Coagulation Corner

COAGULATION CORNER: MAY 2012

2012-05-02T22:41:07.822-04:00

Stents and Stent Thrombosis:

Coronary artery disease (CAD) is the narrowing of the coronary arteries which are the blood vessels that supply oxygen and nutrients to the heart muscle. This is caused by a buildup of fatty material within the walls of the arteries. This buildup causes the inside of the arteries to become rough and narrowed, limiting the supply of oxygen-rich blood to the heart muscle. This causes ischemia which decreases the heart pumping ability. If a coronary artery is completely closed by a blood clot, a myocardial infarction may occur. The blood clot may occur when a plaque (build-up of fatty tissue inside the artery walls ruptures. If the blood flow cannot be restored quickly to the particular area of the heart muscle affected, the tissue dies. Percutaneous transluminal coronary angioplasty (PTCA) is performed to restore coronary artery blood flow when the narrowed artery can be accessed.

During PTCA A special catheter is inserted into the coronary artery to be treated. This catheter has a tiny balloon at its tip. The balloon is inflated once the catheter has been placed into the narrowed area of the coronary artery. The inflation of the balloon compresses the fatty tissue in the artery and makes a larger opening inside the artery for improved blood flow. Plaque can be broken up or cut away to open the artery by atherectomy or it can be vaporized by laser.

One common procedure used in PTCA is stent placement. A stent is a tiny, expandable metal coil that is inserted into the newly-opened area of the artery to help keep the artery from narrowing or closing again .Once the stent has been placed, tissue will begin to form over it within a few days after the procedure. The stent will be completely covered by tissue within a month or so.

TYPES OF STENTS:

Stents are metal mesh tubes inserted during angioplasty to prevent restenosis. Without the use of stents, about 30 percent of arteries become blocked again.

There are two basic kinds of stents: bare-metal stents (BMS) and drug-eluting stents (DES). Bare-metal stents act as simple scaffolding to prop open blood vessels after they're widened with angioplasty. As the artery heals, tissue grows around the stent holding it in place. However, sometimes an overgrowth of this scar tissue in the arterial lining increases the risk that the artery will become blocked again. Drug-eluting stents are coated with medication that is slowly released to help prevent the growth of scar tissue in the artery lining. This helps the artery remain smooth and open, ensuring good blood flow through it. Drug-eluting stents were developed because in some people who get bare-metal stents, tissue growth over the stent eventually leads to re-blockage .Many people with heart problems have been successfully treated with drug-eluting stents, preventing the need for more-invasive procedures, such as coronary artery bypass surgery. The reduced risk of re-narrowed arteries from drug-eluting stents reduces the need for repeat procedures, hospitalization and angioplasty procedures.

These new devices releasing anti-proliferative and immunomodulatory medications have been shown to improve major adverse cardiac events and vessel failure when compared with BMS. A recent meta-analysis (n= 14,500) also showed some improvements in lesion and vessel revascularization for stents releasing sirolimus, paclitaxel, dexamethasone and zotarolimus. In selective populations (such as patients with chronic total coronary occlusion),DES versus BMS have been demonstrated to cause less restenosisas and lesion revascularization rate (respectively 7% vs 13%; HR 0.49,95% CI 0.28–0.86; p = 0.01).

A second generation of DES using more biocompatible polymers has been investigated with promising beneficial results in a follow-up period of about 2–3 years. New materials of biodegradable or non-erodable polymers and non-polymeric compounds and designs have been shown to further influence cardiovascular outcomes after stent implantation in coronary atherosclerotic disease . Revolutionary advances have been recently achieved using nanotechnology in stent material and design. In particular, polyhedral oligomeric silsesquioxane (POSS) appears to be a promising material for coronary stents. Recent microfabrication technologies might further minimize stent dimension and improve biocompatibility.

Additionally, the use of statins in patients implanted with stents in other arteries is largely unexplored. Statin-eluting stents (only tested in pigs) have to be evaluated in other animal models and human beings. A clear recommendation on the use of statins to prevent stent thrombosis is not available and caution should be used. The “pleiotropic” anti-atherosclerotic properties of statins might represent a crucial investigation field to pathophysiologically clarify the role of statins in stent complications.

STENT THROMBOSIS

A standard definition of stent thrombosis criteria for diagnosis of coronary stent thrombosis classifies it as:

definite

confirmation

probable

possible

The time to thrombotic event after stent implantation as a pivotal parameter for the definition of the disease. Stent thrombosis after implantation is classified as:

• early (0–30 days),

• late (>30 days),

• very late (>12 months)

The definite diagnosis of stent thrombosis would require post-mortem examination, its real incidence is likely to be underestimated.

The pathophysiology and risk factors of thrombotic events have been widely described. The placement of a stent in a coronary artery is conducive in the formation of a thrombus. Platelets, white blood cells and red blood cells and coagulation factors are the major players contributing to the thrombus formation. Thrombotic disorders have been proposed as two potential elements underlying symptomatic atherosclerosis from venous thrombosis, arterial thrombosis has been described as rather associated with vessel wall injury and a hypercoagulatory state. Both mononuclear and polymorphonuclear white blood cells might be attracted early within the thrombus by fibrin and its degradation products. After this first phase, the local production of cytokines and chemokines might further favor the accumulation of inflammatory and vascular smooth muscle cells (VSMCs). However, the pathophysiological role of these soluble mediators has not been clarified.

TREATMENT

Platelet-rich thrombus formation in response to stenting is common therefore antiplatelet therapy is utilized. The superiority of dual antiplatelet therapy with aspirin and adenosine diphosphate (ADP)-receptor blockade over aspirin alone or aspirin plus warfarin has been conclusively demonstrated in numerous trials Furthermore, clopidogrel plus aspirin appears superior in safety to ticlopidine plus aspirin.

Data suggest drug-eluting stents—while outperforming bare metal stents—have a slightly higher risk of late stent thrombosis, with the most important risk factor being stopping dual antiplatelet therapy (DAT) (clopidogrel and aspirin) too soon. However, long durations of dual antiplatelet therapy can increase the risk of bleeding.

In the EXCELLENT study, 1,443 patients from 19 centers were randomly assigned to 6 or 12 months of DAT following implantation with a stent that released either everolimus or sirolimus. The hypothesis going into the study was that 6 months of therapy is noninferior to 12 months in terms of target vessel failure at 12 months. The 12-month data are available for 1,428 patients. This is the first presentation of findings that compare 6-month and 12-month DAT. It was demonstrated that six months of antiplatelet therapy is equivalent to the 12-month regimen that guidelines currently recommend for patients with drug-eluting stents, according to data from the EXCELLENT study presented at the American College of Cardiology's 60th Annual Scientific Session. These results provide the first evidence from a randomized controlled trial in support of a shorter duration of treatment.

COAGULATION CORNER: APRIL 2012

2012-04-02T14:17:07.614-04:00

WHAT IS NOT SO HOT!

I am always on the lookout for coagulation topics, and I was doing some research on the internet and I came across HOT TOPICS IN COAGULATION on WIlkepedia! This is an internet encyclopedia, however be careful, the information can be updated by anyone. But it can be a quick source of information for many people. It appears the top viewed topics on this site are: films, gay actors, and upcoming video games!

At a much less rate of hits, they did have a list of hot topics in coagulation from 2010- and the number one hot topic is: Platelets with 6,806 hits, followed by Coagulation (5,025), Hemostasis (1,386), Thrombin (1,006) and Fibrinogen (979)

Here are the rest of the top 20 topics:

6. Medical Laboratory Scientist (897) 13. Tissue factor (418)

7. Von Willebrand factor (879) 14 Factor V (353)

8. Fibrin (766) 15. Factor X (344)

9. Factor VIII (694) 16. Factor VII ( 277)

10. Protein C (653) 17. Protein S (237)

11. Lupus anticoagulant (583) 18. Factor IX (180)

12. Antithrombin (462) 19. Factor XII (177)

20. Thrombomodulin (148)

Rounding out the not so hot topics, the bottom ones included: Cofact (13), (I am not even sure what that means) and the total bottom is Fibrinogenolysis with less than 5 views. So our not so hot topic for this month will be fibrinogenolysis.

So as defined by Wilkepedia: (now with 6 views!)

Primary fibrinogenolysis is a medical condition that appears with abnormal production of fibrinogen/fibrin degradation products (FDP), degradation of coagulation factors V, VIII, IX, XI and/or degradation of the fibrin present in any pre-existing localized thrombi and hemostatic clots.

When bleeding occurs, coagulation results in production of a clot at the site of injury, and subsequent fibrinolysis dissolves the clot as the vessel wall heals. Fibrin degradation products are fragments (polypeptides) produced when either fibrin or fibrinogen is broken down by the enzyme plasmin. There are four principal fibrin degradation products called X, Y, D, and E that are released in various combinations. The fibrinolytic system is highly complex. A deficiency of plasminogen will result in increased risk of thrombosis. Plasmin is inactivated by several proteases, which are enzymes that catalyze the breakdown of polypeptides. A deficiency of one of these can result in spontaneous bleeding. FDPs themselves can neutralize the activity of some coagulation factors and interferere with normal clot formation. In three conditions, disseminated intravascular coagulation, thromboembolytic therapy, and primary fibrinogenolysis the fibrinolytic activity of the plasma is increased. When this occurs, depletion of coagulation factors, including fibrinogen, results in uncontrolled bleeding. Measurement of FDP and D-dimer are used to identify these causes of hemorrhage.

A test for FDP may be requested by a physician when excessive bleeding occurs and thrombosis or other serious disorder in the coagulation mechanism is suspected. The FDP assay measures amounts of the fibrin and fibrinogen split products in the blood and directly indicates the level of activity of the fibrinolytic system. High levels of FDP will indicate increased fibrinolysis. Excessive fibrin degradation products are released into the plasma in three main conditions: disseminated intravascular coagulation (DIC), thromboembolytic therapy, and primary fibrinogenolysis. Fragments X, Y, E, and D are released whenever either fibrin or fibrinogen is broken down by plasmin. This degradation occurs in all three situations resulting in a positive FDP. Therefore, the FDP test is not specific for thrombotic diseases such as DIC or deep vein thrombosis.

When a fibrin clot is broken down by plasmin, the last fragment to be degraded is one consisting of two D and one E subunits. This is split, releasing the E fragment and two D fragments that are covalently linked together. This fragment is called D-dimer, and it is produced from fibrin but not from fibrinogen degradation. Therefore, the D-dimer test will be positive only when fibrin degradation has occurred. Normal blood plasma does not have significant amounts of D-dimer. The D-dimer test is used to diagnose DIC. It is also frequently used to help diagnose deep-vein thrombosis (clots in veins); pulmonary embolism (clots in the lungs); the thrombosis of malignancy; and sickle cell anemia (a form of anemia characterized by bleeding episodes); and to monitor the effects of thrombolytic drugs. Thrombolytic drugs that may increase D-dimer levels are barbiturates, heparin, streptokinase, and urokinase. Levels of D-dimer will be elevated in these conditions.

Primary fibrinogenolysis is a condition in which fibrinogen is broken down to fibrin in the absence of an underlying thrombotic tendency. Unlike DIC, the formation of intravascular thrombi does not occur. However, if severe, hemorrhage can result because the body's supply of fibrinogen becomes depleted. Causes include shock, hypoxia, heat stroke, hemorrhage, surgery, and liver disease. Therefore in Primary Fibrinogenolysis, due to absence of a stable fibrin clot the D-Dimer level in the circulation would be within the Normal range.

I get asked all the time, should I keep running the FDP? It appears the only reason to perform an FDP would be to aid in diagnosing primary fibrinogenolysis. However, using clinical information and the differentiation between a hemorrhage and thrombotic event, the D-Dimer appears to be a better choice.

COAGULATION CORNER: MARCH 2012

2012-03-02T14:15:08.028-05:00

CHANGES IN COAGULATION

Ch-ch-ch-ch-Changes (Turn and face the stranger)
Ch-ch-Changes Oh, look out you rock 'n rollers
Ch-ch-ch-ch-Changes (Turn and face the stranger)
Ch-ch-Changes Pretty soon you're gonna get a little older
Time may change me But I can't trace time
I said that time may change me But I can't trace time
David Bowie 1971

Coagulation has had a rapid growth in the last 30 years, there have been many changes that have brought us to where we are today. Here are a few.

How many people have done a tilt tube and watched clot formation? I hope that almost everyone has said yes- I think it is important for all clotters to be able to evaluate the formation of a clot, with that said I am not suggesting that we revert back to manual PT and APTT’s! The days of reagent in one hand, stopwatch , water bath and a cup of coffee nearby are long gone! So while I take a walk down memory lane, I hope I bring a smile with some memories, and a good riddance to some others. But remember, if it wasn’t for these methods, there wouldn’t be testing that there is today.

Deficient Plasmas:

Want to run a factor assay? You can either use fresh frozen deficient plasma, or deficient plasma that has been immune-depleted that you reconstitute. But it wasn’t always that simple. Until about 1985, we used barium sulfate adsorbed normal plasma and aged normal serum. The barium sulfate absorbed plasma was deficient in factors VII, IX, X and prothrombin, and aged normal serum, deficient in factors V and VIII, prothrombin, and fibrinogen . You would treat a plasma that had a prolonged prothrombin time (PT) or activated partial thromboplastin time (PTT, APTT) with both reagents and observe patterns of correction to conclude which coagulation factor was deficient. This assumes the deficiency is congenital and there is only one factor that is deficient. Talk about a puzzle! Now it is much easier to just know, prolonged PT; look at II, V, VII or X, prolonged APTT, VIII, IX, XI and possibly XII. Think about that the next time you run a factor assay!

WHOLE BLOOD COAGULATION (CLOTTING) TIME (LEE-WHITE)

Now, I am not sure how many people remember a Lee White clotting time, but I could run three at a time, as long as I had that many stop watches!. The whole blood clotting time is a rough measure of all intrinsic clotting factors in the absence of tissue factors. Variations are wide and the test sensitivity is limited. Whole blood, when removed from the vascular system and exposed to a foreign surface, will form a solid clot. Within limits, the time required for the formation of the solid clot is a measure of the coagulation system. In fact during an episode of ER (remember that, George Clooney) , when they needed to measure if heparin was on board, and they had no power, guess what test they ran! Yep, Lee White! So, how did we do this? Three glass test tubes, stop watch and water/dry bath (at 37oC). Collect at least 2 ml of blood in a plastic syringe. Discard this blood.(This prevents tissue thromboplastin from entering the blood sample.) Change syringes. Collect at least 5 ml of blood in the second plastic syringe. Approximately 1 ml of blood is placed in each of the three glass test tubes. (#3 first, then #2, then #1) The stopwatch is started as soon as the blood enters the first tube #3. All tubes are placed into the 37°C water bath. Gently tilt tube #3 (45 angle) every 30 seconds, until the blood in it clots. Thirty seconds after tube #3 clots, proceed with tube #2, tilting every 30seconds, until a clot is formed. Thirty seconds after tube #2 is clotted, tube #1 is tilted until no flow of blood is observed on tilting. The coagulation time is the time required for the blood to clot in the last tube. This range should be between 5 to 10 minutes. The following variables tend to decrease the clotting time: rough handling of the blood specimen, presence of tissue fluids (traumatic venipuncture),frequent tilting of the tube, and unclean tubes. The following variables tend to increase the clotting time: extreme increases in temperature, variation in pH, and performance of the test at room temperature. This test is of value primarily as it was used to follow heparin therapy. Its use as a screening procedure is limited due to its poor sensitivity. The whole blood clotting time is affected mainly by defects in the intrinsic pathway factors and by defects in fibrin and fibrinogen. It is not sensitive to platelet abnormalities. A prolonged clotting time immediately indicates impaired coagulation, but a normal clotting time does not exclude many serious clotting defects. How would you like to do this versus a heparin assay?

Clot retraction:

Normally clot retraction begins as soon as the blood clots and should be almost complete in two hours. Normally there should be no lysis before 72 hours Clot lysis testing is a measure of the presence of the clot after 24 hours (blood fibrinolysis).Clot retraction depends primarily on the number and activity of the blood platelets. This test is reliable only when the concentration of fibrinogen and hematocrit are both within normal limits. Normally a blood clot will begin to retract from the walls of the tube and express serum within one hour.

A freshly drawn blood sample placed immediately into a glass and placed in a 37oC water bath. The clot is visually inspected at 2 and 24 hours after clotting for retraction, quality, and lysis of the clot. Clot retraction is decreased in thrombocytopenia (counts below 100,000 mm3) and thrombasthenia. In vivo, it is normal for a thrombus to retract if normal platelet activity is present, since a smaller thrombus is tougher and serves as a better clot. A clot may also retract poorly with excessive utilization of aspirin. This test is insensitive for blood clot lysis testing except in thrombolytic therapy because natural inhibitors of fibrinolysis will neutralize plasmin as it forms and so prevent the digestion of fibrin. When an abnormal concentration of immunoglobulin is present (multiple myeloma, cryoglobulinemia, or macroglobulinemias), there is interference with the conversion of fibrinogen to fibrin and the clot fails to retract.

Who knew clot retraction could tell so much!

Rumpel-Leede Capillary-Fragility Test

This was also known as a tourniquet test or a capillary fragility test. This was used to determine a patient's haemorrhagic tendency. It assesses fragility of capillary walls and is used to identify thrombocytopenia. Believe it or not, the test is defined by the WHO as one of the necessary requisites for diagnosis of Dengue fever.

A blood pressure cuff is applied and inflated to a point between the systolic and diastolic blood pressures for five minutes. The test is positive if there are 10 or more petechiae per square inch. In DHF the test usually gives a definite positive result with 20 petechiae or more. This test does not have high specificity. Interfering factors with this test are women who are premenstrual, postmenstrual and not taking hormones, or those with sun damaged skin, since all will have increased capillary fragility. A positive finding (more than 10 petechiae or a Score of 2+ to 4+) indicates weakness of the capillary walls (vascular purpura) or a platelet defect. It may occur in such conditions as thromboccytopenia, thrombasthenia, purpua senilis, scurvy, DIC, von Willebrand's disease, vitamin K deficiency, dysproteinemia, and polycythemia vera and in severe deficiencies of factor VII, fibrinogen, or prothrombin. Conditions unrelated to bleeding defects, such as scarlet fever, measles, influenza, chronic renal disease, hypertension, and diabetes with coexistent vascular disease, may also increase capillary fragility. An abnormal number of petechiae sometimes appear before menstruation and at other times in some healthy persons, especially in women over age 40.

BLEEDING TIME

The very first bleeding time that I performed was with a metal plate and a double razor that was placed on a shaved forearm, a blood pressure cuff was pumped to 40mm of pressure. The metal plate was placed on the forearm, the blades placed in the groves, and you pulled back, the patient screamed and so did I. Needless to say there was a scar and lots of blood.

As time progressed, I have had patients who told me they were up all night terrified of this test. The doctor had told them they were coming to the hospital to determine how long they would bleed after they were cut. Doesn’t sound pleasant does it? When they had the procedure, they laughed. I have had residents ask where to put the blood pressure cuff? They thought they were doing a Duke bleeding time (ear lobe), I imagine they thought the cuff should go on the neck? Scary?

So what about this test? I believe it has seen its time. Think about it, if this was a new test, we could never bring it into the laboratory. Unless of course laboratories perform transference of a reference range (that would mean normal ranges every lot?), precision studies (know anyone who would volunteer for that 10-20 repeat tests?), Method comparison, to what? And what about QC? Did I mention, this is a manual procedure, should it be performed in duplicate? Some clinicians say we don’t have anything better, but what do the results really tell us? So many things interfere, and there is so much variation, what are we using the results for?

Think about it!

Thromboplastin Generation Time:

This test was the reason that I hesitated to go into coagulation, only when I found out that is was no longer used, did I consent to become a clotter!

This test was used to evaluate coagulation defects such as hemophilia. It was based on the failure of the normal thromboplastin mechanism,and three components; normal plasma treated with AI(OH)3, platelets, and normal serum, react to form thromboplastin. Further analysis has shown that two essential substances, antihaemophilic globulin and factor V, occur in the Al(OH)3-treated plasma, while serum contains two factors, factor VII and the Christmas factor, both of which are required for thromboplastin formation. These five components-platelets, antihaemophilic globulin, the Christmas factor, and factors V and VII-react together in the presence of CaCl2 to form a labile thromboplastin. A lack of any one of the five produces a coagulation defect associated with abnormal thromboplastin formation.

The test involved pipetting 0.1 ml. of substrate plasma into each of six small. In a further tube in the water-bath at 37' C. is placed 0.3 ml. of alumina plasma diluted 1 in 5, 0.3 ml. of platelet suspension, and 0.3 ml. of serum diluted 1 in 10. To this is added 0.3 ml. of M-40 CaCI2 and a stop-watch started. At intervals of one minute 0.1 ml. of the mixture is withdrawn into a graduated Pasteur pipette and, using the other hand, 0.1 ml. of M-40 CaC12 is withdrawn into a second pipette. The contents of the two pipettes are then discharged simultaneously into one of the tubes containing 0.1 ml. ofsubstrate. The clotting times of the substrate samples are recorded. It is usually not necessary to continue the test for more than six minutes. The clotting times of the substrate give a measure of thromboplastin concentration in the incubation mixture and may be expressed in terms of thromboplastin concentration using a thromboplastin-dilution curve. Thromboplastin Dilution Curve.-A potent preparation of blood thromboplastin, as made in the thromboplastin generation test described above, will usually cause clotting of normal plasma in eight to ten seconds. When made deterioration of the thromboplastin can be delayed by placing the tube in melting ice. The preparation can then be diluted 1 in 2, 1 in 4, 1 in 8, 1 in 16, and 1 in 32 with 0.85% saline. The dilutions are also kept at the temperature of melting ice and tested with normal plasma substrate as described above. A curve relating clotting time and thromboplastin concentration can then be drawn.

Now do you understand why I hated this test, and all of this was used to distinguish hemophilia versus Christmas Disease (hemophilia B), or FIX deficiency. Genuis! Imagine all of that work, to get that information?

Conclusions:

So clearly, the changes in coagulation have evolved over time. Innovation has provided tools to the coagulation laboratory that have not only improved our ability to test for coagulation disorders, but decreased turn around time and enhanced patient care. I am sure 20 years from now, someone will have an article about factor assays using plasma and how they provided minimal amount of information in comparison to whole blood assays. I plan on reading that one on a beach!

COAGULATION CORNER: FEBRUARY 2012

2012-02-02T09:20:29.629-05:00

Medical Errors in the Coagulation Laboratory

Medical errors are still one of the most frequent causes of patient death.
Doesn’t give you a warm and fuzzy feeling to be a patient does it? I admit, I am not the best patient, why, because I ask questions-
What is that?
Why are you giving it to me?
What are the side effects? What tests are you ordering, what are the results?
So, I used to say even on my worst day working in the laboratory, it was still better than being a patient!

An adverse event is defined as any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease temporally associated with the use of a medical treatment or procedure, regardless of whether it is considered related to the medical treatment or procedure. Most common in coagulation would be excessive bleeding due to the administration of anticoagulants.

The study, by researchers from the US Centers for Disease Control and Prevention (CDC), singles out 4 drugs and drug classes as being the most common causes of adverse events: warfarin, oral antiplatelet medications, insulins, and oral hypoglycemic agents. Alone or together, they account for 67% of emergency ADE hospitalizations of adults 65 years and older. Warfarin was implicated in 33%! In contrast, medications red-flagged as high risk or inappropriate by health authorities explained only 1.2% and 6.6%, respectively, of such hospital admissions.

Two-thirds of the emergency hospitalizations were due to four medications, either used alone or in combination:

33,171 emergency hospitalizations (33%) were due to warfarin,
14% of emergency hospitalizations were because of insulin.
13% percent were caused by antiplatelet drugs, such as aspirin and clopidogrel
11% were due to oral diabetes medications (oral hypoglycemic agents)

For clinicians, the take-home message of the study is clear: Improved management of antithrombotic and antidiabetes drugs can keep thousands of seniors out of the hospital.

So with all of the events that occur due to drugs, how scary is it that 2 coagulation drugs: Anti-platelet drugs and an anticoagulant –warfarin, are responsible for most events! And how does the laboratory help to prevent errors:

Errors in the Coagulation Laboratory:

THE ISI and the INR: Okay so we know that warfarin is one of the most dangerous drugs. It is imperative that we ensure in the laboratory that our PT reagents have the correct ISI to reflect the correct INR. Check your analyzers, to ensure the correct ISI provided by the manufacturer is enterd and part of the calculation. Also, using calibrated standardized plasmas can help to harmonize your ISI among analyzers. If your INR is incorrect, you can impact a patients dose of warfarin, causing them to either be over or under anticoagulated and putting them at risk.

Insensitive reagents: It is important to understand how your reagents work and that they correctly reflect a low level of factor activity. A PT and or APTT should be prolonged when levels of factors approach 30%. This is the level in which a patient may bleed. If a reagent is insensitive to a factor, the screening test may be normal and may fail to detect a patient who may bleed.

Adding an APTT on to a sample > 4 hours old: An APTT is stable for 4 hours (CLSI guidelines). An APTT on a patient that is receiving heparin should be performed within 2 hours. If an APTT is added onto a sample that is older than 4 hours old, PF4 will neutralize the heparin in the sample, falsely decreasing the APTT. This can cause a clinician to increase heparin in a patient, putting them at a risk for bleeding.

LMWH and the APTT: You cannot monitor levels of LMWH with the APTT, however, LMWH can prolong the APTT (at levels of about 0.6U/ml). This prolongation is a result of the LMWH and is not due to a factor deficiency. It is important for laboratories and clinicians to understand this may occur so as not to needlessly expose a patient to product.

New anticoagulants: The new direct thrombin inhibitors perform very well, and inhibit the formation of thrombin. However, when patients are on this, all clot based assays will be prolonged, and factor assays will demonstrate the presence of an inhibitor. Additionally, since there are no FDA approved tests for monitoring these drugs, there is no way to determine if a patient is properly anticoagulated.

Anti-platelet drugs: An issue with anti-platelet drugs is that many patients have genetic mutations that impact the drug metabolism. As a result, dosing patients is challenging. Many clinicians exceed the recommended dose to eliminate any issues with patients demonstrating resistance to these drugs. However, this puts a patient at a bleeding risk. Additionally, clinicians question if these drugs need to be monitored, since there really isn’t a gold standard method.

Recombinant FVIIa: Giving activated factor VII is a very effective way to stop bleeding. Conversely, it can also cause a patient to have a thrombotic event. It is important that laboratories are aware, that patients on this drug will present with a shortened PT, which may present itself as an undetectable clot in an optical system, when in actuality the clot formed prior to the lamp reading. This can result in reporting out a prolonged result, when in reality it is a very shortened PT.

So, as you can see there are many issues in the coagulation laboratory that can cause errors in patient results-

Let us not forget the issues that may also occur that we don’t even know about:

Pouring a purple top into a blue top.
Removing the clot from the blue top with a clotted sample.
Testing day old blood that was left at the nurses’ station.
Testing transfused blood.
Testing blood drawn from above an IV line.

Yikes, sometimes too much information isn’t best!

So, put your best foot forward when performing coagulation testing, and try to remember, you aren’t testing an ID number, it is someone’s mother, father, sister or brother. Also, techs have really good instincts; I would rather go the extra mile and investigate, then just give a number. Remember we promise to “do no harm”!

COAGULATION CORNER: JANUARY 2012

2012-01-01T00:00:00.000-05:00

Just wanted to wish all of my readers a very happy and healthy 2012- all the best to you and your family!

January 2012: BLEEDING SCORES IN THE ASSESSMENT OF BLEEDING RISKS

One of the most effective tools in the assessment of bleeding is symptoms and bleeding history, however this is still a subjective evaluation. In order to standardize this quantitative bleeding assessment tools (BAT) have been developed. The issue is there are so many of them, their results may actually be conflicting. Questionnaires on bleeding history have been proposed, and validated as diagnostic tools by comparing data obtained from patients with normal controls.

Von Willebrand Disease

The Vincenza Bleeding Score (BS) has shown good sensitivity and specificity for the diagnosis of type 1 VWD. This can be integrated in a full diagnostic algorithm that also accounts for laboratory and family data and can be correlated with several biological variables, including VWF and FVIII:C levels, platelet function analyzer (PFA-100) closure times, and platelet glycoprotein haplotypes. . Collection of the BS at the time of the diagnosis may be a useful addition in the evaluation of the bleeding patients. In a study 215 patients were evaluated: bleeding symptoms (n = 71), abnormal laboratory clotting test results (n = 105) or family investigation (n = 39). Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) were computed for a predefined bleeding score (BS) cut-off (BS of > 3). Receiver operating characteristic curves were used to establish a diagnostic prediction rule. Results: Assuming the prevalence of mild bleeding disorders (MBD) in the general population to be 1%, a normal BS (≤ 3) had a very high NPV (99.2%). The PPVs in patients referred for hemostatic or family evaluation were estimated to be 71.0% and 77.5% (assuming MDB prevalences of 20% and 50%, respectively, in these settings). Measurement of BS in addition to activated partial thromboplastin time significantly increased the diagnostic efficiency of the BAT instrument (NPV of 99.6%). BAT use improves the evaluation of patients with suspected MBD, and we propose its use in a clinical prediction guide based on BAT and activated partial thromboplastin time for the exclusion of patients with suspected MBD in a low-prevalence setting.

Antithrombotic Therapy:

Warfarin is effective for the prevention of thromboembolism in a variety of conditions, but hemorrhage is a major side effect. Physicians use their clinical judgment to estimate the risk of bleeding in an individual patient. Prediction rules can help physicians more accurately weigh the risks and benefits of warfarin therapy. The Outpatient Bleeding Risk Index (BRI) included patients initiating warfarin upon discharge from the hospital, regardless of their indication for therapy, and prospectively classified patients who were at high-, intermediate-, or low-risk for major bleeding. This index considered age > 65 years, prior stroke, prior GI bleed, and any of four comorbidities (recent myocardial infarction, anemia, diabetes, or renal insufficiency) in order to stratify patients into three risk groups. In the original validation cohort, the index predicted major bleeding better than physicians, who estimated the probability of major bleeding no better than that expected by chance. The cumulative incidence at 48 months was 3% in 80 low-risk patients, 12% in 166 intermediate-risk patients, and 53% in 18 high-risk patients.

The second model was developed by Kuijer using age > 60 years, gender, and malignancy to stratify patients into three risk groups based on a calculation score with the formula (1.6 × age) + (1.3 × sex) + (2.2 × malignancy). They evaluated this score in an initial cohort of 241 patients with venous thromboembolism and, subsequently, in an independent cohort of 780 patients and found that it was possible to identify a subgroup of patients who would be high risk for bleeding, although clear discrimination was lacking in low-risk groups.

Non-ST-segment elevation myocardial infarction (NSTEMI):

The CRUSADE Bleeding Score was developed help clinicians estimate a patient's baseline risk of in-hospital major bleeding during non-ST-segment elevation myocardial infarction (NSTEMI). This initiative was designed to increase the practice of evidence-based medicine for patients diagnosed with non-ST segment elevation acute coronary syndromes (NSTE ACS) (i.e., unstable angina or NSTE myocardial infarction). Data include demographic and clinical information, medical history, use of antiplatelet, anti-thrombin and anti-ischemic therapies and use of invasive procedures, laboratory results, in-hospital outcomes, physician and hospital characteristics, and discharge medications and interventions.

Using data from over 89,000 "real-world" patients enrolled in the CRUSADE Quality Improvement Initiative that presented with NSTEMI. The CRUSADE Bleeding Score was created by assigning a weighted integer to each predictor based on its coefficient in the regression model. A patient's CRUSADE Bleeding Score equals the sum of the weighted scores for the independent predictors (range 1-100 points).

This core considers baseline patient characteristics (female sex, history of diabetes, peripheral vascular disease), admission clinical variables (heart rate, systolic blood pressure, signs of CHF), and admission laboratory values (hematocrit, calculated creatinine clearance) to estimate the patient's likelihood of having an in-hospital major bleed event (www.crusadebleedingscore.org). While treatments increase the likelihood of bleeding, they were not included in the model as they are post-admission variables.

Atrial Fibrillation

A score for predicting bleeding risk in patients with atrial fibrillation (AF), has been developed called HAS-BLED. This is an easy assessment tool used to help doctors make informed decisions.

HAS-BLED stands for hypertension, abnormal renal/liver function, stroke, bleeding history or predisposition, labile INR, Elderly (>65), and drugs/alcholo concomitantly. Each component is 1 point with a maximum score of 9. Drugs include any medications that increase bleeding risks during anticoagulation (aspirin, NSAIDS, steriods).

The score can also be modified. For example, physicians can choose to stop aspirin to reduce bleeding risk or controlling hypertension.

Upper Gastrointestinal bleeding

The Glasgow-Blatchford bleeding score (GBS) is a screening tool to assess the likelihood that a patient with an acute upper gastrointestinal bleeding (UGIB) will need to have medical intervention such as a blood transfusion or endoscopic intervention. Advantages of the GBS include a lack of subjective variables such as the severity of systemic diseases and the lack of a need for esophagogastroduodenoscopy (EGD) to complete the score, a feature unique to the GBS.

In a study 16% of patients presenting with UGIB had a GBS score of "0", considered low. Among these patients there were no deaths or interventions needed and the patients were able to be effectively treated in an outpatient setting. In the validation group, scores of 6 or more were associated with a greater than 50% risk of needing an intervention.

Score is equal to "0" if the following are all present:

Hemoglobin level >12.9 g/dL (men) or >11.9 g/dL (women)
Systolic blood pressure >109 mm Hg
Pulse <100/minute
Blood urea nitrogen level <18.2 mg/dL
No melena or syncope
No past or present liver disease or heart failure

Conclusions:

Clearly BATs come in a variety of shapes and sizes. Their goal, as is any scoring tool, is to remove subjectivity and provide a standardized assessment of a patient risk of bleeding.

The ISTH has established a new SSC standing committee: the ISTH-BAT Standing Committee. They have provided a tool that provides a web based accessible platform to aid in the standardization and collection of bleeding histories. This database will capture bleeding symptoms throughout the world encouraging sharing of de-identified phenotypic and laboratory data. This will enhance statistical power and provide important genotype-phenotype-environment correlations. This tool can be accessed online at:

https://bh.rockefeller.edu/bat/

COAGULATION CORNER: DECEMBER 2011

2011-12-03T00:00:00.000-05:00

COAGULATION 2011: YEAR IN REVIEW

It is hard to believe another year has gone by, as I get older time passes much more quickly. The end of a year is time that we reflect what has happened, review what has been positive or negative and try to rejuvenate to begin the next year.

So what has 2011 brought us in the coagulation world? Where have the advances been, what have been the biggest issues, and what is the future? I think the hottest topic is anticoagulation. For so many years there have been limited options, and now the field has exploded. Have heparin, and warfarin out lived there time? Will they become the 8 track players of coagulation? (for those of you who don’t know what an 8 track player is- google it) I am not about to take that stand. Not until we have more information on the new anticoagulants and how to reverse them, and more importantly how to monitor them. If we have learned nothing else this year, it is that the oral direct thrombin inhibitors and oral Xa inhibitors are not a perfect science. They can place patients at a risk for bleeding, additionally, how is one to know if patients are complying with dosing? It is an oral anticoagulant- compliance is a big issue- and there is no way to check what a patient has on board.

What has happened to thrombin generation? Just two years ago, every other abstract looked at thrombin generation. Okay, so the assay is not ready for prime time, so is this the tape cassette of coagulation, was good for awhile, but we know that something better is out there? Do you know that an APTT only measures 2% of thrombin generated? Thrombotic events are on the rise, we need to not only be able to diagnose patients, but understand who is at risk and focus on prevention.

Ah, platelets, let us not forget out little friends- and maybe learn that they do have a nucleus of sort- and that they roll along, as they gather together! Another huge issue, does aspirin really help? Should dual anti-platelet therapy be used? What about genetic mutations? Increasing doses to overcome them works, but can put a patient at a risk for bleeding- wouldn’t it be easier to test patients for the mutations? Cost, yes it costs, but again, we have the knowledge available, shouldn’t we put it to good use and improve patient outcomes?

Next issue, pregnancy and thrombosis, big issue- why do we not screen women? Why in this day in age, when we have the technology do we still wait for women to have 3 miscarriages before we look for a cause for thrombosis? Again the cost, how much is the cost of treating patients after a miscarriage versus a thrombotic workup? We have so many synergistic issues that put women at risk- pregnancy itself is a thrombotic state, add thyroid disease, homocysteine, folic acid levels, and I haven’t even mentioned genetic mutations- MTHFR, Prothrombin mutation, FV Leiden, PAI-1, as well as sedentary life styles and obesity. Again, technology is there, shouldn’t we use it? On the flip side, excess bleeding during menstruation- your grandmother, your mother, your daughter. This is a pattern, it is not acceptable to say we are all bleeders- when it impacts your quality of life- don’t be a martyr- ask questions- ask to be tested, you might have Von Willebrands disease.

So, that is my soap box issues for the year 2011. I can’t close out the year without some type of holiday cheer- so here is my version of:

Walking in a Winter Wonderland- or
Working with the Waterfall Cascade-

Factors activate are you ready
In a vessel coagulation is steady
But wait there’s a cut-
Platelets do your stuff
Working with the Waterfall Cascade

Gone away is the bleeding
Thrombin is here that’s what your needing
To form a blood clot
As fibrin gets hot
Working with the Waterfall Cascade

But wait that clot is getting much larger
Heparin needs to be given right away
Should we give unfractionated heparin
Or will lovenox be the drug to save the day?

Later on, we’ll switch to warfarin
As we check the PT/INR range to be in
To ensure the dose we gave
Will not be too grave-
Working with the Waterfall Cascade

In the case we need to use another
Now we can give dabigatran
Twice a day to inhibit thrombin
Except that there is no monitoring on hand

When it works, ain’t it thrilling
Preventing clots and halting bleeding
We’ll test and monitor
In the coag lab
Working with the Waterfall Cascade!

Have a wonderful Holiday season, all the best and happiness in 2012-
Be proud of what you do, your patients need you!

COAGULATION CORNER: NOVEMBER 2011

2011-11-04T00:00:00.000-04:00

ORAL ANTICOAGULATION: WILL WARFARIN BE ELIMINATED?

Warfarin has been used for about 50 years in patients requiring long term anticoagulation. It has a long history and has become the gold standard for the prevention of ischemic stroke in patients with atrial fibrillation.

Atrial fibrillation affects roughly 2.5 million persons in North America and is the primary cause of cardioembolic stroke. AF is the most common sustained cardiac rhythm disorder.. In patients with AF, the heart’s irregular heartbeat makes them vulnerable to the formation of a blood clot in the atria, which can travel to the brain, potentially resulting in a stroke. People living with AF are at a five-fold increased risk for stroke compared with the general population. Worldwide, stroke is the second leading cause of death above the age of 60 years, and is the fifth leading cause in people aged 15 to 59 years, responsible for 5 million deaths each year. Stroke leaves an additional five million people permanently disabled annually.

Warfarin is an oral anticoagulant that acts through inhibition of vitamin K-dependent coagulation factors (ie, factors II, VII, IX, and X) and is consistently ranked among the most frequently prescribed drugs in the United States. Warfarin is far from perfect but it has well established efficacy, is reversible in that Vitamin K can be given or fresh frozen plasma if the drug needs to be neutralized. However, a disadvantage is that it requires frequent monitoring, and therapeutic levels (INR 2-3) are achieved only about 60% of the time. The drug also has many interactions with food and medicine, a long half-life and is affected by genetic polymorphisms, specifically in the genes coding for VKORC1 and CYP2CP enzymes. Bleeding is the major adverse reaction with warfarin. In general, patients who have been on long-term established therapy have lower rates of major bleeding. Rare but potentially serious side effects include anaphylaxis, "purple toe" syndrome, tissue necrosis, and limb gangrene. When warfarin is prescribed and used responsibly, however, the benefits have clearly been shown to outweigh risk. Net treatment benefit is greatest in patients with history of previous stroke, those older than 85 years, and others with high stroke risk. Warfarin is available in several generic formulations for less than $10 per month. Associated costs of therapy include laboratory monitoring with PT/INR, which costs approximately $30 per test, along with additional expenses related to office visits for medication management

Emerging anticoagulant therapies now include direct thrombin inhibitors and factor Xa inhibitors. Reported advantages of these therapies include reduced burden of frequent therapeutic laboratory monitoring, simplified dosing, and fewer clinically significant drug-drug interactions.

Oral Direct Thrombin Inhibitor: Dabigatran

Dabigatran etexilate is an oral prodrug that is rapidly converted by a serum esterase to dabigatran, a potent, direct, competitive inhibitor of thrombin. It has an absolute bioavailability of 6.5%, 80% of the given dose is excreted by the kidneys, its serum half-life is 12 to 17 hours, and it does not require regular monitoring. Dabigatran has been approved for stroke prevention in patients with non-valvular atrial fibrillation including patients with previous stroke or transient ischemic attack.

Patients who should NOT receive the drug include those with a severe heart-valve disorder, stroke within 14 days or severe stroke within the last 6 months, a condition that increases the risk of hemorrhage, a creatinine clearance of less than 30 ml per minute, active liver disease, or pregnancy. Patients taking quinidine should also not take the medication because of a significant drug interaction.

The approval was based on the prospective, randomized RE-LY trial recently published in the New England Journal of Medicine that compared the safety and efficancy of two doses of dabigatran (110 mg and 150 mg twice daily) to conventional warfarin (Coumadin) therapy in 18,113 patients: The drug's most common side effect was dyspepsia (GI upset) but liver enzyme elevations were not any different than that seen with warfarin.

Price is another important factor to be considered when selecting which agent to use. Dabigatran has come in at a US "wholesale-acquisition" cost of $6.75 per day for AF. Cost-effectiveness over warfarin has been demonstrated, but the use of dabigatran will be affected by the existence of different payers for medication, INR checks, hospitalizations, and rehabilitation-center/nursing-home stays.

Oral Factor Xa Inhibitors: Rivaroxaban

Rivaroxabanis indicated for the prophylaxis of deep vein thrombosis (DVT), which may lead to pulmonary embolism (PE), in patients undergoing knee or hip replacement surgery. As well as for the prevention of stroke and systemic embolism in patients with non-valvular atrial fibrillation (AF). Dosing is once daily and there is no routine monitoring of INR or other coagulation parameters. In the event of bleeding, rapid reversal may not be required considering that the drug's therapeutic effect begins to diminish within 24 hours after the latest dose This drug should not be used in patients with severe renal impairment in particular in patients older than 65 years of age.

Rivaroxaban should not be taken with other anticoagulants due to increased bleeding risk other than during therapeutic transition period. NSAIDs/aspirin are known to increase bleeding; therefore bleeding risk may be increased when these drugs are used concurrently. Avoid concurrent administration of clopidogrel unless benefit outweighs the risk of increased bleeding.

ADVANTAGES and DISADVANTAGES:

Disadvantages of dabigatran

1. The administration of two doses daily, which becomes a compliance issue.
2. Forgetting more than one dose can put the patient at a prothrombotic risk.
3. Gastric intolerance leading to stopping the medication.
4. There is no antidote to neutralize the action of the dabigatran in cases of bleeding or when acute surgical intervention is necessary.
5. The anticoagulant effect must be controlled and there is no test to assess the effect or levels of therapeutic range.
6. Caution is advised in the case of impaired renal function. Dosing should be reduced in renal failure or the medication discontinued. Nevertheless there are no studies that clearly indicate the dose to use in these circumstances.
7. In phase III studies, patients have shown a small increase in hemorrhages; data related to this risk must be corroborated in phase IV.
8. The instability of the drug once the packaging is opened.
9. There are some drug interactions that must be known
10. As for other anticoagulants, age is an additional risk factor for hemorrhages.
11. How anticoagulant bridging can be done before surgery has not yet been established.
12. Cost.

Advantages of dabigatran, rivaroxaban

1. Fixed dose.
2. No laboratory control (strong arguable).
3. Few drug interactions.
4. No food interactions.

Drawbacks of warfarin

1. Warfarin requires frequent monitoring to maintain the INR between 2.0 and 3.0 and this is achieved, at best, in only 55-60% of patients and has a narrow therapeutic window..
2. Its onset of action is slow and, according to the basal values of vitamin K factors, between 3 and 6 days are needed to reach therapeutic levels.
3. There are many interactions with medications and meals.
4. Polymorphisms exist that could determine increased sensitivity or resistance to warfarin.
5. The suspension of the medication before a surgical procedure is difficult.
6. Warfarin has a very long half-life.
7. Prothrombin time with INR determination is the best method available to control therapy but is not good enough.
8. Age is an additional factor in the risk of bleeding.

Advantages of warfarin

1. Well-established efficacy.
2. Adequate efficacy/safety ratio.
3. Its effect can be reversed by vitamin K.
4. Very low cost.
5. No side effects.

Conclusions

There are several issues concerning the new oral anticoagulants. It is essential that the effect of any anticoagulant can be measured in plasma. But to date, there is no FDA approved test to assess the effect or therapeutic range for the new oral anticoagulants. There is no antidote to neutralize the action of the new drugs in cases of bleeding or when acute surgical intervention is necessary.

So, do I think that warfarin is going away? Not in the distant future. There are still many unanswered questions regarding these new OAC. Until laboratories can aid in the quantitation of levels, and the ability to reverse the drugs, I wouldn’t get rid of the PT reagents just yet!

COAGULATION CORNER: OCTOBER 2011

2011-10-03T10:35:45.711-04:00

THE 10 MOST COMMON COAGULATION QUESTIONS PEOPLE ASK ME!

I have been lucky, I have met techs from all over the world, been privileged to lecture in so many wonderful places and learned so much from so many. Here are some of the most common questions that I am asked.

1. How do I make sure that a sample isn’t contaminated with heparin?

Simple, perform a thrombin time. A thrombin time is one of the most sensitive tests for heparin and will be prolonged when there is as little as 0.1U/ml of heparin. It is a really good idea to routinely perform a TT on all inpatients that have factor assays ordered on them to rule out the presence of an inhibitor. Also, if you get a request for a heparin level on a patient and your result is <0.1 or 0.1 and the clinician insists it should be higher- perform a TT, if it is normal there is no heparin on board, or the sample was drawn too soon after administration.

2. What does it mean if the INR is abnormal and the patient is not on warfarin?

The INR is used to monitor patients on a stable does of warfarin. That means the patients have been on the dose for about 2 weeks. An INR will also be abnormal when you have an elevated PT- makes sense, however, you make the diagnosis based on the PT and the follow up testing, not on the INR. Again, the INR is used to monitor warfarin, period.

3. What ISI should I use for my PT reagent?

Use a PT reagent with an International Sensitivity Index ( ISI )of </=1.2

Using insensitive reagents with high ISI’s will account for more variability in your results. Remember, in the formula for the Internationalized Normalized Ratio (INR), the ISI is exponential, so the higher the ISI, the greater the error in calculating the INR.

4. How many levels of coagulation controls should I run, and what levels?

Well, you know that you need to run 2 levels of controls both a normal and abnormal level. Regarding routine testing, make sure the levels of your controls mimic your patient population and challenge your reagent. If you use your reagents to screen for factor deficiencies, you might want to run just an abnormal level with the normal, but if you rely on your APTT reagent to monitor heparin levels, you might want that control to be an abnormal high level.

5. Should I report out INR > 6.0?

So my initial answer is why? If you have an INR of >6.0 on a patient that is receiving warfarin, there needs to be some course of action – FFP, decrease in dosage, etc. Does an 10.0 tell you more than a 6.0, and what about the formula and the PT results, you have to be greater than 50 seconds, and what does that mean? 50, 69, 100 sec, we know none of them are good. With all of that being said, the American College of Chest Physicians (ACCP) publish guidelines on the INR, dosing, treating etc, and they want those levels about 6.0, so if your clinicians want them, go right ahead and report awa.

6. Should I use a Lupus sensitive or insensitive reagent?

Well now that depends on what you are looking for- just for a review. A lupus sensitive reagent has a lower concentration of phospholipid, and will be prolonged in the presence of a lupus or lupus like inhibitor. That is in contrast to an insensitive reagent which will have a higher level of phospholipid to mask the presence of an inhibitor. Remember, with a lupus anticoagulant or lupus like inhibitor you are at a greater risk of clotting, than bleeding. Many of these inhibitors are transient, and if you don’t want to investigate this phenomena, then your laboratory should use a lupus insensitive reagent.

7. Should I perform precision testing on my assays?

Yes, Yes and Yes and on more than 1 level, at least at high, normal and low. Don’t you want to know what your CV’s for that assay are? At those levels, for your specific instrument/reagent combination? How can you tell if an assay result has changed or is really the same answer and related to the CV of the assay. Little effort, great amount of information. I know coagulation assays are expensive, run at least 10 times, 20 is better, can use controls, but patient pools are even better, want to keep that matrix.

8. How do you determine if a mixing study has corrected?

Okay so not my favorite question, since there are several answers. Here is my take on this- you run your PT or APTT, you run your pooled normal plasma, and you run your 1:1 mix. Now a few things, make sure you PNP is platelet poor, make sure you have a reference range on your 1:1 normal pool- with normal subjects (run at least 20 mixes 1 part PNP: to 1 part normal donor to determine), also make sure that your PNP isn’t frozen too long, it will result in an elevated FVII, and finally make sure that you know the levels of all the factors of your PNP- if they are less than 60% they may fail to correct your 1:1 mix. So I still haven’t answered what is a correction? Some labs use the mix that returns to the normal range, or some labs use the within 3 seconds of the PNP or some combination of the above, most important is to be consistent, set your procedures, determine what is a correction and stick with it!

9. Should I incubate my mixing study and how long?

Second least favorite question, again if you are looking for a weak inhibitor, and if you aren’t sure if your immediate mix has really corrected, your answer may be in looking at an incubated mix. The purpose is to really allow those weak inhibitors to inhibit, so incubating for 1 hour may help answer some of those grey areas, and 2 hours may help even more. Did that help?

10. Should I use an anti-Xa assay to monitor heparin instead of the APTT?

Believe it or not, I think most labs would move to this, even though I know it can be expensive. It seems that the most resistance comes from the clinician since they prefer to use the APTT. Advantages to using this are: you would never have to run another heparin therapeutic range! You wouldn’t have to worry about the sensitivity of your reagents to heparin- and as you know there is no dose response relationship with heparin and the APTT- clearly cleaner answers.

So if you can, go for it!

Any questions?

COAGULATION CORNER: SEPTEMBER 2011

2011-08-27T15:41:05.981-04:00

An unusual cause for a prolonged APTT:

A 65 year old male patient being screened pre-operatively is tested for PT, APTT, fibrinogen and a Factor VIII. Previously, having had bloods drawn for hernia surgery, he was told that he had a prolonged APTT and was diagnosed a moderate hemophiliac. He was sent to have a hemophilia work up. Results were as follows:

PT = 11.2 seconds
APTT= 98.5 seconds
Fibrinogen = 300 mg/dL
FVIII = 52%
vWF activity = 68%
vWF antigen=72%

Based on a normal fibrinogen, FVIII, and vW work up, additional testing was performed:

FIX = 92%
FXI =103%
FXII=3%

Diagnosis: Factor XII deficiency

Factor XII deficiency is a congenital disorder that is most commonly inherited as an autosomal recessive trait and is not associated with a bleeding diathesis. Easy bruising and epistaxis has been reported in an occasional FXII deficient patient.

Factor XII deficiency is the most common cause of an isolated prolongation of the APTT in a non-bleeding child or adult. Most patients are diagnosed as part of a pre-operative screen. Bleeding

The incidence of prolonged screening tests are as follows:

Prolonged PT Normal APTT	Prolonged APTT Normal PT	Prolonged PT Prolonged APTT
	Deficiency of
FVII(1:500,000)	FVIII (1:5000 – 10,000) males	FI
Vitamin K	FIX (1:100,000)	FII (rarest deficiency)
	FXI (1:100,000)	FV (1:1,000,000)
	FXII (1:1,000,000)	FX(1:500,000)
	Contact Factors	Vitamin K

FXII is a single chain beta-globulin serine protease with a molecular weight of 80-84,000 daltons. Factor XII is a coagulation protein which is either autoactivated by contact with a number of artificial or biologic negatively charged surfaces (contact activation) or by proteolytic activation on the surface of endothelial cells by prekallikrein/kallikrein and high molecular weight kininogen. Cleavage is mediated by charged surfaces (glass, kaolin, celite, dextran sulfate, endotoxin, urates, crude collagen, and sufatides. Activated factor XII converts prekallikrein to kallikrein (which activates more factor XII, liberates bradykinin from high molecular weight kininogen, and activates complement components C3 and C5), activates factor XI which eventually leads to thrombin generation via the intrinsic pathway, and also activates C1 esterase, thereby activating the complement system.

Factor XII deficiency has been reported to be a risk factor for the development of arterial and venous thromboembolism. The first patient discovered to have a FXII deficiency was John Hageman who died of a PE. Hence the name Hageman factor. A high incidence of strokes, DVT, MI have been described in this cohort of patients at a rate of 1-8%. Heterozygous individuals have factor XII levels between 20-60%. Homozygotes or double heterozygotes have levels < 0.01%.

In a study that looked at the normal population, of about 300 healthy blood donors seven FXII deficiencies were detected. On the basis of these data the prevalence of severe and mild FXII deficiency in the normal population can be estimated to be 1.5-3.0%. Assessment of FXII antigen levels revealed, that all seven FXII deficient individuals had FXII antigen levels matching the activity. One presented a severe FXII deficiency (1/300, 0.3%) without detectable FXII activity and an APTT prolongation of more than 120 s.

Acquired FXII is seen in nephritic syndrome, endotoxin-induced sepsis and shock, DIC, respiratory distress, polycythemia vera, hepatic cirrhosis and other diseases. A spontaneous increase in FXII occurs in pregnancy and with the use of oral contraceptives.

Activated Factor XII (FXIIa)

Contact activation of negatively charged surfaces which activate FXII affects several pathophysiological processes including hypotension, inflammation, thrombosis and fibrinolysis. Studies have demonstrated a relationship between XIIa and an increased risk of coronary heart disease. Additionally XIIa measured after an MI predicts recurrent coronary occlusive events and Is an independent marker in these patients. It has been recently discovered that FXII exsists in different forms. One of those forms, XIIaA was evaluated to access the long term prognosis of chest pain individuals. It was demonstrated that these levels are predictive of long term mortality and may aid in the risk assessment of patients with acute coronary syndrome and my be of greater value in unstable patients with coronary heart disease.

COAGULATION CORNER: AUGUST 2011

2011-08-05T15:20:24.012-04:00

PLATELET FUNCTION AND DRUGS, VITAMINS AND SUPPLEMENTS – OH MY!

We know that drugs like aspirin irreversibly inhibits cyclo-oxygenase-1 (COX-1), inhibiting platelet aggregation for 7-10 days, while conventional NSAIDs reversibly inhibit COX-1. The drug half-life determines the duration of the antiplatelet effect. Clopidogrel and ticlopidine block ADP receptors, and inhibit platelet aggregation for 4–7 days.

Medications:

What about the action of other drugs on platelet function? Some antibiotics such as penicillins and some cephalosporins bind to platelet membranes impairing platelet aggregation. Theophylline and caffeine affect platelet function in vitro via their effects on phosphodiesterase and adenosine receptors2, however there is no clinical evidence that significant antiplatelet effects or bleeding problems occur.

Antidepressants with high affinity for the serotonin transporter deplete platelet serotonin thus decreasing one of the signals for intracellular calcium release. Calcium antagonists, β-blockers and nitrates have antiplatelet effects in vitro, but information on their in vivo effect is limited. Verapamil and diltiazem have the most consistent evidence supporting an antiplatelet effect. They inhibit platelet aggregation in whole blood. It is important to understand these effects and their impact on patients, in particular elderly patients.

Supplements:

There have been many studies that have looked at the effect of fish oil or the n-3 fatty acids on platelet function. Doses ranged from 1-4 g/d and as a result higher does demonstrated inhibition of platelet function. Such changes have been attributed to a reduced decrease of thromboxane A₂ and an increase of the nonaggregating thromboxane A₃.

Bleeding tendencies can increase when some herbal medicines are taken in conjunction with non‐steroidal anti‐inflammatory drugs (NSAIDs) or acetaminophen. Implicated herbs include ginkgo (Ginkgo biloba), garlic (Allium sativum), ginger (Zingiber officinale), and ginseng (Panax ginseng); known as the “4 G’s”. Others include meadowsweet (Filipendula ulmaria), willow bark (Salix alba), dong guai (Angelica sinensis), turmeric (Curcuma longa), bilberry (Vaccinium myrtillus), and feverfew (Tanacetum parthenium) These are thought to impair platelet aggregation through reducing available intracellular calcium.

It has been demonstrated in patients with vitamin E deficiency, marked platelet hyperaggregability have been observed. The action of vitamin E appears to be at the step of phospholipase A activation, or the conversion of arachidonic acid into the cyclic endoperoxides. Administration of vitamin E appears to normalize this hyperaggregability.

Other:

Studies have been done to asses whether olive oil or its principal component, oleic acid, is capable of affecting platelet function. As far as olive oil is concerned, a daily dosage of 30 to 45 g/d has been assessed in 3 studies and the trials do not support an antiplatelet effect of either olive oil or oleic acid .

Moderate daily wine drinkers (3 to 5 glasses daily) had a lower risk of cardiovascular death compared with nondrinkers. This finding was confirmed by a meta-analysis. Globally considered, the studies inconsistently showed that short- or long-term wine intake is associated with an inhibition of platelet function in humans. Experimental studies suggested a role for the nonalcoholic components of wine in inhibiting platelet functionraised the hypothesis that polyphenols, which are abundantly present in wine, possess an antiplatelet property.

Several sources of polyphenols have been investigated to test their antiplatelet effect in human. Grape juice, berries, pomegranate, apples, garlic, onion, tea, cocoa, tomato, and garlic have been tested to asses whether they possess antiplatelet activity. Data obtained with grape juice and cocoa seem to show an antiplatelet effect more consistently than other sources of polyphenols. An in vitro study showing that 5 to 10 μmol/L polyphenols inhibits platelet recruitment via increase of nitric oxide production. Its mechanism is unknown. Grapes and grape products, especially those made from the skins, seeds, and stems of Concord grapes, are good sources of flavonoids. Compared with white wine, red wine contains a 10-fold increase in flavonoid compounds. Flavonoids may contribute to the cardioprotective effects of grape products, as suggested by several studies associating increased flavonoid intake with reduced risk of coronary events

Tomato extracts can influence platelet activity in vitro and ex vivo. The tomato’s bioactive components inhibit platelet aggregation in response to a range of agonists in vitro and reduce the platelet expression of activation-dependent antigens. The tomato extract is known to contain a wide variety of different types of compounds that have antiplatelet activity in vitro and that affect different mechanisms of activation and aggregation.

Substances that can affect platelet function:

aspartame (NutraSweet, can also reduce the number of platelets)
beer (including non-alcoholic beer)
blueberries
chocolate (dark)
coffee
feverfew
garlic
onions
gingko biloba
ginger
ginseng
green tea
guarana (a dietary supplement)
kiwi fruit
omega 3 fatty acids (hemp seed, fish oil)
pycnogenol (pine bark extract)
quercetin, rutin, and related bioflavonoids
red/purple grape products (grape juice, red wine, raisins, grape seeds)
red wine
tomatoes
vitamin E
wood ear or cloud ear mushroom (Auricularia auricula-judae, used in Chinese cuisine)

So, the list is quite extensive, but if you have ever done aggregation testing, you know how frustrating it can be with that abnormal result and no explanation. The most important take away message is the impact of these products may have on bleeding when used concurrently with known medications that have anti-platelet effects.

So we know, coffee, dark chocolate, green tea and red wine, no sticky platelets, more reasons to enjoy!.

COAGULATION CORNER: JULY 2011

2011-07-10T15:19:49.809-04:00

COAGULATION AND ANTI-THROMBIN

Antithrombin (AT) is a potent inhibitor of the reactions of the coagulation cascade. This is a serine protease inhibitor which primary enzymes it inhibits are factor Xa, factor IXa and thrombin (factor IIa). It also has inhibitory actions on factor XIIa, factor XIa and the complex of factor VIIa and tissue factor. Its ability to limit coagulation through multiple interactions makes it one of the primary natural anticoagulant proteins. The interaction between thrombin and antithrombin could be accelerated by as much as 2000 times by heparin.

Antithrombin is synthesized in the liver and secreted into plasma containing 432 amino acids with a molecular weight of 58,200, with a plasma half life of 3 days. Normal levels are between 80-150%. A 50% reduction in AT levels is sufficient to result in thrombosis. Neonates have reduced level of AT at birth (30%-50%) which rise to 60% of adult levels 1 month after birth.

Five types of antithrombin were classified by Seegers in 1962 with the first family identified by Olav Egeberg in 1965. The most recent research has described novel mutations in the antithrombin gene. This has been described as the most severe of the inherited conditions with an odds ratio for thrombosis of 10-20 fold however the incident of venous thrombosis is ~ 1 per 2000-5000 people. AT deficiency is the most serious disorder of the inherited deficiencies of protein C or S. 85% of people with congenital AT deficiency experience thrombotic episodes by age 55, initially with presentation at between 20-30 years of age having multiple events throughout lifetime

Laboratory Testing:

Patients suspected with AT deficiency will have normal screening tests for PT, APTT, Fibrinogen and D-dimer. First line of testing is activity which is perfomed by a chromogenic assay. The principle uses excess amount of Factor Xa added to sample in the Presence of heparin, portion of enzyme is complexed, and inactivated by AT in sample. Excess uninhibited Xa cleaves a specific chromogenic substrate releasing a dye. The change is measured by increase in absorbance. Result is inversely proportional to inhibiting activity of Antithrombin. If this test is abnormal, it can suggest either a dysfunctional (qualitative) disorder or a quantitative disorder. In order to distinguish between the two types an antigen test should be performed.

AT deficiency can be congenital or acquired. There are 2 types of deficiencies, Type 1 deficiency is a quantitative deficiency where patients present with reduced activity and antigen levels

Congenital Antithrombin deficiency:

Type I antithrombin deficiency are heterozygous mutations which lead to a complete loss of the mutant antithrombin protein result in immunologic and functional levels that are 50% or less than normal. The genetic basis includes major gene deletions or point mutations causing a quantitative reduction in antithrombin synthesis by various processes, including premature termination of translation, aberrant RNA processing, and production of unstable antithrombin molecules that have short plasma half lives. Twenty two novel mutations of which 9 missense mutations resulted in type I deficiency and led to low antithrombin activity and antigen levels. Clinically these mutations were associated with venous thrombosis occurring before the age of 32 years. Homozygous type I antithrombin deficiency (AT deficiency) is almost always fatal in utero.

Type II antithrombin deficiency states are usually the result of single amino acid changes that result in functional deficits in a molecule that is otherwise synthesized and secreted into the plasma in a normal fashion. The variant antithrombin molecules may have abnormalities at the reactive site or the heparin binding site. Most cases of type II antithrombin deficiency are also heterozygous, although rare cases of homozygous type II deficiency have been described.

There also exists a third category of type II antithrombin deficiency in which multiple or "pleiotropic" abnormalities affect the reactive site, the heparin binding site, or the plasma concentration. Type II heparin binding site variants are not associated with a high risk of thrombosis unless the affected individual is a homozygote.

Acquired Antithrombin deficiency:

There are several conditions which can cause an acquired AT deficience

- Liver failure: Since AT is synthesized in the liver, disorders of the liver result in decreased production. The more severe the condition the greater the impact on the levels of AT (such as liver cirrhosis)

- Nephrotic syndrome: antithrombin is in the urine, resulting in reduced plasma levels, and these patients are at higher risk for thrombotic events. Conversely, patients with inherited antithrombin deficiency (AT deficiency) may develop renal failure due to renal vein thrombosis or due to fibrin deposition in the glomeruli.

- Acute respiratory distress syndrome is a known secondary cause of antithrombin deficiency (AT deficiency), and this is a major cause of both morbidity and mortality in the newborn. Extracorporeal membrane oxygenation used in the treatment of respiratory failure can be associated with reduced antithrombin and thrombotic events.

- Pregnancy: Although it is widely believed that no substantial reduction of antithrombin occurs during normal pregnancy, antithrombin levels were lower during the third trimester of pregnancy and in the postpartum period. Pregnancy-induced antithrombin deficiency (AT deficiency) is more likely to be seen in twin and triplet pregnancies.

- Sepsis: antithrombin deficiency (AT deficiency) in the setting of sepsis appears to be a correlation between the severity of illness and the degree of antithrombin reduction. .

- Consumptive coagulopathies such as DIC, thrombotic microangiopathy, and acute hemolytic transfusion reactions are associated with antithrombin depletion.

Treatment:

Patients who have had an episode of DVT and whose antithrombin deficiency has been recognized should receive lifelong oral anticoagulation to protect them from recurrent VTE. Patients with AT deficiency may present with atypical site thrombosis, such as mesenteric or hepatic vein thrombosis, and should be placed on lifelong anticoagulation immediately.

Antithrombin supplementation has been suggested to be useful in patients with the following conditions or those undergoing the following procedures.

Malignancies
Sepsis
Shock
Open heart surgery
Orthopedic procedures

Each unit of FFP obtained from a blood bank contains normal levels of antithrombin. If a patient requires 3000 U of antithrombin, that patient would require 3000 mL of FFP given rapidly to raise the level of antithrombin in the recipient. Volume overload becomes a problem in such patients, FFP replacement is not a reasonable source of repeated antithrombin replacement and a choice only when no concentrate is available. .

Antithrombin is an important part of the coagulation cascade. Although AT is a rare deficiency, consumption of AT is more common. AT testing is important to complete a thrombophia workup.

COAGULATION CORNER: JUNE 2011

2011-05-31T11:16:14.499-04:00

Coagulation and Pregnancy

There are both physiological and chemical changes in pregnancy that affect the coagulation and fibrinolytic systems. Pregnancy is associated with changes in haemostasis, including an increase in the majority of clotting factors, a decrease in the quantity of natural anticoagulants and a reduction in fibrinolytic activity. These changes result in a state of hypercoagulability and are likely due to hormonal changes which increase the risk of thromboembolism. The increase in clotting activity is greatest at the time of delivery with placental expulsion, releasing thromboplastic substances. These substances stimulate clot formation to stop maternal blood loss. As placental blood flow is up to700mlmin, considerable haemorrhage can occur if clotting fails. Coagulation and fibrinolysis generally return to pre-pregnant levels 3–4 weeks postpartum.

Physiological Changes:

Bleeding can occur in the first trimester due to abortions which may be caused by chromosomal abnormalities, or other reasons which will cause cramping and bleeding resulting in a completed abortion or one that requires intervention to remove the remaining tissue. Other conditions such as ectopic pregnancy, or a tubal rupture can result in intrperitoneal bleeding that requires surgery to mange symptoms. Third trimester bleeding is cause by placenta anomalies. Placenta previa occurs when the placenta is improperly implanted in the lower uterine segment. While abruptio placenta is the premature separation of the placenta form the implantation site. It usually occurs after the twentieth week of pregnancy.

Platelet abnormalities

The platelet count decreases in normal pregnancy possibly due to increased destruction and hemodilution with a maximal decrease in the third trimester.This is not a pathological process. It is the most common cause of thrombocytopenia during pregnancy and occurs in 5-8% of all pregnant women. The platelet counts are in the lower range of normal and can be as low as 100x109 L-1. The quantitative decrease in platelets is balanced by enhanced platelet activity. Women with this disorder are not at risk of bleeding. It is a diagnosis of exclusion.

Idiopathic/Immunological thrombocytopenic purpura is an immunological disorder in which antiplatelet antibodies are produced resulting in thrombocytopenia with platelet counts are usually = 50x10⁹ L^-1 there is an increased incidence of bruising, epistaxis and gingival bleeding. It is important for labor and delivery that platelet counts = 50x10⁹ L^-1 for normal vaginal delivery. As there is transplacental transfer of antiplatelet antibodies the neonate may be born with thrombocytopenia. If fetal platelet count is = 50x10⁹ L^-1then delivery should be by a caesarean section.

Coagulation Factors

Factors VIII(FVIII), vonWillebrandfactor(vWf), ristocetincofactor(RCoA) and factors X(FX) and XII (FXII) increase during pregnancy. Levels of factor VII (FVII) increase gradually during pregnancy and reach very high levels (upto1000%) by term. Fibrinogen also increases during pregnancy with levels at term 200% above pre-pregnant levels. Other factors either remain at non-pregnant levels or decrease during pregnancy. Factor XIII(FXIII), increases in the first trimester but by term it is 50% of non- pregnant levels. Factor V (FV) concentrations increase in early pregnancy then decrease and stabilize. Factor II (FII) levels may increase or not change in early pregnancy but are normal by term. There is debate about factor XI (FXI) levels with reports indicating increases or decreases. Similarly,FIX levels are reported as increasing, decreasing or remaining stable through out pregnancy. In one study, 50% of carriers of FIX deficiency had FIX levels 50 IUdl at term.

Anticoagulant Levels

Protein C levels remain the same or are slightly increased during pregnancy while protein S decreases. Antithrombin levels remain normal during pregnancy.

Fibrinolytic Factors

Fibrinolysis is reduced in pregnancy due to decreases in t-PA activity, which remains low until1-h postpartum when activity returns to normal. This reduction is due to the gradual, eventually threefold, increase in plasminogen activator inhibitor-1(PAI-1)and the increasing levels of plasminogen activator inhibitor-2(PAI-2). The placenta produces PAI-1and is the primary source of PAI-2. PAI-2 levels at term are 25 times that of normal plasma. Postpartum, t-PA levels quickly return to normal as PA-1 levels decrease; however,PA-2 levels remain elevated for a few days. Thrombin activatable fibrinolysis inhibitor(TAFI)(an antifibrinolytic which cleaves the C-terminal lysine in fibrin to render it resistant to cleavage by plasmin) levels are increased in the third trimester. D-Dimer levels increase in pregnancy but are not thought to indicate intravascular coagulation as fibrinolysisis depressed. These D-Dimers may originate from the uterus

It is important to know the expected levels of these factors during pregnancy so as not to misdiagnosis patients. Many of these levels indicate the bodies protective mechanisms during pregnancy against bleeding and clotting.

Venous thromboembolism (VTE)

Pregnancy is a hypercoagulable state and certain disorders increase the risk of thrombosis. Patients with inherited thrombophilia have an increased tendency to venous thromboembolism. VTE is the leading cause of maternal mortality in the developed world. The risk of pulmonary embolism and deep vein thrombosis is increased during pregnancy. VTE occurs in 10 per 100,000 women of child bearing age and affects100 per100000 pregnancies. Pregnancy-related hypercoagulation is maximal immediately postpartum, increasing the risk of VTE at that time Thromboembolism is 20 times more likely to occur following caesarean delivery than vaginal delivery. The thromboplilias with the highest risk of thrombosis are AT, PC or PS deficiency. The most frequently identified deficiency are those patients presenting with Factor V Leiden. Patients presenting with antiphospholipid antibody are at the highest risk for adverse pregnancy outcomes. Heparin prophylaxsis is recommended for high risk categories.

Disseminated Intravascular Coagulation

Acquired coagulopathies are due to uncontrolled activation of the coagulation system causing disseminated intravascular coagulation (DIC). Once triggered the uncontrolled activation of procoagulants leads to widespread intravascular coagulation. This leads to a fall in the levels of clotting factors to such a low level that they are insufficient to stop further bleeding.

The most common causes of DIC during pregnancy are abruption placentae, intrauterine death, missed abortion, amniotic fluid embolism, ruptured uterus, eclampsia/pre-eclampsi, throphoblastic disease, saline abortion and sepsis

Conclusions

Pregnancy clearly unbalances the coagulation scale causing it to be tipped back and forth between bleeding and clotting. Many of these processes are natures way to protect mothers from bleeding or clotting. However understanding these mechanisms and when factors are elevated are important when managing mom and baby.

COAGULATION CORNER: MAY 2011

2011-05-03T18:05:39.589-04:00

It is with great sadness that the coagulation community has lost a dear friend Diane Shafer. Diane was a well respected professional who taught so many people coagulation through her lectures. She was a friend and a mentor and will be missed by so many people. Our lives were richer because of her, but will not be the same without her. I dedicate this to her and thank her for the endless hours that we spent together and all that I learned from her- she was truly royalty-

Royal Coagulation

I admit - I was up at 4 am to watch Kate and William get married, just as I watched Diana and Charles many years ago. Bloodlines are very important to royalty- as we are reminded of Kate being from a common background. Well, if you look at the background of “royal coagulation” adding a little common blood may be a good thing!

Hemophilia has been present in many royal families including Great Britain’s Queen Victoria(1819 -1901) who was married to Albert, and it appears passed this disease to Spanish royalty and Russian royalty.

Background:

A deficiency in FVIII clotting activity leads to a common bleeding disorder, hemophilia A. This is an X linked disorder which affects 1 in 5000 males due to mutations in the FVIII gene. This heterogeneous disorder results in variable severity and can be due to gene deletions, exon inversions, translocations, nonsense shifts, premature stops as well as missense point mutations. These all can cause defects in the expression, secretion and the half-life of FVIII in circulation. Some mutations can generate stable but dysfunctional FVIII molecules resulting in various severity of the disease. The different degrees of hemophilia are classified into three categories dependent of the circulating FVIII levels: (1) severe hemophilia A when the level of FVIII in the plasma of the patient is less than 2% of the FVIII found in normal plasma; (2) moderate hemophilia A (2%-5%) and (3) mild hemophilia A (6%-40%). Some patients who lack functional FVIII may demonstrate trace to normal levels of circulating nonfunctional FVIII antigen.

Now back to those families:

Queen Victoria felt that since their children presented with fair hair and blue eyes, their blood was lymphatic, and what they really needed was some more black eyed Princes and Princesses to marry so they could have some strong blood- Really? Ever hear of consanguinity? Besides she was the carrier! Her eighth child Prince Leopold, presented with severe hemorrhages, and died at the age of 31 due to a minor fall- so my guess is he had moderate hemophilia. He was married and had 2 children a daughter and a son. Monarchs arranged marriages to consolidate political alliances and this effort did a good job of spreading this disorder to Spain, Russian and Prussia. So Leopold’s daughter Alice had to have hemophilia- X linked recessive- gets the X from Mom and Dad- had a hemophilic son- Rupert and a boy and girl whose status was unknown.

Victoria’s youngest daughter Beatrice had 1 daughter and 3 sons, 2 of which were hemophilic, so Beatrice was clearly a carrier. Beatrice’s daughter Eugenie married King Alfonso XIII of Spain and had 6 children, 2 of whom were hemophilics- she was also a carrier. One of her children was the father of Juan Carlos the current King of Spain. So explains the Spanish connection-

Now on to Russia. Alice, Victoria’s third child, had 6 children and 3 were hemophilic. Her daughter Irene married her first cousin (not good) Prince Henry of Prussia and gave birth to 2 hemophilic children- so now Prussia royalty is included. Her sister Alix another carrier married Tsar Nikolas and carried the disease into the Russian imperial family. She had four girls, but alas finally had a son, and yes of course he was a hemophilic. But sadly all were murdered in the Russian revolution. DNA testing of the Romanov family remains in 2009 showed that one of the four daughters, thought to be Maria by American researchers and Anastasia by Russian researchers, was a carrier. Grand Duchess Maria was thought by some to have been a symptomatic carrier because she hemorrhaged during a tonsillectomy.

So how did all of this start? Queen Victoria’a father, Prince Edward, Duke of Kent was not a hemophiliac-her mother Victoria, was not known to have a family history of the disease, although it is possible that the mutation began at her conception and was passed down only to Victoria and not to her other children. In the same way, had Queen Victoria herself only had seven children, the mutation would likely be assumed today to have occurred at the conception of Princess Alice, as she was the only known carrier among Victoria and Albert's first seven children.

At least one modern descendant of Queen Victoria has been diagnosed with haemophilia: Ferdinand Soltmann, the son of Princess Xenia of Hohenlohe-Langenberg, born 2005. Xenia is a male-line descendant of Victoria, but the disease did not come from Xenia's maternal family, the Croÿs. If the disease came from Xenia, there are two possibilities. The first possibility is that it would have had to be inherited from her father, Kraft, Prince of Hohenlohe-Langenberg, a descendant of Victoria through the female line. Kraft had some clotting issues, which led the family to believe he may have been a mild haemophiliac. If Kraft was a haemophiliac, then his daughters Xenia and Cécile were definitely carriers. The second possibility is that Xenia or Ferdinand had a spontaneous mutation, as Victoria herself apparently had. Because the last known descendent with haemophilia of Queen Victoria's family tree died in the 1940s, the exact type of haemophilia found in this family remained unknown until 2009. Using genetic analysis of the remains of the assassinated Romanov dynasty, and specifically Tsarevich Alexei, Rogaev et el were able to determine that the "Royal Disease" is actually haemophilia B.

Hemophilia B is an inherited, X-linked, recessive disorder resulting in deficiency of functional plasma coagulation factor IX. Spontaneous mutation and acquired immunologic processes can result in this disorder as well. Severe disease, defined as less than 1% factor IX activity, accounts for 50% of those with hemophilia B.Moderate disease, defined as 1-5% factor IX activity, typically presents in children aged 1-2 years and accounts for 30% of those with hemophilia B. Mild disease is defined as levels greater than 5% factor IX activity and accounts for 20% of those with hemophilia B.

Hemophilia is suggested by a history of hemorrhage disproportionate to trauma, spontaneous hemorrhage, or familial hemorrhage. Concomitant illness may include chronic inflammatory disorders, autoimmune diseases, hematologic malignancies (acquired form), and allergic drug reactions

So what does this mean for today’s Royal family? Since the present royal family of England descended from Edward VII, Victoria’s first son, that lineage is free from hemophilia.

Good news, unless of course Kate is a carrier……

COAGULATION CORNER: APRIL 2011

2011-04-04T15:45:16.844-04:00

Quality Control and Medical Decision Levels in Coagulation

QC ensures better patient care by assuring the accuracy and precision of laboratory resting. Having acceptable QC results validates the reliability of instruments and reagents as well as allowing result to be reported with confidence.

So what do we want from our QC? We want it to detect an error in the coagulation process- the reagents, the analyzer or the operator.

This error can result in a clinical error that may result in a change in diagnosis or treating a patient. For example if a PT result is 12.0 sec versus a 12.5 sec is that a clinical error? No- but it may be an analytical error. It is important for laboratories to understand what their coefficient of variation (CV) for their testing at several different levels in particular at medical decision points. A medical decision point is defined as the concentration of analyte being analyzed, at which some medical action is indicated for proper patient care. There may be several medical decision levels for a given analyte. In coagulation the best way to determine medical decision levels by looking at where intervention may be required, and also to understand at what CV a test that answer becomes relevant versus just being inherent variability within a test result.

CLIA includes criteria for acceptable performance for three coagulation tests in its hematology category:

Prothrombin time Target value +/- 15%
Partial thromboplastin time Target value +/- 15%
Fibrinogen Target value +/- 20%

So what does that tell me? I have acceptable limits that tell me how my test is performing, but then I need to understand medical decisions- remember the PT at 12.0 versus 12.5? Well what happens when that result goes from 12.0 to 13.0? Does that mean that something is happening in my patient? Do I need to watch to see if medication needs to be adjusted, or is that just the variation in my test? How was my QC, was it within limits, but on the high side? Lots of questions here- so how do I answer them?

First- QC- we know that 2 levels should be run, we want a normal level, and what about the second level? There are 2 ways to choose that level- it can be a high level, in particular if your patients are on anticoagulation, and you want to make sure that your reagent works well in that range, or where your total system (analyzer and reagents) are challenged- where product may have to be given, where a patient may bleed. Those levels should be determined by the laboratory director to best reflect your patient population. Okay so step one is we have good control over how our reagents perform.

Second- what is a significant CV? This is such an important question- it helps to understand how your test performs, so how do you know what this is? The best way to demonstrate this is to perform precision testing- at a minimum to look at inter-laboratory variability. Precision is the reproducibility of the result when repeatedly measured in the test system. Running at least 10 repeats ( or 20) at a low, normal and high level, can help to determine if a change in a result is inherent in the testing system, or really a significant change in result. Lets look at a Protein C result in the low range- of 30%, and the clinician wants to know if the level is improving- since the second result is 35%? From precision testing you know that at 30%, the CV is 20%, that means result of 24-36% are probably the same result, and not directly related to an increase in result. That is a lot of information to be able to provide regarding results.

So what about controls on the high side? We know that the performance of analytical methods can be monitored by analyzing specimens whose concentration are known and by comparing them to the observed values with known levels. So what do we expect from our controls? We expect stabile controls with little vial to vial variation and to have the same test matrix as our specimens. We also assume that each control has the same chance of detecting an error, which is not always a valid assumption. Error is some assays occur more readily at higher concentrations than at lower in particular in calibration curves are not linear. It is also important to be aware of reagent degradation and contamination. And no matter how many times it is said, your source of water and pipettes are still an important pre-analytical variable.

So while QC is necessary in the laboratory and can be a painful and time consuming task, we know it is important is how a result is used, and how the physician makes a decision.

COAGULATION CORNER: MARCH 2011

2011-03-01T00:00:00.000-05:00

March is DVT prevention month

Awareness & Prevention

In 2008 the U. S. Department of Health and Human Services issued a call to action to prevent deep vein thrombosis. More than 900,000 people are diagnosed each year in the United States alone with a VTE. Approximately 300,000 of those will die from this condition the remaining 600,000 result in primary care stays. New cases of VTE ranged from 250,000 cases during 1966-1990, with 900,000 cases determined from 2002 data. Signs and symptoms of DVT are non-specific and present in a myriad of non-thrombotic disorders. Most pulmonary embolisms (PE) result from a DVT in about 90% of cases, stressing an essential need for early diagnosis and treatment. A WHO health report states that a health care system should have 3 fundamental objectives which include:

Improving the health of the population they serve
Responding to people’s expectations
Providing financial protection against the costs of ill-health.
The cost and treatment of patients are between 5.8-7.8 billion dollars

The costs and complications of a DVT make prevention one of the best approaches. Long term complications of a DVT can exceed over $4000 in the US and about $2000 in the UK. Long term anticoagulation is an option to prevent additional DVTs however extending therapy can also increase the patients risk of bleeding. A possible solution is to follow initial anticoagulation with lower doses to minimize bleeding risks.

In 2010 a panel of experts met to discuss steps for better surveillance for VTE. The objective was to demonstrate prevalence of DVT and PE, to demonstrate any changes in the incidences and to determine proven an effective preventative measures. The diagnosis of DVT has become standardized using implementation of compression ultrasound imaging and provides good sensitivity and specificity (>95%) for proximal deep veins and major calf veins. There is also a high inter-rater reliability in symptomatic patients. There is no standard method for surveillance of DVT.

Here are some statistics:

The average annual incidence of VTE over the past 30 years is 108/100,000 among the white US population.
Incidence among minorities is limited: African Amercians is 78/100,000, American Indians/Alaskan Natives 33/100,000
VTE increases with age, previous history of VTE, surgery, trauma, cancer, paralysis of the leg, stroke, MI, LA, obesity, pregnancy and postpartum.

The purpose of having a surveillance database will be to demonstrate clinical practices proven effective preventive measures in reducing death and morbidity.

Additional advantages will be in providing a demographic measure of the incidence of DVT and PE in the US population and determine if differences exists within populations. It will also aid in defining risk factors and evaluate whether certain clinical practices are effective in diagnosis and treatment and whether implementing these practices decrease VTE incidences. Additionally, other areas may be identified for future research to reduce the burden of VTE.

Management of Patients:

The management of patients presenting with suspected venous thromboembolism is problematic. Urgent diagnostic imaging is inconvenient, costly, and often not available. Elevated levels of D-Dimer are not specific to venous thromboembolism however; normal D-Dimer concentrations are useful as a negative predictor to rule out DVT and PE. Many studies have demonstrated that negative D-Dimer results in addition to a low pretest probability score can be used to exclude PE and DVT.The most commonly studied pretest probability assessment models were developed by Wells and colleagues.This score combines the risk factors for DVT and clinical examination. Patients are classified into low (0 or less points), moderate (1 or 2 points), or high (3 or more points) probability of DVT. Using this strategy reduced repeat ultrasound testing 28% and demonstrated only a 1% thromboembolic risk at 3 months.

As previously stated there are many conditions in which the D-dimer testing is positive that do not result in a DVT. The validity of this test is in the ability to rule out a blood clot, a positive result alone can mean many other things. Conditions that present with elevated D-dimer levels are liver disease, infections, disseminated intravascular coagulation, pregnancy, malignancies, post surgery and immobilized patients. Many of these conditions are found in hospitalized patients resulting in many false positive D-dimer testing and needless imaging tests. Of 233 inpatients tested and compared to imaging >80% gave a false positive D-dimer result. The NPV is higher in outpatient than in inpatients due to the many comorbidities found in inpatients.

The main purpose of treating DVT is to prevent pulmonary embolism. Proximal DVTs (popliteal and higher) are associated with as high as a 50% incidence of pulmonary embolism if untreated therefore, anticoagulation should be instituted promptly. Distal DVTs propagate less than 20% of the time and usually do so within 1 week. Therefore, distal DVTs can be observed with serial ultrasonography (a second ultrasonogram 1 week later is usually sufficient), with treatment instituted only if proximal propagation is noted. The DVTs that remain confined to the calf are associated with a less than 1% risk of pulmonary embolism and a 2% chance of recurrent DVT.

The public needs to be aware of the signs and symptoms of DVT and VTE’s. It is our duty as health care professionals to start with your family and make sure they know their risks, prevention and treatment options.

COAGULATION CORNER: FEBRUARY 2011

2011-02-01T00:00:00.000-05:00

HEART HEALTHY VALENTINE: COAGULATION, CHOCOLATE AND WINE!

Coronary artery disease (CAD) is among the leading causes of death and reduced quality of life in developed countries. Approximately 67 million U.S. adults have high blood pressure (hypertension), with an additional 85 million Americans classified as having pre-hypertension. Hypertension, atherosclerosis and other inflammatory conditions are major contributors to the development of cardiovascular disease (CVD).

Wine and coagulation:

In 1786, an English doctor noted that wine relieved pains of patients suffering from angina pectoris. In 1970, a cardiologist at the Kaiser Permanente hospital center in Oakland (California) initiated a study on over 100 000 people.The first results where published in 1974, and indicated the fact that the risk of death from coronary diseases (notably myocardium infarct) is lower for moderate consumers (1 to 3 glasses of wine a day): 6,2 to a 1 000 against 8,2 to a 1 000 for people who do not drink wine and 11 to one thousand for people drinking more than six glasses of wine a day.
Okay 6 glasses of wine in the name of science might be a little much and lead to a host of other problems! However, Doctor Rimm of the School of Public Health of Harvard - Cambridge - Massachusetts calculated that the risk of heart disease is reduced from 25 to 45% for people drinking 1 or 2 glasses of wine a day.

So how does this work? Moderate alcohol intake decreases clot formation by multiple additive mechanisms. It reduces platelet aggregation, decreases fibrinogen levels, plasma viscosity, von Willebrand factor, and factor VII. Regular moderate alcohol consumption has no significant effect on fibrinolysis, as opposed to higher levels of consumption. Alcohol consumption up to 14.9 g/d is not associated with increased PAI-1, whereas greater amounts of alcohol intake do result in increased PAI-1. Polyphenolics (ie, catechin, epicatechin, quercetin, resveratrol) found in red wine upregulate both tissue-type plasminogen activator (t-PA) and urokinase-type PA (u-PA) gene transcription. Wine phenolics increase fibrinolytic activity independent of alcohol. Red wine supplementation, in both the Mediterranean-type diet (MD) and high-fat diet (HFD), resulted in decreased plasma fibrinogen and factor VIIc, and increased tPA antigen and PAI-1 antigen. A MD and moderate consumption of red wine has complementary, mostly beneficial effects on hemostatic cardiovascular risk factors.

Prevention and treatment of CAD start with diet and a little wine might not be a bad thing either.

Chocolate:

Cocoa has been around for thousands of years, dating back to the Incas and Aztecs around 1600 BC. The Aztec emperor Montezuma supposedly drank cocoa to fight fatigue. Cocoa was thought to have health benefits, ranging from an aid in digestion, relief of angina to improving kidney and bowel function.

Epidemiological research has shown that a diet robust in fruits and vegetables can prevent or delay the onset of CVD. Many researchers wondered what components of fruits and vegetables are responsible for the possible health effects. Flavanols are abundant in certain fruits and vegetables, leading researchers to focus attention on plants with a higher concentration of flavanols which include cocoa. Cocoa, which is the primary ingredient in finished chocolate, is rich in antioxidant polyphenols, a group of protective chemicals found in many plant foods such as red wine and tea, which have been the objects of scientific investigation for their beneficial influence on cardiovascular health.

Polyphenols are reportedly cardioprotective in two ways. First, they help to reduce the oxidation of low-density lipoproteins (LDL), or so-called ‘bad cholesterol.” Oxidation of LDL is considered a major factor in the promotion of coronary disease, most notably heart attack and stroke. Additionally, polyphenols inhibit blood platelets from clumping together. This clumping process, called aggregation, leads to atherosclerosis, hardening of the arteries. By inhibiting aggregation, polyphenols reduce the risk of atherosclerosis. Since atherosclerosis is a major killer of American adults, the protection provided by the polyphenols in cocoa is of real value.

Cocoa not only inhibits platelet aggregation, but it thins the blood, thus slowing coagulation. In a study of healthy subjects given a strong cocoa beverage, platelet aggregation was reduced and fewer microparticles had formed than normal. Additionally, blood from the subjects took longer to form a clot than blood from control subjects. This study showed that cocoa performs the same beneficial anti-clotting activity as aspirin.

The beneficial effects of dark chocolate (50%-71% cocoa content) were not seen with milk or white chocolate, once again suggesting the dark polyphenols are critically important to this action. The anticoagulant effects of chocolate received additional support by the findings of the GeneSTAR trial which studied genetic differences in the cardioprotective effects of aspirin, however the study also stumbled onto a protective effect of chocolate.About 140 people refused to eliminate chocolate from their diets while participating in the study and so the investigators followed this group separately. They discovered chocolate eaters had slowed blood clotting compared to others. Although the protective effect of chocolate was approximately 1/10th of aspirin, the main action appears to be the same.

Reported cardiometabolic benefits of dark chocolate:

Improves blood pressure
Improves insulin sensitivity
Increases HDL (“good”) cholesterol
Reduces oxidation of LDL (“bad”) cholesterol
Increases total antioxidant capacity
Reduces coagulation of the blood

Conclusion:

In conclusion a nice glass of red wine, and one square (about 6 grams) of dark chocolate can be part of a heart healthy celebration- enjoy!

COAGULATION CORNER: JANUARY 2011

2011-01-03T13:04:36.701-05:00

OVERVIEW OF ASH

HAPPY AND HEALTHY 2011 TO ALL!

The annual American Society of Hematology meeting was held in Orlando Florida. There were many attendees, lots to listen to and record breaking low temperatures in Orlando!

One of the main topics of discussion was the administration of 2 recently cleared US drugs- rivaroxaban (oral direct Xa inhibitor) and dabigatran (oral direct thrombin inhibitor). These drugs are body weight dependent and are said to not require monitoring, however there are instances where they will need to be monitored. They have been in use ex-USA for about a year however clinicians expressed the following concerns. These drugs do not require monitoring clinicians are concerned that they will not know if a patient is being compliant. Patients who are starting long term anticoagulation on these drugs may stop taking the drugs after symptoms of a thrombotic event subsides. Dabigatran requires twice daily dosing making compliance more difficult. Many patients can not commit to this regimen will not be able to take the drug. Warfarin is monitored on a regular basis and compliance issues can be addressed. As a result, people failing to take the drug will be at an increased risk of thrombosis, clinicians will not have a way to determine if the drug is on board or not. They feel that there should be a way to measure levels.

Regarding monitoring of the drugs, Rivoroxaban: may be able to be monitored by an anti-Xa assay, however the curve must be made from the drug. Dabigatran: Anti-IIa drugs prolong all clot based assays. The thrombin time is too sensitive to use to monitor this anticoagulant. The APTT result will be prolonged however depending on the sensitivity of the reagents, the reaction flattens out and increasing doses will not be reflected by the APTT. As a result the clinician may continue to increase the dose of the anticoagulant and place the patient at a risk for bleeding. Also, patients with kidney disease may be at risk for over anticoagulation. There is a definite patient population that will require monitoring.

There were 2 large symposiums on Hemophilia that were well attended, and that addressed laboratory testing of factor VIII (FVIII). The importance of not only diagnosing but also monitoring FVII was discussed in detail. Reagent sensitivity is important at levels of 80% and borderline levels of 40-50%. These levels are important to reflect if the hemophiliac patient FVIII levels are stabile pre and post operatively. Elevated levels of FVIII are also important to detect an excess of infused product which can reflect supra-therapeutic levels of FVIII and place a patient at a risk for thrombosis.

Also recombinant von Willebrand factor is being developed. This will impact how vW assays are run, as well as levels of FVIII from patients who have received this product. At present a new drug Wilate is being looked at for the treatment of VWD. This is an FDA approve drug derived from human plasma, at present most centers use Humate. The take away message from this is that patients who receive this drug may not maintain levels of VWF and FVIII for as long as patients who receive Humate P. This is important for people in the field to know, since clinicians and laboratories may see greater changes (decreased levels as opposed to patients treated with Humate) in repeated measures of FVIII and VWF.

Immune thrombocytopenic purpura (ITP) is a platelet disorder that doesn’t have one specific diagnostic test. Even testing for anti-platelet antibodies may not prove diagnostic since not all ITP patients have antibodies. Since 1913 spleenectomy has with used with a 66% chance of a positive response. Rituxamab has been used with success and a thrombopoeitin receptor, Romoplostin, has been used to stimulate platelets. Also, Ettrombopog, and FDA approved drug has been found to sustain platelets for about 2-3 years, however it is an early drug and there may be future toxicity. 20% of ITP patients have a secondary etiology. H pylori and ITP seem to have a patho-physiological link, when treated for the H. pylori, there was a 50% improvement rate, with increased response rates seen in Japan and Italy, most likely due to the serotypes found in those countries. ITP occurs in pregnancy at a rate of 1:1000, and usually presents early on and is treated with IVIG.

The risk of thrombosis and cancer ranges from 1-30%. The ability to target a patient’s risk will aid the clinician in who should be treated. A scoring system based on 5 independent risk factors include the site of cancer, pre-chemotherapy platelet count, hemoglobin level, pre-chemotherapy WBC and body mass index are evaluated and placed in a low, intermediate or high risk. This can help to improve the risk benefit of anticoagulation. To prevent VTE it is important that all surgical patients are anticoagulated. Additionally many cancer patients benefit from a longer duration of prophylaxis for up to 1 month post surgery. The recommended treatment for cancer-associated thrombosis is LMWH. It is more effective than warfarin and reduces the risk of recurrent VTE by 52%.

This meeting was a wonderful opportunity to learn, network and see old friends. Hope that I have give you a piece of what was experienced-minus the cold weather!

Pre-analytical Variables in Coagulation Testing - You haven't heard anything yet!

2010-12-03T00:00:00.000-05:00

Laboratory medicine is typically divided into three main phases (preanalytical, analytical and postanalytical), Still with all of the advances in laboratory testing that have been made, lack of standardized in procedures for sample collection, patient preparation, specimen acquisition, handling and storage, account for up to 93% of the errors within the entire diagnostic process.

The complete elimination of laboratory testing errors is unrealistic, especially since errors relating to extra-analytical phases are harder to control. When you add additional people to the mix, the difference between in- and out-patients, error rates (0.60% vs. 0.039%) is directly attributed to human factors related to skill in drawing blood and the sheer volume of laboratory tests carried out for inpatients. Non-laboratory personnel seems to account for the majority of errors, (95.2%). Data show that problems directly related to specimen collection are the main cause of preanalytical errors. This further enhances the importance of Good Laboratory Practices and compliance with regulatory agencies as well as the practice of standard operating procedures.

So what defines a standard operating procedure? That is a procedure that is written so well, a technologist running an assay can leave what they are doing, (or fall over) and a new tech can pick up (either them or the procedure) where they left off- there is no room for discussion. Everyone should be on the same page, with that being said, techs should have input on these procedures, they are the one who will be on the bench, they know what works and doesn’t.

Medical errors are the eighth leading cause of death exceeding those from traumatic accidents, breast cancer or AIDS, that is a lot of errors. We all know how complex coagulation testing is, and as many times as it is said, pre-analytical variables account for many of the errors. In fact since instrumentation is so improved, it has really made these pre-analytical errors the main factor influencing the quality of testing- sort of- we are talking about coagulation testing. We have reagents, buffers, calcium, deficient plasmas, standards, dilutions, inactivated factors that need to be activated, cofactors. Need I go on? So needless to say for coagulation testing, it is only amplified.

We know there are patient variables like age, gender, diet, smoking, alcohol intake, exercise, medication, physical & emotional stress those are the normal ones. Lets now add, biological variation, blood type, genetic mutations, and ethnic groups! Ideas on how to control for that?

We also know about Specimen Variables: Order of draw, atraumatic blood draw. tube fill- over/under filled, platelet Poor Plasma and time to perform testing. Lets not forget pouring red tops into blue tops, sending EDTA samples for coagulation testing- just change the tube top, and what about the samples that sat at the nurses station for five hours and now require a stat APTT. Of course my favorite are the removable clots, just take them out- don’t mean much at all. Manuals for that anyone. Forget CSI, coagulation techs have better instincts and problem solving skills, they are true detectives. (CSI people wear better shoes then techs, but what does that tell you?)

So now let skip ahead to an age old topic - hemolysis- what do we do? So here are the issues -

Lab gets a sample, it is hemolyzed? Should we run it, how hemolized? Is it from in-vitro hemolysis (like a device or a bad draw) or is it in-vivo, like a burn patient or DIC? Vascular cell damage that occurs during phlebotomy, is the most frequent reason for specimen rejection, prevalence as high as 3.3%; five-fold more frequent than QNS. In vitro hemolysis traditionally reflects a more generalized process of vascular and blood cell damage that can occur during phlebotomy, which causes cell membrane disruption and leakage of hemoglobin into the surrounding fluid.

Can you get that information? Okay stop laughing- we know you can’t-

So what do you do? Patient result, result may change course of treatment, good result, bad result? What to do?

Lets discuss -

We know to be ruthless about your specimen and results can only be as good as your sample.

The Clinical and Laboratory Standards Institute guidelines for prothrombin time (PT) and activated partial thromboplastin time (aPTT) testing states:

“ samples with visible hemolysis should not be used because of possible clotting, factor activation and interference with endpoint measurement.”

Seems clear, HA - so what do you do when the clinician says, give me the result, I will know what to do with it- clearly they were taught something about hemolysis that we were not- so here is my question:

Does it shorten or prolong results?

Theory: Shorten: may be caused by the exposure of anionic membrane phospholipids during erythrocytolysis which may provide a phospholipid-rich surface to accelerate coagulation reactions.

Theory: Prolong: exposure of membrane phospholipids could compete with thromboplastin for activated factor VIIa (FVIIa) availability and have the converse effect.

So you can pick a theory and make the results be what you would like. Also as you know hemolysis is subjective

Hemoglobin < 20 mg/dL appear clear (nonhemolyzed) and those with a supernatant hemoglobin > 30mg/dL “hemolyzed.”

Between 20 and 30 mg/dL in which the sample is questionable.

I know, I know results some times must be reported, and I know labs use messages stating that the results may be questionable due to hemolysis, suggest a re-draw etc etc ( everything but a skull and cross bones). However, I was at a meeting once and a lawyer/former tech addressed this issue- and the response made a lot of sense- If you were to testify in front of a jury of your peers (which is the operative word here) and you said that the result was sent because a clinician wanted it- well you are supposed to be the “expert” in laboratory testing.

Did you knowingly release a result that was possibly not correct? Makes sense, makes me scared. I have also been on the receiving end of a clinician needing those results -

So, just as laboratories are trying to minimize pre-analytical variables, more are discovered that impact coagulation results. All of this ensures job security.

The solutions that you come up with in your laboratory need to be standard and practiced by all. I know hospitals that reject all hemolized samples, you need for everyone to be on board with that, including your pathologists. Not an easy issue, but remember you want the best result for patients.

I would like to wish you all a happy holiday and a happy and healthy 2011!

Enjoy the season.

LIVER DISEASE AND COAGULATION OUTCOMES:

2010-11-08T00:00:00.000-05:00

The liver plays a central role in the clotting process. Acute and chronic liver diseases are invariably associated with coagulation disorders due to multiple causes including: decreased synthesis of clotting and inhibitor factors, decreased clearance of activated factors, quantitative and qualitative platelet defects, hyperfibrinolysis, and accelerated intravascular coagulation. The liver is the site of production of most coagulation factors, but the response of each factor to liver disease is variable due to differences in biologic half lives and acute phase reactions. The PT is usually prolonged first, then APTT. Factor VII: shortest biologic half life, often affected earliest with largest decrease in plasma level. Factor VII also decreases earliest with warfarin treatment. Factor VIII: may be normal or elevated due to acute phase reactants. Factors XI and XII:have long biologic half lives, and may be normal until liver disease is advanced.

As a result the PT, APTT, TCT, and all factors are prolonged with the possible exception of fibrinogen (I) and Factor VIII which may be increased, decreased or normal since they are acute phase reactants. Also, FDPs: may be increased due to decreased clearance by liver. However the D-Dimer is usually normal which is helpful to differentiate liver disease from DIC. Remember, DIC is a consumptive process in which factors are being consumed, and in this process there is the simultaneous formation of thrombin and plasmin causing increased D-dimers. Platelets will also be decreased, due to aggregation, while in liver disease platelets will be either normal or decreased. The bleeding tendency accounts for increased risk of morbidity and mortality in patients with liver disease undergoing diagnostic or therapeutic invasive procedures.

Since almost all procoagulants and inhibitors are synthesized in the liver, several conditions can cause acquired bleeding disorders. Liver disease can be divided into groups with distinctive pathogenesis:

1. Decreased synthesis of clotting and inhibitor factors: Cirrhosis
2. Decreased clearance of activated factors: Chronic liver disease
3. Quantitative and qualitative platelet defects: Splenomegaly secondary to liver-induced portal hypertension
4. Hyperfibrinolysis : Tumors
5. Accelerated intravascular coagulation: Acute hepatitis

The bleeding tendency accounts for increased risk of morbidity and mortality in patients with liver disease undergoing diagnostic or therapeutic invasive procedures. Coagulation disorders in liver disease are extensive, complex and expensive to treat. Many mechanisms can cause the coagulation changes, but many can be attributed to cytokine activation. Sepsis further impairs hemostasis in patients with liver cirrhosis bleeding from esophageal varices. Thrombotic events, even if rare in cirrhotic patients, occur mainly in the portal and mesenteric veins.

Liver disease can cause both quantitative and qualitative abnormalities in clotting factors. Vitamin K-dependent factors (II, VII, IX and X, Protein S, C and Z) undergo posttranslational gamma-carboxylation, which can be impaired by a deficiency of vitamin K or by lack of blood-clotting enzymes in hepatocellular disease. The presence and level of vitamin K-dependent clotting factors that have undergone incomplete or slight carboxylation are also increased. Levels of factor VIII are typically normal or elevated due to having extra hepatic sites of synthesis. Fibrinogen is synthesized in a large capacity in the liver, and the level is maintained until late in the disease stage. Primary fibrinolysis results from reduced clearance of tPA, impaired by increased synthesis of PAI-1 or diminished synthesis of plasminogen. Conversely, fibrinolysis may be enhanced due to impaired production of alpha-2- antiplasmin.

Measurement of clotting-factor levels in hepatocellular disease often reveals reduction, even when hemostatic-competency screening tests produce normal results, and the extent of reduction in levels of vitamin K-dependent factors correlates with disease severity. However, clotting factor levels are not uniform in liver disease, and the response is not the same in all forms of the disease.

For example, levels of prothrombin and factors VII and X are often severely lowered in liver disease. Because it has a short half-life (VII has a half life of 4-6 hours) and is unaffected by inflammation or DIC, factor VII is an important early marker of significant parenchymal liver disease; it is usually the first vitamin K-dependent factor to reflect a reduced level. In contrast, factor IX levels typically are only modestly reduced until advanced stages of liver disease. The behavior of factor VIII and V can be a cost effective way to help distinguish between DIC and liver disease. Since VIII is also produced by endothelial cells, it is not only dependent on hepatocyte function. Both factors will be consumed in DIC, but in liver disease, V will be low and VIII is either normal or high. Factor V is not a vitamin K dependent factor, as opposed to factors II, VII, IX and X, so it is a cost effective way to look for liver disease. Acquired dsyfibrinogenemia can be seen in patient with cirrhosis and hepatoma. They tend to have “flimsy fibrinogen” with increased carbohydrate content, resulting in reduced functional activity.

In liver disease the platelet count is either normal or decreased. The liver is a source of thrombopoietin which stimulates platelet production . Mild splenomegaly may be present in cirrhosis resulting in platelet pooling. Alcohol intake inhibits the production of platelets by megakaryocytes. Folate deficiency, which may accompany cirrhosis may also cause thrombocytopenia.

Liver Disease is a complex disorder that impacts coagulation testing. It must be diagnosed cost effectively and treated appropriately.

October 2010: COAGULATION AND BREAST CANCER

2010-10-04T00:00:00.000-04:00

National Breast Cancer Awareness Month

To the survivors- your strength takes my breathe away
To those we have lost- we will never stop the fight

The known association between coagulation and cancer dates back to 1865 and Armand Trousseau when the observation was made that patients who had idiopathic thrombotic venous thromboembolism had an underlying occult cancer. Some coagulation factors display a role in tumor progression. Coagulation disorders are a common problem in neoplastic patients and many factors contribute to increase the risk of thromboembolic events in these patients. An hypercoagulable state is induced by malignant cells interacting directly with hemostatic system and activating the coagulation cascade. More sensitive tests to assess a hypercoagulable state in cancer patients have been developed; even though these tests are always altered in cancer patients, none of them possess a clinical significance in terms of predictive value for the occurence of thromboembolism and disease prognosis in the individual patient. The most frequent thromboembolic complications in cancer patients are deep vein thrombosis of the lower extremities and pulmonary embolism; therefore, disseminated intravascular coagulation, thrombotic thrombocytopenic purpura or haemolytic uremic syndrome are special manifestations of neoplastic disease. Diagnosis of idiopathic deep vein thrombosis, in the absence of other risk factors, could indicate the presence of occult malignant disease; however, the need for an extensive work-up to detect malignancy is still controversial.

The most frequent reports on coagulant proteins and cancer interactions include tissue factor; TF TF-factor VIIa, factor Xa, factor IIa (thrombin)-factor II receptors (also called proease-activated receptors (PARs), and factor XIIIa-factor Ia (fibrin). TF and factor VIIa contribute to the extrinsic cascade and possibly to the development of cancer. Other factors from the intrinsic pathway, such as factors XI and XII, have not yet been directly implicated in cancer progression. Blood coagulation cascades can be activated by different mechanisms and to different levels in cancer patients. The alterations range from subtle abnormalities in laboratory tests to clinically overt thrombosis and disseminated intravascular coagulation. Up to 50% of all cancer patients and 90% of those with metastases exhibit hemostatic abnormalities. These abnormalities may be reflected in the dominance of the tumor cell-associated procoagulant pathway, which leads to thrombin generation and hypercoagulation. Similar observations were made using in vitro ovarian cancer cells for the coagulation process. Direct activation of blood coagulation by the induction of thrombin may occur through the activity of tumor cell procoagulation, whereas indirect activation may occur through the production of tumor-associated cytokines that trigger TF production by host macrophages (MAs). The coagulation pathway components may contribute to tumor cell proliferation, invasion, and metastasis although these alterations could also be a consequence of advanced disease.

Tissue Factor (TF) is the cell surface receptor that activates coagulation by binding the serine protease coagulation factor VIIa (VIIa). The activation of the coagulation cascade leads to thrombin generation, fibrin formation and platelet activation which together may aide tumor growth and metastasis. TF is released from tumor cells by shedding or TF comes in contact with coagulation factors when tumor cells enter the blood stream, leading to a hypercoagulable state and its clinical manifestation of spontaneous thrombosis (Trousseau's Syndrome) that occurs in several types of cancer. This provides clear evidence that TF is a frequent marker of advanced cancer. Aberrant TF expression has been detected in various human tumors, including breast cancer but is not usually found in normal tissues from these sites. Elevated expression of TF in tumors has been associated with certain unfavorable prognostic indicators, such as angiogenesis, metastasis, advanced disease stage, and multidrug resistance

Treatment:

Some chemotherapeutic agents used for cancer may induce thrombosis but their biological alterations in the hemostatic system are not well understood. The main mechanisms of thrombogenesis associated with chemotherapeutic agents are: 1) The release of procoagulantsand cytokines from tumor cells damaged by the cell-targeted treatment; 2) A toxic effect directed towards vascular endotheli- In a cooperative group study of 433 breast cancer patients treated with adjuvant chemotherapy it was also to be about a 5% incidence of thromboembolic disease. It has also been reported a 17.6% incidence of thrombosis in159 patients receiving a five-drug chemotherapy regimen for stage IV (metastatic) breast cancer.

Heparins are the most extensively used anticoagulants in clinics. In blood coagulation, unfractionated heparin and low-molecular-weight heparins potentiate the activity of antithrombin III, thus inhibiting the activation of coagulation factors II and X. They also release TFPI, a physiologic inhibitor of the TF pathway that prevents pulmonary embolism and is used to treat deep vein thrombosis. Retrospective and meta-analytic studies of deep vein thrombosis treatment have shown longer survival among cancer patients with thrombosis who were treated with unfractionated heparin and low-molecular-weight heparins than among patients treated without heparin. Thus, the use of anticoagulants might allow them to live longer.

Coagulation factors have a profound effect on tumor cell behavior in both in vivo and in vitro studies. These factors could enhance tumor cell proliferation, invasion, angiogenesis, and metastasis. Hence, targeting activated coagulation factors might provide a viable cancer treatment strategy.

Clinical Studies:

A clinical study that included fifty subjects with breast cancer patients and 25 healthy control was conducted. Routine hematological investigations i.e. HGB, WBC, platelets count were done by hematology analyzer and specific investigations like prothrombin time (PT), activated partial thromboplastin time (APTT) and fibrinogen level were performed by using commercially available kits. Results obtained were analyzed by using Student`s `t` test and level of significance was done. Platelets count and fibrinogen levels were increased in breast cancer patients but PT and APTT were comparable with control group.

A prospective study on platelet-derived microparticles (PMP) and their procoagulant potential was conducted in breast cancer patients. Fifty-eight breast cancer patients and 13 women with benign breast tumors were included in the study. Microparticles (MP) were examined by electron microscopy and FACS analysis using labels for annexin V (total numbers), CD61 (PMP), CD62P and CD63 (activated platelets), CD62E (endothelial cells), CD45 (leukocytes) as well as CD142 (tissue factor). Prothrombin fragment 1+2 (F1+2) and thrombin generation were measured as blood coagulation markers. Numbers of annexin V+-MP were highest in breast cancer patients with larger tumor size and patients with distant metastases versus those patients patients with in-situ tumors, small tumor size and women with benign breast tumor. The difference was statistically significant. . A total of 82.3% of MP were from platelets, 14.6 % from endothelial cells and 0.3% from leukocytes. Less than 10% of PMP showed degranulation markers. Larger tumor size (T2) and metastases correlated with high counts of PMP and with highest F1+2 levels. Since prothrombin levels and thrombin generation did not parallel MP levels, we speculate that MP act in the microenvironment of tumor tissue and may thus not be an exclusive parameter reflecting in-vivo procoagulant activity.

Clearly a long standing association has been documented with coagulation and cancer. Breast cancer, and its treatment can increase the risk of a thrombotic event. Patients should be aware of this and the signs and symptoms that may accompany a DVT or a PE.

So use this month wisely, perform self examination, encourage other people to do so, don’t forget your daughters! Men are also not immune to this cancer and need to be aware of their risks. Get out there and walk, or donate, but don’t give up!

MOLECULAR METHODS IN COAGULATION: FACTOR V LEIDEN

2010-09-03T00:00:00.000-04:00

Okay, so I admit, I come from a time when we still did tilt tube testing, I still believe every coagulation tech should know what a clot looks like in a tube, how is forms and how and if it lasts. You never know when it could come in handy. So molecular testing in coagulation, not even a glimmer in anyone’s eye when I started in the laboratory.

Now, we even have guidelines for molecular testing in coagulation-

CLSI document: H21-45 Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis! It discusses what samples are acceptable, (including buccal smears) what additives may inhibit PCR reactions (like heparin), extraction for DNA samples (up to 922 days with acceptable yields!), DNA isolation, and storage

Molecular genetic markers:

One of the most widely utilized molecular testing is in regard to the detection of the molecular basis of activated protein C (APC) resistance. This was discovered in 1994 by Bertina. Prior to this, many patients thrombophilic states could not be linked to a disorder and were describes as idiopathic. One of the advantages of molecular testing is that patients can be tested while they are on anticoagulation. The range of molecular genetic markers, linked with a clearly documented increased risk of thrombophilia are include mutations of factor V Leiden 506R/Q, of protrombin 20210G/A, MTHFR 677C/T in homozygous form, mutation of PAI-1 4G/5G, mutations of different coagulation inhibitors and finally a range of polymorphisms with still not precisely defined increased risk for thrombophilia (F XIII Val34leu, platelets glycopeproteins, endothelial protein C receptor and thrombomodulin

It is known that VTE is a multifactorial disorder and can impact the incidence by combining genetic risk factors and acquired or environmental conditions such as pregnancy, oral contraceptive use, estrogen therapy, malignancy, stroke with extremity paresis, trauma, surgery, or immobility. The more stimulus that you have the greater risk increases of a clot. Known genetic causes are present in approximately 25% of unselected venous thrombosis cases and up to 63% of familial cases. Factor V R506Q (Leiden), (FVL) causing activated protein C (APC) resistance, is the most common genetic risk factor for venous thrombosis. Protein C is a circulating vitamin K-dependent zymogen, which is activated to APC, the active enzyme, by the thrombin-thrombomodulin complex. APC functions as a natural anticoagulant byinactivating (via proteolysis) procoagulant factors Va and VIIIa in the presence of protein S. Factor V Leiden appears to account for 90-95% of cases of APC resistance. Two rare mutations in the factor V gene have been described and are of dubious clinical significance.

Factor V-Cambridge (R306T) is not strongly associated with venous thrombosis in controlled epidemiologic studies. Factor V-Hong Kong (R306C) has been found in 1-2% of Chinese patients but does not appear to be associated with APC resistance.The R2 allele (H1299R, or A4070G) of the factor V gene, associated with a haplotype known as HR2, is present in 10% of the general population, and early studies indicate that it increases the risk of venous thrombosis in individuals heterozygous for factor V Leiden an additional 3-fold beyond their already 7-fold increased risk.

FVL is present in 5% of Caucasian Americans, it is rarer in Hispanic-Americans, rarer still in African-Americans, and virtually absent in Africans and Asians. It is believed to produce a relative risk of venous thrombosis of about 7-fold in the heterozygous state and about 80-fold in the homozygous state. It is found in 11-20% of individuals of all ages presenting with their first episode of venous thrombosis. If the patient is under 50 years of age, up to 40% present with this FVL mutation is involved, The environmental factor most extensively discussed in this context is oral contraceptive use in women, which produces a 30-fold increase in thrombotic risk when the factor V Leiden mutation is also present.

The following recommendations regarding FVL can be accessed at:
Consensus Statement on Factor V Leiden Mutation Testing Page
http://www.acmg.net/resources/policies/pol-009.asp 5/14/2007

Issue 1: Which methodology should be used: Factor V Leiden DNA testing or functional activated protein C (APC) resistance testing?

Recommendation 1

When appropriate clinical care requires testing for the factor V Leiden allele, either direct DNA-based genotyping or a factor V Leiden-specific functional assay is recommended. Patients who test positive by a functional assay should then be further studied with the DNA test for confirmation and to distinguish heterozygotes from homozygotes. Patients on heparin therapy or with known lupus anticoagulant should proceed directly to molecular testing if the modified functional assay is not used. When relatives of individuals known to have factor V Leiden are tested, the DNA method is recommended.

Issue 2: Who should be tested?

Recommendation 2

Opinions and practices regarding factor V Leiden testing vary. Some physicians advocate testing of all patients with venous thrombosis except when active malignancy is present. Others exclude testing in patients over age 60 in the absence of a family history of thrombosis or a previous thrombotic event. There is growing consensus that testing should be performed in at least the following circumstances (these are the same general recommendations for testing for any thrombophilia):

- Age <50, any venous thrombosis.

- Venous thrombosis in unusual sites (such as hepatic, mesenteric, and cerebral veins).

- Recurrent venous thrombosis.

- Venous thrombosis and a strong family history of thrombotic disease.

- Venous thrombosis in pregnant women or women taking oral contraceptives.

- Relatives of individuals with venous thrombosis under age 50.

- Myocardial infarction in female smokers under age 50.

Testing may also be considered in the following situations:

- Venous thrombosis, age >50, except when active malignancy is present.

- Relatives of individuals known to have factor V Leiden. Knowledge that they have factor V Leiden may influence management of pregnancy and may be a factor in decision-making regarding oral contraceptive use.

- Women with recurrent pregnancy loss or unexplained severe preeclampsia, placental abruption,intrauterine fetal growth retardation, or stillbirth. Knowledge of factor V Leiden carrier status mayinfluence management of future pregnancies.

Random screening of the general population for factor V Leiden is not recommended. Routine testing is not recommended for patients with a personal or family history of arterial thrombotic disorders (e.g., acute coronary syndromes or stroke) except for the special situation of myocardial infarction in young female smokers. Testing may be worthwhile for young patients (<50 years of age) who develop acute arterial thrombosis in the absence of other risk factors for atherosclerotic arterial occlusive disease.

Issue 3: Should testing be offered to individuals with environmental risk factors?

Recommendation 3

Factor V Leiden testing is recommended in women with venous thromboembolism during pregnancy or oral contraceptive use. In contrast to general screening before administration of oral contraceptives, targeted testing of women with a personal or family history of venous thrombosis is advisable. Routine screening for factor V Leiden in asymptomatic women contemplating or using oral contraceptives is not recommended, except for those with a personal history of thromboembolism or other medical risk factors. Those women with a family history of thromboembolism, APC resistance, or documented factor V Leiden mutation should be counseled about their risks and options and considered for testing depending on the overall clinical situation. Women with a history of recurrent late-trimester fetal loss should also be considered for testing. Whether or not the woman smokes would not alter these recommendations. Screening of asymptomatic individuals with other recognized environmental risk factors such as surgery, trauma, paralysis, and malignancy is not necessary or recommended, since all such individuals should receive appropriate medical prophylaxis for thrombosis regardless of carrier status.

Issue 4: Should patients found to be positive for factor V Leiden or APC resistance be tested for any of the other heritable thrombophilic risk factors?

Recommendation 4

Patients testing positive for factor V Leiden or APC resistance should be considered for molecular genetic testing for the most common other thrombophilias with overlapping phenotype for which testing is easy and readily available. At present, only the prothrombin 20210A variant fits these criteria. It is present in 1-2% of the general population, its involvement in venous thromboembolism is well-established, and the DNA test is as simple as that for factor V Leiden (with which it can even be multiplexed). Protein S, protein C, and antithrombin III deficiencies are too genetically heterogeneous for routine molecular genetic testing, but testing by functional coagulation assays may be considered, especially if there is a strong family history of venous thrombosis. Hyperhomocysteinemia should be considered and tested (in most cases by measuring plasma homocysteine levels) as another potential risk factor in those found to be positive for factor V Leiden. Patients with classic homocystinuria are at extremely elevated risk of thromboembolism and should probably be tested for other available thrombophilic risk factors.

Issue 5: Should testing for other heritable thrombophilic factors be performed simultaneously with factor V Leiden testing?

Recommendation 5

Physicians ordering factor V Leiden on a venous thrombosis patient for any of the indications recommended here should also consider the utility of functional, biochemical, and molecular screening for other heritable thrombophilic factors, especially prothrombin 20210A and plasma homocysteine levels.

Issue 6: Are there any other factor V mutations in addition to factor V Leiden which should be tested?

Recommendation 6

The factor V Leiden (R506Q) mutation is currently the only molecular analysis of the factor V gene indicated in the routine workup of thrombotic risk.

Issue 7: What are the recommended methodologies and quality assurance standards for performing these tests?

Recommendation 7

The factor V Leiden mutation test should be performed using any of the accepted technical approaches as long as they have been properly validated by the laboratory, while adhering to current ACMG/CAP quality assurance guidelines for molecular genetic testing.

Issue 8: What are the appropriate pre- and postanalytic procedures to be followed in factor V Leiden testing?

Recommendation 8

Individuals being tested should be made aware that this is a genetic test, that test results have implications about risk in other family members,and that there may be attendant issues of confidentiality and possible insurance discrimination. The laboratory's report should state explicitly the relative risk implications for factor V Leiden heterozygotes and homozygotes,the risk that other relatives may have the mutation, and the recommendation, if indicated, for testing for other inherited hypercoagulabilities. It is important for individuals testing positive for factor V Leiden to understand the risk implications and genetic implications of their result. Patients should be counseled about these implications by their physician or genetic counselor.

FUTURE PROJECTS: Molecular Genetics of Coagulation Disorders from:
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE: NIH

The NIH has a Program Project to explore the molecular basis for selected disorders of coagulation and thrombosis and the role of hemostatic balance in vascular disease pathogenesis. The individual projects in this proposal emphasize the use of new technologies to provide improved biologic insight and develop new treatments for hemorrhage, thrombosis and related cardiovascular disorders. The three individual projects contained in this PPG will: (1) continue a whole genome ENU mutagenesis analysis in the mouse to identify genetic modifiers of factor V Leiden, while also taking advantage of natural murine strain variation to identify additional thrombosis modifiers, as well as modifier genes for thrombotic thrombocytopenic purpura (TTP); (2) continue to explore the critical factors that limit factor VIII expression. The current proposal focuses on the molecular mechanisms responsible for the oxidative stress that results from misfolded FVIII in the ER lumen; and (3) explore the role of bacterial streptokinase (SK) and its interaction with plasminogen in the pathogenesis of Group A streptococcal infection; high throughput chemical screening will be used to develop specific SK inhibitors as a potential new class of antibiotics for this important human infection. The PPG will continue to support 4 cores: (A) the Mouse Coagulation Laboratory, (B) the Genetics Core, (C) the Administrative Core, and (D) the Morphology Core. The PPG will aim to increase interaction and collaboration between individual project participants, as well as among the large number of other laboratories at the University of Michigan already engaged in research on coagulation, thrombosis and vascular disease. We anticipate that the overall program resulting from the combined efforts of all participants will significantly exceed the sum of the individual parts. Relevance to public health: Abnormalities in the control of blood clotting are a critical factor in a number of diseases, including heart attack and stroke (the leadings causes of death in the US), as well as several important infectious diseases. This Project will identify key genes in this system that should provide valuable new diagnostic tools as well as suggest novel approaches to treatment.

BIOMARKERS OF COAGULATION: What, when and where?

2010-08-02T15:18:26.204-04:00

Last month the coagulation community lost a dedicated colleague and a friend. JoAnn Carabello ran the special coagulation laboratory at Temple University. She was also responsible for organizing the annual Temple Coagulation Symposium, which was responsible for educating and updating so many technologists and clinicians on relevant coagulation topics. She will be missed. Please remember her in your prayers. This is dedicated to her.

BIOMARKERS OF COAGULATION: What, when and where?

What is a biomarker- a biomarker is defined as a parameter that is measureable, reflects disease, is reproducible and will be affected by treatment. Biomarkers are biological indicators that have been associated with disease. Certain markers of inflammation, blood clotting, and blood vessel function have been associated with risk of cardiovascular disease, stroke and death. For example, markers of inflammation include: C-reactive protein (CRP), fibrinogen and white blood cells (WBC), with CRP being the most sensitive. It is also known that fibrinogen is an independent risk marker for an MI, higher than elevated cholesterol. It must be determined that the elevated fibrinogen is not a result of an acute phase reactant and is truly a hereditary persistence of an elevated fibrinogen.

Other biomarkers of coagulation include:
Prothrombin time (PT), activated partial thromboplastin time (APTT), and D-dimer, protein C, protein S, and antithrombin, IL-6 , prothrombin fragment F1.2 (F1.2), thrombin–antithrombin complex (TAT), plasminogen activator inhibitor (PAI)-1, thrombin activatable fibrinolysis inhibitor (TAFI), α2-antiplasmin (α2-AP), plasminogen, and soluble thrombomodulin (sTM).

Biomarkers and Sepsis:
In patients with sepsis the coagulopathy is more reflective of activation of coagulation and thrombin generation than of impaired fibrinolysis. Thrombin generation markers TAT and F1.2 are present in most patients versus TAFI, which is an acute phase reactant. The more severe septic patients (higher APACHE scores) may also prevent with elevated baseline D-dimers, PT, APTT, PAI-1, sTM versus decreased PC, PS and AT. A global improvement in coagulation markers has been observed in survivors as compared with nonsurvivors. Markers of ongoing thrombin generation, TAT, F1.2, and D-dimer, improved more rapidly in survivors than in nonsurvivors over time. Of the 13 coagulation markers that correlated with disease severity, PT may be the most clinically useful. Consumption and depletion of endogenous hemostasis factors occurs frequently in patients with severe sepsis, as shown in this and other studies, and may occur before the clinical diagnosis of the first sepsis-associated acute organ dysfunction. The PT is a marker that is easily and readily measurable in most clinical settings.

Coagulation Biomarkers and HIV:
More than 30 million people are infected with the human immunodeficiency virus(HIV), the virus that causes acquired immunodeficiency syndrome (AIDs, HIV infects and destroys immune system cells (including CD4 cells, a type of lymphocyte). The immune system becomes so damaged that HIV-infected individuals begin to succumb to “opportunistic” infections (for example, bacterial pneumonia) and cancers (in particular, Karposi sarcoma) that the immune system would normally prevent. HIV infections cannot be cured but antiretroviral therapy (ART)—combinations of powerful antiretroviral drugs—can keep them in check, so many HIV positive people now have substantially improved life expectancy.
A study looked at the frozen blood samples of patients who had died from HIV. Levels of proteins that indicate the presence of inflammation or increased coagulation (biomarkers) were measured. In this study, an increased risk of death was associated with higher levels at study entry of the inflammation biomarkers high-sensitivity C-reactive protein (hsCRP) and interleukin 6 (IL-6) and of the coagulation biomarker D-dimer. The risk of death among people with hsCRP values in the highest quarter of measured values was twice that among people with hsCRP values in the lowest quarter with an odds ratio of 2. For IL-6 and D-dimer, the equivalent odds ratios were 8.3 and 12.4, respectively. Increases in hsCRP, IL-6 and D-dimer after study entries were associated with an increased risk of death. The findings suggest that HIV-induced activation of inflammation and coagulation increases the risk of death among HIV-positive patients and that interrupting ART further increases this risk, possibly by increasing IL-6 and D-dimer levels. These findings suggest that the development of therapies that reduce the effect that HIV replication has on inflammation and blood coagulation, or that reduce IL-6 and D-dimer levels, might extend the life-expectancy of HIV-positive people.

Coagulation Biomarkers and Cardiovascular Disease:
Inflammation plays a major role in the development and progression of atherosclerosis, including plaque rupture which initiates coronary thrombosis and myocardial infarction (MI) .Activated coagulation, endothelial dysfunction and fibrinolysis have also been associated with risk of coronary heart disease (CHD)
The term CHD covers all forms of atherosclerotic disease of the coronary arteries with MI/coronary death as the most serious manifestation and with angina (unstable or stable) as a specific symptom complex indicating myocardial ischemia. Many studies have shown independent associations between CHD events (MI and CHD death) and C-reactive protein (CRP), interleukin-6 (IL-6), fibrinogen, von Willebrand factor (VWF), fibrin D-dimer and tissue plasminogen activator antigen (t-PA) in both middle-aged and older populations.
The major pathophysiological difference distinguishing MI and other acute coronary syndromes from uncomplicated stable angina pectoris is the rupture of an atherosclerotic plaque with subsequent thrombosis formation, which causes acute coronary events.
A study looked at the relationship between inflammatory and hemostatic biomarkers including CRP, IL-6 plasma viscosity and several markers of activated coagulation, fibrinolysis and endothelial dysfunction [fibrinogen; coagulation factors VII, VIII, and IX; fibrin D-dimer, t-PA antigen, VWF activated partial thromboplastin time (APTT) and activated protein C (APC) ratio]. The results concluded that this relationship differed between older men aged 60–79 years who develop (i) incident MI or CHD death and (ii) incident stable angina, uncomplicated by MI or CHD death. Circulating biomarkers of inflammation and hemostasis are associated with incident MI/CHD death but not incident angina uncomplicated by MI or CHD death in older men.

Inflammatory Bowel Disease and Coagulation BIomarkers
Inflammatory bowel disease (IBD) as a chronic inflammatory condition characterized by a hypercoagulable state and prothrombotic conditions. A study evaluated the abnormalities in coagulation and fibrinolysis status in patients with IBD, and to analyze parameters of altered coagulation and fibrinolysis status which can correlated with and predict inflammatory parameters of disease activity. IBD patients were compared with healthy controls for coagulation and fibrinolysis status. Associations between altered coagulation and fibrinolysis status stratified by gender and inflammatory parameters were analyzed. The mean levels of platelet, platelet distribution width, prothrombin time, fibrinogen, activated partial thromboplastin time were significantly higher in IBD patients than in healthy controls (all P<0.05). Mean platelet volume was lower in male patients with IBD than in healthy controls (P<0.01). Furthermore, multiple linear regression indicated that fibrinogen was an independent predictor of ESR (β=1.316, P=<0.001) and CRP (β=1.233, P=0.015) in male patients with active ulcerative colitis. Platelet (β=0.436, P=0.037) and prothrombin time (β=0.810, P=<0.001) were predictors of Crohn's Disease Activity Index in female patients with Crohn's disease. This study provides characteristics on altered coagulation and fibrinolysis status in active IBD patients. The data suggest that in IBD patients, abnormalities in coagulation and fibrinolysis status were associated with disease activity. Fibrinogen, platelet and prothrombin time were predictors of inflammation.

Conclusion:
Biomarkers play an important role in the diagnosis and management of disease. Identifying coagulation biomarkers and their presence in certain disease states may aid in predicting treatment modalities and rate of survival.

WORLD CUP COAGULATION: JULY 2010

2010-07-08T00:00:00.000-04:00

The 2010 FIFA World Cup is the 19th FIFA World Cup, the premier international association football tournament, which is being held in South Africa. It is the first time the finals of the tournament have been staged in an African host nation. Held every four years since 1930, the previous tournament was held in Germany, while the 2014 finals will be hosted by Brazil. The finals tournament sees 736 players representing 32 teams compete for the World Cup trophy in games held in ten stadiums across South Africa. The qualifying teams were selected through a qualification process that began in August 2007. With a pool of entrants comprising 204 of the 208 FIFA national teams, the 2010 World Cup shares with the 2008 Summer Olympics the record for most competing nations in a sporting event.

So this got me thinking, how is coagulation performed throughout the world? We only know what we are used to and what we know. Different healthcare systems as well as disease prevalence can drive how and what testing is performed. Different countries have limited resources and their diagnostic and treatment testing may be restrictive.

The Coagulation Testing Market: US, Europe, Japan

The growing cost-containment pressures in major industrialized nations, coupled with continued technological advances in chromogenic substrates, monoclonal antibodies, immunoassays, molecular diagnostics, computers and laboratory automation will radically change the coagulation testing practice during the next ten years. New specific and sensitive markers of hemostasis will be increasingly used on automated instrumentation. Coagulation testing will also become more standardized, offering opportunities for quality control products and services.

The following processes may turn out to be the major impacts on global coagulation testing:

-     The status of POC and decentralization in lab medicine
-     Miniaturization and molecularization of devices
-     Geo-economic and demographic influences on markets
-     Non-invasive sampling
-     Greatly expanded market data on PGx and SNP testing
-     More information about more immunoassays
-     World blood banking developments

Coagulation Laboratories in the United States:

US laboratories were questioned to determine which test out of 29 coagulation tests, what were the most frequent tests performed. The 5 most commonly performed coagulation laboratory tests were PT, 100%; with an estimated annual test volume of 65.9 million, and that the second most commonly performed coagulation laboratory test was APTT, with an estimated annual test volume of 45.6 million. Those tests are followed by the bleeding time, 90.0%; fibrinogen, 69.2%; and D-dimer 56.5%. A significantly (P < .05) greater proportion of the large hospitals performed each of the 29 surveyed tests in house compared with the small hospitals.

COAGULATION TESTING IN EUROPE:

in Europe, coagulation monitoring is moving towards point-of-care (POC) testing, which ensures quick generation of results. This may provide better patient care. POC coagulation tests have made life simpler, as they enable patients to be well informed about the disease state. The number of patients undergoing anticoagulant therapy have increased. It is very important to closely monitor the PT and the INR. POC instruments are showing up in emergency units, outpatient wards as well as patient homes to enable self-testing. This reduces the burden on hospitals as well as physician offices and prevents the drastic effects of anticoagulants and other bleeding disorders.

Clearly, the growth of POC is generated by quick turn-around-times and easy-to-use technology. At the same time, laboratory tests continue to be preferred in some European countries since they are cost effective and well reimbursed when compared to POC tests. Presently, laboratory tests generate delayed results and do not support fast treatment processes. Hence, there is a need to produce timely results that will result in improved patient care. With an increasing number of tests moving towards POC, the volume of tests in laboratories is adversely affected. To overcome this challenge, laboratories are slowly adopting high-volume analyzers to perform special coagulation tests, which have not yet reached the POC market.

An accurate detection of Lupus Anticoagulant is of utmost importance in patients suspected of an antiphospholipid syndrome. Even though guidelines have been established by ISTH, testing practices are varied. Based on a European proficient test (ECAT)). Fifty nine laboratories participating in this trial were asked to test for the presence of a LA in the 3 samples submitted. The most frequently used screening tests were the aPTT and the dRVVT. Additional testing included the dilute prothrombin time (dPT), the dilute Russell Viper Venom time (dRVVT) and the Kaolin Clotting time. The present study also shows that many laboratories still rely on poorly responsive screening assays for their LA tests. Other laboratories rely on sensitive and more specific integrated test systems based on a sensitive screening assay with a low phospholipid content and a confirmatory test employing high phospholipid concentrations. The most used integrated system was dRVVT based. However, also here the LA responsiveness was largely reagent dependent. In conclusion, many laboratories still rely on poorly responsive screening assays by which weakly positive LA samples are misdiagnosed.Bottom of Form

Coagulation testing in India:

In the year 2000 an external quality assessment scheme (EQAS) was implemented in India to 25 laboratories. Their participation is critical for ensuring acceptable laboratory performance. Participation in such programs is uncommon for laboratories performing tests of hemostasis in developing countries. There are several reasons, including lack of awareness of its significance, absence of locally administered and easily accessible programs, and costs associated with some of the international schemes. This was converted to a national program in 2003, in association with the Indian Society of Haematology and Transfusion Medicine. Local manufacture of survey samples began in 2004, along with analysis of results. Currently, more than 100 laboratories are registered in the program. They receive samples three times a year for the following tests: prothrombin time, activated partial thromboplastin time, and factor assays. Some surveys also include samples for fibrinogen and von Willebrand factor assays. In recent surveys, 60 to 95% of laboratories had their clotting times and 57 to 77% of laboratories had their factor assays within consensus.

SNAKE BITES!

A problem that occurs in India are snake bites. This has a major impact on the coagulation system. Not to mention the impact on the nervous system!! Experts in Indian snakebites developed a protocol specifically designed for snakebite treatment in India. A training program was implemented in Midnapore Medical College in West Bengal, India, under the direction of the Health Minister to train care providers in the new protocol. After training, data were collected for 839 snakebite victims over a 12-month period and included epidemiological data, ASV volumes administered, and mortality. The results were collated and compared with results calculated from 780 snakebite victims treated during the 12-month period before implementation of the protocol. Treatment prior to protocol implementation was based on knowledge gained by the care providers from western and forensic medicine textbooks.

About 80% of the venomous snakebites in India come from the saw scaled viper Echis carinatus, cobra, krait and Russel’s viper. Recognize these names? Look at your coagulation reagents- snake venom are used in many of the testing. On admission, and at relevant intervals afterwards, doctors will probably check on how well the blood is clotting. The most common tests are simple- the bleeding time, and whole blood clotting time. Sometimes tests like PT and aPTT, kidney function (urine output, blood urea, creatinine and electrolyte levels), and of course the vital signs – pulse, breathing, temperature, blood pressure and the amount of oxygen in the blood (pO2). They may also keep tabs on the patient’s haemoglobin, blood cell counts, and perhaps the blood gases. A minimal amount of testing is conducted.

What happens in Malaria?

Different parameters of fibrinolytic systems like t-PA, PAI, D-dimer, and inhibitors of blood coagulation, i.e., protein C (PC), protein S(PS), and antithrombin III (AT-III), have been studied in cases of acute malaria due to Plasmodium falciparum and plasmodium vivax infection, and these patients were followed up. It was observed that the plasma PAI-1 was very high in cases of P. falciparum malaria infection as compared to normal controls and P. vivax infection. The changes in complicated cases of P. falciparum were remarkable as compared to uncomplicated ones. The PC, PS, and AT-III levels were also low in P. falciparum, particularly so in complicated cases, and were normal in P. vivax infection. The factor VIII R:Ag levels were invariably high in acute malaria. On follow-up of some of these cases the values came back to normal after the antiparasite treatment. The monocyte procoagulant activity was found to be significantly higher in P. falciparum infection as compared to that of P. vivax infection. All these findings therefore contribute towards the production of a hypercoagulable state in P. falciparum infection and partly explain the complications of P. falciparum infection like cerebral malaria.

What about Dengue Fever?

The pathophysiological basis of hemorrhage in dengue infections remains poorly understood. In a large prospective study of 167 Vietnamese children demonstrated only a minor prolongation of the PT and APTT, with severe depression of plasma fibrinogen as well as Protein C, S and AT. While increases were seen in thrombomodulin, tissue factor, and plasminogen activator inhibitor (PAI-!). Increased thrombomodulin suggests endothelial activation correlated with increased shock severity, PAI-1 levels correlated bleeding severity. Dengue can directly activate plasminogen resulting in activating fibrinolysis, degrading fibrinogen directly and prompting secondary activation of procoagulant mechanisms.

I have always said, coagulation is a puzzle, and you diagnosis and treat by putting the pieces together. Expect the unexpected! Understand how different components are affected by different disease states and how testing is impacted by what hospital have available to them. More importantly understand, while things may be different globally, good patient care is something everyone strives for!

Warfarin and what affects It.

2010-06-04T00:00:00.000-04:00

Warfarin: Do we have options?

Do you know how warfarin was made? In the 1950s, some ranchers in Wisconsin were upset that their cattle were bleeding to death. The ranchers sought help at the University of Wisconsin. Researchers discovered that the cattle were bleeding to death as a result of eating spoiled sweet clover. They were able to identify and isolate the substance in the spoiled sweet clover that was causing the excessive bleeding. This substance was a derivative warfarin. It has been modified and standardised in a laboratory to make it safe for use by patients.

Warfarin or coumadin has a half life of between 20-60 hours. The response of oral anticoagulation is very varied, it may be enhanced in obstructive jaundice, hepatitis and cirrhosis due to reduced vitamin K absorption, while foods high in Vitamin K (Beef, pork, green leafy vegetables) will decrease the efficacy of warfarin. Additionally, many medications can decrease the risk of anticoagulation (anti-thyroid drugs, barbiturates, estrogens) and others increase the risk of hemorrhage. Inhibition of coagulation factors occurs 12 to 24 hours after oral administration, but antithrombotic effects may not occur until 2 to 7 days after initiation of therapy.

Coumadin has been the only option for oral anticoagulation. This is a long term or life time therapy that is problematic for many people. It is easy to monitor, but most people do not fall into the therapeutic range, requiring constant dose adjustments. The International Normalized Ratio (INR) helped to standardize results regardless of the laboratory instrument reagent combination, helping to achieve the target range of 2-3.

Warfarin has a narrow therapeutic range
Accounts for 15% of all severe adverse effects
Doses from 1-10 mg/daily
Different ethnicities display varying sensitivity, about 35% of population have genetic variants
Cause warfarin to be metabolized less efficiently
The FDA has labeled warfarin concerning role of genetics in dosing
Genes identified as CYP450 2C9 and VKORC1 (RUO)

One of the downsides of this medication is that it interacts with many different foods and beverages. While there is no specific diet for people taking Coumadin, there are general guidelines that should be followed to help improve the safety of the medication.

The foods that you eat can also affect the way this medicine affects your body. This is a result of the effects of the anticoagulant which depend on the amount of vitamin K in your body. It is best to have the same amount of vitamin K in your body every day. Some multiple vitamins and some nutrition supplements contain vitamin K. Vitamin K is also present in green, leafy vegetables (such as broccoli, cabbage, collard greens, kale, lettuce, and spinach) and some vegetable oils.

There are 4 food and lifestyle interactions with warfarin which include:

Coumadin and Alcohol (Ethanol)

Enhanced hypoprothrombinemic response to warfarin has been reported in patients with acute alcohol intoxication and/or liver disease. The proposed mechanisms are inhibition of warfarin metabolism and decreased synthesis of clotting factors. Binge drinking may exacerbate liver impairment and its metabolic ability in patients with liver dysfunction. The risk of bleeding may be increased. Conversely, reductions in INR/PT have also been reported in chronic alcoholics with liver disease. The proposed mechanism is that continual drinking of large amounts of alcohol induces the hepatic metabolism of anticoagulants. Effects are highly variable and significant INR/PT fluctuations are possible.

Moderate Food Interaction

Vitamin K may antagonize the hypoprothrombinemic effect of oral anticoagulants. The intake of vitamin K through supplements or diet can reverse the action of oral anticoagulants. Resistance to oral anticoagulants has been associated with consumption of foods high in vitamin K content which include green, leafy vegetables, avocados, soy beans, and green tea. Lesser amounts are found in liver, bacon, cheese, butter, cauliflower, and coffee. Snack foods containing the fat substitute, olestra, are fortified with 80 mcg of vitamin K per each one ounce serving so as to offset any depletion of vitamin K that may occur due to olestra interference with its absorption. Large amounts of mango has been associated with enhanced effects of warfarin. The mechanism of interaction is unknown but may be related to the vitamin A content, which may inhibit metabolism of warfarin. discontinuation of mango ingestion for 2 weeks.
Warfarin and cranberry juice can result in changes in the INR and/or bleeding complications. The mechanism is unknown but may involve alterations in warfarin metabolism induced by flavonoids contained in cranberry juice. It is not known if variations in the constituents of different brands of cranberry juice may affect the potential for drug interactions.

Soy protein in the form of soy milk was thought to be responsible for a case of possible warfarin antagonism in an elderly male stabilized on warfarin. The exact mechanism of interaction is unknown, as soy milk contains only trace amounts of vitamin K. Subtherapeutic INR values were observed approximately 4 weeks after the patient began consuming soy milk daily for the treatment of hypertriglyceridemia. No other changes in diet or medications were noted during this time. The patient's INR returned to normal following discontinuation of the soy milk with no other intervention.

An interaction with chewing tobacco was suspected in a case of warfarin therapy failure in a young male who was treated with up to 25 to 30 mg/day for 4.5 years. The inability to achieve adequate INR values led to eventual discontinuation of the chewing tobacco, which resulted in an INR increase from 1.1 to 2.3 in six days. The authors attributed the interaction to the relatively high vitamin K content in smokeless tobacco.

DRUGS:

A total of 680 drugs (3290 brand and generic names) are known to interact with Coumadin (warfarin).

High Blood Pressure (Hypertension)
The use of OAC is contraindicated in patients with malignant or severe, uncontrolled hypertension since they may be at increased risk for cerebral hemorrhage. Therapy with oral anticoagulants should be administered cautiously in patients with moderate hypertension.

High Cholesterol (Hyperlipoproteinemia, Hypertriglyceridemia, Sitosterolemia)

A decreased OAC response may be seen in patients with edema, hereditary coumarin resistance, hyperlipidemia, hypothyroidism, or nephrotic syndrome may exhibit lower than expected hypoprothrombinemic response. Thus, more frequent laboratory (PT/INR) monitoring and dosage adjustment of anticoagulant may be required based on changes in the patient's condition.

Potentiating the Effect of Warfarin

Many antibiotics are reported to potentiate the effect of warfarin. Some examples include: cotrimoxazole, erythromycin, isoniazid, fluconazole, miconazole, and metronidazole. Antibiotics with a lower risk of enhancing warfarin are:mciprofloxacin, itraconazole, and tetracycline Several cardiac drugs had highly probable evidence that they potentiated the effect of warfarin: These included amiodarone, clofibrate, propafenone, propranolol, and sulfinpyrazone. Sulfinpyrazone's effect was biphasic, which means that an initial potentiation of the warfarin anticoagulant effect was noted, followed by inhibition of the effect. Quinidine, simvastatin, and acetylsalicylic acid had probable evidence that they potentiated warfarin Among the anti-inflammatory or analgesic drugs, phenylbutazone, piroxicam, acetylsalicylic acid, acetaminophen, and dextropropoxyphene had highly probable or probable evidence ..
Drugs Inhibiting the Effect of Warfarin
Fewer drugs inhibited the effect of warfarin, Highly probable evidence was reported for nafcillin, rifampin, griseofulvin, cholestyramine, barbiturates, carbamazepine, chlordiazepoxide, sucralfate, high vitamn K content in enteral feeds or in the diet, and large amounts of avocado.. Probable evidence was reported with dicloxacillin. The reported interactions of four other drugs in addition to the consumption of large amounts of broccoli were considered possible evidence
New Oral anticoagulants on the horizon:

The new class of oral anticoagulants has been developed to pinpoint a specific target for controlling the clotting cascade with maximum efficacy and minimum inconvenience.
Nattokinase is a soybean food content, produced by the bacterium Bacillus subtilis (natto) during fermentation of soybeans. It is a 275 amino acid peptide. It is also called "Subtilisin NAT". It is said to have similar clot-dissolving abilities as does plasmin. Nattokinase may have some potential to protect from blood clots. However, it has not been appropriately studied in humans. Nattokinase is not a substitute for warfarin. The FDA concluded in 2002 that there is no "adequate basis to conclude that NKCP [Natto extract] containing 0.01 % of Nattokinase enzyme is reasonably expected to be safe" and that "there is inadequate information to provide reasonable assurance that such ingredient does not present a significant or unreasonable risk of illness or injury". Furthermore, the FDA has warned that unsubstantiated and illegal claims are being published about the effectiveness of NSK-SD Nattokinase .
PRADAX™ (dabigatran etexilate), is a novel oral direct thrombin inhibitor (DTI) for the prevention of venous thromboembolism (VTE) in adult patients who have undergone elective total hip or total knee replacement surgery. Oral direct thrombin inhibitor binds directly to thrombin, blocking interaction with its substrates, thereby preventing the conversion of fibrinogen to fibrin and forming a crosslinked blood clot or preventing thrombin generation, and thus producing a state of anticoagulation. It is given once-daily and does not require extensive anticoagulation monitoring. PRADAX™ is taken orally and no injections are needed. PRADAX™ does not interact with food and has a low potential for drug-drug interactions. They offer the advantages of fixed dosing and preclude the need for routine blood monitoring, making them very attractive alternatives to warfarin
Trials are ongoing to establish the efficacy and safety of dibigatran versus warfarin.
Another agent close to approval is the Factor Xa inhibitor apixaban. Apixaban suffered a setback last year when results of the ADVANCE-1 trial were reported. Apixaban did not meet its noninferiority goal, because of an unexpectedly low rate of events in the trial of 3,195 patients.
Despite the promising outlook for the Factor Xa and thrombin inhibitors in the coming years, the drugs are not without limitations. They do not require the INR monitoring that limits warfarin use, but patients with severe renal insufficiencies will most likely not be candidates for the new agents. There are other potential problems as well. Cost may be a factor, warfarin is a cheap drug.
These new oral thrombin and factor Xa inhibitors have demonstrated rapid onset of action and predictable pharmacokinetics.. These anticoagulants also have a low propensity to clinically relevant drug-drug interactions. They potentially eliminate the need to provide additional follow up services to those on extended prophylaxis who are either unable or reluctant to self inject their low molecular weight heparin prophylaxis. US Food and Drug Administration approval of dabigatran is pending.

Animals and Coagulation

2010-05-12T11:52:50.485-04:00

Lions and Tigers and Bears, oh MY!

When I was a new tech, we had gotten a blood sample on a beached whale. We performed a CBC and differential, it was loaded with eosinophils. I remember the conversation when we gave the results, we were asked if that was normal. Of course we had no idea what was normal for a whale. Ever wonder what coagulation results are on animals? Multispecies testing is fairly widespread. Animals are used in safety testing for drugs, so it is important to understand what is normal to know what is abnormal.

It has been well established that coagulation data differ based on instrumentation and reagents. Animal coagulation values differ from human values, even though they still have some of the same coagulation disorders.

For example:
Dogs present with hemophilia, FVII def., vWF disease, and prekallikrein def. Cats present with hemophilia, and FXII def. While Horses can have hemophilia and pigs can have a Lupus anticoagulant.

Some of the disorders are manifested differently, for example bleeding occurs in animals with a prekallikrein deficiency, but not in humans.

Animals in relation to humans have shorter clotting times for the PT and APTT due to having higher factor concentrations.

Assay	Dog	Cat	Human
PT(sec)	7.5-10.5	8.0-12.0	10.0-15.0
aPTTa(sec)	14.4-19.0	14.5-22.0	26.0-40.0

Coagulation factors are produced in the liver. Concentrations of factors are at different levels.

Assay	Dog	Cat	Rat	Human
FII	100%	100%	70%	50%-150%
FV	900%	500%	140%	50%-150%
FVII	300%	300%	160%	50%-150%
FVIII	800%	800%	400%	50%-150%
FIX	200%	200%	40%	50%-150%
FX	100%	60%	40%	50%-150%
vWF	65%	50%	50%	70%-140%
ProC	20%		2%	50%-150%

The APTT or PT (or both) are prolonged in 50-66% of dogs with liver disease, meaning that the factor activity is <30% of normal. Coagulation tests are often performed before liver biopsy. Severe hepatic diseases can also lead to disseminated intravascular coagulation. Fibrinogen, an acute phase reactant, and von Willebrand’s factor, which is produced extrahepatically, can be increased in liver disease.

Factor Deficiencies:
Factor VIII deficiency and factor IX deficiency are severe defects usually recognized as spontaneous hemorrhages before 10 weeks of age. Both are sex-linked recessive defects; males are clinically affected, whereas females are carriers and clinically normal.

Von Willebrand disease is an autosomal defect , and both males and females are affected clinically. It is the most common inherited coagulation defect with high prevalence in many breeds.
Factor X deficiency has been detected mostly in American cocker spaniels. It manifests as stillborn puppies or neonatal deaths related to internal hemorrhage. It is an autosomal defect with a severe defect in homozygotes, whereas heterozygotes may be clinically normal or have only a mild bleeding tendency.
Factor XI deficiency is a rare defect described in English springer spaniels and a few dogs of other breeds. This autosomal defect is mild with bleeding after surgery or injury.
Factor XII deficiency is fairly common in cats but is rarely detected because no bleeding tendency is associated with the defect.

In Disseminated intravascular coagulation (DIC) the syndrome is characterized by massive activation and consumption of coagulation proteins, fibrinolytic proteins, and platelets. This is the same in animals. Death is caused by extensive microthrombosis or circulatory failure, leading to single or multiple organ failure. If the animal survives the acute DIC event, a chronic form of DIC can exist. Compensatory production of coagulation proteins and platelets by the liver and bone marrow, respectively, can alter the results of coagulation screening tests such that they may be within reference ranges or even shortened, and platelet concentrations may be normal. However, DIC can usually be identified by the presence of at least 3 abnormal coagulation test results. Horses, even in fulminant DIC, most often have hyperfibrinogenemia because their liver can produce much fibrinogen. Treatment should be directed toward correcting the underlying problem. Supportive care is essential. Administration of balanced electrolyte solutions to maintain effective circulating volume is imperative. Administration of heparin is controversial and should be accompanied by administration of plasma to assure adequate antithrombin III activity.

Platelets:
Platelet disorders affect platelet adhesion, aggregation or secretion. Horse platelets are the most responsive to ADP, whereas sheep are the most responsive to Ristocetin. Different responses suggest:

Structural differences which include the composition of the platelet membrane as well as specific receptors to certain agonists.
Agonists that activate pathways with distinct signaling.

Regarding platelet activating factor (PAF) animal species platelets react distinctly different. It appears that sheep and humans have a similar specific membrane receptor accounting for them having a similar response in comparison to other species. Platelet aggregation has been found to be steeper in rats compared to does dependent assays of other species. Bovine platelets respond only to ADP, collagen and thrombin and responses were significantly lower than human responses. While canines are significantly higher than humans.

Canine thrombopathia has been described in Basset Hounds. Affected dogs have epistaxis, petechiation, and gingival bleeding. Results of studies suggest that inheritance is autosomal with variable penetrance. Platelets have abnormal fibrinogen receptor exposure and impaired dense granule release. Basset Hounds with mucosal bleeding and petechiation and normal concentrations of platelets and von Willebrand’s factor should be suspected of having thrombopathia.

Bovine thrombopathia is an autosomally inherited platelet function defect seen in Simmental cattle. Bleeding can be mild to severe in affected cattle and is exacerbated by trauma or surgery. Platelets have impaired aggregation responses.

Thrombasthenic thrombopathia has been diagnosed in Otterhounds. It is autosomally transmitted. Affected dogs have prolonged bleeding times and form hematomas at sites of venipuncture or injury. Numerous (30-80% of all platelets), bizarre, giant platelets are seen on blood smears. Membrane glycoproteins II and III are reduced. Blood from affected dogs does not have normal clot retraction, and the platelets do not aggregate normally after stimulation with ADP, collagen, or thrombin.

Von Willebrand’s disease is caused by a defective or deficient von Willebrand’s factor (also called Factor VIII-related antigen). It is the most common inherited bleeding disorder in dogs (reported in nearly all breeds and in mixed breeds) and has also been reported in cats, rabbits, and pigs. The disorder is relatively frequent (10-70% prevalence) in several breeds of dogs: Doberman Pinschers, German Shepherds, Golden Retrievers, Miniature Schnauzers, Pembroke Welsh Corgis, Shetland Sheepdogs, Basset Hounds, Scottish Terriers, Standard Poodles, and Standard Manchester Terriers. Two modes of inheritance are known. In the less common autosomal recessive pattern of inheritance, homozygosity is usually fatal, and heterozygosity results in asymptomatic carriers. Affected animals may have gingival bleeding, epistaxis, and hematuria. Some puppies may bleed excessively only after injection, venipuncture, or surgery, such as tail docking, ear cropping, and dewclaw removal.

So, as you can see many disorders for animals are similar to humans but may manifest itself differently in animals. My dog had lupus when it was a puppy, it is just a self limiting viral disorder, she had lesions in her ears. She is 13 now and has never had any reoccurrence of the disorder. Understanding how disorders present and how platelets react in animals is important in treatment and in performing studies involving multispecies.

KIDDIE COAGULATION

2010-04-05T00:00:00.000-04:00

Kiddie Coagulation
A Case Study Approach

Coagulation is complicated enough considering the issues with pre-analytical variables, interfering substances and reagent sensitivity. When you have a pediatric sample the puzzle becomes even more difficult. One of the largest considerations is that most tests in the laboratory are based on adult ranges.
How do you interpret pediatric results? How do you treat pediatric patients? Establishing normal ranges on apparently healthy children is a daunting task.
To obtain that patient population is very difficult. It is also difficult to obtain that population to determine dosing of anticoagulants and administering products.
Being a pediatric hematologist requires the expertise to be able to look at each piece of the puzzle before putting it in place to make a complete picture.

A study using retrospective chart review including variables of age and gender in pediatric patients have demonstrated that the most significant assessment of coagulopathies is the presence of symptoms and a positive family history. (SHAH, CLINICAL PEDIATRICS) One of the first studies that looked at developmental hemostasis was conducted by Maureen Andrews MD from Children's Hospital in McMasters University in which age specific ranges were determined for neonates, infants and ranges up to 18 years of age for coagulation parameters demonstrating the differences between adults and children. Reasons for this include difference in the:
1. Concentrations of components in the hemostatic system.
2. Turnover rate of components in the coagulation cascade
3. Rate of synthesis
4. Ability of the coagulation and fibrinolytic systems to generate and
Regulate plasmin and thrombin. (ANDREWS 1989)
Since then a few other studies have been conducted using new coagulation reagents and analyzers. One study looked at 902 healthy children from the age range of 7-17 and found differences in the PT, F VIII and von Willebrand factor versus adult ranges, as well as age dependent differences in FIX and FXI. (RODGERS, CLINICAL CHEM 2005.)

It was also noted that the coagulation system of children has a reduced incidence of thrombosis without an increased risk of bleeding. This supports the concept that patients with thrombophilia usually do not present until early adulthood. Also children rarely present with thrombosis when undergoing prolonged stasis or following surgery. (Mongale et al) However, coagulation parameters of Antithrombin, Protein C and S are reduced in neonates as well as throughout childhood.

Understanding all of these nuances relating to pediatric coagulation samples as well as working with very small volumes all are needed when looking at results and reflexive testing.
Case 1:

A 7 year old boy with chronic sore throats and ear infections is scheduled for a tonsillectomy and removal of adenoids. His coagulation results are as follows:
PT = 19.2 (10.5-12.5 seconds)
APTT= 40.1 (24.5-34.5 seconds)

Coagulation factors are made in the liver, at the age of 7 years, in the absence of disease, the level of factor production should be comparable to an adult.
These levels are clearly prolonged. The next step would be a mixing study.

PT APTT
Result 19.2 40.1
PNP 12.1 30.0
1:1 mix 18,1 38.8

Whatever range you are looking at, this clearly doesn't correct, so it appears this patient has an inhibitor. It is important to understand the type of reagent you are using, is it lupus sensitive or insensitive and what does that mean? In the laboratory, a lupus anticoagulant manifest as an antibody to phospholipids, which constitutes a large portion of coagulation reagents? A reagent that is lupus insensitive is made with a high concentration of phospholipids; this will overtake or mask the antibody. .A sensitive LA reagent, has a lower concentration of phospholipids that allows the antibody to manifest, resulting in a prolongation of the clotting time. This mostly occurs in the APTT reagents, since PT reagents generally have high concentration of phospholipids. Occasionally an inhibitor can prolong the PT. Clinically, if the patient was bleeding, you would look at specific factor inhibitors. This test was done as a pre-operative screen, so now what is next? A dilute Russell Viper Venom test (DRVVT) which alters the concentration of phospholipids and activated the coagulation cascade at Factor X is performed. In this case the results are normal.
Non specific inhibitor, most likely caused by chronic infections, just a phenomenon, and this child should not bleed due to the presence of a non specific circulating inhibitor. Surgery will proceed.

CASE 2:

A 2 year old little girl is an inpatient in the ICU has had 3 prolonged APTTs results, but has no history of bleeding. The results are as follows:

APTT = 54.8 seconds (25.5-35.5 seconds)
``````` 66.1
60.9
The surgeon needs to determine the cause prior to performing a procedure, and requested a full prolonged APTT workup. The first question anyone should ask in a prolonged APTT on any in-patient is if that patient is receiving heparin. If that can not be confirmed, it may be easier to just run a thrombin time before investing time and money on in-patient factor assays. A thrombin time is the most sensitive test to residual heparin. . Most will be prolonged at 0.1U/ml of unfractionated heparin.

The results are as follows:
Thrombin time = 25.1 second (12-19 seconds)
Pooled Normal Plasma = 16.1 (11-18 seconds)

A thrombin time detects abnormalities to fibrinogen. We can use Reptilase to distinguish between heparin and a fibrinogen deficiency. Reptilase is expensive, and fibrinogen deficiencies are rare. Additionally, you would see a prolongation in the PT as well as a decreased fibrinogen. How can we get the clinician a good answer? You can use HEPZYME© (Siemens)This will neutralize the presence of heparin in the sample..
Place 0.5=1.- ml of plasma into HEPZYME for 16 minutes-
The sample can them be re-run for an APTT:
APTT = 33.0
Result is normal; no additional testing is required for factor assays- the samples were most likely drawn through a line.

CASE 3
A 1 month old needed to have an operation for a strangulated hernia. The baby was given unfractionated heparin for ease of use, and the ability to neutralize an overdose of heparin by administration of protamine sulfate. Patient was given heparin and an anti-Xa activity level was performed.
Results:
Anti-Xa 0.15U/ml anti-Xa, (0.3-0.7U/ml) another dose was given and the anti-Xa was 0.2U/ml heparin. Should more heparin be given or am I putting this patient at a risk for bleeding?
First the range is based on adult therapy levels, a target anti-Xa level of 0,3-0.7 may not be appropriate for a child. Additionally the binding site for heparin is antithrombin, and children have decreased levels of AT. It may be helpful to measure their Level-
1 month old AT = 48%-108%--ANDREWS
72-134% (Mongale)
May not be able to get an accurate result - AT is the binding site for heparin.

CASE 4
A 7 year old girl has a history of easy bruising and her mother has always had heavy menses, but so did her grandmother. The child seems to bleed longer when scraped. A PT and APTT were performed and were within normal limits. Since she had a positive bleeding history, factor assays were also performed and were all normal. A CBC result show normal platelet count, however primary hemostasis should be evaluated. A platelet aggregation would require 30 mls of blood and the child is very agitated and will not tolerate that large volume of blood nor could a bleeding time be performed as the mother refused due to scarring and trauma to the child.

A PFA 100 test was performed.
- ADP/Collagen= 59-120 seconds
- EPI/Collagen= 80-160 seconds

Children's Hospital in Toronto:

Healthy children with normal platelets and HGB =
- EPI/Collagen= 83-163
- ADP/Collagen=72-111
- Healthy neonates:
- EPI/Collagen=61-108
- ADP/Collagen=48-65

Screened with EPI/Collagen = 198
Ran ADP/Collagen = 180

Further investigation: vWF antigen and activity
Decreased, TYPE 1 vWF.

Conclusions:
Dealing with pediatric patients and their parents creates anxiety for everyone. Being able to serve that population and understand the limitations and the variability's that influence their results can improve patient care and outcomes.

REFERENCES:

1. Mongale, P., Barnes, C., Ignjatovic, v., Furmedge, J., et al, Developmental hemostasis; Impact for Clinical Laboratories, Thrombosis and Haemostasis, 2006, 95: 362-372.
2. Rodgers, G., Roberts, W., Flanders, M., Crist, R., Pediatric References Intervals for Seven Common Coagulation Tests, Clinical Chemistry, 2005, 9, 1738-1742,
3. Andrew, M., Paes, B., Johnston, M., Development of the Hemostatic System in the Neonate and Young Infant, The American Journal of Pediatric Hematology and Oncology, 1990, 1:95-104.

The Olympics of Coagulation-SCORING SYSTEMS: GOING FOR THE GOLD!

2010-03-03T00:00:00.000-05:00

While watching the Olympics, I noticed all of the complex scoring systems-
Remember when a 10 was the best you could do? Now they have video replays and different measures of excellence. I thought of the scoring that is used in health care. These systems are used to remove the subjectivity and grade a series of symptoms to help predict outcomes. Now that is golden!

SEVERITY of DISEASE:
APACHE score [a cute p hysiological a ssessment and c hronic h ealth e valuation] a widely-used method for assessing severity of illness in acutely ill patients in intensive care units, taking into account a variety of routine physiological parameters.APACHE II was designed to measure the severity of disease for adult patients (> 15 years of age) admitted to the ICU. .
This scoring system is used in many ways:
- Some procedures and some medicine is only given to patients with certain APACHE II score
- APACHE II score can be used to describe the morbidity of a patient when comparing the outcome with other patients.
- Predicted mortalities are averaged for groups of patients in order to specify the group's morbidity.
The point score is calculated from 12 routine measurements (such as blood pressure, body temperature, heart rate etc.) during the first 24 hours after admission, The resulting point score should always be interpreted in relation to the illness of the patient. In order to make this calculation of predicted mortality precise, the principal diagnosis leading to ICU admission was added as a category weight: the predicted mortality is computed based on the patient's APACHE II score and their principal diagnosis at admission.

Venous Thromboembolism

Symptoms of deep vein thrombosis (DVT) includes swelling, redness, or pain in the leg that has been present for 60 days or less. While symptoms of pulmonary embolism (PE) includes sudden onset of dyspnea, sudden deterioration of existing dyspnea(s), sudden onset of pleuritic chest pain without another apparent cause.

Patients who present with these symptoms in an outpatient setting can be scored to determine their pre-test probability of having a DVT or PE. Based on their score they will be classified as low, moderate or high probability. The scoring system takes several conditions into consideration.
Data collected for the Wells model for patients suspected of DVT :
- Malignancy (defined as patients with cancer who are receiving treatment or have received treatment in the last 6 months or patients with cancer that are receiving palliative care) (1.0 point)
- Entire leg swollen (1.0 point)
- Paralysis, paresis, or recent plaster immobilization of the lower extremities (1.0 point)
- Immobilized (defined as bed rest, except to access bathroom, for 3 or more consecutive days during the last 30 days) (1.0 point)
- Localized tenderness along the distribution of the deep venous system (1.0 point)
- Calf swelling at least 3 cm larger than on the asymptomatic side (measured 10 cm below the tibial tuberosity) (1.0 point)
- Pitting edema (greater in the symptomatic leg) (1.0 point)
- Previously documented deep-vein thrombosis (1.0 point)
- Collateral superficial veins (nonvaricose) (1.0 point)
- Alternate diagnosis as likely or more possible than that of DVT (-2.0 points)

The patients will be classified into low (0 or less points), moderate (1 or 2 points), or high (3 or more points) probability of DVT during data analysis.
Data collected for the Wells model for patients suspected of PE :
- Clinical signs and symptoms of DVT (3.0 points)
- Heart rate higher than 100 beats/minute (1.5 points)
- Immobilized (defined as bed rest, except to access bathroom, for 3 or more consecutive days during the last 30 days) (1.5 points)
- Previous DVT or PE diagnosis (1.5 points)
- Hemoptysis (1.0 points)
- Malignancy (defined as patients with cancer who are receiving treatment or have received treatment in the last 6 months or patients with cancer that are receiving palliative care) (1.0 points)
- PE as likely or more likely than an alternate diagnosis (3.0 points)

Patients will be classified into low (less than 2 points), moderate (2.0 -6.0 points), or high (higher than 6.0 points) probability of PE during data analysis.Assessment of clinical probability is now widely accepted as an important initial step in the diagnostic approach of pulmonary embolism (PE), as it allows the identification of patients at lower risk of the disease, who require a less extensive diagnostic workup. For instance, the association of a low clinical probability of PE and a low D-dimer concentration has been shown to safely rule out PE.

ISTH Scoring System for Disseminated Intravascular Coagulation
The ISTH recently proposed a scoring system for the diagnosis of DIC based on 4 laboratory parameters and the presence of a predisposing condition. The ISTH algorithm excludes patients without a recognized, predisposing condition at the time of evaluation.

The ISTH defines DIC as "an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes. It can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction".

DIC is caused by several clinical conditions. The degree is dependent on the etiology and the acuteness.

There are 2 types of DIC: (1) acute hemorrhagic DIC, and (2) chronic or overt DIC.

Acute Hemorrhagic DIC
Acute hemorrhagic DIC develops rapidly-from a few hours to a few days-with a high mortality rate of 54% to 67%. Each patient's presentation varies depending on the etiology and the body's ability to control this coagulopathy. Acute DIC is seen in infections. It is a frequent complication of severe sepsis with a high degree of mortality and multiorgan failure. The disseminated microthrombi decrease tissue oxygenation; this can cause organ infarction and necrosis. It is more likely to occur in conjunction with bacterial infection, in particular gram-negative sepsis. Other causes include obstetric complications, liver malignancy, tissue injury, and necrosis. Excess plasmin formation results in a hemorrhagic state. Patients will present with oozing from sites, large subcutaneous hematomas, deep tissue bleeding, and petechiae. It may occur in patient with endotoxemia, extensive tissue trauma, hypotension or shock, and massive surgery. Treatment depends on the symptoms.

Chronic or Overt DIC
Overt DIC is more difficult to diagnosis than acute DIC, and occurs in 10% to 20% of patients. This is a compensated DIC that occurs when fibrin clot formation and the accompanying fibrinolysis are in a steady state because the liver and bone marrow can compensate for the increased use of coagulation factors and platelets. Additionally, fibrin degradation products (FDPs) can still be cleared. Laboratory tests are minimally abnormal.
Chronic DIC is associated with malignancies, aortic aneurysms, and incomplete abortions. Ten percent to 15% of patients with tumors present with DIC, most likely due to tissue factor expressed on the surface of tumor cells. DIC is also seen in M3-acute promyelocytic leukemia , the granules of the promyelocytes release a thromboplastin-like substance that releases procoagulants.1 Survival in cancer patients with DIC is worse than in cancer patients who do not present with DIC. In these cases, if the underlying disease is appropriately treated, the stimulus for the DIC is removed.

The International Society of Thrombosis and Hemostasis developed a scoring system that improves accuracy in the diagnosis of DIC with 91% sensitivity and 97% specificity. The scoring system follows:
Platelet count: >100=0
<100 = 1
<50 = 2

Fibrin related marker no increase =0
Moderate increase = 2
Strong increase = 3

Prolonged PT <3 sec = 0
>3 sec = 1
>6 sec = 2

Fibrinogen >100 g/dL = 0
<100 g/dL = 1

If the sum is ≥5 the patient status is compatible with overt DIC. It appears to be valid once linked to coagulation activation markers such as the D-dimer and the consumption of inhibitors.

Final Score:
All of these scoring systems have been designed to enhance diagnosis and treatment of patients. They have been developed in relation to outcomes of patients, making them an invaluable tool for clinicians. So, in many cases, it isn't the high score that always wins- but it is the most accurate score that gets the gold!

COAGULATION: DID YOU KNOW THAT?

2010-01-31T00:00:00.000-05:00

I admit it, I am a bit of a trivia nerd, I like knowing things, I am a pretty good jeopardy player (except for American Presidents and Greek mythology).
So it stands to reason, I am also a trivia nerd in coagulation- I like finding old textbooks that have different theories. I feel like if you know where things came from, you have a better understanding of where we are today. So, on the lighter side, this month, I will share with you some coagulation trivia, hey you never know when Alex Tribec might say our category tonight is Coagulation Trivia- you will be ready for the Daily Double!

Most people know that Hippocrates (460-377BC) was the Father of Medicine, (the Hippocratic Oath- which was not written by him, I might add) He made reference to animal blood congealing upon cooling and when shaken fibers would be removed, and the blood would return to fluid. Aristotle (384-322 BC) was known for his "cooling theory", that blood cooled when being removed from the body, He also noted that women's blood was thicker than males, old age blood was scanty and that deer had a coagulation deficiency and it was noted that blood fibers go astray, in contrast the blood of bulls congeal much quicker.
This theory was suggested many times doing the course of history, but it wasn't until 1770 that Hewson demonstrated that blood in a vein kept at body temperature would also clot. The theories that were promoted regarding blood coagulation were: cooling, rest, loss of vital force (dying) and air.
How does that translate into today, we know that cold reduces the amount of blood to an area, and the heat will increase it- we want good circulation and blood cells to be in motion, we know that consumption of cells, activators will cause the generation of thrombin and clotting, and air, well we all know what happens when you get a 'cut' and the role of platelets, etc- so there you have it., basic theories to us, but to evolve to these concepts took many centuries.

Platelets were not even described until 1842! This was done by the French histologist Donne, these small globules as being different from red globules or white globules. It was Bizzozero who gave us the term blood platelet (Blut Plattchen) and described the changes that platelets undergo when they are exposed to foreign surfaces as well as the formation of aggregates and white thrombi. In 1865 Schultze associated platelets with clotting and it wasn't really until the 1950's that platelet work really went into full swing - just a mere 60 years ago!

Did you know- factor facts?Hemophilia and royalty

The first written account of haemophilia occurred in the second century in the Babylonian Talmud. In it Rabbi Judah haNasi, redactor of the Mishneh, wrote: "If she circumcised her first child and he died, and a second one also died, she must not circumcise her third child." This passage refers to both the prolonged bleeding caused by circumcision and to the maternal inheritance of the disease. The first medical professional to describe a disease was Albucasis. In the tenth century he described families whose males died of bleeding after only minor traumas. While many other such descriptive and practical references to the disease appear throughout historical writings, scientific analysis does not begin until the start of the nineteenth century. The royal families Queen Victoria has a well established pedigree of disease that traveled through Europe. The Russian Romanov family also had a child that was a "bleeder". The term "haemophilia" is derived the term "haemorrhaphilia" which was used in a description of the condition written by Friedrich Hopff in 1828, while he was a student at the University of Zurich.
So what about Factor IX, or Hemophilia B- it was noted that when blood from a hemophiliac patient was mixed with another patient with severe bleeding, it corrected the patients defect, therefore one was deficient in VIII and the other in IX. This was called Christmas factor or Christmas disease, which was the surname of the person who had the disease. An article regarding this appeared during Christmas week, and was widely read by people who thought this was caused by Christmas overeating!

Did you know- coagulation therapy?

My grandmother believed that 3 things could cure almost anything:
1. Vicks vaporub- colds, pimples, headaches
2. Milk of magnesia - stomach, pimples, canker sores
3. Aspirin - turns out she was correct-

So we know that aspirin is from the tree of the willow bark, but did you know it was the first actual clinical trial conducted? Edward Stone, an English clergyman, reports to the Royal Society of London (world-renowned scientific group) of his successful experiments involving the use of willow bark to reduce fever in fifty of his patients.

Egyptians used lead and copper sulfates, antimony and verdigris to stop bleeding. The most popular treatment however was blood letting. This was done to remove the "plethora" which is blood that is thickened, serous, viscous or putrid. Interestingly, the saliva of the leech is used in the preparation of Hirudun, a direct thrombin inhibitor, or an anticoagulant.

Some treatments were creative- for exhaustion from excessive blood loss opium and brandy in limited amounts were recommended. Chemically, I am not sure of the pathway, physically, with those drugs being administered, I would imagine you wouldn't care, you would be out cold! Hemophilia was treated with citron juice, opium, iron sulfate, magnesium sulfate and egg whites!

Okay, so that was then, this is now, and since all we hear is in these economic times, I thought this might interest you:

Did you know how much it costs to use heparin?

Drug costs for a six-day course of treatment for a patient weighing 176 pounds would be $712 for low molecular weight heparin Lovenox (enoxaparin) or Fragmin (dalteparin) versus $37 for unfractionated heparin, they reported in the Journal of the American Medical Association.
Seven days of prophylaxis with low-dose heparin cost $15.96 in Canada and $36.54 in the US. A week of enoxaparin cost $56.07 in Canada and $158.20 in the US.
Enoxaparin prophylaxis was associated with equal numbers of symptomatic [deep vein thrombosis] and [pulmonary embolism], 12 additional cases of major bleeding, and an additional cost of $86,050. (Canadian data) and $145,667. (US data) for every 1,000 patients treated.

Did you know how much a stroke costs?
Economic Cost of Stroke
- The total cost of stroke to the United States is estimated at $43 billion per year.
- The direct costs of medical care and therapy are estimated at $28 billion per year.
- Indirect costs from lost productivity and other factors are estimated at $15 million per year.
- The average cost of care for a patient up to 90 days after stroke is $15,000.
- For 10 percent of patients, the cost of care for the first 90 days after a stroke is $35,000.
- The percentage breakdown of the direct costs of care for the first 90 days after a stroke is:
Initial hospitalization - 43 percent
Rehabilitation - 16 percent
Physician costs - 14 percent
Hospital Readmission - 14 percent
Medications and other expenses - 13 percent

Did you know how much factor replacement costs:

The median cost for factor products among haemophilia patients with inhibitors was $55,853/year, $2,760 less than comparable haemophilia patients without inhibitors. The median number of hospitalizations per year was 1.0 for both inhibitor and non-inhibitor patients and the median number of days hospitalized was virtually the same. The largest component of the cost of care is that of factor concentrate, it becomes imperative in the current health care environment to better define the true costs and benefits of treatments designed to eradicate or manage inhibitors.

A review of an institution's experience revealed overdosing of coagulation factors in the majority of patients treated during a 12-month period, at a cost that approached$700,000. However and established mandatory
clinical pathology consultation before releasing such factors. In the subsequent 30 months, 32 adults received 64 courses of treatment. For patients with hemophiliaA, the mean cost per admission was reduced by
approximately 27% (total savings, $61,536). For patients with factor VIII inhibitor, there was an approximate 6% cost reduction (total savings, $47,292). The combined savings was $108,828. The mean plasma factor level achieved during the intervention period was 84% ± 55% compared with 117% ± 58% for the preintervention period (P = .008).Neither the number of treatment (factor transfusion) days nor the number of RBC transfusions changed
significantly. Our data support that pathology consultation yields consistent and appropriate therapy and improves resource utilization

Do you know what the risk of hemorrhage and thromboembolism is in patients on oral anticoagulation?

The risk of hemorrhage increased significantly at high international normalized ratios. Compared with the therapeutic ratio of 2-3, the relative risk (RR) of hemorrhage were 2.7 (1.8-3.9; p < 0.01) at a ratio of 3-5 and 21.8 (12.1-39.4; p < 0.01) at a ratio greater than 5. The risk of thromboemboli increased significantly at ratios less than 2, with a relative risk of 3.5. The risk of hemorrhagic or thromboembolic events was lower at ratios of 3-5 (RR 1.8) than at ratios of less than 2 (RR 2.4 p = 0.10). We found that a ratio of 2-3 had the lowest absolute risk (AR) of events (AR 4.3%/yr, 95% CI 3.0%-6.3%).
The risks of hemorrhage and thromboemboli are minimized at international normalized ratios of 2-3. Ratios that are moderately higher than this therapeutic range appear safe and more effective than subtherapeutic ratios.

Coagulation has not and never will be simple. It has been part of the history of the world from the Egyptians to Hippocrates, to the present day evening news. The historical study of how coagulation has been handled from early times has played an important role in history, and we have only just begun.

Coagulation laboratory and the horrible, no good day!

2010-01-03T00:00:00.000-05:00

Ever hear of the book Alexander and the Horrible No Good Day- If you have children I am sure you have read it- if not it basically goes through a day of a child that just gets worse and worse. The adult version would be oversleep, car needs gas, working with a short staff, sitting in traffic, and of course ending the day with that school project that your child has had for the past month-
Everyone has had those days, but what about in the laboratory? You know those days, controls don't work, reagents are expired, new standard curves need to be made, something is always blinking or beeping. One of my worst days ended up providing me with one of my favorite case studies-

If was a Friday afternoon (no surprise there) and I was fairly new in my position as a technical specialist. My director said, I am leaving, you are in charge, okay now if that isn't a prescription for disaster I don't know what is-
Around 2pm we received a sample from the ED on a 12 year old boy with the diagnosis of hematuria for a PT and an aPTT. The results were as follows:
PT= 35 sec (10.5-13.5 sec)
aPTT= 107 sec (24.5-35.5 sec)
So we had a patient who was bleeding, with prolonged screening tests.
Since the results were critical the physician was contacted and he requested that we add a fibrinogen and a D-dimer (the only d-dimer available in the laboratory was the latex agglutination) results were as follows:
Fibrinogen = 256 mg/dL (150-400mg/dL)
D-dimer = <0.5ug/ml - negative
These were added on to determine if the patient was in DIC (disseminated intravascular coagulation). To complete the picture, we looked up the platelet count which was 30,000- so was this DIC? Both the D-dimer and Fibrinogen was normal, which made it unlikely, but what about the process by which platelets are consumed in DIC? We asked for additional information, and discovered that this boy was a patient that had a history of Idiopathic Thrombocytopenic Purpura (ITP), so the platelets could not be used as a discriminator for DIC. (well so much for leaving on time on a Friday!) Additional studies needed to be performed, in the interest of time a mixing study and factor assays were ordered. A resident was contacted to get us as much information as possible. The following additional information was provided.
12 year old boy with a history of ITP, presented with hematuria,
The patient also had a cold and been taken OTC cold medication.
Had a swollen ankle from a fall off a bike the previous day.
He had previously been on nasal steroids as treatment for his ITP,
But currently was doing well and the pediatrician had removed him from the steroids.

So, was the hematuria a result of ITP? ITP presents with mucosal bleeding so this type was not characteristic of ITP, also the platelet count was fairly good for an ITP patient.
Additional testing was as follows:

Mixing study: 1:1 mix= (not incubated)
PT = 35 sec
Pooled Normal Plasma = 11.1
1:1 mix = 36 sec

aPTT= 107 sec
Pooled Normal Plasma = 34.2
1:1 mix = 111 sec

Now, no matter what "rules" you use to determine if a mixing study has corrected, it is safe to say these results show an uncorrected mix which is suggestive of an inhibitor. Since neither test corrected, the inhibitor would be in the common pathway (I, II, V and X). Now was a time to panic, remember I was in charge-
An inhibitor to a specific factor must be diagnosed quickly, and treated. One thing was for certain the patient was already bleeding, and factor specific inhibitors cause bleeding that needs to be controlled. Also, a Bethesda titer should be performed to determine the strength of the inhibitor. A Bethesda titer is performed against whatever factor is determined to be inhibited. This measures the amount of inhibition present in 50% of the factor and is measured in Bethesda Units (BU). The greater the BU the higher the inhibitor.

At this rate, I was going to be lucky to have even a Saturday!

We went ahead to look at the common pathway factors, I, II, V and X- well we knew that I or fibrinogen was normal- factor V, and X were low 56% and 45% respectively, and factor II was undetectable. At that point I knew what the problem was, but remember we can't order or add on tests in the laboratory, and I was new in my position, so now I had to call the physician. Luckily I worked with really smart pediatric hematologists, who quickly suggested that I perform a Dilute Russel Viper Venom- which was very positive- Outcome this patient had a very strong Lupus Anticoagulant- but wait, people with LA, don't bleed they are at a risk for thrombosis- however- there are several instances in which Lupus patients can bleed-
1. Due to a qualitative or quantitative deficiency of platelets
2. Due to an incomplete abortion
3. And due to the consumption of prothrombin- which was the case of our patient, hence the undetectable factor II.

This was causing the hematuria, and do you remember the swollen ankle, which was determined as a result of a fall off a bike? Ha, that ended up to be a blood clot. Why would this manifest itself now? How come it wasn't previously? The patient had been on nasal steroids against ITP-this was also suppressing the LA,
It wasn't until he had been removed from the steroids that the LA manifested itself. So which came first the ITP or the LA? And what is the deal with the consumption of Factor II?

The estimated prevalence of inherited prothrombin deficiency worldwide is 1 per 2,000,000 population. The prevalence is higher where consanguinity is common. In parts of the world where vitamin K is not routinely administered in the neonatal period, hypoprothrombinemia secondary to vitamin K deficiency is relatively common. The incidence of hemorrhagic disease of the newborn in the absence of active prophylaxis is about 1 in 500 newborns.
Patients with hypoprothrombinemia lupus anticoagulant syndrome (HLAS) may have other symptoms of autoimmune disease. (In this case ITP) As an alternative, they may have a history of a preceding viral infection, usually an upper respiratory infection ( there is another clue!) or gastroenteritis.
HLAS is characterized by a very strong LA and polyspecific antibodies. They bind the epitopes of the anionic phospholipids and of prothrombin, but they do not neutralize prothrombin. The FII activity deficiency is not due to an inhibitor, as suspected, but to an evident factor decrease owing to the higher clearance of the prothrombin-antibody complex in the reticuloendothelial system.

And so ended a very long day, with a very engaged staff going step by step through a very interesting case. There were no breaks, no lunches and everyone left late, but they all will never forget the laboratories horrible, no good, very bad day- and how they helped this little boy, no parent will every know, nor will it be put in a journal, but they all know- and they will never forget it. They even all came back on Monday!

Happy 2010-
May your platelets be sticky when needed,
And your factors stay in balance!
Health and Happiness to all-

DECEMBER COAGULATION CORNER

2009-12-09T00:00:00.000-05:00

GOT THROMBIN?

You know fibrin, and factors and inhibitors and platelets
Protein C, Protein S, APCR and activators
But do you recall, the most powerful coagulant of all-

Thrombin the strongest clotter,
Has a very important role
And for you to understand this
You would have to know where thrombin works

All of the other factors
Thought thrombin was just needed for a final clot
They never realized poor thrombin
Worked throughout the cascade and really did a lot!

Then one time when someone said
It seems things are confused
If we have 2 pathways,
Why doesn't one takeover when the other can't work?

Then all the factors realized,
As they shouted out with glee
Thrombin the strongest clotter
Works through initiation, amplification and propagation, all three!

So I hope Rudolph doesn't get insulted that I borrowed his tune to tout the role of thrombin. Like Rudolph thrombin has been very misunderstood and underappreciated!

The primary role of thrombin in the basic in-vitro coagulation cascade is in the conversion of fibrinogen to fibrin. Additional responsibilities of thrombin includes the activation of Factor XIII ïƒ XIIIa, resulting in crosslinked fibrin and the activation of platelets and its feedback mechanism within this cycle to activate additional factors. However, coagulation does not occur as was previously thought of through a "cascade" or "waterfall" series of interactions. In reality, coagulation is a network of simultaneous interactions with regulation and modulation of these interactions during thrombin generation. Generation of thrombin is in reality the pivotal step of hemostasis.

CELL BASED MODEL OF COAGULATION:
The cell-based model of coagulation suggests that there are "intrinsic" and "extrinsic" pathways in the coagulation process, but it has been discovered that these pathways occur on different surfaces.. The "extrinsic" or tissue factor (TF) pathway consists of the FVIIa/TF complex and the FXa/Va complex. It operates on the TF-bearing cell to initiate the coagulation process. While the intrinsic pathway consists of FXI(a), the FIXa/VIIIa complex,and the FXa/Va complex, which operates on the surface of the platelet and generates of burst of thrombin during propagation. Both pathways are needed and operate on different surfaces.

Step one is INITIATION:
In this phase, the combination of tissue factor (TF) and Factor VII (FVII) leads to the direct activation of FX to Factor Xa (FXa). The reaction, however, is not robust and can be effectively inhibited by Tissue factor pathway inhibitor. (TFPI). The combination of TF and FVII also is capable of activating Factor IX (FIX) to Factor IXa (FIXa), which may help to explain why IX is the only intrinsic factor affected by Vitamin K.

Step Two is AMPLIFICATION:

In this phase, the small amount of FXa produced by TF-rFVIIa interaction leads to a limited amount of thrombin generation. The amount of thrombin produced is inadequate to support normal fibrin generation and in fact can be significantly inhibited by antithrombin. The signal becomes amplified when thrombin binds to platelets and initiates several positive feedback loops. This demonstrates the continuing role of platelets in this process, as well as the beginning of the feedback mechanism of thrombin.

What is the role of thrombin:

- Thrombin (IIa) activates FV->Va as the prothrombin complex which assembles on platelet membranes
- Thrombin releases VIII->VIIIa as a component of a tenase complex which assembles on platelet surfaces
- Thrombin (IIa) activates XI->XIa and IX->IXa
- IIa then leads to platelet activation to set the stage for tenase & prothrombin complex
At the end of the amplification phase, the stage is set for the large burst of thrombin generation that is essential to stable clot formation.

Step three is PROPAGATION:
Then propagation of coagulation occurs including activation of platelets, factors V and VIII. Activated factors VIII and IX combine on the surface of these activated platelets activating factor X, resulting in the generation of large amounts of thrombin fibrin formation and the ultimate process of clot formation.

Thrombin not only plays a crucial role in hemostasis it is also a regulator of many pro-inflammatory processes. Some of these include:
- Leukocyte adhesion molecule expression on endothelium
- Platelet activation
- Leukocyte chemotasis
- Endothelial cell production of prothrombotic factors

Thrombin also is a potent growth factor which can initiate endothelial, fibroblast and smooth muscle cell proliferation as well as up regulating other growth factors.
It has also been found that thrombin plays a central role in atherosclerotic lesion formation.
Thrombin's role truly is the center of coagulation. Identifying many of its processes, better explains many unanswered questions in coagulation

H1N1 & COAGULATION

2009-11-01T00:00:00.000-04:00

H1N1- has infected every area of our lives- and I don't mean in the pure definition of infection- you see signs to be cautious at work- every day on the news, you stay away from people who sneeze, coughing into your arm is common place- these are all good practices. Forget epidemic, we want to avoid pandemic! So stay healthy, wash your hands, drink plenty of fluids and stop asking yourself, how does this H1N1 affect the coagulation system- that is what I am hear for!
The notable characteristic of a pandemic quality flu virus is its ability to kill young adults (ages 20 - 45). It does this by "cytokine storm." It is believed that cytokine storms were responsible for many of the deaths during the 1918 influenza pandemic, which killed a disproportionate number of young adults. Here's how:
When the immune system is fighting pathogens, cytokines signal immune cells such as T-cells and macrophages to travel to the site of infection. ... In some instances, the reaction becomes uncontrolled, and too many immune cells are activated in a single place. ... If a cytokine storm occurs in the lungs, for example, fluids and immune cells such as macrophages may accumulate and eventually block off the airways, potentially resulting in death.
The cytokine storm (hypercytokinemia) is the systemic expression of a healthy and vigorous immune system resulting in the release of more than 150 inflammatory mediators (cytokines, oxygen free radicals, and coagulation factors).

Now mind you the research that I found was in pigs (I know we are not supposed to use swine flu), but the coagulation process is explained.
Remember, this is in pigs.

This virus is a member of the Pestivirus (get it, pesti virus) genus in the Flaviviridae family. Cellular response against virus infections includes production of inflammatory and antiviral cytokines, and the induction of cell death through apoptosis. Strains of high virulence cause an acute disease with haemorrhagic fever, thrombocytopenia and disseminated intravascular coagulation (DIC). Vascular endothelial cells maintain balance of the coagulation process by providing an anti-thrombotic barrier as well as activation by pathogens which express a proinflammatory and procoagulant phenotype to eliminate infection. If this system is not controlled, it results in the appearance of microthrombi, DIC, and fibrinolysis. These changes are thought to occur via several factors one by the induction of blood coagulation factors in endothelial cells.

There have been no reported cases of DIC in human cases of HINI.
However it never hurts to review the process.
Disseminated Intravascular Coagulation (DIC) is an acquired syndrome that occurs as a result of an underlying condition. This disorder leads to an unbalance in the coagulation system characterized by the simultaneous activation and consumption of clotting factors and platelets. Consumption results in patients bleeding. While the activation of intravascular coagulation results in fibrin formation which deposits in the microvasculator causing thrombosis.
DIC results from an inappropriate activation of clotting factors or abnormal release of tissue factor into the circulation. The major mechanisms of the coagulation system are activated. The extrinsic pathway is activated as excess tissue factor is released during tissue necrosis and organ failure. This results in the formation of platelet-fibrin rich thrombi in the vasculature. Some of the triggers of DIC that activate the intrinsic pathway are liver disease, immune disorders, burns, shock, OB complications and the most common trigger is sepsis.

Naturally occurring anticoagulants which are inhibitors of coagulation are also impaired. Antithrombin, the most important inhibitor of thrombin, is reduced due to a combination of consumption, degradation, and impaired synthesis. Protein C system is decreased due to impaired synthesis as well as the reduction of protein S which is a cofactor of protein C. There is also evidence that tissue factor pathway inhibitor (TFPI) doesn't regulate tissue factor sufficiently. This further triggers the activation of coagulation in DIC.

Plasminogen activators are released due to bacteremia and endoteoxima. However, the fibrinolytic system, which is responsible for the dissolution of a clot, is mostly shut off during DIC. This is due to suppressed activity resulting from an increase in plasminogen activator inhibitor (PAI-1).

In DIC, bleeding occurs due to deficiencies in multiple coagulation factors (coagulation factors are consumed in thrombi and digested by plasmin) and thrombocytopenia (platelets are consumed in the thrombi). Additionally, intravascular fibrin deposits contribute to organ failure and mortality. All of these processes cause the simultaneous formation of both thrombin and plasmin, resulting in the clinical presentation of bleeding and clotting.
Thrombosis and the Flu
Pulmonary emboli are not known to be a common complication of acute respiratory distress syndrome (ARDS) or of sepsis syndrome, but both ARDS and sepsis represent hypercoagulable states Pulmonary emboli were not noted in patients hospitalized with novel influenza A (H1N1) virus infection in Mexico . One clinical study did not identify any increased risk for pulmonary embolism with seasonal influenza virus infection . However, a report of two patients with rapidly progressive hypoxemia associated with influenza A (H3N2) virus infection noted that they received a diagnosis of acute pulmonary embolism . Clinicians providing care to patients with novel influenza A (H1N1) virus infection should be aware of the potential for patients with ARDS to develop a hypercoagulable state and for pulmonary emboli to cause severe complications, including fatal outcomes.The majority of patients with H1N1 that undergo chest X-rays have normal radiographs. CT scans
One population that might have heightened concern is the bleeding disorder community, There is no threat to the safety of plasma derived clotting factor products. Influenza is a lipid enveloped virus that is inactivated by several steps in the production of products. Additionally, prospective donors who show symptoms of the flu are prevented from donating blood.

So, that is the update on this pesky bug, stay healthy, and
How relevant is it to end with Porky Pig-
That's all folks!

Women's Issues in Von Willbrands Disease

2009-10-02T00:00:00.000-04:00

In the US menorrhagia occurs in 2-3 million individuals. As a result of that statistic almost 300,000 women undergo hysterectomy when in reality about 20% of these women have an underlying bleeding disorder. The most common disorder is VWD. Women having an undiagnosed coagulopathy results in the health crisis which is characterized by a poor quality of life, loss of work, unnecessary surgery, surgical morbidity, increased health care costs and psychological consequences. Even most publications for gynecologists exploring differential diagnosis of abnormal uterine bleeding may briefly mention, after all other measures have been exhausted, to investigate the possibility of a bleeding disorder. Gynecologists tend to focus on anatomical or hormonal problems and need to be made more aware of this disorder as well as do primary care physicians and internists.
Bleeding disorders in women
A major issue in the assessment of bleeding is the lack of a clinical tool for the objective measurement of abnormal reproductive tract bleeding. This added to the lack of awareness of the potential of bleeding disorders to increase or even cause abnormal bleeding leads to the under-diagnosis and suboptimal treatment of women with bleeding disorders. The prevalence of menorrhagia in women with VWD is 74-92% which increases with the severity of the type of VWD. A systematic review of 988 patients in 11 studies demonstrated 5-24% prevalence of menorrhagia is women with VWD, with an overall prevalence of 13%. A 1% prevalence in seen in the general population, but women are more likely to manifest the disorder due to bleeding from menstruation and childbirth. Menorrhagia is the most common symptom in women with VWD. Menorrhagia is defined as >80 mls of blood loss per menstrual cycle. In 2001 a common opinion issued by the American College of Obstetrics-Gynecologist Practice recommended to screen for bleeding disorders adolescents and adult women with severe menorrhagia before performing a hysterectomy.
In a case controlled study conducted by the CDC, more women with VWD had undergone a hysterectomy (28% versus 9%, p<0.01) at a younger age versus controls, but age was not statistically significant. Of 94 women who had undergone surgery, 61% had bleeding complications, with 46% requiring transfusions (p<0.01) as compared to 66 controls of which 8% required surgery and a transfusion. This study demonstrates the disproportionate incidence of bleeding in women with VWD and the expertise that is required by not only gynecologists to understand VWD but also by hematologists to understand anatomical issues with women.
The least amount of variation in VWF is 5-7 days of the cycle and has become the recommended time point for sampling for VWD studies. It was also noted the lowest level of VWF was in day 1-3 of cycle, samples taken at this time interval would represent the lowest value and be diagnostic value in treating women with DDAVP. VWF levels increase significantly in three situations:
1. supra-physiological dosing for ovarian stimulation in the setting of infertility
2. postmenopausal women deliberately taking high does of estrogens
3. pregnancy in a type 1 vWD patient.

Other confounding issues in VW testing are age and ABO blood type. VWF increases 15-17% per decade. It is not known if this increases until menopause. While in pregnancy, the third trimester brings an increase in VWF, >50%, which can cause a missed diagnosis. Pregnant women should have increased levels, therefore a normal level may mean an abnormal result.
In a study conducted on 123 randomly selected female control subjects ages 18-45, testing for VWD included ABO blood typing. Results revealed that women with blood Type O have significantly lower levels for all tests. When comparing levels for VWF:ag for blood types: normal range for VWF in all controls is 49-203 IU/dL, Type O patients 35.6-157.0 IU/dL, Type A 48.0-233.9 IU/dL, Type B 56.8-241.0 IU/dL, Type AB 63.8-238.2 IU/dL. ABO differences account for 19% of the variance in VWF:ag. This in turn has a large effect in determining ratios for diagnosing the different types of VWD. The ratio for VWF:Rco/VWF:ag were 0.97 in non-type O, versus 0.79 for type O and 0.94 and 0.74 for non-type O. Adding blood type into the mix of race and age only compounds the level of knowledge required when diagnosing women with this disorder.

One issue with implementing change is that women are tolerant of bleeding, it is a part of their lives and they do not seek medical help for this problem, additionally if they have daughters who are also "heavy bleeders" it then becomes transitional or passed on further exacerbating the issue. Women need to be educated and be made aware of this as well as the presence of other types of bleeding from VWD which include gastrointestinal bleeding, bleeding from dental or surgical procedures, epistaxis, petechiae, and purpura.
One of the most effective tools in demonstrating underlying coagulation abnormalities is a good clinical and family history. Utilizing a questionnaire that assesses type and severity of bleeding with a numerical value can provide clinicians with an objective tool. Women with three hemorrhagic symptoms or a bleeding score of five or more provides a 98.6% specificity in determining type 1 VWD. Having gynecologists use this scoring system may be an aid in referring patients to hematologists.
The National Heart, Lung and Blood Institute (NHBI) published their first evidence based guidelines "The Diagnosis, Evaluation, and Management of von Willebrand Disease" in March of 2008. This was aimed at not only hematologists and laboratory professionals, but for primary care physicians, gynecologists, pediatricians and nurse practitioners. A quick reference pocket guide provides a synopsis of these guidelines.
Educating women to become aware of this issue and take charge of pursing this diagnosis is one of the first steps to be taken. Many male gynecologists feel that women inherently complain and tend to ignore when they mention heavy bleeding. One venue to explore is to reach a younger generation of women through magazine ads by having them evaluate their own bleeding by tampon usage, quality of life and become aware of the possibility they have a bleeding disorder.

Microparticles

2009-09-01T00:00:00.000-04:00

MICROPARTICLES: What are they? What do they do? How are they involved in coagulation?

First described 40 years ago as the "coagulant material in minute particle form" in platelet-poor plasma (otherwise termed platelet dust), microparticles have been the subject of considerable debate and controversy over the years, particularly in the context of blood coagulation in healthy individuals
Cell-derived microparticles (MP)are small membranous vesicles released from the plasma membranes of platelets, leukocytes, red cells and endothelial cells in response to diverse biochemical agents or mechanical stresses. They are the main carriers of circulating tissue factor, the principal initiator of intravascular thrombosis, and are implicated in a variety of thrombotic and inflammatory disorders. Circulating MP provide an additional procoagulant phospholipid surface enabling the assembly of the clotting enzyme complexes and thrombin generation. Their procoagulant properties rely on the exposure of phosphatidylserine and on the possible presence of tissue factor, the main initiator of blood coagulation. Microparticles constitute the main reservoir of blood-borne tissue factor. Derived from various cells, most notably platelets, erythrocytes, leucocytes and endothelial cells, circulating MP are detectable in the circulation of healthy subjects. Elevated levels are encountered in diseases with vascular involvement and hypercoagulability such as disseminated intravascular coagulation, diabetes, immune-mediated thrombosis, kidney diseases, acute coronary syndromes or systemic inflammatory disease, where they appear indicative of a poor clinical outcome.There is evidence suggesting that cell-derived microparticles are involved predominantly with microvascular, as opposed to macrovascular, thrombosis. Cell-derived microparticles may substantially contribute to ischemic brain disease in several settings, as well as to neuroinflammatory conditions.
GENERATION OF MICROPARTICLES:
The finding of localized biomechanical generation of microparticles on thrombogenic surfaces is interesting and potentially important and should stimulate further investigation into the relationship between shear and microparticle formation. Platelets in travel through vessels in vitro by sheer stress. The shedding of procoagulant microparticles from the surface of platelets and other cell types involves proteolytic shedding mechanisms linked to cell stimulation by potent agonists or following apoptosis. It remains to be seen whether proteolytic shedding is the predominant mechanism of microparticle generation under lower shear conditions and whether biomechanically derived particles become progressively more important at higher shear. Nonetheless, these findings raise interesting issues and should stimulate further investigation into the precise relationship between the earliest events of platelet adhesion and the subsequent initiation of blood coagulation, a topic that remains as uncertain today as it did when microparticles were first discovered.

SEPSIS:
Elevated levels of MPs appear to play a role in the pathogeneisis of thrombosis in sepsis. Both events of thrombosis and sepsis, can be orchestrated by the interaction between circulating and vascular cells that under activation release microparticles. Circulating levels of microparticles and platelet- and endothelial-derived microparticles were increased in septic patients. Sepsis and trauma lead to a sustained activation of monocytes and endothelium. In the vascular compartment, stimulated cells release microparticles. At sites of endothelium injury, enhanced release or recruitment of procoagulant MP through P-selectin-PSGL-1 pathway could concentrate TF activity above a threshold allowing blood coagulation to be triggered. Converging evidences from experimental or clinical data highlight a role for MP harboring tissue factor in the initiation of disseminated intravascular coagulation. In these settings, the pharmacological modulation of MP levels or biological functions through activated protein C or factor VIIa allows challenging issues.
PLATELETS:
Platelet MPs are submicormeter fragments resulting from the remodeling of the platelet membrane in relation to several conditions. These MP are increased in several prothrombotic and inflammatory disorders including cardiovascular disease, autoimmune and infectious diseases and cancer. Clinically, you can use platelet microparticles for identifying patients with vascular risk and monitoring response to treatment. Little is known about the processes by which platelet microparticles are generated in vivo. However observing video microscopy of live mouse megakaryocytes demonstrated that microparticles form as submicron beads and are CD41+, CD42b+. They express surface phosphatidylserine. To determine whether circulating microparticles are derived primarily from activated platelets or megakaryocytes, markers were identified that distinguish between these 2 populations. CD62P and LAMP-1 were found only on mouse microparticles from activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not microparticles from activated platelets. Circulating microparticles isolated from mice were CD62P-, LAMP-1 and expressed full-length filamin A, indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62P- and expressed full-length filamin A. Cultured human megakaryocytes elaborated microparticles that were CD41+, CD42b+, and express surface phosphatidylserine. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles.

WOMEN AND MENOPAUSE:
The risk for symptomatic atherosclerotic disease increases after menopause, currently recognized risk factors do not identify ongoing disease processes in low-risk women. This study tested the hypothesis that circulating cell-derived microparticles may reflect disease processes in women defined as low risk by the Framingham risk score. The concentration and phenotype of circulating microparticles were evaluated in a cross-sectional study of apparently healthy menopausal women, screened for enrollment into the Kronos Early Estrogen Prevention Study. Microparticles were evaluated by flow cytometry, and coronary artery calcification (CAC). The procoagulant activity of isolated microparticles was determined with a sensitive fluorescent thrombin generation assay. Chronological age, body mass index, serum lipids, systolic blood pressure (Framingham risk score < 10%, range 1-3%), and high-sensitivity C-reactive protein did not differ significantly among women with low (0 < 35; range, 0.3-32 Agatston units) or high (>50; range, 93-315 Agatston units) CAC compared with women without calcification. The total concentration and percentage of microparticles derived from platelets and endothelial cells were greatest in women with high CAC scores. The thrombin-generating capacity of the isolated microparticles correlated with phosphatidylserine expression, which also was greatest in women with high CAC scores. The percentages of microparticles expressing granulocyte and monocyte markers were not significantly different among groups. Therefore, the characterization of platelet and endothelial microparticles may identify early menopausal women with premature CAC who would not otherwise be identified by the usual risk factor analysis.
Cancer:
MP has been found to be markedly increased in patients with both breast and pancreatic cancer who present with VTE. A study looked at various malignancies and their corresponding levels of MP. It was observed that there is a relationship between elevated MP activity and VTE in patients with cancer. It appears that is the contribution of the cancer cells that cause the elevated MPs. It was determined that patients that develop VTE during cancer did not have elevated microparticles. Therefore, the VTE doesn't cause the increase in MP, but the cancer cells.

So, there you have it, microparticles do correlate with several disease states, and appear that increased levels correlate with phosphatidylserine expression. Early identification of this process may aid in the diagnosis and early intervention for several disorders. So stay tuned for more research in this area and outcomes in coagulation disorders.

OVERVIEW OF ISTH: 2009

2009-08-03T00:00:00.000-04:00

INTERNATIONAL SOCIETY OF THROMBOSIS AND HEMOSTASIS
BOSTON - 2009

The British are coming! echoed Paul Revere through the streets of Boston-
Well, during the week of July 11th, Boston was invaded with people interested in the field of coagulation. Not only were hematologists, laboratory and research personnel attended, but there were also cardiologists, trauma surgeons, and ER physicians present. The scope of coagulation has invaded many other disciplines.

The week begins with the Subcommittees that meet and cover topics from fibrinolysis to women's health to lupus anticoagulants. If you want to read there minutes, they are posted on the ISTH website; .and can be a great resource. The only problem with this meeting is that it is like a giant buffet, you have to pick and choose from an extensive menu, and you want to sample everything! Plus there are the exhibits, and posters and you never want to miss the flavor of the city!

The first subcommittee was Factor VIII and Factor IX and a discussion on the 1 stage versus 2 stage assay for factor VIII. One-stage is the assay used by laboratories. Most routine labs do not use the 2 stage assay, so they compared it to the chromogenic assay. There are certain mutations that will cause the one stage versus chromogenic assay to have a ratio > 2.0, which means you can miss a mild hemophiliac. There have been 28 mutations identified. These patients clinically bleed. This is important to understand, and depending on your patient population, may need to also have a chromogenic assay on board.

Ever popular are the D-dimer sessions. Everyone is still trying to predict how you can tell who will have a recurrent thrombotic event and what factors may contribute to it.
It was found that gender did not play a role in provoked VTE; however males had an increased risk of occurrence in unprovoked incidences, also being a male under the age of 50 also increased the risk. Patients, who had a negative d-dimer a VTE, had a lower recurrence rate, as opposed to those who had a positive d-dimer. Patients having a positive d-dimer 1 year out can result in a recurrence rate of 12 versus 2.2 in patients with a negative d-dimer. If after you remove a patient from oral anticoagulation and the d-dimer is elevated 1 month post, you still have a 99% risk of reoccurrence. Let us not forget that the d-dimer increase about 10%/ decade.

The subcommittee on DIC had a great lecture on trauma and coagulation. Hemorrhage accounts for 40% of death in trauma. There are issues with dilution of factors because of the fluids and plasma rapidly give and consumption of factors. In as study that looked at 5000 trauma patients it was noted that patients that presented with a PT of > 22 seconds resulted in increased mortality. Also decreased Protein C levels are associated with shock, with an increase in Activated Protein C. It is important to look at a patient's blood smear to look for microangiopathic anemia.

Cardiovascular genetics looked at increased levels of LDL (remember this is lousy cholesterol, and should be low) versus HDL (which is happy cholesterol and should be high). The in a 15 fold increase in cardiovascular disease (CHD) in the US versus pre-industrial countries. Every mg/dl decrease in LDL decreases the risk of CHD by 1%, therefore lowering LDL from 271 to 236 lowers CHD by 26%. Understanding the genetic traits for LDL, as well as the gene metabolism that is involved in LDL production can aid in developing preventative medicine and improve public health.

Heparin-induced thrombocytopenia or HIT is an immunologic adverse effect of heparin therapy causing antibody mediated platelet activation and thrombin generation. That is the drug that is supposed to prevent clotting has now caused a clot. Patients should be diagnosed based on the negative predictive value of clinical tests and the clinical picture. Patients will have a 50% drop in platelet count and a new thrombosis. On a first exposure to heparin this will occur on the fourth day, however, if a patient had been previously exposed to heparin, even in small doses it will happen in 1-2 days. Thrombocytopenia occurs from intravascular platelet activation. The pathogenic immunoglobulin isotype is IgG. Anti-PF4/heparin antibodies bind to PF4 via their F(ab) domains. The lead to formation of large immune complexes that cross=link the platelet FcyIIa receptors causing platelet aggregation. The negative predictive value of assays is a good tool in ruling out HIT. Using polyspecific IgGAM assays have a specificity of about 50-75%, while using an IgG Elisa can enhance up to 90%. A good tool in patient assessment is using the 4T scoring system: Thrombosis, Thrombocytopenia, Time and oTher. Combining the score and the laboratory test for HIT provides the highest predictive value.

Now, on to platelets and how to inhibit them! Aspirin has been around for 110 years and works by acetylating platelet cyclooxygenase (COX-1) inhibiting thromboxane production. The trick is to use the lowest dose that is effective without causing a GI bleed. Aspirin reduces major vascular events by 12%, however, it was noted that aspirin seems to be less effective in the diabetic patient. The new P2Y12 blockers ware ADP receptors that work by inhibiting ADP induced platelet aggregation, which has been shown to decrease the risk of arterial occlusion. Thienopyridines are a class of drugs which irreversibly inhibit P2Y12 function lasting about 8-10 days, or the lifespan of a platelet. Since its effect is irreversible it presents a problem in patients requiring emergent coronary bypass surgery. Clopidogrel treatment up to 4-5 days prior surgery has been associated with increased bleeding and hospital stays. Prasugrel a new thienopyridine is detected in plasma within 15 minutes and reaches maximum concentration within 30 minutes. The difference between these drugs is Prasugrel is more effective in converting from a pro-drug to and active metabolite. A phase III multicenter clinical trial is in progress to determine the safety and efficacy of the 2 drugs and their ability to reduce the risk of cardiovascular death, MI or stroke.

So, as you can see this is just a small insight of what goes on at a congress. There are lectures and posters and exhibits that can satisfy every coagulation pathway that you choose- whether it be platelets, factors, new research, von Willebrand disease or just the interaction among colleagues. It is truly an amazing experience one that I have been privileged to attend- just to mark your calendars, in 2011 ISTH meets in Kyoto, Japan!

ANTI-PLATELET DRUGS

2009-07-01T00:00:00.000-04:00

ANTI-PLATELET DRUGS:

You can't open a journal today without seeing an article about anti-platelet drugs. It is overwhelming- and confusing. So this month I will try to sort out the differences among the choices out there.

No article on anti-platelet drugs would be complete without mentioning the queen of all anti-platelet drugs- aspirin. Almost everyone is aware of how it works and the advantages of this product. Medical historians give 1897 as the date Aspirin was developed, however there are recipes that date back over 3,500 years ago when Egyptians used myrtle leaves to relieve pain. Hippocrates, the father of all doctors prescribed a juice from the bark of the willow tree for fever and pain. The active ingredient in that juice is salicylic acid, which is derived from the Latin word for willow Salix. This is also contained in the myrtle leaves used by the Egyptians. It was used throughout history but in 1897 Dr. Felix Hoffman purified acetylsalicylic acid and in 1899 the first clinical publication referred to this compound as Aspirin.

So how does Aspirin work?

Aspirin is rapidly absorbed from the proximal gastrointestinal tract and irreversibly binds to cyclooxygenase-1,or as it is referred to a COX-1 inhibitor. This impairs thromboxane A2 (TXA2) synthesis and platelet aggregation in response to collagen, adenosine diphosphate (ADP), and thrombin.
The most studied and best-established antiplatelet treatment is aspirin. Based on the time-honored balance between efficacy and safety, lifelong aspirin treatment is recommended for all patients with atherosclerotic vascular disease; exceptions are patients at high risk of bleeding and those with aspirin allergy

It is very effective. If you are a responder it will affect platelet function within 45 minutes. If someone is having a heart attack, crush the aspirin it will work even quicker. Aspirin can have a 25% risk reduction of nonfatal MI, stroke or death. Carry some at all times!

What about Aspirin Resistance? Some people believe it is just non-compliance, others inadequate dosing, or can occur in people concurrently taking NSAIDs. Anti-inflammatory drugs may interfere with the irreversible inactivation of platelet - COX-1 by competing with Aspirin for a common docking site- Naproxen can also compete. Aspirin resistance is difficult to define, but with pharmacokinetics, we have learned that there are many genetic polymoprphisms that can cause either a hyper or hypo response. Approximately 20-25% of the population can fall into this category. It has also been demonstrated that females need higher doses than males for cardio-vascular protection. This may be due to the process in which estrogen up-regulates prostaglyandin production by receptor redirection.

Clopidogrel is a prodrug metabolized in the liver by cytochrome P450
isoenzyme A4 (CYP3A4) after its intestinal absorption. Clopidogrel is an Adenosine 5'-diphosphate (ADP) receptor antagonist indicated for the reduction of atherosclerotic events including stroke or myocardial infarction. (ADP) is an important physiological and pathological platelet agonist. Upon vascular injury, ADP is released into the bloodstream from damaged cells and activated platelets, and in turn acts on other platelets. ADP induces a number of platelet responses, including shape change from disc to sphere, aggregation and secretion of granule contents. These ADP-induced platelet responses contribute to hemostasis, pathological thrombus formation and vascular occlusion. These responses are considered to be mediated by the interaction of ADP with specific binding sites on the platelet membrane designated as P2Y12 (or P2T) receptors. This is a selective and irreversible inhibitor of ADP-induced platelet aggregation, but does not interfere with ADP platelet induced shape changes. It inhibits the binding of fibrinogen without affecting the GPIIb/IIIa complex. Dosage is 75mg 1/day, with a half life of 7-8 hours and will affect platelets within 2 hours. Clopidogrel has transient effects on thrombin and collagen aggregation. The active thiol metabolite irreversibly binds to the P2Y12 receptor and inhibits ADP-induced platelet aggregation, ADP-stimulated P-selectin and CD40L expression, as well as platelet-leukocyte aggregate formation.

Concurrent therapy:
Aspirin and clopidogrel affect different platelet activation pathways, so they are frequently used in combination. Dual antiplatelet therapy may be most beneficial in patients with acute events, There are sever conditions in which combined therapy offers no benefits over monotherapy such as in primary prevention of coronary or cerebral events in patients at high risk.. Aspirin,is the preferred treatment for primary prevention; clopidogrel alone is useful in patients with an aspirin allergy] Likewise, combination therapy is inappropriate in patients with a recent stroke or transient ischemic attack because it increases the incidence of major and minor bleeds without offering any therapeutic benefit over clopidogrel alone. The most appropriate indications for the use of combined clopidogrel and aspirin therapy are the treatment of acute coronary syndromes and the prevention of coronary events after placement of a stent.

Prasugrel is a new prodrug-type antiplatelet agent that acts as a P2Y12 receptor antagonist via its active metabolite R-138727. Plasma levels of the active metabolite of prasugrel were also 10 times higher than those of the active metabolite of clopidogrel, suggesting that prasugrel's greater potency may be due to a faster metabolization rate.
The JUMBO-TIMI 26 (Joint Utilization of Medication to Block Platelets Optimally - Thrombolysis in Myocardial Infarction 26) was a multicenter, double-blind, randomized, phase II clinical trial that evaluated the effects and safety of prasugrel. A total of 904 patients undergoing elective or urgent percutaneous coronary intervention were given the standard clopidogrel regimen above or one of three prasugrel regimens (loading doses of 40 or 60 mg, followed by 7.5, 10 or 15 mg/day) for 29-34 days, starting after the diagnostic angiogram. Prasugrel-treated patients showed a lower incidence of major adverse cardiac events than clopidogrel-treated patients, although the difference among study regimens did not reach statistical significance. Only 0.7% of patients had major bleeding, whereas 1.1% had minor bleeding and 2.4% experienced minimal bleeding in all study groups combined. No significant differences among study treatments were found in the incidence of an endpoint combining major and minor hemorrhage unrelated to coronary artery bypass graft. Hemorrhage rates were lower than those expected from historical controls (Wiviott, S.D. et al., Circulation 2005, 111(25): 3366).
Glycoprotein IIb/IIIa Inhibitors:
These IIb/IIIa antagonists receptors are critical to the process of platelet thrombus formation and work by blocking fibrinogen binding to the receptor. It serves as the final common pathway for platelet aggregation. Effectiveness has been established for precutaneous coronary intervention (PCI).It is administered by IV Bolus,and infused for 20-24 hours. Bleeding is an adverse event, but, no increased risk of intracranial hemorrhage, Thrombocytopenia can occur 0.4-1% of the time.Due to the risk of possible thrombocytopenia, platelet counts should be monitored at 1, 2 and 12 hours. ABCIXIMAB - (REPRO) is a chimeric monoclonal antibody that binds non-specifically to the Gp IIb/IIIa receptor.and is cleared between the RE system. The anti-platelet effect can last several days. Non-specific action can also bind to vitronectin and MAC 1-receptors, as a result may have an anticoagulant effect.
EPTIFBATIDE is a cyclic heptapeptide that selectively binds to the GpIIb/III a receptor. The effect is short acting, 50% of platelet function returns within 4 hours of cessation of infusion. Triofiban hydrochloride is a short acting non-peptide derivative of tyrosine that binds selectively to the GpIIb/IIIa receptor.

So, there are several options regarding antiplatelet drugs. Aspirin is still considered as one of the best anti-platelet drugs, however their are, several others that can provide options and decrease function. The challenge in using these agents is to maximize their effectiveness while minimizing the risk of bleeding.

Antiplatelet drugs
Glycoprotein IIb/IIIa inhibitors
Abciximab - Eptifibatide - Tirofiban

ADP receptor/P2Y12 inhibitors
(Thienopyridines) Clopidogrel - Ticlopidine - Prasugrel

Prostaglandin analogue (PGI2)
Beraprost - Prostacyclin - Iloprost - Treprostinil

COX inhibitors
Acetylsalicylic acid/Aspirin # - Aloxiprin - Carbasalate calcium

Other
Ditazole - Cloricromen - Dipyridamole - Indobufen - Picotamide - Triflusal

THROMBOSIS AND GENETICS

2009-06-03T00:00:00.000-04:00

THROMBOSIS AND GENETICS:

Thrombosis and genetics is a complex mix. Not only do genetics play a role but so do environmental factors. It is like a domino effect- combine genetic risks, environmental factors the greater the stimulus the greater the risk. So, if you have a genetic risk and you add things like stasis, obesity, pregnancy, smoking, cancer, hormones, you don't add, but multiply your risk of a thrombotic event. Did you know that in the Genetic Analysis of Idiopathic Thrombosis family-based study estimated a heritability of 60% for thrombosis suggesting that genes would represent the largest single underlying cause of this disease? Yikes! That is more than or equal to Type 2 diabetes, gallbladder disease, alcoholism, and obesity.

This field has really made headway since 1994 when the Factor V Leiden mutation was identified. FV Leiden is an abnormal mutation, a substitution of arginine by glutamine but doesn't interfere with the activity of Factor V it was named after the city in the Netherlands where it was identified. The gene is not susceptible to cleavage resulting in more factor Va is available within the prothrombinase complex increasing the generation of thrombin. This mutation identified a fair amount (about 40%) of people who were previously deemed as having idiopathic thrombosis. It accounts for about 6% of thrombosis in the general population, with a high prevalence in Greece, Sweden, and Lebanon, with almost no incidence in African Blacks, or Asian population. FV Leiden, discovered in the Netherlands, was said to have been a mutation that developed as a protection from the cold weather and the risk of bleeding during childbirth, by making them mothers more hypercoaguable.

The prothrombin G20210A mutation was discovered in 1996, it is a substitution of guanine to adenine. It is present in about 2.3% of the population is also common in the Caucasian population, with a prevalence in southern Europe and Spain, rarely in the Black or Asian population. This is through to be due to the mutation most likely occurring after the divergence of Africans from non-Africans and of Caucasians from Mongoloid subpopulations. These patients may also present with elevated FII levels greater than 115% as opposed to controls. .

What about the risks of increased factor VIII and von Willebrand Factor (vWF)? These are also acute phase reactants, which mean that they are elevated during times of inflammation and stress, or they can be elevated due to a hereditary persistence. How can you diagnosis that? Well, lets look at the most sensitive marker of inflammation C Reactive Protein, if this is tested and is normal , and you still have an elevated FVIII (>150%) or vWF, you may be at risk for venous and arterial thrombosis. So, it would make sense since FVIII is a cofactor of FIXa this can also cause thrombosis, and it increases with age (Yippee). Did you know that von Willebrand levels are also blood group dependent, and people with Type O blood have the lowest normal levels of vWF? This is said to have a protective thrombotic effect on people with blood type O. Let us not forget fibrinogen, which is the same acute phase reactant, but can also have a hereditary persistence for elevation. This is a marker that has a higher risk for an MI than elevated cholesterol.
Other studies have also demonstrated thrombotic risk factors in elevations of Factors VII, IX, XI and XII.

A relatively small population of people (3-5% of the thrombotic population) has deficiencies of Protein C, S or Antithrombin. The basis for looking into genetics of thrombophilia (which is a genetic tendency towards thrombosis) was the occurrence of a thrombotic event at an early age (35-50). However, with the discovery of so many new mutations, that is no longer the case. Patients with FVL may not have an event until their system is challenged. This is called the "double hit theory". They may have a mutation, but they may not have an event until they have surgery, or become pregnant, etc.

Hypercoagulable testing is expensive, and you should screen with a functional assay. If this is abnormal, and it is not abnormal due to an acquired deficiency or because of consumption, you will then proceed to antigen testing. This will distinguish between a qualitative (dysfunctional) or quantitative deficiency.
Also, because you get an abnormal in one test, doesn't mean you won't get an abnormality in another test- so you might want to keep testing. In a study it was demonstrated many patients have more than one defect, putting them at a higher risk for thrombosis increasing their odds ratio to 20!

When do you test these patients? Not and I reply Not when they are having an event, there is consumption occurring, and it the result will not be accurate. . Let's face it; if someone has a clot, they have to be treated, with heparin, either with unfractionated or low molecular weight. The cause is not important, they need to be treated, to prevent further thrombosis, and not overly ant coagulated and has them at a risk for bleeding. Most likely a course of anticoagulation will be followed for about 6 months. However, if you know they have more than one abnormality, it may change the course and length of treatment and involve a course that is life long. When do you test them? They should be removed from anticoagulation for a period of time (2 weeks) and then testing should be performed.

What is the best way to test? Decide what your population is- and the prevalence in your testing. The most common inherited disorder is FV Leiden, but not the highest risk of thrombosis, and remember all of these can be acquired through many disorders such as liver disease, during DIC and from many medications. A test menu should begin with those functional assays and your physicians should begin with a complete patient history as well as taking into consideration environment factors that can enhance risks

Quality in Coagulation

2009-05-04T00:00:00.000-04:00

"Quality means doing it right when no one else is looking"
Henry Ford

We have Quality Assurance, Six Sigma, Quality Control, Proficiency Testing, CAP, JCAHO, Quality Management and we have our conscience. April brought the Quality Testing workshop sponsored by the Mayo Clinic. Here experts from the coagulation field get together to present the latest updates on issues that have been in our field for years. How do you handle issues, but more importantly how do you improve them? What do you use? Tell me - what's in your lab? What processes do you use so you know you can ensure quality when no one else is looking? Surprise inspections are the way it is supposed to be- can an inspector walk into your coagulation laboratory today and be assured you are performing quality results? Let's face it; everyone has that expired box of 'something' somewhere in their laboratory, that doesn't hinder quality, unless of course you use it... So where are the hot spots in the coagulation laboratory?
We know we need quality, where do you get help?

Guidelines: These are the best things since sliced bread, use them! CLSI guidelines are a How to for laboratories. Want to know how to perform a reference range? Method comparison? Issues with the PT and APTT? Platelet Aggregation? These published guidelines are established by input from both industry and hospitals to help standardize and ensure established processes. Use them! Read them-

Proficiency testing: Great stuff, lots of work, this is established material that is peer evaluated, sometimes by method, others by reagents, as well as all methods/reagents. Having instrument trouble, not sure if your factors are running high, get some of that material and run it, will give you a ton of information. When you get back the results, read them, carefully, look to see what is the most frequent result? If this is a standard, how close are your results? Does your test run higher? Is the material patient plasma or adulterated material? Remember with the complexity of coagulation testing and antibodies used, you may have a different answer than other methods with adulterated material, how do you compare with your own method? Are the CV's tight? This is great stuff! But remember - this doesn't test the quality of the laboratory, or pre- and post analytical steps, it doesn't test laboratory efficiency. But, what it does do it help laboratories to improve quality in detecting their own errors and have a mechanism to correct those errors.

Quality Control: So tell me what are you doing here? How many controls do you run? Do they reflect your patient population? Do you know the defined use of run for coagulation controls are every 8 hours? (Unless of course you have to make new reagents) but you really only need to run them once a shift. And while it is very nice to run the controls for the next shift, I think it is important for each shift to run their own controls so they know how the analyzer is performing. More importantly, know what those results mean. To get the most quality out of your QC have established rules, and know the action to take when they fail those rules. When should they be run, or when they are acceptable, there is your quality.

Are you rolling your eyes yet? I know this I have 3 children (Happy Mother's day, by the way to all moms!) So what about in the coagulation laboratory? How do you keep the bar high when you are dealing with pre-analytical variables, unstable enzymes, complex testing and overworked technologists? Wine or whine? Both work. You do your best to control what you can and have good processes in place to catch what you can't. Let's discuss-
So you are running QC, most are lyophilized in coagulation testing; this is the first step of your testing and can make or break your day. How is that water you are using? Was it sitting on a lab bench or is it fresh? Type 1? Or distilled? Remember you are beginning a shift, this can make or break your day, and you can control it, go get fresh water. Should not sit in a beaker more than 1 hour, pour it on the plants. What about the pipette- been calibrated, sure 6 months may be okay for other labs, but lets face it, something that gets used every day, to check the QUALITY of your analyzers might warrant a 3 month check.

Loading that calibrator? Are you sure it has the correct calibration value? I know the analyzers read the barcodes, etc, but techs are smart, and analyzers at times have a mind of their own, takes 2 seconds, verify that you have the correct lot, and calibrator value.

Reagents, are you running factors? Might want to use fresh ones, may be a waste, but sometimes the costs saved in time is more important. Can you use ones that are on board, absolutely - but again if you are having trouble every morning, its not your calibrator, controls work on other assays, might be those reagents.

Running von Willebrand assays? First do not refrigerate whole blood for testing, it can cause in vitro proteolysis of vWF and result in falsely decreased vWF activity. Also, when these samples come into the laboratory, if you batch test them, get them spun and frozen ASAP. If they sit the activity can decrease, the antigen be stable and look like a Type II deficiency (decreased activity and normal antigen) This is a tough assays, automation helps, still- if running frozen samples, run controls FIRST, make sure your assay is working, then thaw the samples at 37 degrees C for 5 minutes, Mix the samples well, and DO NOT PUT THE SAMPLES on ice, keep at room temperature. Remember, you can falsely decrease vWF, possibly due to cold induced binding of vWF to platelets. Now you may test. Seems like a bit of a project, trust me the time you invest will surpass the time you save.

Proficiency testing for coagulation, this is complex stuff- your variables are only the reagents, factor deficient plasma, calibrators, analyzers, buffers and even the calcium chloride! What do you do? Treat these samples the same as patients, I know that is hard to do, but what are we looking for- we are looking to see how we perform with peer analysis. Have different techs perform the assays, make sure that you run patients with the proficiency testing. Use these as blind samples to assess competency. Review the outcomes, READ the summaries, see how your lab performs, how your assay performs with other people using that assay and with the total group. How is that for quality?

Learning about quality is important, but having the tools to help give you that quality is key!

Development of Inhibitors - and I don't mean LUPUS!

2009-04-03T00:00:00.000-04:00

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.

What does that test result mean?

2009-03-02T00:00:00.000-05:00

What does that test result mean?

Coagulation is a puzzle; you need all of the pieces to make a complete picture.
That is a difficult task in the laboratory - you need the test result, the patient history, what type of therapy or anticoagulant they are on, or off of, or being transitioned on too-.Let's face it that never happens in the laboratory- you are lucky to get anticoagulant information. So you have this test result, is it normal, is it abnormal- well it depends on your patient and the situation. What might be normal for one patient may be abnormal for the next.

Let's discuss:
Patient goes to her OB/GYN, she is pregnant, and complains of shortness of breath. This is her third pregnancy. Pregnancy unbalances the haemostatic scale, and can place you at a risk for a bleed or thrombosis. Compounding this with a hereditary predisposition, makes a pregnant women a ticking timebomb. Knowing this along with the symptom of shortness of breath, the OB orders several blood tests, in particular a thrompophilic workup. The results are as follows:
Protein C = 89% (80-120%)
ATIII = 107% (70-130%)
APCR = 2.5 (>2.1)
Lupus = negative
Protein S = 34% (65-135%)
He informs the patient they are Protein S deficient, and both the baby and the mother are at risk. So what are the pieces to this puzzle? This is her third pregnancy, she has not had any previous event, it would be unusual for this to not have been manifested previously- so what does this test result mean?
This is a normal test result for pregnant women. Her thrombophilia work-up is normal for being pregnant. Protein S is decreased in pregnant women, contributing to their hypercoagulable state. So this abnormal result is normal for this patient. Her shortness of breath is just an occurrence of pregnancy. If the clinician had acted on this result, the outcome could have been disastrous.

Okay, next result; we have a mom in labor and delivery they have sent down three fibrinogen levels on this patient. All results are in the normal range-
We have a 289 mg/dL, 250 mg/dL and 245 mg/dL. They have all been sent in the last six hours, how many do they intend to send? They are all normal, correct? Well not exactly - what are the pieces to this puzzle? .Fibrinogen is an acute phase reactant; this means this factor is elevated in times of inflammation, stress, trauma and pregnancy. One would expect this result to be elevated in a pregnant women, this result is in the normal range (150-450,000). What does this mean? It means that this might be being consumed, as in disseminated intravascular coagulation (DIC). So this normal result, is not normal at all for a pregnant women. This result needs to be acted on to prevent the progression of DIC- which is the simultaneous formation of thrombin and plasmin. This condition has a high degree of mortality for both the mother and the fetus. So a normal fibrinogen in this patient is not normal at all.

What about when a clinician wants to add on an APTT to an existing PT sample. You know the routine, the sample arrives in the lab with the morning bloods, and a PT is ordered. They really needed an APTT so they call 6 hours later to add it on. Now we know that an APTT is only stable for 4 hours. This was a pediatric draw, the result is important and the sample was difficult to obtain. So you run it off line, and it is 35.1 sec (27-37.5 sec). It is normal, so what is the harm, you give it to the doctor. That was so nice of you! However, the problem is that, this pediatric patient was on heparin. The result should have been about 55 seconds. So now what? Well based on the result from the laboratory, the patient needs to have their heparin increased. Remember this is a pediatric patient, but the treatment is based on a result that is 4 hours old. Why is the APTT stable for only 4 hours, because when plasma sits on red cells, the PF4 from the platelets neutralize heparin, if you don't believe this, run a heparinized sample from a previous shift and compare the results. So, as no good deed goes unpunished, your "normal" result, now put that patient at an increased risk for a bleed- most likely his level APTT reflected an appropriate level of heparin, and based on this result, the clinician may increase it- not good. Be careful, stick to the guidelines- they are there for a reason. And as we know, sometimes normal is not the correct answer.

What about the anti-Xa assay that comes out to be 0 U/ml? What is that, and what does it mean?- Your controls are normal - so that means your reagents are okay right? What if you are not sure? Run a previous patient, or a CAP sample with known results, that will validate that your assay is working. (Keep all proficiency samples, and you should keep a stock of patient results, to check your assays). But you still have a 0, and the clinician is telling you that is impossible. How can you determine if there is heparin in that sample? The best test for residual heparin is a thrombin time- it should be prolonged with both unfractionated and low molecular weight heparin. If that comes out normal, there is no heparin in that sample. Either the sample wasn't drawn at the correct time, or there is no heparin on board. Find out how long after heparin was given, was the blood sample taken. A repeat sample may give more accurate results.

So, based on the limited information that we get in the laboratory- it is important to understand what test results mean - or better yet, what they can mean. The more pieces to the puzzle you have, the clearer the picture becomes.

Coupon Cutting in the Coagulation Laboratory

2009-02-03T00:00:00.000-05:00

You can't open a newspaper without seeing "in these economic times", or turn on the TV with getting some financial report. People are cutting coupons, not buying expensive coffee, so why not coagulation costs? Okay, personnel is not an issue, there isn't enough of us to go around on a good day! And I am not talking about splitting gauze into pieces. Coagulation testing is expensive- should you "make" or "buy" a test? What about the impact on patient care? All questions that need to be looked at.

Remember when the laboratory used to be a revenue generating center- some of you might not- but tests were done whenever, at a cost set up by the laboratory- no questions asked. With the implementation of Diagnostic Related Groups (DRG's) where ordering tests became relative to diagnosis - ICD-9 codes, the laboratory must have a CPT code for a test in order to charge. For an in-patient laboratory tests are "bundled" for diagnosis- so inpatient testing- if out of the designated scope- the testing the laboratory does is pretty much minimally reimbursed- (trust me this is a watered down version of the complexities of coding etc- it is very intricate)

Therefore the goal is to keep expensive inpatient testing down while not sacrificing patient care - so do more outpatient testing! HOW? restrict inpatient testing- HOW?
make clinicians get approval for coagulation testing- you will eliminate doing factor XIII's when you really have VIII's. Getting a factor V, when they really want an APCR, running factor assays on heparinizned patients,(NOTE: all inpatients that have a factor work-up should get a thrombin time PRIOR to doing that workup to determine if heparin is on board or not. The thrombin time is the best test for residual heparin- and will let you know if you need to cancel that workup, and why your mix didn't correct- save time and money not doing a Lupus workup) or on patients on Direct Thrombin Inhibitors, What about doing factors II, and VII on patients with a prolonged APTT- should I stop here, or my favorite- doing a full thrombophilia workup on an anticoagulated patient or a patient with a clot-
These results are inaccurate- it is a waste- the reality is the patient MUST be treated, so do it- then bring the patient back to find the cause- when the event is over, and the patient can be safely removed from anticoagulation. Okay should we do molecular testing for Factor V Leiden, MTHFR, or Prothtombin mutation? Sure, but not as an inpatient, only as an outpatient. So there, we have not compromised patient care, we have helped to control costs, and given better answers.

Due to the complexity of coagulation testing, interpretaion is very helpful to the clinicians, as well as a great learning experience for the technologists and the residents. This is a coded test, therefore it is billable. Time well spent and everyone profits, patient, students and laboratory.

What about the testing itself- do you make or buy a test? I am very much in favor of having testing in house, available for patient care. Testing should reflect your patient population, maybe slimmed down a bit- for example if you have a coumadin clinic- might want to offer factor II, and VII- less call for V and X. If you want to run one test for thrombophilia- go with APCR, it is the most common disorder. Personnel is always an issue so the best thing is to automate, automate, automate. Tests like ristocetin cofactors, take a long time, but they can be automated. Bleeding times you lose a tech for up to an hour, get an analyzer that screens for primary hemostasis like the PFA 100. It can be coded as you would for a platelet aggregation, and no one has to leave the lab. When you are looking at ELISA based assays see if you can get them on an automated system, or this may be a test that is worth sending out- do your homework.

Batching is also a cost saving measure. Try to accomadate your clinic schedules, this way if certain clinics meet on certain days, their wait time for results will be shorter, so your highest volume will have the quickest turn around time- happy customers- Also, look for mulipurpose controls. Being able to use controls on many analyzers avoids making up different ones, for testing, it is a waste of time and money.

Remember the shortest distance between 2 points is a straight line- so have good testing algorithims. Being able to guide your clinicians on just what should be ordered in what progression will give everyone more bang for their buck!
Better results, shorter response time, direct route, outcome cost savings for everyone.

So look at your spending, use your coagulation coupons and minimze output to maximize your input- everyone wins!

Mixing Studies-To correct or not correct: that is the question

2009-01-06T00:00:00.000-05:00

Ah yes, one of the simplest test in coagulation that can give a clinician a tremendous amount of information is a mixing study.
However, simple and coagulation in the same sentence is like an oxymoron. There is nothing simple when it comes to coagulation.
Okay, back to the mixing study- Here we have a test that can tell the clinician quickly is there is a factor deficiency or an inhibitor. But like any test, in particular coagulation tests, if it is not performed correctly, your answer can affect every aspect of patient care. So how do we give a good answer- lets discuss-
First- what is a mixing study and how does it work- We know that if a mixing study corrects it represents a factor deficiency, that is the factor in the pooled normal plasma (PNP) replaces the factor that is missing in the patients sample. But, if there is not a correction, it suggests there is something present that is "inhibiting" the correction. Seems simple enough- well it is anything but!
A normal PT or APTT tells the clinician that there is at least 30-40% of factor levels present. Any less than that and a patient may bleed-
So what are we doing, we are taking pooled normal plasma (PNP) and adding 1 part of PNP to 1 part of patient plasma ( which represent a 1:2 dilution). Very simple, but there are a few things that all labs need to be aware of-
You should NOT use a control to mix with the patient plasma. A control contains buffers and stabilizers- and has a different matrix than fresh frozen PNP. That is what should be used to mix with the patient's plasma.
All of the factor levels should be known- okay so why is that important- well lets say we have a PNP with a factor VIII level of 60%- (text book levels of normals for factors are 50-150%) We make the 1:1 mix, the 60% now becomes 30%- this may NOT be enough to correct a factor VIII deficiency, so it gives the appearance of an inhibitor. That is why it is important to make sure you have sufficient levels of factors in your PNP.

One of the biggest unanswered questions is: Is your reagent sensitive to all the factors, that is will your PT or APTT be prolonged when factor levels reach 30-40%? Some reagents are insensitive to certain factors, which means you can have a level <30% deficient =" %" mix =" 30" mix =" 50" formula=" %" correction =" Patient">75% correction = Factor Deficiency
APTT < correction =" Inhibitor"> 75% correction = Factor Deficiency
PT <70% correction = Inhibitor
(Cheng, S, American Journal of Clinical Pathology, 2002; 117.)

Rosner Index: Looks at the Clotting time of the Patient =A
Clotting time of the 1;1 mix =B
Clotting time of PNP =C

INDEX = B - C
_______
A

High Index suggests an inhibitor
Low Index suggests a factor deficiency

Cutoff's must be determined in each lab- Most common cutoff is 15.

Conclusion: So how do we tell if there is a correction? You need to use all of the available clinical information and compare to patient results. Make sure that all testing that is done in the laboratory is consistent and have good standard operating procedures. At present this is the best way to ensure standardization within your laboratory- hope this helps!

T'was the night before....

2008-12-01T00:00:00.000-05:00

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 Fibrinolytic System: Testing and Disorders

2008-11-05T00:00:00.000-05:00

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.

Oral Anticoagulation: Positives, Pitfalls and Polymorphisms

2008-10-07T00:00:00.000-04:00

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.

Maximize your time and minimize your problems!

2008-09-03T00:00:00.000-04:00

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!

HISTORY OF BLOOD COAGULATION

2008-08-04T00:00:00.000-04:00

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?

2008-07-10T00:00:00.000-04:00

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

2008-06-04T00:00:00.000-04:00

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?

2008-05-02T00:00:00.000-04:00

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!