universally dropped the descriptive names (also eponyms like Stuart-Prower) in favor of the Roman numerals these days.
Ginger Weeden, Bio-Rad Laboratories asks what heparin-induced thrombocytopenia with thrombosis (HIT) testing is available in the US?
George responds that all the C14 serotonin release assay methods (washed platelets) available from reference labs are laboratory-developed tests. Here are the manufacturers who provide immunoassays designed to detect anti-platelet factor-4 (PF4)-heparin antibodies that are associated with HIT:
- Akers Biosciences PIFA Heparin/PF4 Rapid Assay (Two-minute lateral immunoassay)
- Gen-Probe LIFECODES PF4 (formerly GTI Laboratories)
- HYPHEN BioMed Anti-(h)-PF4, available through Aniara
- Instrumentation Laboratory HIT-Ab (PF4-H, RUO)
- Sekisui Diagnostics (formerly American Diagnostica) IMMUCLONE Platelet Factor 4 ELISA (RUO)
- Stago Asserachrom HPIA (ELISA)
A question from Farah Quresh in follow-up to a presentation on antithrombotics that George made May 24 during “Bioconference Live”: What are the conditions in which Pradaxa is preferred over warfarin?
George equivocates that this is a clinical judgment call that a physician may use based on economics, ability of the patient to comply, and availability. In the USA, dabi is cleared only for prevention of stroke in atrial fibrillation. From anecdotes, it seems that most physicians retain their patients on warfarin if they are using home-testing or have ready laboratory access and are having no trouble remaining within the therapeutic INR of 2-3. If they are encountering difficulty staying in range, dabi may be the answer. Also, physicians appear to be starting all their new afib patients on dabi.
Posted Wednesday, May 23 on Pat Letendre’s Medlab list: We have a patient with lymphoma and an IgG peak. The antibodies are interfering in the coags: INR >5.0, clinical challenges cause no bleeding. Is there any way to remove IgG from samples? I am currently trying a heterophile blocking tube (HBT) hoping that it will neutralize the antibodies. Does anyone know of any other method to remove unwanted IgG from a sample, but have it remove little else? Thanks in advance for any suggestions. Linda Stang MLT, University of Alberta Hospital.
She later adds the HBT tube did correct the INR somewhat, but only from 4.9 down to 3.9 (3.4 if I were to use 2 HBT tubes), and the appropriate controls did not vary much with the HBT treatment. The INR is consistently 4.8-6.0 with different reagents/analyzers; PTT is 48 or 80, depending on the PTT reagent. I am considering purchasing a protein G column, but am a little scared of how the coag will be affected. Surgery is on hold, with us trying to verify that the underlying coags are normal.
May 30: Have you tried mixing studies with normal pooled plasma (NPP) to rule out factor deficiency/liver impairment? Running serial dilutions of the patient sample in NPP with and without pre-incubation will tell you if fast or slow reacting antibodies exist against coagulation factors (parallelism/non-parallelism rule). Removing normal human IgG from plasma per se doesn’t significantly affect coagulation result. I have done it before. However, if we are dealing with Ab against targets involved in coagulation, then you have to make sure other than Ab no active pro/anti-coagulant proteins remain on the column. Dr. Ali Sadeghi-Khomami, PrecisionBioLogic
May 30, “Jlow:” What is the lupus anticoagulant status? Some thromboplastins may give anomalously high results, for instance, Innovin and other recombinant thromboplastins with some patients only. Could be IgG or IgM in our experience. Is this a paraprotein? What is the thrombin time?
June 5, “Sidsham:” Lymphomas with IgM antibodies are more common than IgG-secreting lymphomas; unless you are talking about plasma cell neoplasms where IgG secretion is common. IgM lymphomas manifest like any other cold agglutinin disease. So how about trying the conventional method of incubating the sample at 37 degrees for 15 minutes before performing the test to see if there is any correction?