Introduction to Coagulation Testing

Laura Worfolk, Ph.D. Scientific Director, Hematology Quest Diagnostics Nichols Institute, Chantilly, VA

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
Normal Hemostasis
Absence of overt bleeding/thrombosis

Bleeding
2

Thrombosis

Interested Specialties
Anesthesiology
Anticoagulant Management HIT

Vascular Surgery
Graft Occlusion PAD

Cardiology
Premature CAD

Primary Care
Practice patterns vary

Bleeding & Thrombosis

Hematology
Hemophilia Thrombophilia

OB/GYN
Fetal loss, Infertility, Menorrhagia

Nephrology
AV Graft Occlusion

Neurology
Stroke
3

Hemostasis Statistics
#1 cause of death is CVD (includes heart attack & stroke)* ~1-2% of population w/ von Willebrands disease ~18,000 Americans w/ hemophilia ~600,000/year w/ venous thromboembolism
~ with long-term health consequences; ~60,000 fatalities ~5-8% of population w/ thrombophilia
*WHO. CDC www.dvt.org..

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

vWF, von Willebrand factor.

5

Coagulation Cascade
XII XI XIIa
HMWK/Prekallikrein

XIa VII IXa

VIIIa VIII

Injury

IX X

TF TF/VIIa
V

Xa
Va

XIII

Prothrombin Fibrinogen

Thrombin XIIIa Fibrin (soluble) Fibrin (insoluble)

Cascade Simplified
Activation/Injury

Intrinsic Pathway
(XIIa, XIa, IXa, VIIIa)

Extrinsic Pathway
(TF, VIIa)

Common Pathway
(Xa, Va, IIa, Fibrinogen)

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)

Defects in regulatory mechanisms: thrombosis

Fibrinolytic Pathway
Clot lysis vital in prevention of vessel occlusion
uPA, tPA PAI-1

Plasminogen

Plasmin Fibrin Clot

Alpha-2 AP

Fibrin(ogen) Degradation Products

Defects: bleeding or thrombosis

Hemostasis Balance
Thrombin Generation (ie, Factors II XII, cells) Plasmin Generation (ie, tPA, uPA, cells)

Coagulation

Healing

Fibrinolysis

Thrombin Regulation

Plasmin Regulation

(ie, PC/PS, AT, TFPI, cells)

(ie, PAI-1, cells)

Cellular contribution: platelets, endothelium, monocytes

10

Alteration of Balance
Laboratory testing indicated if
Factor deficiencies Acquired inhibitors Anticoagulant therapy Consumption (DIC) Dysfibrinogenemia Platelet defects von Willebrands disease

11

DIC, Disseminated intravascular coagulation

Alteration of Balance
Laboratory testing indicated if
Inhibitor deficiencies
Acquired inhibitors (eg, lupus anticoagulant) DIC Heparin induced thrombocytopenia

12

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???

13

Case #1: Lab Testing

Screening assays
aPTT: assesses intrinsic & common pathways
PT: assesses extrinsic & common pathways Fibrinogen: hypo- or dysfibrinogenemia? CBC: platelet count

von Willebrands disease (vWD) evaluation

Multiple tests required to classify vWD type
Antigenic and functional assays

14

Case #1: Test Results

Test aPTT Platelet count Factor VIII activity* vWF antigen*
*Acute phase proteins.

Result (Ref. Range) 33.7 sec

(25.3 35.8)

Comment Detects intrinsic/common pathway factor deficiency Rule out thrombocytopenia Rule out FVIII deficiency

231 K/L
(130 400)

85%
(50 150)

40%
(50 150)

Consistent with vWD

15

Case #1: Test Results

Test ABO blood type Result AB+ Comment Type O: levels of vWF Ag

vWF Functional Assays Test Ristocetin cofactor activity

Result
(Ref Range)

Comment If abnormal activity:antigen ratio, suspect qualitative (ie, Type II) defect

22%
(50 150)

Platelet aggregation

No aggregation Indicates abnormal vWF function

16

Case #1: Multimer Analysis

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

17

Case #1: Summary

Probable diagnosis von Willebrands disease type 2A (bleeding disorder)

18

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??

19

Inherited Thrombophilia Risk Factors

Condition
APC resistance/FV Leiden mutation AT deficiency Protein C deficiency

% in Healthy
5 0.020.17 0.3

% in VTE
21 1 3

RR (%) of Thrombosis
37 15 40 5 12

Protein S deficiency
Prothrombin (FII) 20210GA mutation Hyperhomocysteinemia

0.7
2 510

2
6 10 25

4 - 10
23 3-4

VTE, venous thromboembolism; RR, relative risk; APC, activated protein C; AT, antithrombin.

20

Case #2: Lab Testing

Test Result (Ref. Range) Comment

Protein C activity 80% (70-180%)

Protein S activity 95% (70-150%) AT III activity 110% (80-120%)

Activity assays detect qualitative or quantitative deficiencies

APC Resistance 1.1 (< 2.0)

Positive; suggestive of FV Leiden mutation; genetic testing for confirmation

Prothrombin gene mutation

Not detected

21

Case #2: Summary

Probable diagnosis Thrombosis caused by APC resistance/factor V Leiden mutation

22

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.

23

Case Study #3
40 y/o woman with iron deficiency anemia due to menorrhagia; hysterectomy delayed due to prolonged screening test
aPTT: PT: Fibrinogen: 47.8 sec (elevated) 13.0 sec (normal) 300 mg/dL (normal)

No history of bleeding or bruising; no family history Differential diagnosis??

24

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)

25

ISTH Criteria for Lupus Anticoagulants

1. Prolongation of a phospholipid dependent clotting assay (ie, aPTT) 2. Evidence of inhibition in mixing studies 3. Evidence that inhibition is phospholipid dependent 4. Lack of specific inhibition by any one coagulation factor or other circulating inhibitor
(ie, FVIII inhibitors, heparin)

26

Case #3: Lab Testing

Test aPTT mixing studies Result No correction Comment Differentiate factor deficiency from inhibitor

Lupus Anticoagulant Testing Test dRVVT screen & confirm Result Positive Comment Consistent with presence of lupus anticoagulant

Hexagonal phase Positive confirm

27

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.

28

Role of Laboratory Testing

Assist in diagnosis of bleeding and thrombotic disorders; for example:
Screen for von Willebrands 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

29

Role of Laboratory Testing

Monitor replacement therapy
Factor levels (ie, FVIII, vWF)

Pre-op screening Risk assessment

30

Pre-analytical Considerations
Proper specimen handling, processing, and storage is critical for accurate and precise results General specimen requirements available
www.questdiagnostics.com (click on Test Menu)

www.nicholsinstitute.com (click on lab information

specimen requirements)

Quest Diagnostics Nichols Institute Directory of Services (contact your local representative)

31

Resources
Laboratories performing routine, specialty, and esoteric hemostasis testing Consultative services available @ Quest Diagnostics Nichols Institute
Mervyn Sahud, MD San Juan Capistrano, CA 949-728-4794 Jeffrey Dlott, MD Chantilly, VA 703-802-6900, x7259

Quest Diagnostics Interpretive Guide: http://www.questdiagnostics.com/hcp/intguide/hc p_ig_main.html

32

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: http://www.nicholsinstitute.com/Coagulation/Default.htm.

33

References
ACOG committee opinion. von Willebrands disease in gynecologic practice. Int J Gynaecol Obstet. 2002;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.

34

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: http://www.questdiagnostics.com/hcp/intguide/jsp/sh owintguidepage.jsp?fn=CF_Thrombophilia/CF_Thro mbophilia.htm. Accessed March 21, 2007.

35

Thank you.