2012 Cheat Sheet

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.

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?

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!

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 (mesoscale.com), and capture beads with analyte specificities and fluorescence detection by flow cytometry (bdbiosciences.com). Currently these assays are RUO, however it is only a matter of time until assays geared towards measuring inflammation become optimized for diagnostic use!

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.

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.

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.

An unidentified contributor asks about the means for monitoring clopidogrel efficacy, first posted February 18, 2014.

“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.

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.