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Prothrombin time (PT):
Performed by adding tissue thromboplastin (tissue factor) to plasma. Measures VIIa-->Xa-->Iia-->CLOT. Prolongation of the PT only is indicative of factor VII deficiency. Combined prolongation of PT and APTT is indicative of either factor X, II, or V deficiency. Main clinical use of PT is to monitor warfarin therapy. Since different laboratories use different reagent, best way to monitor is to use INR.
Evaluation of an elevated PT: Is it the sole abnormality? If so then this indicates factor VII deficiency and means no additional risk of bleeding since one needs only factor VII levels of over 5 - 10% to support hemostasis. Most common acquired cause is vitamin K deficiency due to warfarin or inadequate vitamin K intake. Liver disease is the next most common acquired cause. In combined elevations of the PT and aPTT the differential is either the rare factor V, X or II deficiency or multiple acquired defects. Antiphospholipid antibodies can cause elevation of the PT by two mechanisms which can be told apart by a 50:50 mix. The first is by the antibodies interfering with the phospholipid in the test reagent (50:50 mix does not correct). The other is by depletion of prothrombin which can be associated with severe bleeding (50:50 mix does correct).
Activated Partial Thromboplastin Time:
Performed by adding dirt (clay) to plasma. The aPTT measures the contact pathway (XII, kallikrein, XI)-->IXa+VIIIa-->Xa+Va-->IIa-->CLOT. The are four major diagnostic considerations when the aPTT is elevated:
1. Factor Deficiency. aPTT does not elevate until one single factor is below 30%. However only mild decreases (60-70% range) in multiple factors will prolong the aPTT.
2. Lupus Inhibitors (Antiphospholipid Antibodies). Antiphospholipid antibodies (APLA) are antibodies which react with certain phospholipid in the body. They will also react with the phospholipid in the test reagent for the aPTT. Thus they will artifactually prolong the aPTT.
3. Factor Inhibitors. These are antibodies to coagulation factors such as factor VIII. Usually found in hemophiliacs or acquired states such as the elderly and after pregnancy. Almost always associated with bleeding, often severe with large ecchymosis.
4. Heparin. Heparin, even in minute amounts, can prolong the aPTT. This most often occurs when blood for the aPTT is drawn from lines. This occurs up to 30% of the time in certain laboratories.
How to tell 1-4 apart. Simplest way to avoid heparin contamination is to always draw blood from peripheral sites. Tests have shown that discarding the first 10-50 cc of blood from a line is not helpful (and wasteful of the patents blood!). The 50:50 mix will differentiate the rest. This is done by making a mixture of the patients and normal pool plasma and performing aPTT on it. Then the mixture is incubated for 30, 60 and 120 minutes and aPTT's are performed at various times. Each of the three major diagnostic considerations will perform differently on the 50:50 mix:
1. Factor Deficiency. aPTT will correct to normal at time 0 and stay in the normal range on each of the time points.
2. APLA. Does not correct to normal (may partially correct) at time 0 or any time point. May actually prolong further. (Lupus cofactor effect).
3. Factor Inhibitors. Corrects to normal at time 0 but then prolongs with further incubation.
TIME 0 30 60 120
NORMAL 30 32 33 34
PATIENT'S 50 52 55 53
50:50-DEF (1) 30 32 33 34
50:50-APLA (2) 40 38 42 39
50:50-INHIB (3) 30 34 40 45
Bleeding time:
Once standard screening test, now very controversial. Best way to view it as being sensitive but not specific. If a patient has a normal bleeding time then their risk of bleeding with a procedure is low. Unfortunately a prolonged bleeding time does not guarantee bleeding with a procedure. Not usually in otherwise asymptomatic patients before procedures.
TESTS FOR DIC
Simply put, DIC is inappropriate activation of thrombin (IIa). This leads to 1) conversion of fibrinogen to fibrin, 2) activation of platelets (and their consumption), 3) activation of factors V and VIII 4) activation of protein C (and degradation of factors Va and VIIIa), 5) activation of endothelial cells, and 6) activation of fibrinolysis.
There is no one test that will diagnosis DIC, rather one must match the test to the clinical situation.
Screening Tests: The PT and aPTT are usually elevated in severe DIC but may be normal or shorted in chronic forms. One may also see a shortened aPTT in severe acute DIC due to large amounts of activated II and factor X "bypassing" the contact pathway. aPTT's as short as 10 seconds have been seen in acute DIC. The platelet count is usually decreased but may be normal in chronic DIC. Serum fibrinogen is decreased in DIC but again may be in the "normal" range in chronic DIC.
"Specific Tests": These are a group of test that allows one to deduce that abnormally high concentrations of IIa are present.
Ethanol gel and Protamine test: Both of these tests detect
circulating fibrin monomers. One sees circulating fibrin monomers when IIa acts on fibrinogen. Usually the monomer polymerizes with the fibrin clot but when there is too much IIa, these monomers can circulate. Detection of circulating fibrin monomer means there is too much IIa and, ergo, DIC.
Fibrin Degradation Products: When plasmin acts on the fibrin/fibrinogen molecule it cleaves it in specific places. Thus levels will be increased in situations of increased fibrin/fibrinogen destruction (DIC, fibrinolysis).
D-Dimers: When fibrin monomers bond to form a thrombus, factor XIII acts to bond their "D" domains. This bond is resistant to plasmin and this degradation fragment is called the "D-dimer". Elevated levels of D-dimer indicates that 1) IIa has acted on fibrinogen to form a fibrin monomer which bonded to another fibrin monomer and 2) this was lysed by plasmin.
Other tests that are sometimes helpful:
Thrombin time: Performed by adding IIa to plasma. Elevated in 1) DIC (FDP's interfere with polymerization), 2) low fibrinogen levels, 3) dysfibrinogenemia and 4) heparin (very sensitive).
Reptilase time: Same as thrombin time but performed with a snake venom which is insensitive to heparin. Is elevated is all condition as the thrombin time except heparin. Thrombin time and reptilase time are most useful in evaluation of dysfibrinogenemia.
TESTS FOR ANTIPHOSPHOLIPID ANTIBODIES (APLA)
APLA's are important to detect because in certain patients they are associated with a syndrome that includes a hypercoagulable state, thrombocytopenia, fetal loss, dementia, strokes, optic changes, Addison disease, and skin rashes. There are two main tests for APLA's: testing for presence of antibodies to cardiolipin and the coagulation based tests for APLA.
Coagulation Based Tests: As you recall, APLA react with phospholipid. The phospholipid provide a surface for the coagulation reaction to take place on. The basis for all these tests is that if there are antibodies on the phospholipid, it will prolong the coagulation reactions and the test time. Once an elevated aPTT is found one must verify it by demonstrating it does not correct with a 50:50 mix. To ice it then one adds phospholipid derived from platelets. APLA do not react with phospholipid from platelets and this will correct the coagulation tests. To summarize, one screens for APLA with coagulation based test to see if any are prolonged. If one is then one uses a 50:50 mix and show's it does not correct. Then one uses platelet to correct it.
aPTT. Only sensitive to 30% of APLA. One can increase sensitivity by using different reagents.
Dilute Russell Viper Venom Time (dRVVT). This test is very sensitive to any interference with phospholipid and is very sensitive to APLA. It is performed by initiating the coagulation cascade with Russell Viper Venom.
Kaolin clotting time. This test uses no added phospholipid and is the most sensitive test to APLA.
Platelet Neutralization Test. This test takes a reaction that is prolonged by plasma which does not correct with a 50:50 mix and adds extracts of platelet. The platelet phospholipid "soak up" the antiphospholipid antibody and corrects the aPTT. If it corrects to normal this is very specific for APLA.
Hexagonal Phospholipid Neutralization Based on the same principle as the Platelet test except using hexagonal phospholipid which are more specific for antiphospholipid antibodies.
Anticardiolipin Antibodies: This is an ELISA test for antibodies to cardiolipin. Therefore it can be performed on plasma which has been anticoagulated. Tests results at OHSU is reported in standard deviation with >3SD abnormal. Tests are also reported as specific isotype (IgG, IgA, IgM).
Approach to the patients suspected of having APLA. Unfortunately there is no one test that can screen a patient for APLA. One must do the whole panel on patients suspected with APLA. This would include 1) Anticardiolipin antibodies, 2) Kaolin clotting time 3) dRVVT 4)"Lupus Inhibitor Screen" (different aPTT reagents). At OHSU the dRVVT is done along with the "Lupus Inhibitor Screen".
HYPERCOAGULABLE STATES
Definition
A hypercoagulable state is one in which due to an inherited or acquired disorder there is an increased propensity to form thrombosis. This state is manifested clinically by an increase in number of thromboses, thrombosis at an early age, a familial tendency toward thrombosis, and thrombosis at unusual sites.
ETIOLOGIES
Congenital
Hereditary resistance to activated protein C (HRAPC) a recently described defect in factor V which renders it unable to be degraded by activated protein C. Associated with primarily venous thrombosis. VERY common: 40 - 60% of hypercoaguable states, 20% of first DVTs, and 2 - 8% of the normal population. Dramaticly increased the risk of thrombosis with pregnancy or estrogen containing oral contraceptives (30x).
Prothrombin Gene Mutation: A recent described defect in the prothrombin gene (nt20210 G-->A). Present in 1-2% of normal population but 18-20% of patients with hypercoagulable states. Early reports indicates it might be involve in arterial disease also. Mechanism of action unsure.
Protein C-a protein which when activated by thrombin degrades factors V and VIII. Deficiencies primarily causes venous thrombosis. Risk of thrombosis ranges from 0.5 - 2.5%/year. (5-10% of hypercoagulable patients, incidence 1 in 16,000).
Protein S-a cofactor for Protein C. Exists in bound and unbound form. Deficiencies of total protein S and of unbound protein S (more common) can lead to the hypercoagulable state. Risk of thrombosis may be up to 3.5%/year. Primarily cause venous thrombosis but has been associated with CVA's in young women. (5-10%, 1 in 16,000)
Antithrombin- inhibits activated clotting factors. Primarily venous thrombosis. It usually NOT associated with heparin resistance. (3%, 1 in 2000).
Dysfibrinogenemia-defective fibrinogen which forms clots difficult to degrade by fibrinolytic agents. Can be both venous and arterial thrombosis. (1%)
Fibrinolytic disorders-defective TPA or an increase in TPA-inhibitor can lead to increase thrombosis (0-40%?). Role in congenital hypercoagulable states controversial.
Homocystinuria-endothelial damage leading to increased arteriosclerosis. Very common risk factor for both venous and arterial disease. Modest elevations (>11.0 nmol/L) associated with increased risk of atherosclerosis. Levels over 18 nmol/l associated with increased risk of venous thrombosis. High homocystine levels are synergist risk factor with factor V Leiden.
Lipoprotein (a)-Increases risk of arteriosclerosis.
Acquired
Pregnancy- DVT and its sequelae pulmonary embolism is the most common cause of maternal death. The incidence during pregnancy is 1 - 5/1,000 pregnancies (includes postpartum). In women with previous DVT, the risk of thrombosis is 12 - 35% and rises up to 75% in women with antithrombin deficiency. Etiologies include decreased levels of protein S and "free" protein S, decreased levels of antithrombin and venous stasis. Increased levels of factor VII, VIII and fibrinogen are also seen.
Inflammatory disorders-Inflammatory bowel disease, infections, etc. Due to inflammatory mediators, monocytes procoagulants, decrease in free protein S. Spectacular large vessel thrombosis (IVC, carotid) have been reported in Crohn's disease.
Myeloproliferative disorders-Thrombosis is the leading cause of death in myeloproliferative diseases. ? platelet defects, increased viscosity. Thrombosis can predate the appearance of the myeloproliferative syndrome by months to years.
Antiphospholipid syndrome ("lupus anticoagulant")-Although associated with thrombosis, exact mechanism is unclear. Can be associated with arterial and/or venous thrombosis, thrombocytopenia, miscarriages, livedo reticularis, neurological disorders (strokes, TIA's, delirium) and myocardial infarctions.
Post-surgery-inflammatory mediators, venous stasis, decrease in fibrinolytic activity.
Nephrotic syndrome-inflammatory mediators, decrease in free protein S.
Estrogens/BCP-increased procoagulant protein synthesis, decreased free protein S.
Malignancy-production of procoagulant substance by the tumor, tumor tissue factor production, venous obstruction, inflammatory mediators.
Paroxysmal nocturnal hemoglobinuria-abnormal platelets?
Testing
One should be suspicious a hypercoagulable state is present if there is an increase in number of thromboses, thrombosis at an early age, a familial tendency toward thrombosis, and thrombosis at unusual sites. Contrary to popular belief, over 50% of thrombosis in patients with document hypercoagulable states occurs with "provocation" (surgery, pregnancy).
The key to diagnosing the exact cause of an hypercoagulable state is to try to determine if it is an acquired or inherited disorder. The key is a good family history. Most patient with an inherited disorder will have several relatives afflicted with thrombosis. A good thrombotic family history is mandatory on any patient with thrombosis. Studies on families with protein C and protein S deficiency show that deficient individuals can have their first thrombosis as late as age 70. In order to obtain the best history one must sit with the patient and inquire specifically about each relative. Repeated thrombosis spread over several decades is another clue or thrombosis in unusual location such as the mesenteric vein. Acquired cause of thrombosis can present with a "flurry" of clots or have thrombosis in associated with other disorders. Recurrent thrombosis while the patient is on warfarin is a strong clue towards malignant hypercoaguable states.
Testing
HRAPC In unanticoagulated patients one tests for HRAPC by performing a coagulation based assay to determine how long the aPTT prolong with the addition of activated protein C. A ratio of under 2.6-2.7 is abnormal. Given the gene mutation is constant (ARG506GLN) one can perform a DNA assay via PCR (OHSU DNA diagnostic lab x503-494-7821). The DNA assay is useful in borderline cases or in patients who are anticoagulated.
Prothrombin gene mutation- Diagnosed via PCR based test via OHSU DNA Diagnostic service.
Protein C and protein S-Since these are vitamin K dependent proteins their levels will be reduced by warfarin therapy. Thus one should draw blood for these proteins before starting warfarin or wait 2-3 weeks after stopping therapy. In patients who require life-long therapy one can do family studies to pick up the deficiency or temporally halt therapy for 2-3 weeks to determine the levels. One should specifically ask for "free protein S" levels since a deficiency in free protein S is much more than total protein S deficiency. Free protein S can be decreased to low levels during a normal pregnancy. Both protein S and protein C may be low in acute thrombosis and illness.
Antithrombin-Acute thromboembolism and rarely heparin therapy can lowers levels. Thus an normal antithrombin levels drawn in these circumstance effectively rule this out as a cause of a hypercoagulable state. Low antithrombin levels performed in acute setting should be repeated before labeling the patient antithrombin deficient.
Antiphospholipid Antibodies (APLA):
Coagulation Based Tests: APLA react with phospholipid. The phospholipid provide a surface for the coagulation reaction to take place on. The basis for all these tests is that if there are antibodies on the phospholipid, it will prolong the coagulation reactions and the test time. Once an elevated aPTT is found one must verify it by demonstrating it does not correct with a 50:50 mix. To ice it then one adds phospholipid derived from platelets or hexagonal phase phospholipids. The added phospholipid "soaks up" the APLA and this will correct the coagulation tests. To summarize, one screens for APLA with coagulation based test to see if any are prolonged. If one is then one uses a 50:50 mix and show's it does not correct. Then one uses phospholipids to correct it.
aPTT. Only sensitive to 30% of APLA. One can increase sensitivity by using different reagents.
Dilute Russell Viper Venom Time (dRVVT). This test is very sensitive to any interference with phospholipid and is very sensitive to APLA. It is performed by initiating the coagulation cascade with Russell Viper Venom.
Kaolin clotting time. This test uses no added phospholipid and is the most sensitive test to APLA.
Platelet Neutralization Test. This test takes a reaction that is prolonged by plasma which does not correct with a 50:50 mix and adds extracts of platelet. The platelet phospholipid "soak up" the antiphospholipid antibody and corrects the aPTT. If it corrects to normal this is very specific for APLA.
Hexagonal Phospholipid Neutralization Based on the same principle as the Platelet test except using hexagonal phospholipid which are more specific for antiphospholipid antibodies. Very useful because this is the only coagulation based test which can be performed on patients receiving anticoagulants and is the most sensitive test for antiphospholipid antibody.
Anticardiolipin Antibodies: This is an ELISA test for antibodies to cardiolipin. Therefore it can be performed on plasma which has been anticoagulated. Tests results at OHSU is reported in standard deviation with >3SD abnormal. Tests are also reported as specific isotype (IgG, IgA, IgM).
Approach to the patients suspected of having APLA. Unfortunately there is no one test that can screen a patient for APLA. One must do the whole panel on patients suspected with APLA. Traditional, this would include 1) Anticardiolipin antibodies, 2) Kaolin clotting time 3) dRVVT 4)"Lupus Inhibitor Screen" (different aPTT reagents). At OHSU the dRVVT is done along with the "Lupus Inhibitor Screen". In patients on anticoagulants one should obtain an hexagonal phospholipid and anticardiolipin antibody. A simplified approach would be to obtain anticardiolipin antibodies and the hexagonal phospholipid test.
Dysfibrinogenemias-Most patients with dysfibrinogenemia will have a prolonged thrombin time. One can confirm this diagnosis by measuring a functional level of fibrinogen and an antigen level. In dysfibrinogenemia these will be discordant.
Malignancy-In patients without obvious malignancy it is unclear how aggressive a workup should be undertaken. One should do a good history and physical, stool guaiac, chest x-ray and perhaps an abdomen CT. Further studies should be undertaken only if clues appear from the initial workup. Most common tumors associated with hypercoagulable states are mucin secreting adenocarcinomas-lung, gastrointestinal and pancreas.
Arteriosclerosis. In patients with early arteriosclerosis the workup is less clear. Most patients smoke and this is the primary risk factor for early arterial disease. Abnormalities of lipid metabolism such as raised LDL, apolipoprotein B and lowered HDL are treatable and may be worth pursuing. Raised blood levels of homocysteine and lipoprotein(a) are also associated with premature arteriosclerosis. One of the strongest predictors of future vascular events is an elevated fibrinogen.
Myeloproliferative syndromes. Certain hypercoagulable patients, especially those with Budd-Chairi syndrome, may have an "occult" myeloproliferative syndrome. Although they may not have evidence of any hematological disorder on the peripheral smear or bone marrow aspirate there is an established clonal proliferation of abnormal hemopoietic cells in these patients. A sensitive test for myeloproliferative disorders is the endogenous erythropoietin colony assay. This test depends on the ability of the abnormal clone to grow in culture in the absence of erythropoietin. A positive test can predate the onset of an overt myeloproliferative disorders by months to years. Up to 25-50% of patients with "idiopathic" Budd-Chairi syndrome will have a myeloproliferative disorders.
Paroxysmal nocturnal hemoglobinuria- The acid hemolysis or sucrose hemolysis test was used to diagnose this entity. Currently the test of choice is flow cytometry to detect cells missing CD59 and other abnormalities of the cell membrane.
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TEST ........................................HEPARIN..........WARFARIN
ANTITHROMBIN........................YES.......................YES
PROTEIN C................................YES.......................NO
PROTEIN S.................................YES....................... NO
ANTICARDIOLIPIN Abs............YES....................... YES
LUPUS INHIBITOR ...................NO....................... NO
HEXAGONAL PHOSPHOLIPID
.....................................................YES....................... YES
HRAPC COAGULATION ASSAY
......................................................NO....................... NO
FACTOR V PCR ........................YES......................YES
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