Coagulation Corner

T'was the night before a thrombosis and all through the cascade,
All the factors were quiet, not even tissue factor was made.
The red cells were flowing through the vessel by shear stress
While white cells were circulating getting ready for a mess.
.
The platelets were nestled on the endothelium ready to go-
While factor VII was waiting, ready for activation you know
And the Weibel-Palade bodies with von WIllebrand molecule
Had just settled by Protein C, which inactivates VIII as a rule

When out from the blood vessel there arose such a jolt
The platelets sprang from their resting, even thrombin started to bolt!
Away came vW factor and collagen promoting platelets to clump,
Arachidonic acid, ADP and cyclooxygenases started to jump-

The platelets began to roll along and adhere as they should-
Gave their granules the stimulus to release because they could.
When what then happened but platelet aggregation would go
Forming the phopholipid basis for secondary hemostasis you know

With activation beginning so lively and quick,
The platelets had done their job, they all were able to stick
More rapid than platelets the factors they came-
Tissue factor activating cofactors and enzymes by name-

Now FVII to VIIa and F X to Xa, calcium and phospolipid too,
On FVa and FII becomes IIa and fibrinogen you knew-
To the extrinsic pathway and the feedback with thrombin-
Now clot away, clot away, the outcome is fibrin

As amplification continues to proceed-
When now secondary hemostasis considers what it needs.
So on to the intrinsic pathway and how this one reacts
With factor IX is also activated by VIIa, that the facts.

And then platelets and cofactors FV and FVIII gets going-
The large scale thrombin generation begins flowing-
Then on to propagation, thrombin generated on the surface of a platelet-
Leading to activation of XI to XIa where explosive thrombin formation is met.

But what about contact factors? They are XII, HMWK and Prekallikrein,
This may be involved in Initiation where XIIa can activiate IX.
The IXa forms a complex with FVIIIa
In which together with phospholipid and calcium it's the complex tenase.

The final phase of making fibrin from insoluable to a clot
Required FXIII to crosslink the whole lot-
However there is still thrombin active in vivo
Thrombogenic material might breakoff and go-

This clot now so formed and solid as such-
Had finished its clotting, we don't want it too much-
So now the fibrinolytic system begins to kick in
To get rid of the clot from the RE system.

Plasmin becomes plasminogen via t-pa
So the clot can can be dissolved and go away
So the system can finish and complete it's run-
All the platelets and factors have a job that's well done.

The system is quiet and has done its best
Now factors, cofactors, platelets will all need a rest.
But do not fear, because we all know-
They will be ready to react, and ready to go-

We all hope this happens only when it is needed
And not inappropriately, and it message is heeded
Keep healthy and active and help your system work right-
Happy holidays to all-and to all a good night!

The one system that gets minimal attention is the fibrinolytic system. This pac man system is responsible for the dissolution of clots. During the appropriate activation of the hemostatic system the outcome expected is the cessation of blood, and the formation of a clot. That clot then must be removed from the system. Spontanous dissolution of clots from living and dead people was noted as early as the time of Hippocratis. It was also noted that once it had liquefied, it could no longer clot again. In 1905 Morawitz concluded that fibrinolysis was probably enzymatic. It wasn't until about 1945 that the terms plasminogen and plasma were utilized, But it wasn't until 1959 that it was discovered in the presence of a clot, the plasminogen is absorbed to the clot so that when the plasminogen converts to plasmin, the plasmin is already in place in the clot.

The fibrinolytic system is responsible for the dissolution of a clot. Fibrin clots are not intended to be permanent, their purpose is to stop the flow of blood until the damaged vessel can be repaired. The presence or absence of hemorrhage or thrombosis depends on a balance between the procoagulant and the fibrinolytic system. The key components of the system are plasminogen, plasminogen activators, plasmin, fibrin, fibrin/FDP and their inhibitors. Fibrinolysis is the process by which the hydrolytic enzyme plasmin digests fibrin and fibrinogen resulting in progressively reduced clots. This system is activated in response to the initiation of the activation of the contact factors. This produces a proteolytic enzyme plasmin. Plasmin is capable of digesting either fibrin or fibrinogen as well as other factors in the cascade (V,VIII,IX and XI). Normal plasma contains the inactive form of plasmin in a precursor called plasminogen. This precursor remains dormant until it is activated by protelolytic enzymes , the kinases, or plasminogen activators. Fibrinolysis is controlled by the plasminogen activator system. Plasmin digests fibrin and fibrinogen to produce smaller fragments. This occurs the same time healing occurs and the cells of the mononuclear phagocytic system phagocytize the products.

An activator tissue-plasminogen activator t-PA results in the activation of plasminogen to plasmin resulting in degrading fibrin. The fibrinolytic system includes several inhibitors. Alpha-2-antiplasmin is a rapid inhibitor of plasmin activity and alpha-2-macroglobulin is an effective slow inhibitor of plasmin activity. This system is in turn controlled by inhibitors to t-PA and plasmin-plasminogen activator inhibitors (PAI-1) and alpha 2-antiplasmin.


Laboratory Testing:

The principle of the Euglobulin Clot Lysis time is used to evaluate increased fibrinolytic activity and is isolated from plasma by precipitation with 1% acetic acid. This euglobulin fraction is relatively free of fibrinolytic inhibitors. The precipitate is redissolved and calcium is added to form a fibrin clot. The resulting clot serves as a substrate for plasmin which is generated from plasminogen by the plasminogen activators. The clot is incubated at 37 degrees C, and examined at 30 minute intervals for evidence of lysis. The euglobulin lysis of the clot is the time required for complete degradation of the clot. Conditions associated with increased fibrinolytic activity are DIC, liver disease, surgery, certain malignancies and women receiving oral contraceptives or during menstruation. A Euglobulin Clot Lysis is decreased in pregnancy due to an increase in fibrinogen, Plasminogen Activator Inhibitor and plasminogen.

Prolongation can be due to a decrease in Plasminogen activator (PA) and an increase in PA- PAI complex, or even a plasminogen defect. Lysis times of less than 30 minutes indicate a hyperfibrinolytic state.

If fibrinogen is greater than 600mg/dl, the fibrin clot formed provides excessive substrate for the formed plasmin and time for full lysis would be increased. However, if fibrinogen values are decreased, the results may be difficult to interpret, since normal fibrinolytic activity may dissolve a small clot somewhat more rapidly than normal.

Clot Lysis time is shortened in Factor XIII deficiency since the fibrin clot formed is poorly crosslinked and dissolution more rapid. Platelets prolong the lysis time due to their antiplasmin activities, therefore, platelet poor plasma must be used.
The lower the pH of the acid-plasma mix, the more prolonged the lysis time is.
The test may be performed on patients receiving heparin since it is removed during the precipitation process. One of the problems with this test is the lack of standardization. The buffers are made from scratch, and determination of the status of the clot is subjective. All persons performing the test should demonstrate concordance in determination of the clot. A normal and abnormal control must also be run when performing the test. Also, since the test is manual, it must be performed in duplicate.


Alpha-2-antiplasmin assay provides a quantitative measure of activity in human plasma by a chromogenic assay. This assay uses excess plasmin that reacts with alpha-2-antiplasmin in the patient sample to from an inactive complex. Residual plasmin activity is then determined by hydrolysis of a plasmin-specific chromogenic substrate which releases p-nitroaniline when cleaved. The absorbance is inversely proportional to the alpha-2-antiplasmin.


Plasminogen is the proenzyme of plasmin, a proteolytic enzyme which lyses fibrinogen/fibrin as part of the fibrinolytic system. Under the influence of tissue or plasma activators, plasminogen is converted into plasmin. The main role of plasmin is to degrade fibrin and secondarily fibrinogen,leading to the production of degradation products of stabilized or non-stabilized fibrin and fibrinogen, respectively.

The test uses a chromogenic substrate to measure biologically active plasmin which can differ from the concentration of immunoreactive plasminogen depending upon the patient population. Measuring the activity as opposed to the antigen, can be an aid in evaluating some fibrinolytic disorders and responses to therapy.
In this assay, plasminogen in the sample forms a complex with the streptokinase . The active plasminogen concentration is determined by measuring proteolytic activity against a synthetic substrate, resulting in an increase in absorbance at 405nm.
This assay is insensitive to plasma inhibitors and fibrin or fibrinogen degradation products. During thrombolytic treatments it is of interest to measure the plasminogen level to monitor the hepatic regeneration level and to control and adjust the perfusion rate if plasminogen is being given to the patient. The plasminogen level in newborn babies is usually low, particularly if they are premature. Falsely low plasminogen activity may be obtained in patients undergoing treatment with Aprotinin.


Congenital Disorders:

Plasminogen Activator Inhibitor-1:
Congenital plasminogen activator inhibitor-1 (PAI-1) deficiency is an extremely rare disorder characterized by a bleeding diathesis that begins in childhood due to hyperfibrinolysis as a result of decreased PAI-1 activity. Deficiencies of PAI-1 inhibitor leads to excessive tissue plasminogen activator activity resulting in excessive plasmin activity. Patients have a history of recurrent episodes of subcutaneous bleeding beginning in early childhood. These episodes are characterized by abnormal prolonged bleeding after trauma, tooth extraction, and surgical procedures, as well as by rebleeding following initial hemostasis. The euglobulin lysis times can be shortened as compared with those in normal control subjects. The Euglobulin Clot Lysis time is used to evaluate increased fibrinolytic activity. The euglobulin fraction of plasma consists of fibrinogen, plasminogen and the activators of plasminogen. This factor is isolated from plasma by precipitation with 1% acetic acid. This euglobulin fraction is relatively free of fibrinolytic inhibitors.
Alpha-2 antiplasmin:
Alpha-2-antiplasmin is also known as alpha-2-plasmin inhibitor or antiplasmin. This is a single-chain glycoprotein with a molecular weight of 70,000 which is produced in the liver. When alpha-2-antiplasmin is absent or present at low concentrations, excessive bleeding may occur. The fibrinolytically active enzyme plasmin with which it extremely rapidly forms an irreversible, inactive complex. Diminished activities of alpha-2 are found in hyperfibrinloysis, which can occur as a complication of disseminated intravascular coagulation (DIC) or in operations on organs with a high content of plasminogen activators. A deficiency may be due to a disturbance in synthesis (for example severe liver cell damage) as well as the additional assessment of problematic cases in fibrinolytic therapy. Homozygotes will have <10% of activity and present with mucosal membrane bleeding particularly in the GI tract, subcutaneous hematomas, spontaneous bruising and severe bleeding in trauma. Heterozygotes are usually asymptomatic but may have mild bleeding tendencies. Inhibiting plasmin results in excess fibrinolysis and causes lysis of fibrin thrombi at sites of vascular injury.


The fibrinolytic system is an important part of hemostasis. This complex process can not be overlooked in it's role regarding the balance between bleeding and clotting.

Do you know how frequently patients are in a therapeutic range on warfarin?
How does your reagent affect their results?
How does this drug work?
Are there alternatives for oral anticoagulants?

In 1936 cattle grazing in a field developed a bleeding disorder from eating spoiled sweet clover hay. The hay contained a chemical agent (dicumarol). Warfarin was discovered by the Wisconsin Alumni Research Foundation or WARF and was found to inhibit vitamin K synthesis.
We know that we measure oral anticoagulation in the laboratory with the Prothrombin time (PT). This renders the Vitamin K dependent factors (II, VII, IX, and X, Protein C, S ) non-functional, so it works to prevent clotting. Warfarin works by enabling the liver to
inhibit the y-carboxylation step of clotting and the Vitamin K dependent factors, rendering them non-functional. This will, as a result, impair fibrin formation. Sounds like a great system!

However, there are issues- first and foremost this is an oral anticoagulant (OAC) drug, therefore you might deal with non-compliance- when the patient takes the drug, the amount that they take, and when they decide to adjust the dose on their own. People are placed on warfarin for extensive periods of time, even lifetime. Warfarin has a half life of between 20-60 hours, this varies in individuals. Dosing must be individualized and the patient must be considered for hepatic and cardiac function, age, nutrition, concurrent therapy and the clinical situation. Patients are started on 2-10mg daily for 2 days, doses are adjusted according to INR results with maintenance doses from 2-10mg daily.
There are several inherent variables with warfarin use including the addition or discontinuation of other medication or changes in diet. The response to oral anticoagulants may be markedly enhanced in obstructive jaundice, hepatitis and cirrhosis due to reduced vitamin K absorption. Foods high in vitamin K such as beef and pork liver, green tea and leafy green vegetables will decrease the efficacy of warfarin. Many drugs can decrease the risk of anticoagulation (anti-thyroid drugs, barbiturates, estrogens, aluminum hydroxide), while others can increase the risk of hemorrhage (quinidine, indomethacin, adrenal corticosteroids). There are also over 80 drugs that interfere with OAC.
We also then have our reagents which are phospholipid based with inherent variabilities. In the 1940 and 50 the initial thrombolplastin was made with human brain, yes that is human brain where tissue factor was extracted and formulated into reagents. In the 1960's commercial reagents came into play using rabbit brain etc. The outcome was a reagent that was not as responsive to oral anticoagulation.

The combined rate of a major hemorrhage or recurrent thromboembolism is 15 percent per patient year of therapy. This presents with a risk of being either excessive or sub therapeutic They wanted to come up with a mechanism for standardizing the monitoring of the drug, so people could be monitored anywhere, and end up with the same result regardless of the instrument reagent combination. This mechanism was called the International Normalized Ratio (INR), that is, sort of a correction factor to harmonize PT results, so that regardless of the result in seconds, the INR should be the same. The way the correction factor is derived is compare a manufactures reagent against the "Manchester reagent", or one made with human brain thromboplastin, which is very sensitive, with an International Sensitivity Index of 1!
In 1995, it was published that "Oral anticoagulant therapy should only be monitored with thromboplastin reagents with an ISI of 1.2 or lower" In addition , the imprecision of the PT that occurs with a higher ISI will be magnified. The lower the ISI, the less the variability resulting in a lower CV of about 6-8%, while a higher ISI will result in inter-laboratory variations of 15-26%. The lower the ISI the better able you are to assess bleeding potentials, and is more sensitive to factor deficiencies as well as liver disease and vitamin K factors. Since the PT will have a longer range, these entities will demonstrate a wider range of clotting times, which will allow for finer adjustments in dosages of warfarin.

The PT's are performed by a manual tilt tube method ( how many people remember how to do that!!! Raise there hand- mine is up) versus the PT on the manufacturer's analyzer with their reagent, hence they come up with a "corrected" sensitivity the ISI of the manufacture's reagent. Sounds pretty good, don't get too excited yet!


ISI
The formula is: INR = Patient PT
Geometric mean of PT
Normal range
.
First, you must make sure you collect a good normal range, normal controls should be used, no ED or pre-op patients, too many acute phase reactants. Remember, coagulation is inversely proportional- when factors are increased, clotting times will be shortened. Second, make sure you use the geometric mean. Your greatest variable is the ISI, if there is a miscalculation of the ISI, it is exponential, and will cause the greatest error. That in itself is a different article- The question I am asking is: How frequently are patients in a therapeutic range while on OAC-
The answer about 50-80%- average about 64% of the time, not so hot-

This brings me to the focus of this - which would be pharmacogenetics of warafin.
This area is becoming more and more prevalent in medicine to determine how people metabolize drugs.
There have been several components that affect warfarin dosing variability identifying clinical, demographic and genetic components. Three common single nucleotide polymorphisms (SNPs) account for between 40-55% of dosing variability. Two SNPs in the cytochrome P-450 (CYP2CP gene are associated with impaired metabolism. A glutamine to arginine SNP in the promoter region VKORC1 results in decreased messenger RNA transcription and increased sensitivity to warfarin. Other factors influencing dose include body mass index, age, interacting drugs, and indication for therapy. In August of 2007 the FDA changed the labeling of warfarin to include a description of the use of lower doses for patients with certain CYP2C9 and VCORC 1 SNPs.
Allelic frequencies for these variants differ considerably among different ethnic groups: Caucasians carry the 2C9*2 and 2C9*3 variants (8% to 20% and 6% to 10%, respectively) more frequently than Asians do (0% and 2% to 5%) while polymorphisms of cytochrome P450 CYP2C9 do not seem to play an important role in sensitivity to dicoumarol in the Spanish population.
There is a growing interest in the variation of genetic populations and how they respond to OAC. It is believed there are several mutations that contribute to this occurrence. I will keep you posted!

What about alternatives to warfarin?
To date there has not been any other oral anticoagulant FDA approved.
Ximelagatran: (EXANTA) is an oral agent that acts as a competitive inhibitor of thrombin. It is converted to the active form of meletagran which inhibits clot-bound thrombin. It was being evaluated for use in acute venous thrombosis and prophylaxis, but there were problems with liver enzymes, so the drug was not approved. There is no interaction with drugs, food or alcohol, or Vitamin K, therefore it can be administered without coagulation monitoring. This drug has a half life of 3 hours, excreted by the kidney, is dosed twice/day. Pharmacokinetics differs due to differences in renal function.

Warfarin is one of the most commonly given drugs with many inherent problems. Defining pharmacogenetics may be a wonderful option to help patients minimize bleeding events and maximize dosing capabilities without forming a clot.

The MAYO conference in August was dedicated to the memory of Dr. Walter Bowie. He passed away in March of this year. He was a pioneer in coagulation and a very gentle soul. Dr. Bowie loved limericks- so I dedicate this as well as this month's column to him:
Dr. Bowie was dedicated to the science of clots
To hear him lecture, you would end up knowing lots
He had a passion for Lupus and Protein C's
Dr. Bowie taught with a cascade that was made up of fleas!

Maximize your time and minimize your problems in the coagulation laboratory:
Troubleshooting tips:

I have had both the honor and the privilege of lecturing to clinical laboratory scientists and visiting their laboratories from New York, to Jackson Hole Wyoming, to Ponce in Puerto Rico, and even Vincenzia in Italy. Technologists all struggle with the daily routine of balancing work, dedication, conflicts with turn around time and producing quality results. The complexity of coagulation is difficult enough, it is compounded with trying to get qualified personnel, training them (which I might add takes 2 years), and retaining them. From all corners of the earth, laboratories are faced with this dilemma. So, what we need are options, we need to maximize time and minimize problems!

You know those days - someone calls in sick, the phone hasn't stopped ringing, the floor needs a factor VIII NOW, your QC is due and it isn't even 8am. Well, you can so forget about lunch!
So what do you do to keep problems to a minimum? These are the days you want to cut corners, but in fact you need to slow down, take a deep breath, and proceed with caution. Here are some tips that can be used on any day to maximize your time and minimize your problems.

1. Make sure all your reagents are fresh. That one day old bottle of APTT reagent, don't take a chance, get a new bottle.
2. Make sure your maintenance is done on your analyzer. Check all you consumables, your reagents, and your water - make sure they are filled. There is nothing worse than losing precious time because you need to load some type of consumable, worst yet- reinitialize!
3. Get those controls cooking - let them sit for the correct amount of time- or they might just compound your problems. Make sure you use GOOD, FRESH, Type I or sterile water; you can halt many problems by this one little good laboratory practice.
4. Organize your work, ELISA testing can wait, things like factor assays, heparin levels, HIT assays need to be performed. For the staff that has braved the day, put your strongest person in their strongest testing place - and really schedule those breaks, you are going to need them, walking away for a cup of coffee sometimes makes the difference between finding a problem and going for a hammer!
5. You can always do the "Lets Make a Deal" - if a physician wants a full hypercoagulable workup, try to work through a rational plan with them, maybe the Protein C is more important then the Protein S, or even worse the patient is on heparin! The results of these assays are going to be affected by anticoagulation, besides the expense to the patient, and the time it will take, the answer really isn't reflective of what may be the root cause.
6. What if everyone wants factor VIII's right away? Can you give them 1 point with the rest to follow, as a preliminary, knowing if they suspect an inhibitor, it might be higher, and it is not likely to be lower if you run it at a 1:10 dilution. They might need to if it is just greater than 50%. Only use this when the clinician is fully aware of the implications of getting 1 point, and the rest will follow shortly.
7. Okay, so even before you get that far, your controls for the factor assay are out- what do you do? Lots of people just go to running a new curve, it may actually take less time in the long run - Lets take a minute to look and see what is "out"-
a. Did you run both PT and APTT based assays? Was just one- say factor IX out, if just one level is out, I would rerun that control.
b. If both levels are out on the Factor IX only, I would cut to the chase and rerun the curve.
c. If all APTT based assays are out, I would discard APTT reagents, and start again. (Once I was helping and reconstituted a reagent with 5mls instead of 10 mls, not much of a help!}
d. If all the controls are out, I would get new controls. (maybe new reagents, even new buffers!)
e. If you can't get anything to work, power down, restart, say a prayer, and maybe it will reset.
f. If you run a curve and controls are still out-, look at the points on the new curve, how do they compare to the previous curve. Sometimes only 1 point needs to be re- run, this will save a lot of time.
g. Then there are the times where the points look exactly alike- and the controls still won't work. You can try different types of controls, run them as patients so they don't mess up your QC files, MAKE SURE YOU HAVE DOCUMENTATION, or you can use proficiency testing material in the control ranges. This will also help you to troubleshoot, and if you need to call service, it will be good information to give to the company. Left over proficiency samples are gold, they are peer evaluated material, and you know how they ran in your laboratory, so they can really provide good information. Don't have any PT material, what about previous patients that are in the range needed? Again these were performed on your instrument/reagent combination and can give great insight as to what is going on.




Sometimes in the laboratory investing a little time, can help to decrease time in the long run- and with inspections now a surprise, everything always needs to be perfect, not that is wasn't before... some of my best tips are:
1. Color code shorted dated reagents by expiration date with a big DOT and a corresponding log: for example
Red dot for reagents that expire in October
Blue dot for reagent that expire in November
When you have to check inventory or discard expired reagents- just a quick
visual will let you know what has to be removed
2. Make sure everyone is aware of changes, post changes, and document it, because if it isn't documented, it wasn't done.
3. Use your proficiency tests to help establish competency, great way to do it, and to accomplish 2 tasks at once.
4. Try to minimize the controls that you use, they should be multipurpose and stable, make up more than 1 vial if you need it for more than one analyzer, sometimes spending a little more on utilization of a control, will save more time than waiting for a control to be run on one analyzer, then another and holding up test results.
5. Listen to the techs, allow all techs to have input in the running of the laboratory, if they can make suggestions as to a worksheet that will make it easier, or a computer function that will improve how they work and minimize errors, or they can improve a procedure, look at it, and listen to them. Allowing people to "own" processes make them more willing to adhere to standard operating procedures.


So, you have survived yet another stressful day in the coagulation laboratory. No patient will even know what you did, or how dedicated you are to their care. Yes, we will still continue to be portrayed on TV as the ones with the lost samples, or the ones having coffee, someone has to be blamed! However, you will know what you have done, other techs know what you have done, and occasionally even a physician will know what you have done. And guess what, you will come back tomorrow, and do it again- you love it, you know you do!

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!

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:

1. Rerun the sample in question.
2. 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.
3. Look at running independent controls to see where they are running.
4. Determine if there was a change in reagents, plasma or a new standard curve was performed.
5. What about maintenance on the analyzer, was one due?
6. Water can always be a problem, try using sterile water.
7. 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.
8. 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.

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:

1. Ambulatory
2. Critical Access Hospital
3. Home Care
4. Hospital
5. Long Term care
6. 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.