An Algorithmic Approach to Coagulation Testing

An Algorithmic Approach to Coagulation Testing Kimberly Klein, MD, S. Kate Hartman, MD, Jun Teruya, MD, DSc, and Shiu-Ki Rocky Hui, MD Coagulation Testing The complexity of coagulation testing often makes the interpretation daunting. Basic knowledge of the coagulation cascade is not enough. Knowledge of the test’s mechanistic features and functional limitations is required. Although the acquisition of such knowledge is important, the clinical scenario may not afford the luxury. With the aid of a clinical pathologist, understanding can be achieved quickly in the most costeffective way. The basic questions one is faced with include the following: What test do I order? What does this result mean? Is my workup complete? We are often faced with explaining unexpected results or are sometimes plagued by deciding what to do with the new information at hand. At times, we question how the specimen is to be collected, the validity of use of new coagulation tests, and occasionally the validity of the results. Understanding coagulation testing principles and consulting a pathologist are essential to answering these daunting questions. In this section, we accomplish the following tasks: first we give a brief overview of coagulation testing. Next we discuss 3 commonly encountered clinical examples and provide an algorithmic approach to evaluate each. Finally, we discuss the role of the pathologist in assisting clinicians with coagulation test selection and interpretation. Like the coagulation cascade, selecting and interpreting coagulation tests can be intimidating. There are several laboratory tests available and each functions to assess different aspects of hemostasis. Primary hemostasis can be assessed by evaluating platelet adhesion and aggregation using platelet function assays (PFAs), such as platelet aggregation studies, ristocetin cofactor activity, and von Willebrand factor (vWF) antigen. Secondary hemostasis, also known as the coagulation cascade, is most commonly evaluated by measuring prothrombin time (PT) and activated partial thromboplastin Dis Mon 2012;58:431-439 0011-5029/2012 $36.00 ⫹ 0 http://dx.doi.org/10.1016/j.disamonth.2012.04.006 DM, August 2012

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time (PTT). These tests function as general coagulation screening tests and allow for the assessment of the extrinsic, intrinsic, and common pathways. More specific analysis of these pathways can be achieved by ordering factor-specific assays and mixing studies. Factor assays determine the presence of a factor deficiency, whereas mixing studies can detect the presence of factor inhibitors. The presence of lupus anticoagulant (LAC) can be identified by assays specific for identifying LAC such as: STACLOT/Diluted Russell’s Viper Venom Time (DRVVT), whereas anti-␤2 glycoprotein I (␤2GPI) positivity identifies patients at risk for developing antiphospholipid syndrome. Fibrinogen, which is catalyzed by thrombin to form fibrin, is measured in conjunction with thrombin time (TT) or PT to test for the ability to form clot. The final stages of hemostasis—fibrin cross-linking and fibrinolysis— can be analyzed with a thromboelastogram (TEG), factor XIII assay, fibrinolysis markers, such as tissue plasminogen activator, plasminogen activator inhibitor-1, and ␣2-antiplasmin activity.1,2

Incidentally Prolonged of PT and/or PTT Conceptually understanding the function of each of these laboratory tests provides some guidance as to what test to order. However, the clinical scenario dictates which studies should be ordered and the most efficacious manner of doing so. Take for example the case of an asymptomatic patient whose baseline coagulation status is being evaluated as part of a presurgical workup (Fig 1). In this case, it is not uncommon to encounter an incidentally prolonged PT or PTT. So what do you do now? Is this significant? How does this affect patient management? Reviewing the patient’s medical history is essential, and the pertinent clinical information to collect includes a comprehensive list of medications, personal or family history of bleeding/bruising, dietary history, recent history of exposure to bovine-derived “fibrin glue,” and a history of lupus or liver disease. It is also imperative to measure the fibrinogen level, especially in patients with liver disease, treated with plasma exchange chronically or those taking l-asparaginase. Fibrinogen, also known as factor I, is the final step of the common pathway and its presence indicates clot formation. Thus, hypofibrinogenemia or dysfibrinogenemia can lead to prolongation of PT and PTT and needs to be assessed before ordering additional costly tests. If a decreased fibrinogen level does not account for the abnormalities observed in PT or PTT, the following algorithm should be performed. With a minimally prolonged PT, less than 2 seconds, factor assays II, V, VII, and X should be ordered as they allow for assessment of factor 432

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Prolonga
on of PT, PTT or both

Clinical History and Check Fibrinogen Level

↑ PT only, Normal Fibrinogen

PT > 2 sec

PT < 2 sec

Mixing Studies

F2, F5, F7 & F10

↑ PTT only, Normal Fibrinogen

↑ Thrombin Time

No. Thrombin Time

PTT Heparinase

PTT > 3 Sec

PTT < 3 Sec

↑ PT & PTT, No. Fibrinogen

↑ Thrombin Time

No. Thrombin Time

↓ Fibrinogen, ↑ PT &/or PTT

Afibrinogenemia Acquired Hypofibrinogenemia Dysfibrinogenemia L asparaginase

F2, F5, F7, F8, F9, F10, F11, mixing studies & STACLOT/DRVVT Consider Correc
ng Fibrinogen and Re-assess

Heparin STACLOT / DRVVT

Lupus An
coagulant

Liver disease, Vitamin K antagonist, FSP, Extrinsic Factor deficiency/ inhibitor

PTT Heparinase Mixing Studies

F8, F9, and F11 Heparin

STACLOT/ DRVVT

If + consider

Vitamin K deficiency, Lupus An
Coagulant or Mul
ple Factor deficiency/ inhibitor

Intrinsic Factor deficiency/ inhibitor, VWD

If F8↓

Consider An
- beta 2-

glycoprotein 1 assay

If + consider

Lupus An
coagulant

VWF:Ag VWF:Rco

Mul
mers Distribu
on

FIG 1. Algorithm for workup of prolonged PT and/or PTT. (Color version of figure is available online.)

deficiencies in the extrinsic and common pathways. Mixing studies typically are not performed as they are generally not helpful. Factor deficiencies may be due to liver disease, vitamin K deficiencies, or antagonists.2,3 Prolongation of the PT greater than 2 seconds should raise the possibility of a factor inhibitor, which can be evaluated with 1:1 mixing studies in conjunction with the previously mention factor assays. If the PT corrects with mixing studies, an inhibitor is not likely. However, if the PT fails to correct with mixing studies, then an inhibitor is likely present. Factor inhibitors are sometimes observed in patients exposed to bovine thrombin, patients on certain medications, and those with a medical history of amyloidosis or systemic lupus erythematosus.2-5 The presence of an inhibitor in mixing studies should be followed by DRVVT/STACLOT testing to evaluate for the presence of a LAC. Both tests include a coagulation-based screening test and a confirmatory test containing high levels of phospholipids as recommended by the International Society on Thrombosis and Haemostasis. If LAC is identified by either of these 2 methods, the diagnosis of antiphospholipid syndrome is likely but should be repeatedly positive in 3 months. A test for DM, August 2012

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anti-␤2GP1 and cardiolipin antibodies can be ordered at the clinician’s discretion.6-9 Evaluation of an isolated prolonged PTT is similar to the isolated prolonged PT workup mentioned above with the exception of a few variances. First, TT, which functions to assess fibrin formation, is ordered before factor assays and mixing studies to rule out the possibility of heparin contamination.2,3 Because heparin acts as a thrombin inhibitor, specimens contaminated with heparin will have a prolonged TT. This issue can be resolved by ordering a PTT with heparinase. Correction of the PTT after hepzyme treatment is diagnostic of heparin contamination.3 However, if TT is not prolonged and the PTT is prolonged less than 3 seconds, assays for factors VIII, IX, and XI should be performed. In the case of decreased factor VIII, von Willebrand antigens and ristocetin cofactor should be ordered to differentiate between a hemophilia A and von Willebrand disease (vWD). If the PTT is prolonged more than 3 seconds, mixing studies and the above-mentioned factor assays should be performed. Once again these studies help to differentiate between the presence of factor deficiencies and inhibitors.2,4 Similar to the above algorithm, if mixing studies demonstrate an inhibitor, testing for LAC should be performed with or without subsequent anti-␤2GPI and cardrolipin antibody testing.6-8 Simultaneously prolonged PT/PTT is evaluated for factors II, V, VII, IV, X, and XI. Elevated PT and PTT are most commonly caused by vitamin K deficiency.4 This is because factors II, VII, IX, and X are vitamin K– dependent factors. These factors are components of the extrinsic and common pathways. Thus, when a patient has a mild vitamin K deficiency, PT may be the only affected value. However, because some the vitamin K— dependent factors listed above are involved in the common pathway, severe cases of hypovitaminosis can cause prolonged PTT as well. Some common causes of vitamin K deficiency include dietary intake deficiencies and disorders in absorption/metabolism owing to cholestasis and/or treatment with antibiotics. In addition to vitamin K hypovitaminosis, LAC is also a common cause of prolongation of both the PT and the PTT as are multiple factors deficiencies/inhibitors, liver disease, disseminated intravascular coagulation, and even heparin. Factor V level can help to distinguish between vitamin K deficiency and decreased liver synthesis.2,3,6-9

Coagulation Testing for a Bleeding Patient Aside from presurgical evaluation, coagulation studies are also ordered to manage and diagnose bleeding disorders. Similarly, assessment of PT 434

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Abnormali
es of PT, PTT, or Fibrinogen in a Bleeding Pa
ent with a Normal Platelet Count

Abnormali
es of PT or Fibrinogen

Isolated prolonged PTT which corrects 1:1 mixing studies

No Abnormali
es

See Chart Number One

Clinical history or symptoms sugges
ve of VWD

Clinical history or symptoms not sugges
ve of VWD

Platelet Func
onal Assay (PFA)

Abnormal PFA

Acquired Plt Dysfunc
on

Medica
ons

VWF:Ag, VWF:Rco , Factor 8

Normal PFA

Unknown

Mul
mers Distribu
on

Connec
ve
ssue Disorders VWD

Uremia Monoclonal Gammopathies

Platelet Aggrega
on

Abnormal aggrega
on with ristoce
n only

Glycoprotein IIB/IIIA

MPD Normal platelet aggrega
on to ristoce
n only

Glanzmann Thrombasthenia

FIG 2. Algorithm for workup of possible platelet disorders or von Willebrand disease. (Color version of figure is available online.)

and PTT in a bleeding patient is critical. Other tests that should be ordered simultaneously include fibrinogen and platelet count. Collectively, these studies help to discriminate between primary and secondary abnormalities in hemostasis.2 As illustrated above, disorders of secondary hemostasis manifest with isolated prolongation PT or PTT. However, this is not always the case (Fig 2). Patients with vWD, a disorder of primary hemostasis, can also present with an isolated prolonged PTT. In this situation, ristocetin cofactor and von Willebrand antigen may be performed concurrently with mixing studies and factor assays. This demonstrates how the clinical scenario can modify one’s approach to coagulation testing. Bleeding patients without abnormalities in the screening coagulation studies are approached differently. When all parameters are within normal limits, a thorough clinical history should be performed. Although not always possible, the emphasis should be to differentiate patients with vWD. The pertinent clinical information to collect includes family/ personal history of bleeding, current bleeding symptoms (mucosal vs deep tissue bleeding/hemarthrosis), presence of anatomic sources of DM, August 2012

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bleeding, renal function, medications, and assessment other confounding medical conditions, such as lymphoproliferative disorders, essential thrombocythemia, Wilms tumor, and hypothyroidism.2,10-12 When the index of suspicion for vWD is high, assays for vWF antigen, ristocetin cofactor, and factor VIII should be performed. vWF activity less than 30 IU/dL is diagnostic of vWD. However, because vWF antigen varies with blood type and can be elevated in the acute phase, activity levels less than 80% are problematic and require further evaluation. Subsequent multimer analysis and evaluation of the antigen to risoctein cofactor ratio help confirm and subclassify the disease. When the clinical suspicion for vWD is low, a platelet function assay 100 (PFA-100) should be performed. The PFA-100 is a screening test that assesses the functionality of platelets. It measures both platelet adhesion and aggregation. In this test whole blood is drawn through an aperture that is coated with either collagen and epinephrine or collagen and adenosine diphosphate (ADP). The closure time for the aperture is then measured. A prolonged closure time indicates acquired and/or inherited platelet dysfunction or low vWF factor.12 Identifiable causes of platelet dysfunction include medications (aspirin, certain antibiotics, nonsteroidal anti-inflammatory drugs, clopidogel), uremia, monoclonal gammopathies, vWD, and myeloproliferative diseases.2,3,9,12 Abnormal PFA results without an identifiable cause should subsequently be evaluated by platelet aggregation studies in addition to vWF evaluation. When performing this study, platelet aggregation is evaluated in the presence of a specific agonist (collagen, epinephrine, ADP, arachidonic acid, and ristocetin). If an abnormal platelet aggregation in response to all agonists except with ristocetin is observed, Glanzmann’s thrombasthenia is likely.2,13 Confirmation of this diagnosis is performed with platelet glycoprotein IIb/IIIa studies. Abnormal aggregation with ristocetin only is suspicious for BernardSoulier or vWD. In this situation, vWF antigen, ristocetin cofactor, factor VIII, multimeric analysis, and glycoprotein Ib studies should be performed. Assessment of these laboratory tests is necessary to differentiate, confirm, and subclassify these diseases.2,11,12 As previously discussed, some patients with vWD do not demonstrate evidence of inhibition on PFAs. Thus, bleeding patients even with a normal PFA-100 result should be evaluated with repeat PFA testing and screened for vWD and vascular causes of bleeding, including hereditary hemorrhagic telangiectasia, Elhers-Danlos, and scurvy.2,12 436

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Thromboelastogram (TEG)

Reac
on
me (R)

Maximum amplitude (MA) + G

Lys 30

Time to clot forma
on

Maximum clot strength and Clot Elas
city

Clot Lysis at 30 mins

↑R

↓R

↑MA and G

↓ MA and G

↑ Lys 30

Hypocoagulable state

Hypercoagulable state

Strong Clot

Weak Clot

Hyperfibrinolysis

Hyperfunc
oning Platelets Increased Fibrinogen Thrombocytosis

Hypofunc
oning platelets Thromombocytopenia Decreased Fibrinogen

Heparin Factor deficiency

Protamine

An
-Fibrinoly
c agents -

E-aminocaproic acid

-

Tranexamic acid

TEG with Heparinase Platelet and / or Cryoprecipitate

FFP

FIG 3. Interpretation of thromboelastrogram (TEG) and management. (Color version of figure is available online.)

Assessment of Intraoperative Bleeding Figure 3 depicts how to assess the dynamically changing coagulation status of trauma and surgical patients using a TEG. This instrument rapidly provides the following information: time to clot formation (R-value), maximal clot strength (MA), clot elasticity (G), and clot lysis at 30 minutes (Lys 30). It does so using a potentiator to accelerate the speed of clot formation. Whole blood is dispensed into a disposable cup and loaded onto an analyzer. Initially, the cup oscillates freely around a stationary pin, but as clot forms, the pin imitates the motion of the cup. This movement is graphically recorded, and the resultant TEG tracing allows for the global assessment of coagulation.13,14 The time to initial fibrin formation is the measurement represented by the R-value. An increased R-value is reflective of factor deficiencies, the presence of heparin, or dysfunctional factors. Heparin contamination can be differentiated by normalization of the R-value on TEG with heparinase. Heparin effect and factor deficiencies/dysfunction can be reversed with protamine and fresh frozen plasma, respectively.2,13,14 MA and G are reflective of clot strength and elasticity. The MA reflects the plot of a maximally oscillating pin, whereas G is a calculated value. DM, August 2012

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Together these values demonstrate platelet aggregation and fibrinogen. Decreased MA and G indicate thrombocytopenia, decreased platelet function, or hypofibrinogenemia and are corrected with platelets and/or cryoprecitate transfusion. Lys 30 represents clot retraction, graphically represented by decreased amplitude of the TEG tracing. An increased Lys 30 is representative of hyperfibrinolysis and can be treated with Amicar or cryoprecipitate. Collective analysis of the above values allows for global assessment of coagulation and can effectively change the management of emergently bleeding patients.14

Conclusions Each of the above examples illustrates the importance of laboratory testing. From treatment to diagnosis, laboratory testing is an invaluable tool, if used appropriately. As companions of the laboratory, pathologists are an untapped resource. From specimen adequacy to assay limitations to interpretation of unusual results, pathologists are as essential as the tests they perform. Consulting pathologists can take the mystery out of coagulation testing and aid in patient care.

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The Diagnosis, Evaluation and Management of Von Willebrand Disease. Bethesda, MD: National Heart, Lung, and Blood Institute, 2007. Mascelli MA, Worley S, Veriabo NJ, et al. Rapid assessment of platelet function with a modified whole-blood aggregometer in percutaneous transluminal coronary angioplasty patients receiving anti-GP IIb/IIIa therapy. Circulation 1997;96:3860-6. Chitlur M, Warrier I, Rajpurkar M, et al. Thromboelastography in children with coagulation factor deficiencies. Br J Hæmatol 2008;142:250-6. Caprini JA, Traverso CI, Arcelus JI. Perspectives on thromboelastography. Semin Thromb Hemost 1995;21:91-3.

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