2012 Cheat Sheet
George was asked, “How long does a patient need to be off warfarin or heparin before LA testing can be done?”
George suggests waiting ten days, representing four prothrombin half-lives (240 hours): the PT and PTT should no longer be affected by heparin or warfarin. Contributor Dave McGlasson, who checked with Dr. John Olson, confirms this, though neither Dr. Olson nor Dave know of a reference documenting vitamin K factors’ return to normal levels after discontinuing warfarin. Heparin is of little concern, as it disappears within 48 hours. Dave suggests assaying factor X, which has the second-longest half-life, 30 hours, and when normal, proceed.
If considering a complete thrombophilia workup, the effect of warfarin on protein S may last up to six weeks (reference provided). It is also necessary to wait at least six weeks after the resolution of a thrombotic event to ensure all coagulation and control factors have returned to baseline.
Dr. John Olson wrote: I read with interest the brief piece on an RI for the INR (from George’s 11.4.11 comment to Patti Richardson and Dr. Manjula Balasubramanian, in which he suggested the INR RI was optional). When I (Dr. Olson) was at the University of Iowa, we reported the INR and the PT in seconds, the idea being that the INR would be used only to monitor VKA. When I would ask the residents or fellows what the PT was, I would uniformly get the answer in INR no matter what the etiology; most patients were not on VKA. Thus, there is a need for the reference interval for the INR to be published with the result, as well as the therapeutic interval. In my mind, the only exception would be the INR performed at POC in the anticoagulation clinic. In that setting, the only application would be evaluation of VKA and the reference interval may not be useful. In all other settings, patients will be evaluated for all conditions that may require a PT and the upper limits of normal are necessary. The CAP does currently require that the INR have a reference interval (this is being challenged) and I believe that the reference interval is important. The query asked if the interval can be derived from the reference interval for the PT in seconds and the answer is yes. It is, after all, just math.
From Kathy Jacobs, Chrono-log Corp: As a follow-up about a lab experiencing difficulties with ristocetin testing (Kerri Klem, 11.9.11), that lab is now using our older lot # B-1186-8, which is working nicely. Here is the information on new lot # B-1186-9 compared to the old, using both WBA and LTA. First number is new lot; second number is old:
1. WB @ 1.0 mg/mL = Average 20.5 vs 17 ohms
2. PRP @ 1.0 mg/mL = Average 76.3 vs 78.3%
3. PRP @ 1.25 mg/mL = Single tests 78 vs 87%
4. PRP @ 1.5 mg/mL = Single tests 86 vs 87%
5. PRP @ 0.9 mg/mL = Single tests 2 vs 2%
6. PRP @ 0.75 mg/mL = Single tests 0 vs 1%
So, it looks like the new Chrono-log ristocetin is maintaining its viability.
Also, we had some issues with reagents stored frozen in aliquot tubes losing some or all of their volume. We did a 3-month study with ristocetin using a wide variety of tubes. We will be recommending the following tubes: Free-standing polypropylene tubes (natural or amber) with caps + O-ring seal available from www.denvillescientific.com, part # C19053 and C19046 (natural) or C19053-A and C-19048 (amber). Tube volume is 0.5 mL. We do not recommend using tubes less than 0.5 mL as we saw huge shifts in test results over a 3-month period using tubes of 0.3 mL. We also weighed the tubes before storing and after thawing and saw more weight loss with this size tube.
From Lisa Reid-Fifoot, Precision BioLogic: An institution performs several dilutions on the patient’s sample (1/5, 1/10, 1/20, 1/40, 1/80, 1/160) for the clottable protein S (PS
) assay. They find they are not getting good agreement amongst the dilutions.
George responds, there are no documented instances of PS inhibitors, thus the assay is performed on a single dilution. When results are outside the linearity limits, either low or high, the specimen is rediluted and assayed again. Multiple dilutions are likely to produce non-parallel results, as the least dilute and most dilute aliquots are likely to be outside of linearity.
On October 21, George posted a conversation with Steve Duff, Precision BioLogic Inc about establishing the PTT
therapeutic range for UFH
therapy. George speculated that with the arrival of all the new anticoagulants, the ex vivo Brill-Edwards curve is becoming a thing of the past. Steve challenged George’s statement, prompting this Quick Question:
How do you produce your PTT therapeutic range for monitoring UFH ?
a. Prepare a Brill-Edwards ex vivo curve using anti-Xa as the reference method: 54 respondents, 57%
b. We bypass the PTT and use the anti-Xa for heparin monitoring: 25 respondents, 26%
c. Spike a normal plasma with measured UFH and perform PTTs : 15 respondents, 16%
d. UFH has been largely replaced, we do little UFH monitoring: 1 respondent, 1%
George had to throw in the sponge! In an attempt to save face, he noted that at least a significant minority have moved to the chromogenic anti-Xa assay. Also, for those who are spiking normal plasma with heparin, this method, owning to poor linearity at the high end, is not recommended by the laboratory accrediting agencies and probably should be abandoned.
From the ASCLS Consumer Web Forum: Where would you draw blood from a hemodialysis patient to obtain an ACT ? I was taught to draw from the venous port to prevent clotting and blood loss.
George responds that as a general rule, line draws are discouraged because vascular access devices tend to cause hemolysis. Nevertheless, we often use line draws for convenience and patient comfort and can prevent hemolysis by withdrawing slowly. The venous port is the correct port to collect from. First flush with 5 mL saline, then withdraw and discard a volume of blood that is 2X the volume of the device, typically 5 mL. Then collect the specimen. Reference: Ernst D. Blood Specimen Collection FAQs, Center for Phlebotomy Education.
Laura Lum at Seattle Children’s Hospital couldn’t follow the mathematical calculation for the 4:1 mix mentioned in the Lupus Anticoagulant Part 2 audio module. The normal plasma is assumed 100%; the patient was 20%. The calculation was [100 + (20 x 4)] divided by 5. The answer was given as 32 but by Laura’s calculation, it should be 36 (180/5 = 36). Where did I go wrong? George responded that Laura didn’t go wrong, there is an error in the slide, due to be corrected.
Heidi Lawson wonders why MM specimens do not spin down well in SSTs or red top tubes. She called BD and they gave her an excerpt of information stating that MM patients, due to myeloma proteins, inhibit the stages of fibrin formation.
George first thought that the high plasma specific gravity associated with M-protein causes the gel to float, rather than position itself between the serum and the RBC layer. This is a problem, as the gel particles plug instrument probes. However, specific gravity does not account for poor clotting in the red-closure tube.
A BD Tech-talk article indicates that M-protein partially blocks thrombin’s action on fibrinogen, slowing formation of fibrin monomers. The monomers, in turn polymerize, slowly. To resolve the problem, let the tube stand for an hour until clotting is complete. If the analyte you wish to measure is not heat sensitive, incubate at 37°C. Also, inject a small aliquot of topical thrombin into the tube.
Added comment from Vilas Hiremath: Clotting occurs less than 1 min, never separate serum even after 12 hours. Rinse the syringe with heparin and draw sample. I had one patient M band positive, bone marrow showed plasma cells. Addition of thrombin cleared gelification.
Laura Lum, Seattle Children’s Hospital, asks, What is the ristocetin cofactor assay for, how is it performed, and can it be done on an STA Compact?
George responds that the VWF :RCo tests for VWF activity. The assay began as platelet aggregometry in which patient plasma is mixed with preserved reagent platelets such as those provided in Chrono-Log Corp Ristocetin Cofactor Assay Kit. Ristocetin is added and aggregation is recorded; the degree of tracing deflection estimates VWF activity. Siemens Healthcare Diagnostics, Inc offers an automated form, their BC von Willebrand reagent, designed to work on Siemens’ instruments. Several US operators have successfully adapted Siemens’ reagent to Stago equipment, and you can obtain assistance from you account manager or the technical support folks at Stago if you would like to adapt the assay to your instrument.
From “Joyce:” Other than Siemens BC von Willebrand reagent, is there any other company that offers this assay (automated)? George asserts that Siemens provides the only automated VWF :RCo assay.
From “rhe25:” When the D-dimer is very high, but the soluble fibrin monomer complex (SFMC) is negative, does that indicate primary fibrinolysis in an amyloidosis patient who is bleeding? Could the very high D-dimer be a lab artifact? Thanks!
From George: The SFMC, similar to the thrombus precursor protein (TPP), is the first product of the action of thrombin on fibrinogen. In normal coagulation, SFMC rapidly assembles to form insoluble fibrin polymer, however SFMC may accumulate during thrombotic events.
The assay for SFMC or TPP is an ELISA or hemagglutination assay that employs a polyclonal antibody. In rapid thrombosis, characteristic of venous thromboembolic disease, myocardial infarction, and stroke, the SFMC becomes elevated. The SFMC is also elevated in DIC , and is more specific for DIC than D-dimer, which rises during any acute inflammatory event or chronic inflammation. Although George finds no published evidence, he speculates that D-dimer could be elevated as a result of the severe chronic inflammation inherent in amyloidosis, whereas the SFMC is probably unaffected.
From our knowledge of fibrinolysis physiology, it seems as though D-dimer should not rise in primary (systemic) fibrinolysis (PF). PF is a rare condition in which fibrinolysis is triggered in the absence of coagulation. Since D-dimer arises subsequent to factor XIIIa crosslinking, it theoretically should be absent. This is not true, however.
While PF is rare, it is a consistent component of thrombolytic therapy. In 1997 Gordon Ens, CEO of Esoterix Coagulation, experienced a mild heart attack and had the presence of mind to request that blood be collected at serial intervals during TPA administration. The results demonstrated that fibrinogen dropped to immeasurably low levels while D-dimer rose. Apparently, D-dimer rises in PF, perhaps as a marker of inflammation, or perhaps because some fibrin crosslinking does occur.
Comment from “Morer:” For SFMC detection, the FS test is not an immunological reagent but fibrin monomer coated on erythrocytes. To check if it’s a laboratory artifact it will be of interest to know if some other immunological tests have been affected.