Almazov Federal Medical Research Centre

St.Petеrsburg, Russia

Laboratory tests in blood coagulation

disorders

Vavilova Tatiana

September 18, 2014Moscow

Conflict of interests is absent

Thrombosis

Disorders of haemostasis

Bleeding

• hereditary• acquired

• hereditary• acquired

DIC

Basic principle in diagnostic and laboratory assessment of hemostatic disorders

None of the components can be prioritizedClinician + Laboratory staff = correct diagnosis

and benefits for patient

Clinical since

Laboratory testing

Individual and family history of thrombotic disorders

The investigation of haemostasis disorder requires a stepwise approach

Clinical decision

Clinical signs

Laboratory results

Anamnesis

Anamnesis

Anamnesis

START

Laboratory results

Laboratory results

Clinical signsClinical signs

Essential aspects of haemostasis diagnosis

1

2

3

4

4

4

plasma factors testing

THROMBUS

Vessel wall Blood cells

Plasma factors

Coagulation Platelets and vessel wall –

microcirculation, small arteries Plasma factors – veins, cardiac

chambers

Anticoagulation Fibrinolysis

ADP, serotonin

Adgisive proteins

Mitogenic Factors

Coagulation factors

Proteases inhibitors

β -ThromboglobulinPlatelets factor 4…CD62P (P-selectin),CD40L – exposureon membrane

Jurk K,Kehrel BE // Sem Thromb Hemost (2005) 31, 381-92

Platelet receptors

Preanalitical phase

➙ The blood sampling procedure for haemostasis test is a critical issue.

➙ Avoiding the prolonged application of a venous cuff can reduce artifacts.

➙ Blood should be taken carefully into the tube without foam formation and the tube should be gently inverted in order to completely mix the citrate and blood.

➙ Samples in whith incorrect ratio of blood to anticoagulant or samples with visible fibrin strands, must not be used for testing because the results will be inaccurate.

➙ The main screening tests need to be performed within 4 hours

Platelet Function Assays

Traditional Evaluation of Platelets

www.clot-ed

www.clot-ed

www.clot-ed

www.clot-ed

Functional assays for platelet activation1. Measuring of spontaneous and induced

aggregation of platelets in PRP and whole blood (light transmission, impedance measuring)

2. Laser light dispersion for aggregate size measuring in PRP

3. Flow cytometry (microparticles, platelet/leucocytes aggregates, platelet receptors …)

4. …

Platelet Aggregometry

www.clot-ed

Platelet aggregation testing assess: platelet adherence (Glycoprotein Ibα [GPIbα]), secretion (secondary wave or increase in

luminescence), aggregation (αIIb/β3).

Agonists are used to activate platelets via various receptors:

1. arachidonic acid (thromboxane pathway via cyclooxygenase),

2. collagen (integrin α2/β1 & GPVI receptors),3. ADP (receptors P2Y1 & P2Y12), 4. epinephrine (α2-adrenergic receptor),5. ristocetin (GPIbα), 6. thrombin receptor activating peptide (Protease

Activated Receptor [PAR] 1 and 4).

Standard aggregometry (light transmittion)

Born GVR. Nature 1962; 194: 927-929Born GVR, Cross MJ. J Physiol 1963; 168:178-195

Manufacturers• Bio/Data, www.biodatacorp.com (PAP-4)• Chrono-Log, www.chronolog.com• Labitec, Germany www.labitec.de (APACT)• Solar (Belorussia)

Understanding Platelet Aggregation Tracings

Standard aggregation

Application:• Detection of platelet function defects

• Visualization of aggregation• Widely available• Use >40 years• Very flexible• Continuous monitoring “Real Time”

• Poor reproducibility• Need of platelet preparation• Sample processing time • Low sensitivity

+ -

5.0

2.5

1.25

0.630.31

0.16

5.0

2.5

1.250.63

0.31

0.16

LT R

Laser aggregometry (light dispersion)

Manufacturers: Biola, Moscow, Russia; www.biola.ru (LA220, LA230);

Kowa, Japan; www.kowa.com (AG-10, PA-20, PA-100, PA-200)

Miyamoto S et al. Thromb Haemost 2003; 89(4):681

Microaggregates and coronary risk factors

Plasma markers of the platelet activity

Requirements:

Must be specific marker for platelets

Must be resistant to preanalitical artifacts

Must be measured by cheap, reproducible, simple laboratory technique, such as ELISA, immunoturbidimetry or latex aggregation

Possible candidate molecules:

Substances that are released from the platelet granules

Platelet factor 4 (PF 4) β-thromboglobulin

(β-TG)

Molecules that are exposed on, and then shed from, platelet surface

P-selectin

Secreted metabolic molecules Thromboxane B2

Laboratory tests for plasma coagulation, anticoagulation and

fibrinolisis

• Haemostasis starts with the interaction between TF and FVIIa on the surface of subendothelial cells.

• The small amount of thrombin generated during the amplification phase activates platelets locally on whose surface the subsequent reactions take place.

• The resulting thrombin burst results in the formation of a stable clot.

TF : VIIa IX

XIXa

Xa : Va

IIa (thrombin)

Activated platelet

vWF : VIII → VIIIa V → Va

X

Xа

VIIIa : IXa Va : Xa

II

IIa (thrombin)

FIBRINOGEN FIBRIN

I phase

II phase

III phase

VII

XI

XIainitiation

amplificationpropagation

thrombin and fibrin generation

Laboratory coagulation tests and technological principals

1. Clotting tests

2. Tests with chromogenic substrates

3. Immunochemical methods

4. Methods of molecular genetics

The most common coagulometers principles – clotting assays

mechanical (steel ball) turbidimetry nephelometry/light scatter

Immunochemical methods

The principle of latex enhanced immunoassays (antibody coated particles agglutinate in the presence of antigen)

The principle of the enzyme linked immunosorbent assay (ELISA)

Screening assays for either the extrinsic or

intrinsic pathways are performed in order to get

an overview of the enzymes, cofactors and

inhibitors, involved in the respective pathway,

and also of the influence of drugs or

autoantibodies.

The most important tests are prothrombin time

and aPTT.

TF : VIIa IX

XIXa

Xa : Va

IIa (thrombin)

Activated Platelet

vWF : VIII → VIIIa V → Va

X

Xа

VIIIa : IXa Va : Xa

II

IIa (thrombin)

FIBRINOGEN FIBRIN

VII

XI

XIa

aPTT

PT

The principle of the aPTT

red arrows = positive feedback reactions

A prolonged APTT is found in:Contact activator,

phospholipids

Ca Cl2

Heparin sensitivity of different aPTT reagents

Prolongation of aPTTNumber of platelets, PT and

BT are normal

1:1 mixt testaPTT

Correctionof aPTT

No correction of aPTT

Factors deficiency Presence of inhibitors

FVIII, FIXactivity

FVIII, FIXactivity

FXII, HMWK, PKactivity

FXII, HMWK, PKactivity

HemophiliaHemophilia FXII, HHMWK, PKInsuff. (clinical

negative)

FXII, HHMWK, PKInsuff. (clinical

negative)

Detection ofinhibitors to FVIII

Detection ofinhibitors to FVIII LА/аPLLА/аPL

Inhibitor-dependentHemophilia

Inhibitor-dependentHemophilia

APS (if clinical andlab. symtoms are

combined)

APS (if clinical andlab. symtoms are

combined)

The prothrombin time assay

An abnormal PT is found in:

red arrows = feedback reactions

Tissue factor, Ca2+

The calibration of thromboplastins in the ISI/INR system

The INR is calculated

according to:

INR = PRISI

WHO reference thromboplastin(human, rabbit or bovine)

House standard thromboplastin

ISI determination(instrument specific)

Thromboplastin lotWith assigned ISI value

Determination of • Normal PT• Clotting time patient

Calculation of INR

Ind

ust

ryL

abo

rato

ry

“International Normalised Ratios (INR) –the First 20 Years”(Poller L.// JTH 2004; 2: 849-60)

Benefits

Decreasing of oral anticoagulants dosage

Decreasing the number of bleeding complications

Improvement anticoagulant therapy - became more safety

Possibility to computerize the management of anticoagulation

Problems

Heterogeneous of referent materials

Complexity of calibration process

ISI determination must be instrument specific

Differences in INR level because of reagent and equipment distinction

Vitamin K reductase(VKORC)

Vit K-OHVit K=O

CarboxylaseCarboxylase

Warfarin

PIVKA – proteins

induced in vitamin K absence

(Factors II, VII, IX, X;protein С, protein S)

Proenzymes (Factors II, VII, IX, X;

PС, PS)

ActiveFactors

(II, VII, IX, X;PС, PS)

CYP2C9

Example of a patient with high anticoagulation variability

Markers of coagulation activity(hyprcoagulability, prethrombotic stage)

Prothrombin Fragment 1+2 Thrombin-antithrombin

complex

Fibrinopeptide A Fibrin-monomer Fibrinogen/fibrin

degradation products D-dimer

Research Clinical practice

Fibrinogen vWF FVIII activity D=dimer

Step 1

Thrombin converts fibrinogen to soluble fibrin by cleaving the fibrinopeptides A and B. The fibrin monomers polymerize spontaneously

Step 2

Active factor XIII links tow D-domains and generates a solid fibrin clot. A new plasmin-resistant antigenic determinant (D-Dimer) is produced

Step 3

Thus, fragments containing D-Dimer are formed during the degradation of the fibrin clot by plasmin

An Introduction to D-Dimer A summary of the D-Dimer origin

47

Se

nsiti

vity

, %(f

requ

enc

y o

f tru

e p

osi

tive

res

ults

)

1 – specificity, %(frequency of false positive results)

Specificity, %

1 -

se

nsiti

vity

, %

Optimal division point cut-off

VTE suspition

Unlikely Likely or non indicated DD

DD

DD < 500 mg/l DD > 500 mg/l

STOP

CT Scan

PE? DVT?

US

STOP

« Neg » Prox TVP

Clinical probabilityccording Well’s score

1st Trim: 139 – 602 mg/l2nd Trim: 291 – 12313th Trim: 489 - 2217

Delivery, D1-3: > 500Utility first 4W PP?

DD: pregnancy?

Chabloz, 2001 – Epinay 2005

0

20

40

60

80

100

120

0 5 10 15

Time (min)

Th

rom

bin

Lag-time

Time to peak

Area Under the Curve'ETP'

Peak Hight

Source: Lawrie A, Béguin S, Hemker H C, Henckel T, Samama M, Woodhams B, Gray E. (2005). The Thrombin Generation Test (TGT); On behalf of the International Society on Thrombosis and Haemostasis (ISTH) Scientific and Standardization Committee (SSC) Working Group on Thrombin Generation Tests. www.blood.com 2005.

Thrombin Generation Test

Calibrated Automated Thrombogram - CAT

Invented by the “father” of TGT - Prof C Hemker (Maastricht)

The “Gold Standard Method” A Fluorogenic Assay Uses PPP or PRP

Thank you for your attention!