December 2012 Cheat Sheet

Dave Easler contacted participant Dave McGlasson through the Medlab_L list to ask if he recommends to not use a POC INR method for patients bridging from LMWH to Coumadin. Dave gained the impression that the recommended method for testing INRs in this class of patients is with a laboratory analyzer rather than POC.

McGlasson answered, when bridging from heparin or a DTI to Coumadin the recommended test is the chromogenic factor X (ten, CFX). It is impervious to the other anticoagulants. POC PT/INR assays are clottable assays that can be affected by heparin or DTIs. Measure the CFX until it gets to be <30% activity. The CFX is just as easy to perform as an anti-Xa assay.

Tania Puro asks to explain why the PTT and PT are normal in dysfibrinogenemia.

George answers that dysfibrinogenemia is a qualitative or functional fibrinogen deficiency associated with moderate to severe liver disease. The liver produces normal or even elevated fibrinogen levels, but the molecule is coated with excess sialic acid and functions poorly. Liver disease is associated with soft tissue (anatomic) bleeding owing to deficiencies of several coagulation factors and to dysfibrinogenemia. The thrombin and reptilase clotting time are prolonged, and clot-based Clauss fibrinogen assay results are reduced. Because dysfibrinogenemia is a functional disorder, fibrinogen assays that rely on nephelometry, such as estimates provided by coagulometers in the performance of a PT , may be normal or elevated.

PT and PTT reagents are moderately insensitive to hypofibrinogenemia and dysfibrinogenemia, and only become prolonged when fibrinogen concentration or functional levels are below 100 mg/dL , which explains why the PT and PTT may be normal in dysfibrinogenemia.

Mohamed Bashir asked, what is the guideline for thrombophilia testing, how to test AT , PC , and PS ?

George responded that thrombophilia profiles are developed locally to meet population needs, however most include AT , PC , and PS activity, APCR , prothrombin G20120A mutation, and LA testing. Some include ACA , anti-b-2-GP 1, fasting homocysteine, and coagulation factor VIII.

If the patient has experienced a recent thrombotic event or is currently on Coumadin, the tests that remain reliable are the FVL mutation, which substitutes for the APCR , prothrombin G20210A, ACA , anti-b-2-GP 1, and fasting homocysteine. The AT , PC , PS , APCR , and LA are reliable only when the subject is not receiving Coumadin and has not had a recent thrombotic event.

Owing to the false positive rates of most assays, we avoid screening healthy individuals. Thrombophilia profiles are chosen for patients who have experienced an unprovoked thrombotic event, an event in an unusual location or before age 60, or who have first-degree relatives with thrombotic risk factors. A positive APCR ratio is confirmed using the FVL mutation, and low AT , PC , or PS values are repeated after 12 weeks for confirmation. Low AT , PC , and PS activity assays are then followed up using AT , PC , and PS antigen assays to distinguish qualitative from quantitative deficiencies.

Ann McConnell asked, why are factor activity assays reported in the range of 50%–150%? “I am having a difficult time wrapping my mind around the concept that something can be more than 100% ‘active.’”

George responded that in fact the correct way to express factor activity is in international units, with 1 IU = the mean factor activity of normal plasma. Thus 150% would be 1.5 units. From a pure mathematical standpoint, percent is an incorrect expression, however, we’ve been using it to express factor activity since the mists of time, so, given normal human inertia, we probably won’t change!

Mahnaz Sairi asked, what is the highest limit we can report for FVIII ? And how we determine this? Is it by clinical significance or by instrument linearity? Also, should we still do LA testing if both the PT and PTT are normal?

George suggests to begin with a calibrator of known factor activity such as CRYOcheck™ Normal Reference Plasma and prepare several ascending dilutions using the required buffer. Compute the anticipated activity for each dilution, then assay each dilution and prepare a linear graph of anticipated activity versus assay values. By inspection you will find where linearity is lost at the top and bottom of the curve; select the points just inboard to establish your limits.

The limits of linearity vary among instruments and reagents, however, when preparing dilutions, just as a rule of thumb, develop a range of 1%–150%. Some instruments use two curves, the second of which is designed to provide linearity at the low range, for instance, 0.1%–10%, in this case it is necessary to test linearity in both ranges by preparing and testing the appropriate calibrator dilutions. The instrument automatically selects dilutions and curve based upon the initial assay.

Specimens that are out of linearity, for instance, FVIII activity levels over 150%, are diluted, dilutions are re-assayed, and the result of the assay is multiplied by the dilution factor. High-end coagulometers automatically make the appropriate dilutions, and it is not unusual for FVIII levels to exceed 150%, as FVIII is an acute-phase reactant. Because chronically elevated FVIII is a thrombosis risk factor, laboratory directors may choose to report levels as high as 250%.

For the second question, the PTT reagents employed to monitor UFH or to screen for coagulopathies are formulated with high phospholipid concentrations, rendering them relatively insensitive to LA. These are employed to reduce the frequency of detection of transient, clinically innocent LAs. See Fritsma GA, Dembitzer FR, Randhawa A, et al. Recommendations for appropriate activated partial thromboplastin time reagent selection and utilization. Am J Clin Pathol 2012;137:904–8. If there is clinical suspicion of an LA , for instance, an unprovoked thrombotic event, it is necessary to test using low-phospholipid PTT reagents such as PTT-LA, and to test in parallel using a separate platform such as CRYOcheck™ LA Check™ (dRVVT screening reagent).

Lisa Ledford asked, Would you be willing to share your ∂-check values for PT/INR , PTT , fibrinogen, and D-dimer?

George checked with Warren Varden at UAB, who doesn’t use ∂-checks. Our special coagulation staff carefully reviews results and communicates with our inpatient and outpatient units and are able to discern when there is a change in therapy or a faulty specimen collection.

Joanna Carroll is running automated D-dimers in a clinic at a veterinary school and recently got a result of 0.00 ng/mL. Have you seen a value reported this low? Would it be better to report out the number as less than our lower limit of the reference range?

George has never seen a D-dimer value of 0.0 ng/mL , and assumes this was performed on a human. He suggests reporting as less than the lower limit; particularly as a result this low would have no clinical significance.

Added 12.7.12: Dave McGlasson has worked with a number of animal specimens, and says the monoclonal antibodies in the quantitative D-dimer kits are specific for humans and do not detect animal D-dimers.

Scott Miller wrote: We will soon be validating our PTT heparin therapeutic range by using a comparison between an anti-Xa assay and PTTs run with our new lot of PTT reagent. I have always understood that when calibrating an anti-Xa assay for this procedure, one should always make the calibrator by dilution of the same type of heparin used at the institution. Yet the kit we received from IL includes a box of WHO-certified calibrators. Should these be used instead of the homemade stuff?

George definitely recommends using the provided calibrators provided. Not only are they traceable to WHO international UFH and LMWH standards, they also enable you to construct a hybrid curve, one that may be used to report both UFH and LMWH results from the anti-Xa assay without have to change curves. Plus, home-brew curves tend to flatten out at the top end. George also suggests using the anti-Xa directly to report heparin results.

Kelly Townsend received a letter stating that BD Vacutainer is recalling a lot of their 2.7 mL sodium citrate tubes due to lithium heparin contamination. Has anyone seen issues with this and how are folks investigating?

Elaine Benoit sent an FDA alert regarding a heparin label change: “This label change will require manufacturers of Heparin Lock Flush Solution, USP and Heparin Sodium Injection, USP to clearly state the strength of the entire container of the medication followed by how much of the medication is in 1 milliliter (mL). These modifications will eliminate the need for health care professionals to calculate the total amount of heparin medication in a product containing more than 1 mL, thereby reducing the risk of miscalculations that may result in medication errors.”