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Coagulation Corner

Monday, November 6, 2017

November 2017 - Coagulation Reagents - NOW WHAT?

Written By Donna Castellone, MS, MT (ASCP) SH | LinkedIn

Everyone knows there are issues with coagulation reagents- just look at a proficiency testing survey for factor assays - results are all over the place. There are different activators - (ellagic, silica, micronized silica and kaolin). Different types and concentrations of phospholipids. And that is just the reagents! When you are performing factor assays- you have deficient plasma (immune-depleted, chemically depleted and congenital), people even use different buffers to make dilutions as well as different methodologies.

To further complicate the reagent issues- there is factor sensitivity, heparin sensitivity and lupus sensitivity. A reagent is deemed as being factor sensitive if it can pick up a factor deficiency in the screening test of the PT and aPTT when there is <30% of that factor level. For example, if I have a factor IX levels of 25%, that should reflect an abnormal aPTT. This abnormality will alert the clinician to investigate further factor levels.

For example:
Factor IX Sensitivity: APTT = (29.5-33.5)

Normal plasma + Deficient plasma = % Activity APTT
500ul 0ul 100% 29.0
250ul 250ul 50% 31.0
125ul 375ul 25% 33.0
62.5ul 437.5 12.5% 36.0
This reagent does not reflect an abnormality until 12.5%

A lupus sensitive reagent is one that will detect a lupus inhibitor. These reagents contain a decreased concentration of phospholipids. In-vitro a LA is manifested as a antibody to phospholipids. Having a reagent with a lower concentration of phospholipids will allow the antibody to manifest and prolong your results as compared to a reagent with a higher concentration of phospholipids which will overwhelm the antibody resulting in a normal result. Many laboratories choose to use an insensitive reagent so they don't have to deal with many transient non-specific inhibitors. These patients do not bleed from factors (they may bleed due to a platelet disorder).

Next we have heparin sensitivity- that is if you use the aPTT to monitor unfractioned heparin, at what levels does the aPTT correspond to the UFH therapeutic range (0.3-0.7 U/mL). This study has to be performed at least once, where samples from patients on UFH are tested for anti-Xa levels and compared to their corresponding aPTT results. These are then graphed and the best fit line is drawn (which is not easy, since there is no dose response relationship in patients on heparin). The seconds that coreespond to a 0.3-0.7 U/mL range becomes your heparin therapeutic range for that lot of reagents. Subsequent years, you can compare the aPTT in seconds between the old lot and new lot of reagents on patients that are on UFH. If the difference is less than 5 seconds,you can accept the previously established range that was verified by the anti-Xa study. It is a lot of work, but at least it doesn't have to be done every year.

CAP recommends that each laboratory establishes its own heparin therapeutic range for each new lot of aPTT reagent based on reagent/instrument combination

Heparin Anti-Xa Method

    50-60 plasma samples collected from patients treated with UFH Mix of samples should span the therapeutic range No more than two samples on the same patient The PT/INR should normal (INR >1.3)
    Assay aPTT, anti-factor Xa (UFH), and PT/INR
    Plot the aPTT on the y-axis and the anti-Xa units on the X-axis
    Draw a line through the middle of the points
    The UFH therapeutic range is equivalent to 0.3-0.7 units/mL Anti-Xa Heparin assay

Heparin Therapeutic Range

Based on this chart the therapeutic range for this lot of reagents that correspond to 0.3-0.7 U/mL would be 70-120 seconds.

So that would seem to be enough, wouldn't you think, well that's not all folks! Now we have the impact of some new replacement therapies for Factor VIII and IX that are reagent dependent. Some reagents over recover, some under-recover- and of course they are different for VIII and IX! So now what else do we need to know and understand in the coagulation laboratory?

There are several new extended half-life products on the market to treat hemophilia A and B. They have the ability to increase efficacy of the FVIII products by 1.4-1.6 fold and the FIX products by up to 3.5 fold. They use 3 molecular modifications for extension: fusion to either albumin or the Fc portion of IgG1; covalent attachment of a hydrophilic protein such as PEG of modification of single chain FVIII covalently bonded heavy and light chains with increased affinity for vWF binding. These modifications can greatly improve the quality of life for these patients, decrease infusions and also help to prevent inhibitor formation. However there is a problem.

Certain modified recombinant FVIII and FIX replacement products demonstrate variable and clinically significant differences in post-infusion recovery (that is, the amount of factor measured vs. the actual concentration present), based on the aPTT reagent and the assay methodology that can either over-estimate or under-estimate the true value by up to about 50%! This of course can greatly impact patient care. The worst case scenario is, product can be under-dosed and the patient can be put at a risk for bleeding. While overdosing a patient can put the patient at a risk for inhibitors.

What is most disturbing is that, a reagent that may work for an extended half-life factor IX product, may not work well for an extended FVIII product. And again, do you know what product your patients are getting- are the clinicians aware of this issue? Do they even know the reagent you are using? Okay let's face it, do they know where the lab is?

The solution is to perform a chromogenic assay which seems straightforward...not so fast! There are issues with the assays- first, there are only 2 FDA approved FVIII chromogenic kits, and only one for use on coagulation analyzer, AND if they don't happened to be the one you are using, that will mean a full validation and no support from your analyzer manufacturer- meaning where do you get the settings? Also, the kits are expensive and not very stable- you can aliquot, but that means you need to prove stability. Now for factor IX, there are no FDA approved chromogenic kits- that means you can't charge for the test, and you need to state in the reporting of the results that the results cannot be used clinically. So it is for sure complicated!

How do products versus reagents perform? Here is an overview:

Products vs Reagents

Products vs Reagents

Coagulation just keeps getting more and more complicated. It is so important to be aware of not only how your reagents work, but now how they work in conjunction with these new products. So please investigate what your patients are getting, what your clinicians are using and how to handle it so your patient are getting the best results. Because at the end of the day, it is all about the patient!



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