1. Introduction to Coagulation Testing
Laura Worfolk, Ph.D.
Scientific Director, Hematology
Quest Diagnostics Nichols Institute, Chantilly, VA
The goal of today’s presentation is to provide a general overview of hemostasis and how laboratory testing contributes to the diagnosis and management of patients with bleeding and thrombotic disorders. Since this is a rather large topic, I’ve chosen several case studies that highlight the contribution of laboratory testing to patient care. Please keep in mind that we are just going to skim the surface of this topic and it is not my intent to provide an in-depth discussion on bleeding and thrombotic disorders.

2. Absence of overt bleeding/thrombosis
Hemostasis
Intricate system maintaining blood in fluid state
Reacts to vascular injury to stop blood loss and seal vessel wall
Involves platelets, clotting factors, endothelium, and inhibitory/control mechanisms
Highly developed system of checks and balances
Bleeding
Thrombosis
Normal Hemostasis
Absence of overt bleeding/thrombosis
Hemostasis is the process by which the body maintains blood fluidity in the vasculature under physiologic conditions but is primed to react to injury in an explosive manner in order to stem blood loss by sealing the defect in the vessel wall. It is a normal process that leads to cessation of blood flow from a damaged vessel. The process involves platelets, clotting factors, endothelium and inhibitory control mechanisms that are finely balanced and regulated. Derangement of any portion of this complex process can result in hemorrhagic or thrombotic clinical disorders.
Thrombosis is a pathologic process which may occur if the hemostatic stimulus is unregulated either because the inhibitory pathways are impaired or overwhelmed by the procoagulant stimulus.

3. Interested Specialties
Anesthesiology
Anticoagulant Management
HIT
Vascular Surgery
Graft Occlusion
PAD
Cardiology
Premature CAD
Primary Care
Practice patterns vary
Bleeding & Thrombosis
Hematology
Hemophilia
Thrombophilia
It is not only the hematologist that is presented with patients affected by bleeding and thrombotic disorders. For example the ob/gyn physician may be presented with a patient experiencing menorrhagia or recurrent fetal loss, the neurologist with a stroke patient, the anesthesiolgists faced with the challenge of anticoagulant management for the patient with heparin induced thrombocytopenia. The hematologist is often consulted in these cases…..
What to say???
OB/GYN
Fetal loss, Infertility,
Menorrhagia
Nephrology
AV Graft Occlusion
Neurology
Stroke

4. Hemostasis Statistics
#1 cause of death is CVD (includes heart attack & stroke)*
~1-2% of population w/ von Willebrand’s disease†
~18,000 Americans w/ hemophilia†
~600,000/year w/ venous thromboembolism‡
~½ with long-term health consequences; ~60,000 fatalities†
~5-8% of population w/ thrombophilia†
The prevalence of hemostatic disorders may be under-appreciated. Summarized on this slide are some statistics worth mentioning. The #1 cause of death worldwide is cardiovascular disease, a group of disorders that includes heart attack and stroke. On the bleeding side of the scale, approximately 1-2% of the general population is affected with von Willebrand’s disease, an inherited bleeding disorder of varying intensity. The CDC estimates that athere are ~18,000 Americans living with Hemophilia, another inherited bleeding disorder that is associated with significant morbidity and requires life-long management. On the thrombotic side of the scale, it is estimated that ~600,000 individuals in this country will experience a venous thromboembolism, which generally presents as a clot in the leg. ½ of these individuals will suffer from long-term health consequences, with 60,000 fatalities per year. Thrombophilia, an acquired or inherited defect which increases an individuals risk for developing a venous thromboembolism affects ~ 5-8% of the US population.
So, the bottom line, these are not rare disorders and many different medical professionals may encounter a patient with a hemostatic defect.
Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels and include:
Related links
Each year, at least 50,000 and perhaps as many as 200,000 patients will die from blood clots that obstruct blood flow to their lungs (pulmonary embolism).
:: The Atlas of Heart Disease and Stroke World Health Organization, :: Avoiding Heart Attacks and Strokes: Don’t be a victim, protect yourself World Health Organization, :: Health topic: Cardiovascular diseases
Coronary heart disease – disease of the blood vessels supplying the heart muscle
Cerebrovascular disease - disease of the blood vessels supplying the brain
Peripheral arterial disease – disease of blood vessels supplying the arms and legs
Rheumatic heart disease – damage to the heart muscle and heart valves from rheumatic fever, caused by streptococcal bacteria
Congenital heart disease - malformations of heart structure existing at birth.
Deep vein thrombosis and pulmonary embolism – blood clots in the leg veins, which can dislodge and move to the heart and lungs.
On warfarin. Atrial fibrillation is the most common sustained heart rhythm disorder and increases the risk for heart disease and stroke, both leading causes of death in the United States. An estimated 2.2 million adults in the United States have been diagnosed with atrial fibrillation
*WHO.
†CDC ‡

5. Primary Hemostasis Platelet role:
Adhesion (via vWF), post injury to vessel wall
Activation: shape changed, contents released
Aggregation, ie, “plug formation”
Formation of surface for coagulation reactions - “fibrin glue”
Before I get into the case studies, I’d like to briefly review hemostasis in the next several slides. Primary hemostasis is defined as the formation of the primary platelet plug, which occurs immediately after the injury. This process involves platelets, the blood vessel wall and von Willebrand factor.
Platelets adhere to the site of injury, mediated by von Willebrand Factor and other proteins. Platelets then become activated, they undergo a dramatic change in shape and release contents of their granules, molecules which further promote clot formation and wound healing. Other platelets are recruited to the area, resulting in a platelet aggregate which serves to ‘plug up’ the injury and provide the surface area for fibrin formation which resulting in stabilization of the platelet plug.
As a general rule, abnormalities in primary hemostasis result in hemorrhage from mucosal surfaces (epistaxis, melena, hematuria), petechial or ecchymotic (bruising) hemorrhages, and prolonged bleeding after venipuncture or wounds. However, if the defect is severe, bleeding more typical of disorders of secondary hemostasis, can result, e.g. intracavity hemorrhage. Therefore, an patient presenting with clinical signs typical of a defect in primary hemostasis may have abnormal platelet number or function, abnormal von Willebrand factor or defects in the blood vessel wall (the latter are very rare). The normal endothelium prevents hemostasis by providing a physical barrier and by secreting products which inhibit platelets, including nitric oxide and prostaglandin I2 (prostacyclin). Following injury to the vessel wall, the initial event is vasoconstriction, which is a transient, locally-induced phenomenon. Vasoconstriction not only retards extravascular blood loss, but also slows local blood flow, enhancing the adherence of platelets to exposed subendothelial surfaces and the activation of the coagulation process. The formation of the primary platelet plug involves platelet adhesion followed by platelet activation then aggregation to form a platelet plug. Click here for a visual representation of primary hemostasis. Platelet adhesion: The first event in hemostasis is the adhesion of platelets to exposed subendothelium. In areas of high shear rate (in the microvasculature), this is mediated by von Willebrand factor (vWf), which binds to glycoprotein Ib-IX in the platelet membrane. In areas of low shear rate (e.g. aorta), fibrinogen mediates the binding of platelets to the subendothelium (by attaching to a platelet receptor - the integrin, glycoprotein Ia/IIa).
Platelet activation: The adhesion of platelets to the vessel wall activates them, causing the platelets to change shape, to activate the collagen receptor on their surface (an integrin receptor called glycoprotein IIb/IIIa) and to undergo the release reaction (release alpha and dense granule constituents). In addition, upon activation, platelets synthesize and release thromboxane A2 (TXA2) and platelet activating factor (PAF), which are potent platelet aggregating agonists and vasoconstrictors.
Platelet aggregation: Thromboxane2, PAF, ADP and serotonin (ADP and serotonin are released from dense granules) are platelet agonists, causing the activation and recruitment of additional platelets, which bind to the adhered platelets. This activation is enhanced by the generation of thrombin through the coagulation cascade; thrombin being an important platelet agonist. Platelet aggregation is mediated primarily by fibrinogen (vWf has a secondary role), which binds to glycoprotein IIb/IIIa on adjacent platelets. This aggregation leads to the formation of the primary platelet plug, which must be stabilized by the formation of fibrin.
Platelets also contribute to secondary hemostasis (coagulation cascade) by providing a phospholipid surface (this used to be called PF3) and receptors for the binding of coagulation factors.
vWF, von Willebrand factor.

6. Coagulation Cascade XII XIIa Injury XI XIa VII IX IXa TF X Xa TF/VIIa
HMWK/Prekallikrein
VII IX
IXa
VIII TF
VIIIa X Xa
TF/VIIa Va V
XIII
Prothrombin
Thrombin
Secondary hemostasis involves a series of reactions leading to the formation of a stable fibrin clot. Platelets also contribute to this process by providing a PL surface and receptors for the binding of coagulation factors required for fibrin formation. The series of reactions leading to fibrin formation is referred to as the coagulation cascade. A common theme for the reactions leading to fibrin formation is ca-dependent complex assembly on a PL membrane surface, the complex consisting of an activated cofactor and protease. The requirement for PL membrane binding serves to localize the reactions to the site of injury and is one control mechanism. Another regulatory mechanism is that proteins necessary for fibrin formation, circulate in an inactive form and must be activated for maximal proteolytic activity.
The key enzyme in the coag cascade is thrombin. There are two pathways leading to thrombin generation. Following injury, TF ( asubendothelial protein) is exposed to flowing blood. FVII binding to TF will autoactivate and the TF/FVIIa complex will activate FX. (there is also a small amount of FVIIa circulating.)
Following injury, TF, a subendothelial protein is exposed to flowing blood, TF will
Platelets also contribute to secondary hemostasis (coagulation cascade) by providing a phospholipid surface (this used to be called PF3) and receptors for the binding of coagulation factors
Series of reactions leading to fibrin formation
Zymogen & cofactor activation
Complex assembly binding to phospholipid membrane surfaces
Localizes & amplification of reactions
Critical enzyme: thrombin
XIIIa
Fibrinogen
Fibrin
(soluble)
Fibrin (insoluble)

7. Cascade Simplified Activation/Injury Intrinsic Pathway
(XIIa, XIa, IXa, VIIIa)
Extrinsic Pathway
(TF, VIIa)
Common Pathway
(Xa, Va, IIa, Fibrinogen)

8. Defects in regulatory mechanisms: thrombosis
Thrombin Regulation
Activity and formation tightly controlled
Antithrombin III
Inactivation of IIa and other enzymes involved in its formation
Protein C and protein S pathway
Inactivation of cofactors Va and VIIIa
Tissue factor pathway inhibitor
Turns off extrinsic pathway (TF, VIIa)
Thrombin is a potent enzyme. Activity and formation tightly regulated.
Defects in regulatory mechanisms: thrombosis

9. Defects: bleeding or thrombosis
Fibrinolytic Pathway
Clot lysis vital in prevention of vessel occlusion
uPA, tPA
PAI-1
Plasminogen
Plasmin
Alpha-2 AP
Fibrin Clot
Clot lysis vital for hemostasis & prevention of vessel occlusion
Fibrin(ogen) Degradation Products
Defects: bleeding or thrombosis

10. (ie, Factors II – XII, cells) (ie, PC/PS, AT, TFPI, cells)
Hemostasis Balance
Thrombin Generation
(ie, Factors II – XII, cells)
Plasmin Generation
(ie, tPA, uPA, cells)
Healing
Coagulation
Fibrinolysis
Thrombin Regulation
(ie, PC/PS, AT, TFPI, cells)
Plasmin Regulation
(ie, PAI-1, cells)
Cellular contribution: platelets, endothelium, monocytes

11. Alteration of Balance Laboratory testing indicated if Thrombosis
Factor deficiencies
Acquired inhibitors
Anticoagulant therapy
Consumption (DIC)
Dysfibrinogenemia
Platelet defects
von Willebrand’s disease
Bleeding
Thrombosis
DIC, Disseminated intravascular coagulation

12. Alteration of Balance Laboratory testing indicated if
Inhibitor deficiencies
Acquired inhibitors
(eg, lupus anticoagulant)
DIC
Heparin induced thrombocytopenia
(acquired or genetic)
Bleeding
Thrombosis

13. Case Study #1
21 y/o female with vague family history of bleeding disorder; evaluated prior to taking scuba diving lessons
Has nose bleeds following aspirin ingestion
Differential diagnosis?
Role of laboratory testing???
Vague family history.
Vwd, platelet disorder, fibrinolytic defect, hemophilia carrier.

14. Case #1: Lab Testing Screening assays
aPTT: assesses intrinsic & common pathways
PT: assesses extrinsic & common pathways
Fibrinogen: hypo- or dysfibrinogenemia?
CBC: platelet count
von Willebrand’s disease (vWD) evaluation
Multiple tests required to classify vWD type
Antigenic and functional assays
PT/APTT global coag screening assays, sensitive but non-specific.
Based on patient history, vWD evaluation pursued.

15. Case #1: Test Results Test Result (Ref. Range) Comment aPTT 33.7 sec
(25.3 – 35.8)
Detects intrinsic/common pathway factor deficiency
Platelet count
231 K/L
(130 – 400)
Rule out thrombocytopenia
Factor VIII activity*
85%
(50 – 150)
Rule out FVIII deficiency
vWF antigen*
40%
Consistent with vWD
*** acute phase proteins. Can’t rule our vWD Type I.
*Acute phase proteins.

16. Case #1: Test Results Test Result Comment ABO blood type AB+
Type O:  levels of vWF Ag
vWF Functional Assays
(Ref Range)
Ristocetin cofactor activity
22%
(50 – 150)
If abnormal activity:antigen ratio, suspect qualitative (ie, Type II) defect
Platelet aggregation
No aggregation
Indicates abnormal vWF function
Should I change the RiCoF values? This case study may be confusing to the viewer. Should I lower the values to less than 50%?

17. Case #1: Multimer Analysis
Normal
Type 2A
Type 1
Shown is representative gel
of normal and type 1 and 2A
vWF deficiencies
Patient results demonstrated
absence of high and
intermediate molecular
weight multimers consistent
with type 2A vWD

18. Case #1: Summary Probable diagnosis
von Willebrand’s disease type 2A (bleeding disorder)

19. Case Study #2
38 y/o Caucasian man admitted for evaluation of portal hypertension; history of recurrent thrombosis (>10 years)
PT, aPTT, fibrinogen: normal
Positive family history; father and sister with venous thrombotic episodes, but no laboratory investigation
Differential diagnosis??
Made up case! Inherited thrombophilia???
Father: elderly when experienced DVT. Should we insert slide with frequency of mutations? (PS, PS, AT, II gene???) Yes.

20. Inherited Thrombophilia Risk Factors
Condition
% in Healthy
% in VTE
RR (%) of Thrombosis
APC resistance/FV Leiden mutation 5 21
3 – 7
AT deficiency
0.02–0.17 1
15 – 40
Protein C deficiency
0.3 3
5 – 12
Protein S deficiency
0.7 2
4 - 10
Prothrombin (FII) 20210GA mutation 6
2 – 3
Hyperhomocysteinemia
5–10
10 – 25
3 - 4
VTE, venous thromboembolism; RR, relative risk; APC, activated protein C; AT, antithrombin.

21. Case #2: Lab Testing Test Result (Ref. Range) Comment
Protein C activity
80% (70-180%)
Activity assays detect qualitative or quantitative deficiencies
Protein S activity
95% (70-150%)
AT III activity
110% (80-120%)
APC Resistance
1.1 (< 2.0)
Positive; suggestive of FV Leiden mutation; genetic testing for confirmation
Prothrombin gene mutation
Not detected
Family studies suggested?
Add summary slide for this case. Use Quest comment for FV Leiden.

22. Case #2: Summary Probable diagnosis
Thrombosis caused by APC resistance/factor V Leiden mutation

23. Value of Thrombophilia Testing
Testing does not affect management of acute events
Test results may influence decisions
How long & how intensively to treat
Prevention of recurrence
Prophylaxis during high-risk procedures
Need to evaluate family members
Estimate future risk (ie, risk associated with HRT)
HRT, hormone replacement therapy.

24. Case Study #3
40 y/o woman with iron deficiency anemia due to menorrhagia; hysterectomy delayed due to prolonged screening test
aPTT: sec (elevated)
PT: sec (normal)
Fibrinogen: 300 mg/dL (normal)
No history of bleeding or bruising; no family history
Differential diagnosis??
Differential diagnosis: Most likely LA or fib defect??

25. Lupus Anticoagulants
Antiphospholipid antibodies (APA) are directed against proteins bound to phospholipid membrane surfaces
Lupus anticoagulants (LA) are a type of APA
Associated with thrombosis & recurrent fetal demise
Characterized by prolongation of phospholipid dependent clotting assays (ie, aPTT)

26. ISTH Criteria for Lupus Anticoagulants
Prolongation of a phospholipid dependent clotting assay (ie, aPTT)
Evidence of inhibition in mixing studies
Evidence that inhibition is phospholipid dependent
Lack of specific inhibition by any one coagulation factor or other circulating inhibitor (ie, FVIII inhibitors, heparin)

27. Lupus Anticoagulant Testing
Case #3: Lab Testing
Test
Result
Comment
aPTT mixing studies
No correction
Differentiate factor deficiency from inhibitor
Lupus Anticoagulant Testing
dRVVT screen & confirm
Positive
Consistent with presence of lupus anticoagulant
Hexagonal phase confirm
Treated with prednisone (don’t know if was treated with prophylaxis heparin).
Hysterectomy performed, DVT 1 month post-op. Indefinite oral anticoagulation.
Add the ISTH criteria before this. Rule out other coagulopathy.
Add Quest slide with comments for LA.

28. Case #3: Summary Probable diagnosis: Lupus anticoagulant
LA may be asymptomatic or associated with thrombotic
events or recurrent abortion. A bleeding history
requires other coagulopathies be excluded. Since LA
may be transient, international consensus guidelines
suggest waiting at least 12 weeks before retesting to
confirm antibody persistence.
J Thromb Haemost. 2006;4:295.

29. Role of Laboratory Testing
Assist in diagnosis of bleeding and thrombotic disorders; for example:
Screen for von Willebrand’s disease in patients with menorrhagia (ACOG recommendation)
Test for thrombophilia risk factors in patients with recurrent spontaneous abortion or thrombotic events
Monitor anticoagulant therapy
Oral anticoagulants, heparin, thrombin inhibitors
Anything else?

30. Role of Laboratory Testing
Monitor replacement therapy
Factor levels (ie, FVIII, vWF)
Pre-op screening
Risk assessment
Cardiovascular diseases (CVDs) are a group of disorders of the heart and blood vessels and include:
Related links
Each year, at least 50,000 and perhaps as many as 200,000 patients will die from blood clots that obstruct blood flow to their lungs (pulmonary embolism).
:: The Atlas of Heart Disease and Stroke World Health Organization, :: Avoiding Heart Attacks and Strokes: Don’t be a victim, protect yourself World Health Organization, :: Health topic: Cardiovascular diseases
Coronary heart disease – disease of the blood vessels supplying the heart muscle
Cerebrovascular disease - disease of the blood vessels supplying the brain
Peripheral arterial disease – disease of blood vessels supplying the arms and legs
Rheumatic heart disease – damage to the heart muscle and heart valves from rheumatic fever, caused by streptococcal bacteria
Congenital heart disease - malformations of heart structure existing at birth.
Deep vein thrombosis and pulmonary embolism – blood clots in the leg veins, which can dislodge and move to the heart and lungs.
On warfarin. Atrial fibrillation is the most common sustained heart rhythm disorder and increases the risk for heart disease and stroke, both leading causes of death in the United States. An estimated 2.2 million adults in the United States have been diagnosed with atrial fibrillation

31. Pre-analytical Considerations
Proper specimen handling, processing, and storage is critical for accurate and precise results
General specimen requirements available
(click on Test Menu)
(click on lab information specimen requirements)
Quest Diagnostics Nichols Institute Directory of Services (contact your local representative)

32. Resources
Laboratories performing routine, specialty, and esoteric hemostasis testing
Consultative services Quest Diagnostics Nichols Institute
Quest Diagnostics Interpretive Guide: p_ig_main.html
Mervyn Sahud, MD
San Juan Capistrano, CA
Jeffrey Dlott, MD
Chantilly, VA
, x7259
??????

33. Case-Oriented Symposium on Bleeding & Thrombosis
October 11-12, 2007, Renaissance Hotel, Washington DC; topics:
Pediatric hemostasis issues
Thrombophilia
Platelet disorders
Thrombotic thrombocytopenia purpura
FVIII Inhibitors
Point of Care testing
New technologies & more
For more information on this CME approved symposium, go to:

34. References
ACOG committee opinion. von Willebrand’s disease in gynecologic practice. Int J Gynaecol Obstet ;76:336.
Brandt JT, et al. Laboratory identification of lupus anticoagulants: Results of the Second International Workshop for Identification of Lupus Anticoagulants. On behalf of the Subcommittee on Lupus Anticoagulants/ Antiphospholipid Antibodies of the ISTH. Thromb Haemost. 1995;74:1597.
Miyakis et al. International consensus statement on an update of the classification criteria for definite APS. J Thrombo Haemost. 2006;4:295.

35. References
Press et al. Clinical utility of FV Leiden testing for the diagnosis and management of thromboembolic disorders. Arch Pathol Lab Med. 2002;126:1304.
Sadler et al. Update on the pathophysiology & classification of von Willebrand disease: a report of the Subcommittee on von Willebrand Factor. J Thrombo Haemost. 2006;10:2103.
Thrombophilia: Laboratory support of risk assessment and diagnosis. Available at: owintguidepage.jsp?fn=CF_Thrombophilia/CF_Thro mbophilia.htm. Accessed March 21, 2007.

36. Thank you.