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Learning Center
Coagulation Corner
Friday, April 3, 2009
Development of Inhibitors - and I don't mean LUPUS!
Happy National Medical Laboratory Week!
I would like to wish all of my readers a happy NMLW (week of April 19th)-
And congratulate them on the wonderful and important role that they play in patient care. Even if most people don't know what you do, or clinicians don't respect your knowledge, or nurses yell at you for taking to long to get a result, we know-
We have a large body of knowledge, one that is constantly challenged and as soon as we learn something, it is on its way to becoming obsolete. A test may take a long time because we make sure answers are both accurate and precise- there are controls and curves and the intuition of a tech, that just knows this isn't correct. So you should be proud of what you do, who you help, and take pride in your work. Thank you from the millions of patient who will never know- but it is important that you know!
DEVELOPMENT OF INHIBITORS- and I don't mean LUPUS:
We have learned that when a mixing study doesn't correct, that the next course of action is to look for an inhibitor. There is something in the patient's plasma is inhibiting something in the pooled normal plasma and preventing the test from correcting. Most commonly we then look for a lupus anticoagulant. These patients do not bleed (for the most part, but that is another session) but are more prone to thrombosis. When patients develop antibodies to factors the difference in these patients are that they will bleed, and they bleed a lot!
Hemophiliacs develop alloantibodies, as opposed to antibodies that are acquired or non- hemophiliacs develop autoantibodes. Hemophilia A is caused by a defect in the clotting of factor VIII, hemophilia B results from a defect in the clotting protein of factor IX. FVIII activity <1% are classified as severe, 1-5% moderate, and 5-25% as mild disease.
The formation of alloantibodies were found to be 10 fold higher in Hemophilia A than B, four times more frequent in severe than in non-severe hemophiliacs and higher in children aged under 5 years. There are a number of risk cofactors that should be taken into account with regard to genetic, environmental and conditions of treatment administration.
1. Non-white patients are a higher risk: African-Americans 51.9%,
Caucasians 25.8%
2. The Relative Risk is 3.2% if a sibling presented with one.
3. Patients who undergo treatment at young age are at a higher risk
4. The type of causative mutation, phenotype, as well as polymorphisms
of the gene coding for cytokines and immune regulatory molecules.
5 Patients with FVIII genes with nonsense mutations, large deletions and
inversions show the highest incidence, while mis-sense mutations and
small deletions exhibit a lower incidence.
6. Other causes can be infections or vaccinations. However there is not
unequivocal explanation.
Autoantibodies are formed in elderly patients and spontaneously in pregnant women. Patients who receive topical thrombin can also develop antibodies to Factor V due to the bovine thrombin. Did I mention that these patients bleed?
Autoantibodies are treated with steroids to suppress the antibody causing the inhibitor. Chemotherapy can also be used, as well as IgG. Diagnosis is important so treatment can begin.
How to test:
The detection of inhibitors includes:
1. Screening for a prolonged activated partial thromboplastin time (APTT) or prothrombin time (PT)
2. Mixing study that doesn't correct - PT you would look for VII, APTT test VIII, IX and IX, both fail to correct II, V and X
3. Levels of factor assays should be less than 25%, many will be undetectable.
4. Only when a factor is less than 25%, the next line of testing is an inhibitor or Bethesda This assay will quantitate the amount of inhibitor that will inhibit 50% of the factor that you are testing. This is done by creating several dilutions of patient plasma diluted in imadazole buffer, and then mixed with a pooled normal plasma, This is incubated for 2 hours at 37 degrees to allow the inhibition to occur. (This is where you get the 50%, you are making a 1:2 dilution) This is compared to a residual amount by taking a 1:2 dilution of the pooled normal plasma and comparing the activity of that mix.
Sound complicated, well it is, it is an involved test that takes a long time, lots of calculations but when performed correctly can give the clinician an huge amount of information. A few suggestions:
1. Perform the assay in imadazole buffer, it helps to keep the factors stable. Even better you can purchase the buffer premade from ANIARA.
2. Make sure that when you bring in a new reagent you validate it for your Bethesda assays.
3. Make sure you validate your range, some centers use <0.5, others use <1.0. A good way to determine what is negative is to run samples between 0 and 1.5 and see how they repeat. We found that samples <0.6 would repeat always below 0.6, and ones above that would repeat >0.6, so we felt comfortable with that level.
4. Always run both a negative and a positive control. Either purchase a positive or rerun a positive patient.
Why are inhibitor levels so important?
Patients that have low titer FVIII inhibitors, < 5BU can be treated with more frequent doses for FVIII to saturate existing antibodies and provide sufficient factor for hemostatis. The goal is to achieve factor levels of about 30%. Those with BU >5 have to be treated with bypassing agents that will provide hemostasis independently of FVIII and therefore are insensitive to inhibitors. Activated prothrombin complex such as FEIBA was found to be effective. Possible side effects included thrombo-embolic events, and DIC. This was attributed to the presenece of activated coagulation proteins. Recombinant VIIa (rVIIa) and activated prothrombin-complex such as FEIBA are most widely used.
HOW DOES rVIIa WORK?
We know that FVIIa is important in the initiation of hemostasis. After vascular injury, FVIIa binds to its receptor tissue factor (TF) on the surfaces of TF-bearing cells. This complex along with FVa generates small amounts of Xa and thrombin. This thrombin then activates platelets to the site which serve as a template for IXa, VIIIa, Xa and Va, which in turn generates a thrombin burst. rVIIa- Manufactured using recombinant technology, a chromatographic purification process in the absence of human serum or proteins. It has a very short half life (remember factor VII has the shortest half-life), and in children it is cleared faster, so higher doses may be required.
So what do we see in the lab, when patients are treated with rFVIIa? Since the PT is exclusive for Factor VII and you are adding an activated factor it stands to reason that your PT will be shortened. That is correct, however be very careful, it may be so short, that the sample may clot before your analyzer has a chance to read it, resulting in what looks like a prolonged result. It is important to check all "no clot" PT's, they may be very short PT;s. At present there is no routine laboratory method to measure rVIIa.
Inhibitors are complex, the testing is complex and when a spontaneous inhibitor forms the bleeding is significant. The information provided can aid in the diagnosis and treatment of these patients.
Donna Castellone
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About the Author
Donna Castellone,
MS, MT(ASCP)SH
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