Cheat Sheet for January 14-31 and February, 2011

Quick Question Results: PFP (January 14)
In a December 2, 2010 post, Bob Gosselin at UC-Davis in Sacramento suggests that the term platelet free plasma (PFP) should be reserved for plasma with a platelet count of zero, achievable only by filtration; and that we should call plasma with a platelet count less than 10,000/mcL platelet poor plasma (PPP).
George posted a December Quick Question and got the following results:
How do you define platelet free plasma (PFP)?
1.    Plasma with a platelet count less than 10,000/mcL: 39 (60%)
2.    Plasma with a platelet count less than 5,000/mcL: 9 (14%)
3.    Plasma with a platelet count of zero, prepared by centrifugation: 6 (9%)
4.    Plasma with a platelet count of zero, prepared by filtration: 11 (17%)
There are a substantial number of respondents like Bob whose definition fits the terminology. There are also a number who set the platelet count at 5,000/mcL, which is Dr. Douglas Triplett’s recommendation. It looks like the PFP-PPP terminology issue is unresolved.
Top Ten Coag Problems (January 14)
Cara Calvo, director of the Medical Technology program at the University of Washington reminded George of a workshop he presented with Lynn Quarles of Diagnostica Stago entitled “The Top Ten Problems in the Coagulation Laboratory.” George asked for a list of current top ten problems.
Joe Lamb’s Top…Seven (January 15)
Joe Lamb, St Francis Hospital; “Are you sure you want these? I have some:”
1.    Orders for a factor V when they really wanted a factor V Leiden.
2.    Orders for platelet antibodies when they really want an HIT screen/profile.
3.    Orders for fibrin split products, we don’t do those anymore.
4.    Orders for a thrombotic profile, i.e. protein C and S and other tests, right after an event and/or while the patient is on heparin/coumadin.
5.    Orders for a factor V Leiden AND an APCR.
6.    Mixing studies when the PT/PTT is not prolonged.
7.    Nurse draws under-filled tubes or the blood is left in the syringe too long.
Falsely Elevated Anti-Xa? (January 17)
Is there any explanation for a patient having a measurable anti-Xa level, when the physician insists that the patient has been on no anticoagulants for several months? (value was 0.2)
This question generated a series of six responses from Herb Crown, Joe Lamb, and Gary Bullard.
Here is George’s initial response: I know of no interference that would result in an unexpectedly positive chromogenic anti-Xa heparin assay. Marked hemolysis or lipemia can interfere, but with the effect of falsely reducing the anti-Xa result. One colleague speculate that perhaps  elevated plasma antithrombin could account for this. I would begin with the patient; was the blood collected through a vascular access device like a “peripherally inserted central catheter (PICC line),” and was the line flushed with heparin before collection? Was the specimen collected in the proper tube, and not, for instance, a heparinized tube? Was there a miscommunication; for instance, is the patient receiving low molecular weight heparin, a danaparoid, or a pentasaccharide not specifically identified as heparin? Be sure to read Herb, Joe, and Gary’s comments about the anti-Xa standardization curve and dextran sulfate contamination.
Change in Vorapaxar Study (January 17)
George linked an announcement from theheart.org indicating that Merck has discontinued a portion of a phase III trial of vorapaxar, an oral antiplatelet drug that blocks the thrombin membrane receptor protease activated receptor-1 (PAR-1).
Russell Viper Venom Time (January 20)
George asked what is an older name for the original Russell viper venom (RVV) assay and what was its original purpose? Joe Lamb answered Stypven time, which was used when there was a prolonged PT. If the Stypven time was normal, the PT was prolonged by a factor VII deficiency, since the Stypven activates at the factor X level.
POC Assay for Dabigatran (January 20)
Terry Black at Orlando Regional Medical Center is getting a lot of questions about monitoring Pradaxa (dabigatran) and it keeps coming back to the ecarin clotting time (ECT). She asks, “Do you know of any POC testing kit or instrument that could be used to monitor this drug’s effect?”
George provides a link to Hyphen Biomed’s new Thrombin Inhibitor assay for direct thrombin inhibitors, which Hyphen claims is linear for dabigatran measurement. He knows of no point of care instrument available for dabigatran monitoring. There are responses from Jonas Kingo, of Aniara, which markets Hyphen products, and Paul Riley, Diagnostica Stago, describing their chromogenic ecarin time.
Reptilase Time and Heparin (January 21)
From Debbie Moffitt, Queens Medical Center, Honolulu: A hematologist is asking for the reptilase time test to distinguish when prolonged PTT and TT results are due to heparin. When heparin is suspected, we use Hepzyme to try to correct the TT or PTT . We also review the patient’s clinical condition, medications, and if the specimen was drawn through a heparin line. Does anyone have experience with reptilase, and what vendors do you use?
George responded: Stago offers a reptilase kit. Because reptilase cleaves only fibrinopeptide A from fibrinogen, as opposed to thrombin, which cleaves fibrinopeptides A and B, it is not prolonged by heparin, so when the thrombin time is prolonged and the reptilase time is normal, you’ve confirmed heparin. There is no other current use for the reptilase time and thus no reason to expend the resources to set up the assay. Your approach, neutralizing with Hepzyme plus checking the chart, is the way most of us do it, and if that doesn’t offer enough proof, you can offer the chromogenic anti-Xa heparin assay, which is definitive.
Here is Debbie’s follow-up conclusion: We have used Hepzyme for years. We always keep it on hand although we don’t have to use it often. It has been useful when there is a question of whether a sample contains heparin in cases of disputed contamination. Reconstitute  with 1 mL of patient plasma, then re-test the plasma. It neutralizes up to 2 units of heparin per vial. We use commercial heparin controls and test PTT pre and post neutralization.
PT Specimen Clotting: Cold Agglutinin? (January 23)
“Pathous222” describes a 64 YO lady on coumadin for patent foramen ovale who had a PT. The blue top clotted so they recollected, collecting two tubes. One clotted and the other appeared to be OK but the PT was >109 sec, platelet count 56K; the patient usually runs between 250-300K. They made a smear of the blue top and there was some fibrin at the feathered edge. Suspecting a cold agglutinin, they recollected and put the tube in a warm block for delivery. This tube was not clotted grossly but once again the PT was >109 sec, platelet count is 86K and platelet clumps on the smear. Are there any options to get an acceptable specimen to perform a PT for this patient?
George wasn’t convinced this was a cold agglutinin, as there were no RBC clumps. Platelet satellitosis may be part of the picture, as it is occasionally seen in sodium citrate anticoagulant (blue-top), though platelets more often form satellite patterns in EDTA (lavender). However, satellitosis does not account for partial clotting, evidenced by the fibrin that accompanies the platelet clumps and the prolonged PT.
George guessed the patient is receiving a treatment that triggers in vitro coagulation: one of the pegylated drugs or a plasma expander could be the culprit. Alternatively, she may have developed a protein imbalance such as an M-protein spike interfering with the PT. Meanwhile, to monitor coumadin, switch to the chromogenic X assay, whose therapeutic range is approximately 20-40% factor X.
Here is a further response from January 27: There is no evidence of platelet satellitosis or RBC agglutination, and the patient is not on any of the medications described. We will evaluate for a possible paraprotein.
Shortened PTT and Mixing Studies (January 23)
“Plovejoy” asks is there a reason to perform a PTT mixing study on a patient whose initial PTT result is slightly shorter than the reference range? Since a short PTT would appear to indicate there isn’t a factor deficiency or a circulating anticoagulant, might there be another reason?
George responds that nothing can be gained by performing a standard mixing study on a shortened PTT. Mixing studies are designed to detect inhibitors or factor deficiencies that prolong the PTT, and are unlikely to provide any information for a shortened PTT. Short PTs and PTTs are probably the consequence of a specimen management error, however Lippi G, Salvagno GL, Ippolito L, Franchini M, Favaloro EJ. Shortened activated partial thromboplastin time: causes and management. Blood Coagul Fibrinolysis 2010;21:459-63 speculates on the possible clinical and physiological causes. While a mixing study doesn’t help, there may be an argument for factor assays that detect, for instance, elevated factor VIII, which could be associated with thrombophilia.
Any Relationship Between RVV and D-dimer? (January 23)
Vilas Hiremath, United Labs, spiked normal pooled plasma with crude Russell viper venom (RVV). The D-dimer is negative. Your comments please.
George knew of no reason to anticipate a positive D-dimer from a clotting assay on normal plasma, be it RVV, PT, or PTT. The fibrin that is the endpoint of a clotting assay is not cross-linked, as there is no opportunity for factor XIIIa to act, so it would not produce D-dimer, even if there were time for in vitro fibrinolysis.
More on False Positive Hex-phase Phospholipid Tests (January 24)
On November 29, G Pihan of Beth Israel Deaconess Medical Center in Boston wrote: “What is the incidence of false positive hexagonal phospholipid neutralization tests in the presence of a strong (400-800 BU) factor VIII inhibitor?”
Marlies Ledford-Kraemer, MBA, BS, MT(ASCP)SH, referred to Triplett DA, Barna LK, Unger GA. A hexagonal (II) phase phospholipid neutralization assay for lupus anticoagulant identification. Thromb Haemost 1993;70:787-93. They saw one presumed false positive in a patient sample with a high titer FVIII inhibitor, saying: “The patient was a severe hemophiliac who had been exposed to a wide variety of factor VIII replacement therapy. Although the patient result is presumed to be a false positive, we cannot rule out the presence of an underlying LA in this sample.” Later in the article they write, “A potential source of a false positive Staclot LA in the setting of a factor VIII inhibitor may be due to greater decrease of VIII activity in the diluent tube compared to the hexagonal (II) phase PL tube.” This, of course, still fails to answer G Pihan’s question, however it is clear this concern existed early in hexagonal phase phospholipid neutralization tests.
PTT Prolongation Limit and Mixing Studies (January 24)
Dr. Robert Gay at Greensboro Pathology Associates, Greensboro, NC wrote: “It makes sense not to perform a mixing study when the PTT is just above the reference range but what is commonly used as a cutoff? Is there a way that individual labs can establish what their limit should be?
George responded that this was posted as a Quick Question back in July, and came up again in an October 11, 2011 discussion of PT and PTT reference intervals. It seems there is no firm answer, although 43% of the responders simply chose “Any non-heparin PTT that is prolonged beyond the upper limit of normal.” It is helpful to realize that 28% also responded they solicit clinical information before performing mixing studies. At the UAB lab we use 5 seconds above the upper limit of the reference interval as the cutoff for performing mixing studies, however we have no statistical validation for this, just convention.
Back to the Falsely Elevated Anti-Xa (January 24)
George was intrigued by the comments related to dextran sulfate added by Gary Bullard and Herb Crown to the falsely elevated anti-Xa discussion. Though both Gary and Herb indicated that dextran releases heparin from endothelial cells, George wondered if dextran’s polysaccharide structure could directly interact in the anti-Xa reaction. He could find no reference linking dextran to heparin or to an elevated chromogenic anti-Xa heparin assay nor could the techs at UAB confirm this. George asked Gary, Herb, or anyone else with experience to point us to a publication linking dextran to falsely elevated anti-Xa heparin results.
Answers to RVV Questions (January 24)
Back to our January 20 discussion of the old RVV test. Joe Lamb came up with the name, Stypven time. With improvements to the PTT and the ready availability of factor assays, the RVV has grown obsolete. To learn where “Stypven” came from, George turned to Mary Ann McLane, PhD, MLS, University of Delaware. She reports that “styptic” originated from the Latin and Greek words meaning “to contract”… as in styptic pencils used to stop bleeding from minor wounds (which are made of alum). “Ven” was simply a contraction for “venom.”  She speculates that “Stypven” was a trademark develop by Merck in the 1950s or 1960s.
Still More on False Positive Hex-Phase Phospholipids (February 1)
Dr. Jay Lozier of NIH Clinical Center Department of Laboratory Medicine wrote “I have recently stumbled upon your forum, which has provided me with many interesting things to consider in my position as Senior Staff Clinician who supervises the hemostasis laboratory component of our hematology laboratory at NIH. I would point out that in your discussion of the prevalence of “false positive” lupus anticoagulants in patients with factor VIII inhibitors, it seems to me quite likely that the patient could have a lupus anticoagulant, particularly one that is transient. In my experience with DVT patients and acutely ill patients with inflammation there is a very high rate of transient lupus anticoagulants in our acutely ill patients that disappear as things calm down and the impetus for inflammation subsides.
PT Mix Fails to Correct in Dysfibrinogenemia (February 1)
Here is another thought-provoking email from Dr. Jay Lozier at the NIH: “I run the coag lab and see a fair amount of storage pool deficiency that we diagnose on the basis of aggregations and electron micrographs. Just diagnosed a dysfibrinogenemia that manifests itself as an isolated prolonged PT that did not correct with a mix. Fibrinogen activity (Stago, Clauss method) was 87 mg/dL, but fibrinogen antigen was >400 mg/dL (Mancini radial immunodiffusion). Had not seen that presentation before; no bleeding.”
George responds with this non-answer: Yes, this is a good question to put out there. Why should a dysfibrinogenemia fail to correct in a PT mixing study?
Herb Crown, SLU Hospital Coagulation, recommends repeating on a newly acquired specimen. If the repeat is the same, this is potentially a real issue. “I would like to see the fibrinogen antigen assay on further dilutions to the point that I have an actual number to work with rather than a “greater than.” I would also serially dilute the plasma and run the fibrinogen on the Stago instrument. The only thing I have seen that causes the PT inhibitor screen to not correct is a Factor V inhibitor. I recommend the factor V activity and a bovine thrombin time (the Stago uses human thrombin).
Update on Dysfibrinogenemia and the PT (February 3)
Relative to the February 1 discussion, here is an update from Dr. Jay Lozier: “I thought there would be fibrin split products that are preventing coagulation from occurring normally, but these turn out to be negative. I am getting some help from my folks at my alma mater (UNC) where Susan Lord’s group is interested in fibrinogen.”
George responded: It would also be interesting to learn if Beckman/IL’s QFA Fibrinogen® assay would return a more accurate result. Their reagent employs 100 units/mL thrombin instead of the 30–50 units used for the standard Clauss assay. Dave McGlasson’s January 8 discussion addresses this. Dave’s discussion goes back to Kim Kinney’s question about argatroban and the fibrinogen assay posted January 5. Finally, since Debbie Moffitt recently brought up the reptilase time test, it may be interesting to see what kind of results this test would generate on the patient’s plasma as well.
Bethesda Titer “Prozone?” (February 3)
From Vilas Hiremath, Sc MLT, United Labs: “I have a factor VIII inhibitor titer on a hemophilia A patient earlier treated for inhibitor. At lower dilutions there is appears to be no inhibitor, but at 1:16 and 1:32 the titer shows residual factor at around 50%. Is this type II kinetics, and is that possible in a hemophilia patient?”
George attempts an answer: “I thought of the ‘prozone’ effect in immunoassays, where the antibody titer is so high, it fills the reactive sites on the target antigens so no indicator reaction can occur at the stronger dilutions. However the logic doesn’t seem to fit with the Bethesda titer, plus I’d never seen it. I contacted Laura Taylor and Patti Tichenor at the UAB Special Coagulation laboratory, they had never seen it either.” UAB manages a few acquired anti-factor VIII patients with type II kinetics and the pattern is one in which the percentage hovers around 65 to 70% for several dilutions in a row before dropping low enough to establish an end point Dr. Larry Brace of Edward Hospital in Naperville, IL has seen a similar type II pattern in factor assays of patients with acquired factor VIII deficiency.
Dave McGlasson, Wilford Hall USAF Medical Center once saw this issue with a patient who was an acquired hemophiliac with SLE with a strong lupus anticoagulant (LA).  When the dilutions got out around to 1:32 you started seeing an effect like Vilas described.
More on Transient LAs in Inflammation (February 3)
From Dr. Jay Lozier, NIH: The data in a paper I am writing about DVT and its treatment with tPA showed that 19 of 30 acute DVT patients had positive LAs at the time of presentation, of which 11 had resolved by 6–8 weeks of follow-up, and 2 were documented to be persistently positive 6 months later. The balance were positive at 6–8 weeks and negative at 6 months or lost to follow up. We  see LAs come and go in some patients with inflammatory disorders or cancer at the NIH Clinical Center, so transient LAs can certainly occur.
Saw-scaled Viper Venom (February 12)
From Vilas Hiremath: “I have spiked crude saw-scaled viper (SSV) venom into a normal donor sample in different concentrations and performed TEG. The higher the dilution, the greater the maximum amplitude (MA), from donor baseline of 53 to 73. Also the angle (slope, α) increases from 59 to 74. Further, SSV enhances thrombin-generated platelet aggregation. Does MA increase because of fibrinogen binding to its platelet receptor? The same sample tested on Multiplate using thrombin receptor antagonist peptide (TRAP-6) shows no effect.
George wrote, “Fortunately, I can call on Mary Ann McLane, PhD, University of Delaware.”
She answers: “The SSV is Echis carinatus. Is Vilas is spiking with whole crude venom? The picture seen by the TEG is as complicated as it would be in vivo, with both platelet aggregation and inhibition happening at the same time. This is critical since the venom contains not only fibrinogenases and disintegrins, stopping aggregation; but also thrombin-like enzymes, which would stimulate clotting. His question about the relationship between clot strength (MA) and fibrinogen binding to αIIbβ3 is difficult to answer since clot strength is related to how much fibrinogen has been converted to fibrin and then become crosslinked (a non-αIIbβ3 phenomenon). Using TRAP-6 only isolates the thrombin-activated receptor aspect of platelet aggregation, without including the other realities happening when whole crude venom is used.”
Dr. McLane added: “In AACC’s December Clinical Laboratory News, Kroll, MH wrote about TEG: “MA measures the strength of the clot, which is an overall reflection of the structural interactions and fibrinogen, interlaced with fibrin polymers. Platelet function and aggregation have the greatest effect on MA.” So it looks like the answer to Vilas’s question is “yes”!
George also received this from Dave McGlasson: “The rotational thromboelastometry (ROTEM) machine, a powerful technique for assessment of blood coagulation disorders, is similar to TEG. The ROTEM detects and monitors direct thrombin inhibitors. ROTEM’s ECATEM cuvette contain ecarin and is similar to the Ecarin clotting time.”
Dave said that Haemoscope personnel said they knew of no current studies on the TEG using Echis carinatus. He also contacted personnel who are marketing the Multiplate, who said they had no current protocols for Echis carinatus or the ecarin time.
In a follow-up comment, Vilas wrote: “Crude venom was diluted serially 10 fold: 10 mg/mL,1 mg/mL, 100 ug/mL,10 ug/mL. SSV crude venom on TEG suggests role of fibrinogen binding to GPIIb/IIIa receptor. Is there no defibrination syndrome in SSV bite patients ? It is not clear to me about normal TRAP-6 response in SSV. TEG also shows decreased R suggestive of enzymatic hypercoagulability, what is the cause?
Alteplase and Fibrinogen Assay (February 14)
From Carla Purvis MT (ASCP) SH, Parkview Health System, Fort Wayne, IN: “How does Alteplase (Activase, synthetic tissue plasminogen activator, TPA) therapy affects fibrinogen assays? Are there any guidelines or references for time of collection for coagulation testing when Alteplase is in use?
George found no published guidelines that provide timing for fibrinogen assays after TPA therapy but attached the package insert, which states that Alteplase clears with a half-life of five minutes, implying that you should be able to get a valid measure of fibrinogen less than an hour after completion of therapy.
However, George goes on to recall Molk B. Acute myocardial infarction in an unsuspecting male. Clinical Hemostasis Review, February, 1997, Page 14. The “unsuspecting” patient asked one of the lab’s professional staff to accompany him and collect timed specimens, and 24 hours after his TPA, his fibrinogen had not returned to admission concentration. Based on this, George concludes you need to wait at least 24 hours after TPA therapy to resume fibrinogen measurements. This response generated follow-up comments from Gordon Ens, the unsuspecting patient, and Flo Newlin, the professional staff person (and vice-president of Colorado Coagulation Consultants) who drew the specimens. They describe the incident in personal detail.
Bleeding Time Test (February 16)
“Slacey” asks, is the BT outdated? I would like to see this practice stopped as it is very subjective and leaves the patient with a small scar.
George confirms the BT is obsolete and should be discontinued, it cannot predict surgical bleeding and has limited efficacy in screening for platelet disorders. Phlebotomy and laboratory personnel have trouble convincing surgeons to stop ordering the test, but they are more comfortable when they can read clinical proof. George provides four references that provide data proving the poor efficacy of the BT.
Platelet Response to Epi and ADP Suppressed
Dr. Gerard Donnelly describes an elderly female patient who was off aspirin for 1 month and had a depressed secondary light transmittance aggregation response to epinephrine (16%, range 60-100) and ADP (26%, range 60-100), but responses to collagen (69%), arachidonic acid (75%), and ristocetin (92%) were normal. Are these findings specific for anything? The patient had an unimpressive bleeding history, had been on aspirin for years with no bleeding episodes even with invasive procedures. However she was seen by another physician for what looks like senile purpura of subacute onset, which had not improved after aspirin was discontinued.
George apologizes for what turned into a lengthy answer: “Perhaps the laboratory findings are random analytical error, given that light transmittance aggregometry is equal parts art and science. I’d disregard the epinephrine finding, as there is considerable variation among normal healthy adults. I’d repeat the ADP aggregation in consultation with the laboratory director. The ADP response is concentration-dependent and somewhat idiosyncratic among patients, so it would be valuable to run the aggregation at a series of concentrations, perhaps 5, 10, and 20 uM. Additionally, switch to whole blood impedance lumiaggregometry. This is a standard clinical approach which is not available everywhere, but is more reliable than LTA.
Another approach to this is to find a laboratory that performs closure time assays on the Siemens PFA-100, a sort of substitute for the old discredited bleeding time and are reasonably reliable at disclosing a platelet disorder. A competitor, DiaPharma’s Multiplate analyzer, is currently in FDA review and may be available soon. Should the aggregation results turn out to be normal, the senile purpura could simply be secondary to progressive loss of vascular integrity, a condition we all face.
There is one more interesting possibility: glucosamine. An attached poster reports platelet aggregation suppression in someone whose platelets were suppressed by glucosamine.  Dave McGlasson, author of the poster asserts that the subject had no bleeding tendency when taking aspirin and glucosamine together.
Why is PT Stable 24 Hours?
Madan Verma asked: “Why are we allowed to add a PT up to 24 hours after collection, given the short half-life of factor VII?”
George provides several references and responds that factor VII’s in vivo plasma half-life is 6 hours, perhaps owing to its tendency to become activated and consumed as it binds tissue factor. In vitro, however, factor VII is stable, accounting for the stability of the PT. He contrasts the PT to the PTT, which is stable for only four hours in vitro. The PTT depends upon an intact intrinsic coagulation pathway, which includes factor VIII, whose in vivo half-life is 12 hours but whose in vitro activity deteriorates by approximately 10% per hour. Factor VIII’s deterioration accounts for the progressive lengthening of the PTT during storage. George concludes that specimens for PT and PTT should be stored at room temperature, 18°C–24°C. Refrigeration activates factor VII and platelets and causes large VWF multimers to precipitate, whereas storage above 24°C hastens factor VIII deterioration.

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