Laboratory diagnosis of coagulation disorders

Basic coagulation tests Monitoring of anticoagulant therapy Testing congenital and acquired thrombophilia
va Ajzner MD. PhD.

screening: PT, APTT, TT

screening: Bleeding time, PFA-100 Platelet count

Humoral System disorders

Hemorrhagic diathesis

cellular System disorders

Vascular lesions

screening: Rumpel Leede test Bleeding time

THE COAGULATION CASCADE In vitro

PK: prekallikrein PL: phospholipids HK: HMW Kininogen

Screening test of coagulopathies

Prothrombin time (PT) Activated partial thromboplastin time (APTT) Thrombin time (TT)

Prothrombin time

Thromboplastin:
Tissue factor - apoprotein - phospholipid Phospholipid Calcium

Sodium citrated plasma

Activated factor: FVII Tested cascade phase: extrinsic pathway

What prothrombin time measurement is good for?

Monitoring of oral anticoagulant therapy

Screening for inherited and acquired coagulopathies Determination of the activity of extrinsic pathway factors Lupus anticoagulant diagnostics (diluted prothrombin time assay)

Expressions of prothrombin time

1. Seconds 2. INR= International Normalised Ratio

Same sample measured by different ways can give very different PT values in seconds
- Reagent source - Coagulometer type (mechanical, optical, electromechanical) cal
KC 8.48 8.48 9.49 Coag-a-Mate 9.30 15.08 13.31 ACL 7.27 6.79 6.64

Patient A Patient B Patient C

INR
INR: International normalized ratio - was established by the WHO and the International Committee on Thrombosis and Hemostasis for reporting the results of prothrombin tests. - All PT results are standardized by this calculation:

INR= ( Patient PT / Controll PT)ISI ISI= International sensitivity index

- Given by the manufacturer for each particular thromboplastin reagent and instrument combination - Reflects the reagent sensitivity to different vitamin K dependent factor deficiencies

Prothrombin time prolongs..

Extrinsic pathway factor deficiencies (FII, V, VII, X)
Inherited or acquired Consumption (DIC) PIVKA factors in cumarin therapy

Specific inhibitors against FII, V, VII, X Rare situations, when

hypo- afibrinogenaemias Dysproteinaemia, Too much citrate

Activated partial thromboplastin time

Reagent: Surface activator: silica caolin ellagic acid PL Calcium Activated factor: FXII Tested cascade phase: intrinsic pathway
PL + Contact activator + Ca++

Citrated plasma

What APTT measurement is good for?

Monitoring non-fractionated heparin therapy

Lupus anticoagulant diagnosis Screening for inherited nad acquired coagulopathies

APTT prolongs..
1. Intrinsic pathway factor deficiencies (FXII, XI, VIII, IX, HMWK, prekallikrein ) - Inherited or acquired

- Consumption (DIC) - PIVKA factors in cumarin therapy

2. Specific inhibitors against FXII, XI, VIII, IX, HMWK, prekallikrein 3. Lupus anticoagulant 4. Non-fractionated heparin therapy 5. Rare situations: - Severe FX, V, II deficiency
- hypo- afibrinogenaemia, dysproteinaemik - Too much citrate

Thrombin time

Thrombin (Ca++)

Citrated plasma

Cleaved factor: fibrinogen Tested cascade phase: fibrinogen - solubile fibrin

What TT measurement is good for?

Inherited and acquired hypofibrinogenaemias, dysfibrinogenaemias & afibrinogenaemia Non fractionated heparin therapy Fibrin degradation products

TT prolongs..
1. Hypo- afibrinogenaemia 2. Dysfibrinogenaemia 3. Non fractionated heparin th 4. Fibrinogen/ fibrin degradation products 5. Rare situations
dysproteinaemias severe hypoalbuminaemia

Laboratory monitorisation of anticoagulant & thrombolytic therapy

Goal: Effective treatment Without bleeding

Thrombolytics
Streptokinase urokinase (u-PA) Recombinant tissue type plasminogen activator (t-PA) prourokinase plasminogen-streptokinase-activator complex plasminogen activators short halflife non perfect fibrinspecifity (fibrinogen cleavage also) bleeding complications

Laboratory monitoring of thrombolytic therapy

1. Before thrombolysis:
Screening for bleeding disorders (PT, APTT, TT, platelet count, bleeding time)

2. During thrombolysis:
to confirm thrombolysis: TT (FDP/fdp) to predict bleeding complications

3. After thrombolysis:
to establish the timing of switching to heparin (TT is measurable)

Anticoagulant therapy: oral (cumarin) parenteral - non fractionated heparin - fractionated heparin

1. Conventional= non fractionated heparin therapy iv Na-heparin infusion iv Na-heparin bolus sc Ca-heparin prevention Small dose sc Ca-heparin 2. Fractionated= LMWH therapy prevetion

Non fractionated heparin

heparin-AT-III complex cleaves: thrombin & FXa FXIIa, FXIa, FIXa

Laboratory monitoring of conventional heparin therapy

Test: APTT Baseline APTT, before the therapy starting Therapeutic range: 2-3x prolongation of normal APTT Timing of sampling: i.v. infusion: 0.5-1 hour after start of infusion
i.v. bolus: 2-3 hours after start of infusion s.c. therapy: 4-6 hours after injection

Sample handling:
separate plasma in 1-2 hours
(prevent PF4 heparin - inhibitory effect)

Other clotting tests: PT: not sensitive TT: too sensitive

Heparin-induced thrombocytopenia (HIT)

1. Incidence:
I.v. heparin: 0-30% Mini dose s.c. heparin: no HIT LMWH: less frequent than with conventional heparin

2. Clinical forms:
Type I. HIT (moderate, first 4 days, plt >100G/L, spontaneously improves during heparin therapy) Type II. HIT (4-14th day, plt <100G/L, improves within 1 mont thromboembolic complications)

3. Mechanism:
Type I. HIT: heparin-induced direct platelet aggregation Type II. HIT: immune mechanism (heparin-PF4 complex, anti-heparin-PF4 antibodies bound to Fc receptors of platelets)

Low molecular weight heparin (LMWH)

1. Preparation:
Controlled depolimerization of conventional heparin 4000-6000 daltons

2. Molecular weight: 3. Half-life:

Longer than conventional heparin as it is eliminated via kidney only (s.c. 4 hours, i.v. 2 hours)

4. Dose: 1x daily 5. Advantages:

orthopedics: thrombosis prophylaxis less frequent bleeding complications and HIT

LMWH

Inhibition of FXa: 5-sacharid units (plenty) Inhibition of FIIa: 18-sacharid units (few) Inhibition rate FXa /FIIa: 1.8-3.5

Switching from heparin to coumarine

Proposed timing: ~ 5th day 36-48 hours after starting coumarine : PT, APTT Every 2nd day until achieving therapeutic effect: PT, APTT
Overlapping therapy needed to avoid the coumarine necrosis.

Oral anticoagulant therapy

Drug: acenocumarol (Syncumar) warfarin (Waran) Test for monitoring: PT expressed in INR (APTT is also prolonged)

OH CH3 protein CH2 K vitamine reductase OH

Vitamin K-H2

R Glutamic acid

CH2 C OH O

O CH3 Vitamin K R O Vitamin K-epoxid Vitamin K epoxide reductase O CH3 O R cumarin O

O2 CO2 carboxilase protein CH2 O C OH CH C OH O

gamma-carboxiglutamic acid

Proposed therapeutic ranges for monitoring of oral anticoagulant treatment (International Society of Thrombosis and Haemostasis)
1. Pre-, and perioperative oral anticoagulation: hip replacement other surgeries 2. Primary and secondary prevention of venous trombosis 3. Active venous thrombosis, pulmonary embolism, prevention of recurrent venous thrombosis 4. Prevention of arterial thromboembolism (eg. arteficial heart valve) INR 2.0-3.0 1.5-2.5 2.0-3.0 2.0-4.0 3.0-4.5

The higher the INR, the stronger the coumarine therapy. too high bleeding too low thrombotic events

Long-term oral anticoagulation

(coumarin)

Weekly, then monthly PT (APTT) More frequent monitoring is indicated: change in dosage
instable results change in other medications other co-morbid conditions

Laboratory diagnostics of congenital and acquired thrombophilias

Thrombophilia definition
The predisposition/propensity to develop thrombosis due to abnormalities in the haemostasis system, which can be either genetical or acquired or both.

Why is important to diagnose thrombophilia?

1.primary prophylaxis: for carriers without symptoms 2.Secondary prophylaxis : patients 3.It has influence on thrombosis therapy: combined thrombophilias 4.Effective treatment available: hyperhomocysteinaemia

Whom?

Whom we should test for thrombophilia?

Screening of the total population: NO! Patients after venous/arterial thrombosis Children of patients with thrombophilia in puberty Siblings of those thrombophilia patients who suffered their first thrombotic event in very early ages.
Tripodi A Clinical Chemistry, 2001 Brenner BR Arch Pathol Lab Med 2002

Which tests should be done?

Thrombophila
Inherited Acquired Combined

When should we suspect inherited thrombophilia? Deep vein thrombosis in early ages (<55 v) Familiar occurence
Recurrent thrombosis Thrombosis atypical localisation (not on the lower extremities/limbs)

Inherited thrombophilias
Deficiencies of the inhibitory system
Antithrombin deficiencies

Deficiencies of the inactivator system

Protein C, Protein S deficiencies

Elevated or abnormal procoagulant factors

Activated protein C resistance (FV Leiden) Prothrombin 20210A allele Dysfibrinogenaemia with thrombosis Fibrinogen, FVIII, FIX, FXI Decreased fibrinolytic aktivity: no evidence

Others: Hyperhomocysteinaemia

Relative occurences of genetic reasons between venous thrombophilias

10% 40% 35% 10% 5% APC resistance - FV Leiden Rare thrombophilias AT, PC, PS deficiencies Prothrombin 20210 A allele Unknown

APC resistance & Leiden mutation

Phenotype: APC resistance APTT with APC < 2.0 APTT without APC

Genotype: FV-Leiden FV-A1691G

Hungary: 10%

Activation & inactivation of faktor V

R709 R1018 R1545

A1

A2

A3 C1C2

Thrombin
B B A1 Ca2+ A3 C1C2 A2

APC

9 6 06 R50 R67 R3

A1

A3 C1C2

Ca2+

A2

Pathomechanism of thrombophilia in FVLeiden

1. FV-Leiden is is worse substrate to APC than FV FV-506Qa Thalf 2. FV-Leiden is bad cofactor to APC in FVIIIa inactivation

A FII 20210A allel prevalencies in Europe

Average: 1,7%
50 N latitude

Average: 3,0%
Hungary: 2,7%

Prothrombin 20210A allele

Mechanism:
In 3 nontranslated region of FII gene Guanine-Adenine change increased efficiency in protein translation mRNA is more stabile plasma FII level

DVT relative risk 1,8-4,1x Responsible for 10% of familial thrombophilias

Mild hyperhomocysteinaemia: Risk factor for arterial occlusive disorders

Homocystein ref. range: 5-15 mol/L Hyperhomocysteinaemia: >12.5 mol/L
5 mol/L increase in total homocystein level results in 40%

increase in relative risk for arterial occlusive disorders. This has same increase in relative risk for arterial occlusive disorders than 0.5 mmol/L increase in total cholesterol level.
Metaanalysis of 27 studies published before 1994

Inherited hyperhomocysteinaemias
tetrahidrofolat 5,10-metilntetrahidrofolt MTHFR 5-metil tetrahidrofolt Homocisztein Szerin B6 Cisztation -szintetz Betain Metionin N,N-dimetil glicin

Metil-B12

Cisztation B6 -ketobutirt Cisztein

What shall we test: either homocystein or MTHFR C677T?

Thrombophilia risk factor is the hyperhomocyteinaemia total homocystein level should be investigated MTHFR polymorphism should be checked only in cases with increased homocystein level Vitamine B12, folic acid

Acquired thrombophilias
1. 2. 3. 4. 5. 6. 7. 8. Postoperative status Pregnancy, oestrogen therapy, oral anticoncipients Malignant tumors Immobilisation Artificial heart valves Varicosus veins Atrial fibrillation Anti-phospholipid syndrome

Laboratory criterias of APS (Consensus Workshop 1998)

Lupus anticoagulant (LA): LA positivity twice within 6 weeks intervals (Using criterias established by ISTH SSC LA/PLdependent antibody subcommitte) Anticardiolipin antibody (ACA): IgG &/or IgM isotype twice within 6 weeks intervals (Using standardised 2 glycoprotein I-dependent ELISA)

LA diagnostic tests
1. Phospholipid dependent tests increase: APTT, APTT-LA, rarely PT 2. The prolongation is increased by the presence of inhibitors: mixing studies 3. The inhibitor targets phospholipids: hPT, hexagonal PL tests, PNP

ELISA for detection of the auto-antibody against 2-glycoprotein I

Reaction mix
Anti-2-glikoprotein I Peroxidase-labelled antibody anti-human (patient serum) immunglobulin 2-glycoprotein I (serum)

Reaction

2-glycoprotein I On plate

When should be thrombophilia investigation done?

When should we take sample for thrombophilia tests?

Genetic test: any time but tests should be done once in a lifetime Acute thrombosis: NO, wait min. 3-6 months Anticoagulant treatment: syncumar/cumarin: PC, PS, LA, tests are influenced long-term heparine treatment: AT consumption Inherited thrombophilia dg based on non-genetical type assay should be repeated twice in different samples

Which tests to order?

Thrombophilia markers with strong evidences

venous markers: AT, PC, PS, FV-Leiden, FII20210, APC resistance, dysfibrinogenaemia, hyperhomocysteinaemia, FVIII, LA&ACA arterial markers: FV-Leiden, FII-20210 APC resistance, dysfibrinogenaemia, hyperhomocysteinaemia, FVIII, LA&ACA

Where the thrombophilia investigations should be done?

In such laboratories where all listed factors can be tested and the results are also interpreted