Cheat Sheet for Jan-Feb 2012

How Long Do You Wait to do LA Testing? (January 10, 2012)

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.

CAP Requires INR Reference Interval (January 11, 2012)

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.

New Ristocetin Lot and Storage Requirements (January 11, 2012)

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, 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.

Clot-based Protein S Dilutions (January 11, 2012)

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.

Quick Question: Heparin Therapeutic Range (January 13, 2012)

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.

Collecting Blood From Hemodialysis Patients (January 16, 2012)

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.

Calculation Error in Lupus Anticoagulant Module 8 (January 20, 2012)

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.

Faulty In Vitro Clotting in Multiple Myeloma (January 21, 2012)

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.

Ristocetin Cofactor (January 21, 2012)

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.

High D-dimer, Low Fibrin Monomer in Amyloidosis (January 22, 2012)

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.

Fondaparinux Assay (January 27, 2012)

From Crystal Azevedo: Would you have a list of laboratories performing the anti-Xa assay for Fondaparinux (pentasaccharide, Arixtra)? Do you recommend, when calibrating the assay, to use commercially available material or spiked normal pooled plasma (NPP)?

George responds that Machaon Diagnostics, Quest and LabCorp provide the assay; you can find it listed under Fondaparinux in their catalogs. Further, you’ll find the assay at a nearby academic medical center or tertiary care facility. Many of the labs that offer the heparin anti-Xa assay have set it up as “Fonda” use is growing.

You can definitely spike NPP with Fondaparinux from your pharmacy; several labs are doing that successfully. Alternatively, HYPHEN BioMed distributes a set of Biophen® calibrators and controls designed to be used with their anti-Xa kit. Stago indicates their STA®-Rotachrom assay may be used for monitoring Fondaparinux, however they do not provide calibrators or controls.

PT and PTT for Preoperative Screens (February 1, 2012)

From Steve Marionneaux (Lab manager and UMDNJ graduate student): Medicare no longer regards PT and PTT as medically necessary screens for patients with no history of coagulopathy. No compelling evidence shows that PT/PTT screening predicts bleeding with no history of a bleeding/clotting problem. Surgeons continue to insist. However, in these cases the patient is required to sign an advanced beneficiary notice (ABN), which is an agreement that the patient would pay for the tests.

Comment from another UMDNJ grad student: Coding is an issue. Not only does Medicare no longer pay for these tests for pre-operative purposes, same for several major private payers including BCBS and United Healthcare. We still are able to get the tests covered using v58.61 (long-term anticoagulant use) for those patients who have been on aspirin, Plavix, coumadin, etc. and 782.7 (spontaneous ecchymoses) for those with a history of easy bruising. These two typically cover about 40% of our patients who have PT/PTT pre-surgical orders. All others we have sign the ABN. At least PT/PTT are relatively inexpensive. We never tell the patient that the test itself is medically unnecessary, just that their insurance company dictates that they are. Yes, it is the laboratory’s place to review a doctor’s order, especially when there is no evidence to support the ordering of the test for the patient’s condition, however, to tell the patient that the test their physician ordered is medically unnecessary is potentially telling the patient their physician is incompetent and can result in the physician no longer utilizing your particular laboratory’s services. Telling a patient that their insurance will not cover the test is much different that telling them that their physician ordered a test that is not medically necessary (regardless if it is or not).

Comment from “Morer.” A story on the usefulness of PT/PTT for potential hemorrhagic detection: A 17 yo female was admitted for appendectomy with no history of bleeding. PT/PTT were performed and PT was prolonged. The story ended with liver replacement because PT was linked to early hepatitis. So now tell the surgeon not to perform PT/PTT!

Inflammatory Markers (February 1, 2012)

From another UMDNJ grad student: Some current technologies focus on biomarkers, which include cytokines, vascular markers and growth factors, associated with inflammatory responses. I have been focusing on two methods, a multiplex format that employs capture probes with distinct specificities and electrochemiluminescence detection (, and capture beads with analyte specificities and fluorescence detection by flow cytometry ( Currently these assays are RUO, however it is only a matter of time until assays geared towards measuring inflammation become optimized for diagnostic use!

PRP Gel, Platelet Substitute (February 1, 2012)

From yet another UMDNJ grad student: It appears that platelet rich plasma (PRP) gel has been around for more than two decades. One article details platelet substitutes under development, which include infusible platelet membranes (IPM), thrombospheres, and lyophilized human platelets. Does anyone know if there have been any more recent updates regarding these technologies?

Serotonin Release by Immunoassay (February 7, 2012)

Vilas Hiremath asked, for HIT SRA, can a  serotonin ELISA sensitive to HIT be used?

George went to experts, Drs Larry Brace and Jon Geske: Theoretically, a serotonin ELISA could be used to measure serotonin released from platelets activated by antibodies from HIT patients. Unlike the gold standard 14C-serotonin release assay, however, one would have to differentiate between serotonin present in the patient’s sample vs. serotonin released from the activated platelets. Controls would have to be run (low vs. excess heparin levels) similar to the 14C assay to detect PF4/heparin antibody-dependent platelet activation. Further, the ELISA method for serotonin quantification is complicated because many such assays require serotonin acylation prior to running the ELISA. Having cleared those hurdles, however, an ELISA could be used to detect serotonin levels.

Herb Crown (SLU) responded on February 17: An SRA using ELISA methods could eliminate all of the special handling, training and disposal of radioactive waste. In principle one would still need to incubate donor platelets with serotonin, perform the usual steps for a 14C SRA (without the 14C) and then use the ELISA serotonin to pick up the released serotonin from the platelets. The last test system we used for platelet serotonin was from IBL and it included an overnight incubation, though their website shows a few shorter versions now. The SRA is a complicated test process that demands strict attention to detail. Meanwhile, there are current ELISA HIT antibody test kits that are adequate for most circumstances.

Normal Plasma Storage (February 15, 2012)

From Vicki Lanigan: “I am looking for a reference for storage of PNP.” George responds that buffered control platelet-free plasma products that are frozen and prepared for commercial distribution are Class II IVD “devices” whose stability data are based on extensive studies. Conversely, storage of locally prepared platelet-free PNP adheres to the CLSI Guideline H21-A5, which indicates that specimens for analysis demonstrate less than ±10% variation when stored for up to three months at -24°C or for up to 18 months at -70ºC. The reference given in the post provides stability data for prothrombin time, PTT, thrombin time, all the procoagulants, antithrombin, protein C and S, plasminogen, and D-dimer. Specimens and controls are thawed at 37ºC, mixed well, and tested immediately or stored at 1-6ºC for up to two hours before testing.

Management of transfusion service plasma products is different. Fresh frozen plasma (FFP) is donor plasma that is placed in a -70ºC freezer within six hours of collection. The more commonly prepared frozen plasma (FP-24) is donor plasma frozen within 24 hours of collection. According to the AABB Standards for Blood Banks and Transfusion Services, FFP may be stored up to seven years and FP-24 for up to one year at -70ºC. Anecdotally, most donor services set the expiration date for both at one year. Both FFP and FP-24 are thawed at 30-37ºC in an FDA-cleared device and once thawed, may be stored at 1-6ºC (or transported at 1-10ºC) for up to 24 hours prior to administration, according to AABB.

This information was provided by Stephen Duff and Dr. Jon Geske, Precision BioLogic Inc, Dave McGlasson, and Margaret G. Fritsma, MA MT (ASCP) SBB.

More on PTT Specimen Storage Time (February 17, 2012)

On November 11, George replied to Joe Lamb about RT storage of specimens for PTTs sent to reference laboratories. Joe was concerned that his reference lab claimed PTT results were valid on specimens up to 24 hours old, whereas CLSI Guideline H21-A5 sets the limit at four hours. Actually, H21-A5 permits for room-temperature whole-blood storage beyond four hours provided the local institution provides in-house supporting data.

PTT Mix Prolonged Beyond Patient PTT (February 23, 2012)

Danagah:” asks, have you where the 1:1 immediate mix of a PTT mixing study is longer than the patient’s initial PTT?

George describes an anecdotal phenomenon called “lupus anticoagulant cofactor.” The “cofactor” is associated with a PTT mix that is actually prolonged beyond the patient PTT alone. The prolongation was evident when using a kaolin-based PTT reagent but disappeared when using an elagic acid-based reagent.

QQ Summary: How Long to Discontinue Warfarin? (February 23, 2012)

Our January, 2012 Quick Question was, “How long do you recommend a patient wait after discontinuing warfarin before performing a thrombophilia profile?
a. 48 hours: 10 (16%)
b. 7 days: 19 (30%)
c. 10 days: 21 (32%)
d. 6 weeks: 14 (22%)

No agreement among responses; it appears we are looking for guidelines. The typical thrombophilia profile includes the vitamin K-dependent control proteins S and C. The half-life of protein C is close to three days, and the half-life of protein S is ill-defined, but probably longer. We should wait at least 6 weeks before collecting blood for a complete thrombophilia profile.

DIC Marker Recovery Time in Therapy (February 25, 2012)

Siddhartha” asks, in DIC, what is the minimum time for the coagulation parameters D-dimer, FDP and fibrinogen to return to normal after FFP? Is it worthwhile to do these tests in a case of DIC who has already received FFP?

George found no studies addressing DIC marker return to normal, though there are many that list the markers used to identify DIC upon the appearance of symptoms. These include platelet count, prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen, D-dimer, FDPs, and fibrin monomer.

George speculates that, because fibrinogen, FDPs, and D-dimer are acute phase reactants, their return to normal is too slow and variable to be of any value in tracking therapy. Their variation relates to the source of DIC, for instance, markers are likely to remain higher for extended periods in sepsis but return to normal sooner in trauma. However, the more generalized assays fibrin monomer, and even PT, PTT, and platelet count, which are less sensitive to inflammation, are probably the best markers for return to normal coagulation.

Look-back for Positive APAs (February 29, 2012)

Pam Owens asks: We are wondering if other labs research their patients’ positive APAs such as LLI, ACL, and B2GPI results to differentiate between persistent and new diagnoses. If so, how far back do they look for a positive? For example, if a patient had a positive result in 2000, 2002 and 2004 but was negative in 2005 and 2010 and is now positive, is she persistent or new, given the eight- year gap? Does the overall diagnosis become a factor, such as if the patient has lupus or rheumatoid arthritis (RA)? We were thinking 5 years was a good look back, but what are others doing?

George checked references and contacted some APA experts and is awaiting answers. Based on one referenced case, we can at least propose that for some patients, the APA waxes and wanes. More to come on this question.

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