Coagulation Corner

HISTORY OF BLOOD COAGULATION

2008-08-04T07:06:00.000-07:00

The study of blood coagulation can be traced back to about 400BC and the father of medicine, Hippocrates. He observed that the blood of a wounded soldier congealed as it cooled, as well as bleeding from a small wound stopped as skin covered the blood. If the skin was removed bleeding started again. Aristotle noted that blood cooled when removed from the body which initiated decay resulting in the congealing of the blood. If fibers were removed, there was no clotting. It wasn’t until 1627 that Mercurialis observed clots in veins that were at body temperature. In 1770 William Hewson challenged the cooling theory and believed that air and lack of motion were important in the initiation of clotting. Hewson described the clotting process demonstrating that the clot comes from the liquid portion of blood, the coagulable lymph, and not from the cells, disproving the cooling theory. It was Paul Morawitz in 1905 that assembled coagulation factors into the scheme of coagulation which demonstrated that in the presence of calcium and thromboplastin, prothrombin (II) was converted to thrombin which in turn converted fibrinogen (I) into a fibrin clot. This theory persisted for 40 years until Paul Owren, in 1944, discovered a bleeding patient that a four factor concept of clotting could not apply, thus factor V was discovered. Owren also observed a cofactor that was involved in the conversion of prothrombin to thrombin. In 1952 Loeliger named this factor VII. Factor VIII was identified as classic hemophilia prior to the identification of VII in 1936-1937) In 1947 Pavlovsky reported that the blood from some hemophiliac patients corrected the abnormal clotting time in others. In 1952 this was called Christmas disease, after the family in which it was discovered, or Factor IX. Factor X deficiency was described in 1957 in a woman named Prower and a man Stuart, where there blood clotting when mixed with factor VII deficient plasma; hence a new factor was defined. Factor XI was described in 1953 as a milder bleeding tendency. In 1955 Ratnoff and Colopy identified a patient John Hageman with a Factor XII deficiency that died from a thrombotic event not a bleeding disorder. In 1960 Ducker described patients that had a bleeding diathesis and characteristic delayed wound healing. This fibrin stabilizing factor was called Factor XIII. Prekallikrein (1965) discovered from four siblings in the Fletcher family demonstrated no bleeding tendencies, as well as High-Molecular-Weight Kininogen (1975). These were both identified as contact activation cofactors that participated in the activation of factor XI by factor XII. (1) In 1882 platelets were recognized as being different than white and red blood cells by Bizzozero, but its relationship in coagulation didn’t become important until 1970. Each platelet makes 14,00 trips through the bloodstream in its life span of 7-10 days.
Testing of blood plasma factors and platelets depended on seeing the clotting process directly or microscopically. The first whole blood clotting time was done in 1780 by William Hewson who noted that blood taken from healthy people clotted in 7 minutes while some disease stated took from 15-20 minutes up to 1 ½ hours.
In 1897 Brodie and Russel begin observing the process on a glass slide. A drop of blood was placed on a glass cone, in a temperature controlled glass chamber agitated by an air jet. Blood no longer moved microscopically but clotted in 3 minutes and was completed at 8 minutes. In 1905 Golhorm used a wire loop attached to a glass tube. In 1910 Kottman observed an increased viscosity in clotting blood in a Koaguloviskosimeter. Blood was rotated at 20 degrees 12-15 time/minute. In 1936 Baldes and Nygaard added photoelectric tracings called a coagelgram depicting shape change by light transmittance.
In the 1960’s BBL introduced the Fibrometer. This instrument provided mechanical registration of clots that allowed more reproducible timing and an expression of the clotting process. (2)
1. Owen, Charles, A., “A History of Blood Coagulation”, Mayo Foundation for Medical Education and Research, Rochester, Minnesota, 2001.

2. Hougie, Cecil, “Fundamentals of Blood Coagulation in Clinical Medicine”, McGraw-Hill Book Company, New York 1963.

So what do you know?
We know that coagulation is a system of checks and balances that relies on a series of enzymatic reactions, naturally occurring anticoagulants and inhibitors. This waterfall theory is based on activation of factors and the initiation of coagulation. This occurred through two pathways, the extrinsic and the intrinsic pathway. The extrinsic pathway relied on the interaction of tissue factor and factor VII, while the intrinsic pathway utilized factor XII and the contact factor. These meet at the common pathway where they generate factor X to Xa and the conversion of prothrombin to its active form thrombin which then allows the conversion of fibrinogen to fibrin. This process follows laboratory based testing hence we have the in-vitro process. It allows us to follow the logic (not that you usually see the word logic and coagulation in the same sentence) of our testing- so what do we know? From this we can report the following:
1. Patient with just an abnormal PT, no meds, factor VII deficiency
2. With just an abnormal APTT, no meds, bleeding VIII, IX and XI, no bleeding XII
3. Both PT and APTT, look at the common pathway for factors I, II, V and X.
This schematic has served the laboratory well in testing and diagnosing disorders. But like all of the history before us, there have always been questions. For example:
1. Why is Factor IX a vitamin K dependent factor?
2. How come some factor XI patients bleed, while others don’t?
3. Why don’t people with a XII deficiency bleed?
4. Why can’t one pathway take over when there is a deficiency in the other?
5. What is the relationship of platelets in this process? They aren’t measured in the process.
6. How does this testing account for the variation in hemophilia, and how does recombinant VIIa work?
7. Just how important is thrombin?
This cascade, while is very important in laboratory based coagulation testing,, does not reflect what happens physiologically.

The in vivo model is a cell-based model of coagulation. This relies on 3 stages of coagulation and how they work.

INITIATION - Tissue Factor (TF) activates VII to VIIa which in turn activates FX to FXa and also activates FIX to FIXa

AMPLIFICATION: FXa starte to generate thrombin, which binds to platelets and begins the feedback mechanism of thrombin.

Thrombin is the most powerful coagulant. It activates V and VIII, but as thrombin increases it also destroys V and VIII by proteolysis. Factor Xa enhances fVII, but through a reaction with tissue factor pathway inhibitor, it prevents further activate. Thrombin feedback activation of IX can help to explain whay the intrisinc pathey can occur in the absence of contact factors. Since TF is expressed following an injury, we know it forma a complex with VIIa, then activaes X and IX, it is further amplified by V, VII and XI leading to intrinsic pathway activation. This feedback theory can help explain why patient with fador XII deficiencies don’t bleed.

PROPAGATION: activated platelet and thrombin formation continue to make large amount of thrombin and allows for fibrin formation.
The extrinsic pathway is based on tissue bearing cells to initiate and amplify coagulation
The Intrinsic pathway operates on an activated platelet surface to produce the burst of thrombin to stabilize the clot.

So now what do we know – using the cell based model of coagulation:

1. The APTT reflects factors in the intrinsic platelet surface pathway
2. Hemophilia is a failure of platelet surface thrombin generation
3. Factor Xa can’t tell the difference between a tissue factor bearing call and platelet surface
4. As a result one pathway can’t make up for deficiencies in the other- because they produce complexes on different cell surfaces
5. Using rVIIa in hemophiliacs with inhibitors was though to boost tissue factor pathways, but to work in hemophilia you need to generate Xa to thrombin on platelet surfaces. The way rVIIa works is that is binds to activated platelets and activates X on platelet surfaces, high doses are neede because rVIIa binds to platelets with a low affinity.

It seems as though coagulation has just touched the tip of the understanding and holds keys to the many mysteries of the working and disease process in man.

So what do we know, is that we have a lot more to learn!

Standardization in Coagulation- Or the lack of it- What’s A Laboratory to do?

2008-07-10T11:29:00.000-07:00

Where do we get our guidelines in coagulation? There are several agencies that provide guidelines. The Clinical Laboratory Standards Institute (CLSI –formally NCCLS – National Committee for Clinical Laboratory Standards.) provides many publications regarding everything from the PT, APTT, Factor Assays, Fibrinogen, How to validate reference ranges and instruments, collection of specimens and platelet aggregations. The College of American Pathology (CAP) provides checklists that cover everything from adequate space to proficiency testing, heparin and direct thrombin inhibitor contamination, performing heparin therapeutic ranges and criteria for factor assays. Let us not forget CLIA and JCAHO providing safety initiatives, quality control and validation guidelines. With all of these institutions and their regulations, it is still difficult to decide by a standardized format if a mixing study has corrected!

Coagulation itself provides many challenges. Regardless of the guidelines and attempts at standardization, there are many variables. Pre-analytical variables account for up to 64% of errors, many of which are not controllable by the laboratory. (blood collection, pouring from one tube into the next, heparin contamination, tubes floating around the pneumatic system, and of course the sample that sits on a desk unlabeled and then gets a label smacked on it- any of these sound familiar?) The next issue is the instrument/reagent combination – there are many possibilities, just look at a CAP survey! Reagents have different sensitivities, dilutions can be made in saline, or owrens buffer, lyophilized plasma or fresh frozen deficient plasmas can be used, and what about the standard that is used? It is important that a standard be calibrated against a WHO standard. You should also know how that standard is tested. Is it on a different analyzer; with a different reagent? You might want to test it several times on your reagent/analyzer system to determine a more comparable value for your system.
Also, running independent standards as a patient, can give insight as to how your analyzer is performing. If you feel that you have an instrument bias that you can’t seem to resolve on a test, you can use a trusted standard to implement a correction factor. For example, lets say you always run slightly high on your factor VIII’s, this is confirmed by your proficiency testing. If you run a standard, several times and note that you are consistently higher that the value, even after making all the attempts to correct this ( new reagents, maintenance, water, standard curve, and whatever other tricks you have up your sleeve!) it might be helpful to adjust those values. If the standard is assayed at 85% and you are getting 100%, you are too high, and need to bring those values down.

You would apply the following formula:

85% (Value of the Standard)
100% ( Value Obtained) =0.85 would be the multiplier to bring the value down.

If your result was 120% x .85=102%. You need to really make sure this is the issue, you need to have everyone on board to do this, or your values will be all over the place. You should verify this over a period of time.

So what about correcting that mixing study, well there are no clear cut guidelines. If you ask 10 labs you might get all different answers. Some labs use a correction into the normal range, others use within 1-2 seconds of the pooled normal plasma (PNP), while still others use a formula called the Index of Rossner. Other non standardized practices include: Do you perform an incubated mix, how long do you incubate, do you use a 1:2 dilution or a 1:4 dilution? Too many questions- so how does a laboratory judge what is best? It is important to define what you use and to implement standard operating procedures, so that the work that comes out of your laboratory is consistent, and becomes standard for your coagulation laboratory and the clinicians can count on that when they evaluate patient results.

With all of these issues how do you ensure good results? The coagulation laboratory needs to minimize and control what they can to provide good outcomes. Relying on proficiency testing and peer group evaluations from quality control groups provide laboratories with a lot of knowledge as to how they are performing. So what can you do if your results are not within the state limits?

You have several options:

Rerun the sample in question.
Rerun old proficiency test samples to see how the results are in compared to what they were assayed at, this material is gold and provides a ton of information.
Look at running independent controls to see where they are running.
Determine if there was a change in reagents, plasma or a new standard curve was performed.
What about maintenance on the analyzer, was one due?
Water can always be a problem, try using sterile water.
Pipettes, are they calibrated? Regulations state to calibrate within a stated period of time, but if they are used frequently and are important in your assays, twice a year may not be sufficient.
Run a precision study in the range in question, this may demonstrate carryover or an analyzer drift.

Another issue that plagues coagulation laboratories is setting acceptable limits between dilutions for factor assays. This is important in determining if an inhibitor is present or if the dilutions need to be rerun. First, it is very important for all results to be on the curve; an extrapolated result should not be used. After this criteria is met, an allowable acceptable limit between dilutions should be determined by the laboratory. If the manufacturer has guidelines, they should be adhered to, if not, keeping good clinical practices in mind, the laboratory should determine their own. Classifying an inhibitor can be tricky, what information are you giving the clinician? If the value is within the normal - high range, and is a slight inhibitor, other than being a nuisance to the physician, might be better averaging the results. A true inhibitor going from the abnormal to the normal range can be reported out at the low dilution, and the comment of the increasing percentage of activity at the highest dilution.

The International Society of Hemostasis and Thrombosis subcommittees meet each year. For 2008, they will meet in Vienna and discuss everything from Lupus anticoagulants to thrombin generation. They publish the minutes from their meetings and their progress to implement standardization. Their website is www.isth.org and can provide insight to their ongoing mission for standardization.

Adhering to good clinical practice and standard operating procedures within your own laboratory and striving to provide clinicians with good results is the first step in standardization. The ultimate goal is to provide good consistent patient results.

Heparin, Safety and the Laboratory's Role:

2008-06-04T12:15:00.000-07:00

With all of the attention to heparin contamination, and the amount of people in the hospital exposed to the drug, it is no wonder that the Joint Commission (JCAHO) came out with a directive this year concerning patient safety and heparin utilization. Heparin comes in as number 3 on the dangerous drug list- Insulin being number 1, and morphine number 2. This drug is widely used with a high risk of patient injury including bleeding, heparin induced thrombocytopenia (HIT) and osteopenia. Errors include the need for dose adjustments, and frequent laboratory monitoring Because of heparins poor bioavailability, there is no dose response relationship. This means that if 2 different patients are given a bolus dose of heparin, there is no correlation as to how similar the APTT results may be. Heparin is absorbed through the GI tract, has a half life of about 90 minutes and is excreted by the kidneys, additional problems such as liver disease, lupus anticoagulants and elevated factor VIII's add to the challenge of monitoring this drug.

So what did JCAHO come up with?
They defined patient safety goals- which is a system design or intervention that has demonstrated the ability to prevent or mitigate patient harm stemming from the processes of health care. Several safety goals for 2008 apply to the laboratory. However, this particular goal regarding patient safety 3E states: "Reduce the likelihood of patient harm associated with the use of anticoagulation therapy." This was directed to the following departments:

Ambulatory
Critical Access Hospital
Home Care
Hospital
Long Term care
Office Based Surgery

Anyone see anything glaringly missing?
Ah yes, the laboratory - anyone think of us? That is where the testing is performed that gives them the value which provides the information to allow them to decide what course of action to follow. You would think that it would be important for us to be part of that decision process. Just in case you weren't aware this directive has a one year phase in period that includes: "defined expectations for planning, development and testing (milestones) at 3 months (April 1, 2008- have you been aware of this in your institution?) 6 months (July 1, 2008) and 9 months (October 1, 2008), with full implementation by January 2009." I am sure you have nothing else on your plate, this should be a piece of cake!

There are several implementation expectations that will be much more effectively understood, utilized and effective with input from the laboratory. Either the director of coagulation, or a supervisor should sit on this committee. The directives include: (Bolded information are questions you might want to consider as part of the committee.)

(M) C 4. The organization uses approved protocols for the initiation and maintenance of anticoagulation therapy appropriate to the medication used, the condition being treated and to the potential for drug actions.

QUESTIONS TO CONSIDER:

ARE THE CLINICIANS PROVIDED WITH A CORRECTLY OBTAINED HEPARIN THERAPUTIC RANGE THAT REFLECTS THEINSTRUMENT/REAGENT COMBINATION USED IN THE LABORATORY? DO THEY UNDERSTAND THE VALIDITY OF THAT PROCESS OR ARE THEY STILL USING 1.5-2.5 TIMES THE MEAN OF THE NORMAL RANGE?
ARE DIRECT THROMBIN INHIBITORS (DTI’s)USED IN THE HOSPITAL?
DO YOU KNOW HOW YOUR REAGENTS PERFORM – RESPONSE IS REAGENT DEPENDENT?
DOES YOUR REAGENT FLATTEN OUT WHEN THE DOSE IS HIGHER, THEREFORE UNABLE TO DETECT A DIFFERENCE IN THE HIGHER RANGE?
DO YOU HAVE A WELL DEFINED CURRENT NORMAL RANGE, AND WAS THE GEOMETRIC MEAN USED FOR THE INR?

(M) A 5: For patients started on warfarin, a baseline INR is available and for all patients receiving warafin therapy a current INR is available and is used to monitor and adjust therapy.

QUESTIONS TO CONSIDER:

WHERE DID THAT BASELINE INR COME FROM, WAS IT FROM A CLINIC, DOES THAT CORRELATE WITH YOUR INSTRUMENT?
WHEN YOU ARE TRANSISITIONING A PATIENT FROM A DTI TO WARFARIN, THE INR MAY NOT BE ACCURATE, A CHROMGENIC X ASSAY IS BETTER.

(M) C 8: The organization has a policy that addresses baseline and ongoing laboratory tests that are required for heparin and low molecular weight heparin therapies.

QUESTIONS TO CONSIDER:
CAN YOU PROVIDE ANTI-Xa ASSAYS ON PATIENTS THAT REQUIRE MONITORING ?

LUPUS
PREGNANCY
EXTREME THIN OR OBESE
PEDIATRIC PATIENTS
ORTHOPEDIC
SEVERE TRAUMA

CAN YOU TEST FOR LUPUS?
HIGH FACTOR VIII’S WHICH CAN SHORTEN YOUR APTT, MAKING MONITORING DIFFICULT?
CAN YOU PROVIDE ANY TESTING FOR DTI’s? HOW DO YOU HANDLE THEM?

(M) C 9: The organization provides education regarding anticoagulant therapy to staff, patients and family.

QUESTIONS TO CONSIDER:

HAS ANYONE EVEN SEEN THE LABORATORY?
DO THEY UNDERSTAND THE ROLE THAT WE PLAY IN THIS PROCESS (AND THAT WE WERE ELIMINATED FROM INPUT BY JCAHO!)
DOES YOUR STAFF UNDERSTAND ABOUT THE INR?
AN IN REFERENCE TO PRE-ANALYTICAL VARIABLES- REMEMBER THAT AN APTT IS ONLY GOOD FOR 4 HOURS, AND IF A PATIENT IS ON HEPARIN, IT SHOULD BE SPUN WITHIN 2 HOURS. IF THE PLASMA SITS ON THE RED CELLS, THE PF4 FROM THE PLATELETS WILL NEUTRALIZE THE HEPARIN, AND FALSELY DECREASE RESULTS.
DO STAFF UNDERSTAND WHEN A SAMPLE SHOULD BE DRAWN AFTER A DOSE IS GIVEN?
THAT A THROMBIN TIME CAN BE DONE IF A PHYSICIAN IS INSISTING THAT A HEPARIN LEVEL SHOULD BE ATTAINABLE, IF THE THROMBIN TIME IS NORMAL THERE IS NO HEPARIN IN THAT SAMPLE, IT IS THE BEST TEST FOR RESIDUAL HEPARIN?

Let's hope that institutions realize how complicated this process is, but if communication between departments can be optimized, and the laboratory and the knowledge of the technologists be utilized. Patients will be assured to have safe and accurate results.

What's in your lab?

2008-05-02T12:56:00.001-07:00

So you have this coagulation laboratory that provides testing for a community, what is in your laboratory? Surely there are instruments, samples, reagents, consumables and last but not least technologists. How do you use all of these to provide optimum health care for your patients?

Coagulation testing give clinicians insight into how patients may respond during surgery, how their anticoagulation is performing or if they have some type of coagulapathy. A large problem in coagulation testing is pre-analytical variables, over 64% of errors are caused by this! In your lab, you need to have standard guidelines to minimize errors:

Accepting samples that have a 90% fill rate, keeping that 9 to 1 ratio of blood to anticoagulant.
Making sure that you have platelet poor plasma, platelets are phospholipids and they will falsely shorten samples especially if they are freeze/thawed and used for lupus testing.
Making sure that you don't add APTT's onto samples that have been sitting on the cells longer than four hours. The PF4 will be released and neutralize any heparin in the sample.

So we have some basic pre-analytical criteria in place. Next thing is to make sure that you have good standard operating procedures. Everyone should be on the same page, there is a lot to be said in just observing how techs perform. Also before procedures are put into place, they should be edited by the people who are going to use them, what may be obvious to them, may not be to the person who is writing the procedure. Also, make sure that when a procedure is updated, you have a form that demonstrates that all personnel are aware of the changes. This must be dated and signed.

Next, review the test menu. Instrumentation has greatly improved, and most have the capability to run not only clot based testing, but chromogenic and immunological testing. What should laboratories be running? The PT and APTT are very important. These screening tests give the clinicians a tremendous amount of information, from the presence of a factor deficiency, to an inhibitor, or to monitor anticoagulation. Understanding how your reagents perform is very important. Is your reagent lupus insensitive? That means it has a high concentration of phospholipids and will mask the presence of an antibody. Therefore, the presence of a lupus inhibitor will not prolong your APTT. Also, is your reagent heparin sensitive, will just a small amount of heparin greatly prolong your APTT? This will be important in determining your therapeutic range. In regard to your PT reagent, do you want a high or low ISI. CAP recommends that you use a reagent with an ISI <1.5. This is all important in diagnosing and treating patients. And that is just your basic screening tests. Most labs also perform fibrinogen testing; this test will mostly be decreased that is consumed in DIC. Fibrinogen deficiencies are rare; however, fibrinogen is an acute phase reactant, meaning it is elevated in times of inflammation and stress. Also, pregnant women will have an elevated level. This is very important to know, because you should expect fibrinogens from labor and delivery to be increased, a normal level might just be an indication that women is going into DIC. Some people have a hereditary persistence of elevated fibrinogen. It can be confirmed by running a CRP, the most sensitive marker of inflammation. If this is normal, the elevated fibrinogen is not due to an inflammatory condition. Elevated levels of fibrinogen are considered independent risk factors for myocardial infarcts, more so than cholesterol.

A thrombin time should be among your screening tests. This test is the best test for residual heparin, and can detect up to 0.1U/ml! It can be helpful in determining if a sample is contaminated with heparin and be very cost effective. If a sample contains heparin, additional workups should not be performed, Factor assays will look like inhibitors, and many other assays will be affected. Also, when samples are drawn for Anti-Xa assays or Heparin levels, you sometimes will see a result of 0 U/ml, and worry if your analyzer is working correctly, you can run a thrombin time to see if any heparin is present. The sample may have been drawn at the wrong time, reflecting an undetectable level.

Another important test to have on board is the d dimer. Most instruments can provide this test 24/7. The validity of the test lies in the negative predictive value, the ability to rule out the presence of a clot. A positive d dimer means many things. Be careful when using the latex agglutination test for d dimer, it is really more specific in determining DIC then the presence of a clot. Get them on the analyzer and out the door! As far as FDP's, they are polyclonal antibodies and very non-specific, the only advantage is to distinguish between fibrinolysis and fibrinogenlysis- you can do a fibrinogen test for that, it really isn't cost effective to keep both on board.

What about more specialized testing? Many laboratories feel it is too expensive, but in reality it may be more expensive to send it out, or not perform it and a patient have a delay in diagnosis. A mixing study can help to determine an inhibitor or a factor deficiency. Pooled normal plasma must be used in the mix, not a control or a lyophilized plasma. It is important that the matrix is the same and that all of the levels of the factors are in the normal range. Make sure that you have these values; it will be something an inspector wants to see.

Heparin levels are important. Low Molecular Weight Heparin (LMWH) can not be monitored by an APTT. It's dose is determined by body weight, but should be monitored in pediatric patients, pregnant women, and severe trauma patients. This is an important test to have available.

Hypercoaguable testing is expensive and can be performed in stages. If the laboratory wants to offer one test, it should be the clot based test for Activated Protein C Resistance. This is the most common hypercoaguable disorder, and affects up to 3% of the general population. It is easy and can be put on any analyzer.

Providing these tests can give the clinician the tools required to make informed decisions and provide optimum patient care. So maybe it is time to not only see what is in your lab, but what can be in your lab!