laboratory diagnosis of coagulation disorders

7/30/2019 Laboratory diagnosis of coagulation disorders

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LaboratoryLaboratory diagnosisdiagnosis ofofcoagulationcoagulationdisordersdisordersBasicBasic coagulationcoagulation teststests

Monitoring ofMonitoring ofanticoagulantanticoagulant therapytherapyTestingTesting congenitalcongenital andand acquiredacquired thrombophiliathrombophilia

va Ajzner MD. PhD.va Ajzner MD. PhD.


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Hemorrhagic

diathesis

HumoralHumoralSystemSystemdisordersdisorders

cellularcellularSystemSystemdisordersdisorders

VasculVasculararllesionsesions

screeningscreening::PPTT, APT, APTTT, T, TTT

screeningscreening::

BleedingBleeding timetime, PFA, PFA--100100PlateletPlatelet countcount

screeningscreening::

RumpelRumpel LeedeLeede testtestBleedingBleeding timetime


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THE COAGULATION CASCADE

In vitro

PK: prekallikrein PL: phospholipids HK: HMW Kininogen


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ScreeningScreening test oftest ofcoagulopathiescoagulopathies

Prothrombin time (PT)

Activated partial thromboplastin time(APTT)

Thrombin time (TT)


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ProtProthhrombinrombintimetime

Activated factor: FVIITested cascade phase: extrinsic pathway

Thromboplastin:Tissue factor

- apoprotein

- phospholipidPhospholipidCalcium

Sodium citratedplasma


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WhatWhat pprotrothhrombinrombin timetime measurementmeasurement

isis goodgood forfor?? Monitoring of oMonitoring of orralal antianticoagulantcoagulant therapytherapy

ScreeningScreening forfor inheritedinherited andand acquiredacquiredcoagulopathiescoagulopathies

DeterminationDetermination ofofthethe activityactivity ofofeextrinxtrinssiiccpathwaypathway factorsfactors

LupusLupus anticoagulananticoagulantt diagnodiagnosticsstics

((diluteddiluted prothrombinprothrombin timetime assayassay))


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Expressions of prothrombin time

1.1. SecondsSeconds

2. INR= International2. INR= International NormalisedNormalised RatioRatio


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Same sample measured by different wayscan give very different PT values in seconds

-- ReagentReagent sourcesource-- CoagulometerCoagulometer typetype((mechanmechanicalical,, optiopticalcal,, eleelecctromechanitromechanicalcal)

Patient A

Patient BPatient C

KC

8.48

8.489.49

Coag-a-Mate

15.08

9.30

13.31

ACL

6.79

7.27

6.64


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INR

INR: InternationalINR: International normalizednormalized ratioratio-- waswas establishedestablished byby thethe WHO andWHO and thethe InternationalInternational

CommitteeCommittee onon ThrombosisThrombosis andand HemostasisHemostasis forfor reportingreportingthethe resultsresults ofofprothrombinprothrombin teststests..-- AllAll PTPT resultsresults areare standardizedstandardized byby thisthis calculationcalculation::

INR= (INR= ( PatientPatient PPTT//CControllontroll PPTT))ISIISI

ISI= International sensitivity indexISI= International sensitivity index- Given by the manufacturer for each particularparticularthromboplastinthromboplastin reagentreagent andand instrumentinstrument combinationcombination- Reflects the reagent sensitivity to different vitamin K

dependent factor deficiencies


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ProtProthhrombinrombin timetime prolongsprolongs....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


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AActivatedctivated partialpartial thromboplastinthromboplastin timetime

Reagent:Surface activator:

silicacaolinellagic acid

PLCalcium

Activated factor: FXIITested cascade phase:

intrinsic pathway

PL+

Contact activator+

Ca++

Citratedplasma


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Monitoring non-fractionated heparin therapy

Lupus anticoagulant diagnosisScreening for inherited nad acquired

coagulopathies

WhatWhat APTTAPTT measurementmeasurement isis goodgood forfor??


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APTTAPTT prolongsprolongs....

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, prekallikrein3. Lupus anticoagulant4. Non-fractionated heparin therapy5. Rare situations:

- Severe FX, V, II deficiency- hypo- afibrinogenaemia, dysproteinaemik

- Too much citrate


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TThhrombinrombin timetime

Cleaved factor: fibrinogenTested cascade phase:

fibrinogen - solubile fibrin

Citrated plasma

Thrombin (Ca++

)


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Inherited and acquired hypofibrinogenaemias,dysfibrinogenaemias & afibrinogenaemia

Non fractionated heparin therapy

Fibrin degradation products

WhatWhat TTTT measurementmeasurement isis goodgood forfor??


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1. Hypo- afibrinogenaemia

2. Dysfibrinogenaemia

3. Non fractionated heparin th

4. Fibrinogen/ fibrin degradation products5. Rare situations

dysproteinaemias

severe hypoalbuminaemia

TTTT prolongsprolongs....


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LaboratoryLaboratory monitorisationmonitorisation ofofaantinticoagulantcoagulant && thrombolyticthrombolytic

ttherapyherapy

Goal: Effective treatment

Without bleeding


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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


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Laboratory monitoring of

thrombolytic therapy1. Before thrombolysis:

Screening for bleeding disorders(PT, APTT, TT, platelet count, bleeding time)

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

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

measurable)


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Anticoagulant therapy:

oral (cumarin) parenteral

- non fractionated heparin- fractionated heparin


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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


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Non fractionated heparin

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


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Laboratory monitoring of

conventional heparin therapyTest: 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


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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, spontaneousl

improves during heparin therapy) Type II. HIT (4-14th day, plt


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1. Preparation: Controlled depolimerization of conventional heparin

2. Molecular weight: 4000-6000 daltons

3. Half-life:

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

4. Dose: 1x daily

5. Advantages: orthopedics: thrombosis prophylaxis less frequent bleeding complications and HIT

Low molecular weight heparin (LMWH)


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

LMWH


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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.


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OralOral antianticoagulantcoagulant tthhererapyapy

Drug:acenocumarol (Syncumar)warfarin (Waran)

Test for monitoring:

PT expressed in INR(APTT is also prolonged)


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OH

CH3

R

OH

O2

CO2

Vitamin K-H2

O

CH3

R

O

O

Vitamin K-epoxid

Vitamin K

K vitaminereductase

Vitamin Kepoxide

reductase

cumarin

CH2

CH2

C O

OH

carboxilase

Glutamic acid

O

CH3

R

O

CH2

CH

C C

OO

OH OH

gamma-carboxiglutamic acid

protein

protein


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Proposed therapeutic ranges for monitoring of

oral anticoagulant treatment(International Society of Thrombosis and Haemostasis)

1. Pre-, and perioperative oral anticoagulation: INR hip replacement other surgeries

2. Primary and secondary prevention ofvenous trombosis

3. Active venous thrombosis, pulmonary embolism,prevention of recurrent venous thrombosis

4. Prevention of arterial thromboembolism

(eg. arteficial heart valve)

The higher the INR, the stronger the coumarine therapy.

too high bleeding

too low thrombotic events

2.0-3.01.5-2.5

2.0-3.0

2.0-4.0

3.0-4.5


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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


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Laboratory diagnostics ofcongenital and acquired

thrombophilias


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Thrombophilia definition

The predisposition/propensity to developthrombosis due to abnormalities in the

haemostasis system, which can be either

genetical or acquired or both.


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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


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Whom?


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Whom we should test for

thrombophilia?

Screening of the total population: NO!

Patients after venous/arterial

thrombosis

Children of patients withthrombophilia in puberty

Siblings of those thrombophiliapatients who suffered their first

thrombotic event in very early ages.Tripodi A Clinical Chemistry, 2001 Brenner BR Arch Pathol Lab Med 2002


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Which tests should be done?


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Thrombophila

Inherited Acquired

Combined


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When should we suspectinherited thrombophilia?

Deep vein thrombosis in early

ages (


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Inherited thrombophilias

Deficiencies of the inhibitory systemAntithrombin deficiencies

Deficiencies of the inactivator systemProtein 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


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APC resistance - FV Leiden

Rare thrombophilias

Unknown

Relative occurences of genetic

reasons between venousthrombophilias

AT, PC, PS deficiencies

Prothrombin 20210 A allele

40%35%

10%

10% 5%

APC i t & L id t ti


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APC resistance & Leiden mutation

Phenotype:APC resistance

APTT with APCAPTT without APC

Genotype:FV-Leiden

FV-A1691G

HungaryHungary: 10%: 10%

< 2.0

Activation & inactivation of faktor V


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Activation & inactivation of faktor V

B

B

R709 R1018 R1545

A1 A2 A3B C1C2

A1 A2

A3 C1C2

Ca2+

A1 A2

A3 C1C2

Ca2+

R306 R5

06R6

79

Thrombin

APC

h h i f h b hili i


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Pathomechanism of thrombophilia in FV-

Leiden

1. FV-Leiden is

is worse substrateto APC than FV

FV-506Qa Thalf

2. FV-Leiden is

bad cofactor to

APC in FVIIIainactivation

A FII 20210A ll l l i i E


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A FII 20210A allel prevalencies in Europe

50 N latitudeAverageAverage: 1: 1,,7%7%

AverageAverage: 3: 3,,0%0%

HungaryHungary: 2: 2,,7%7%

h bi ll l


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Prothrombin 20210A allele

Mechanism:In 3 nontranslated region of FII gene Guanine-Adenine

changeincreased efficiency in protein translation

mRNA is more stabile

plasma FII level

DVT relative risk 1,8-4,1x

Responsible for 10% of familial thrombophilias


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Mild hyperhomocysteinaemia:

Risk factor for arterial occlusive disorders

Homocystein ref. range: 5-15 mol/LHyperhomocysteinaemia: >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


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Inherited hyperhomocysteinaemias

Metionin

Homocisztein

Cisztation

SzerinB6

-ketobutirt Cisztein

B6

Betain

N,N-dimetilglicin

5-metiltetrahidrofolt

tetrahidrofolat

5,10-metiln-tetrahidrofolt

Metil-B12

MTHFR

Cisztation -szintetz

Wh h ll


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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

A i d th b hili


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1. Postoperative status

2. Pregnancy, oestrogen therapy, oral anticoncipients3. Malignant tumors

4. Immobilisation

5. Artificial heart valves6. Varicosus veins

7. Atrial fibrillation

8. Anti-phospholipid syndrome

Acquired thrombophilias

Laboratory criterias of APS


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Laboratory criterias of APS

(Consensus Workshop 1998)

Lupus anticoagulant (LA):

LA positivity twice within 6 weeks intervals(Using criterias established by ISTH SSC LA/PL-

dependent antibody subcommitte)

Anticardiolipin antibody (ACA):

IgG &/or IgM isotype twice within 6 weeks intervals

(Using standardised 2 glycoprotein I-dependent ELISA

LA diagnostic tests


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LA diagnostic tests

1. Phospholipid dependent tests increase:APTT, APTT-LA, rarely PT

2. The prolongation is increased by thepresence of inhibitors: mixing studies

3. The inhibitor targets phospholipids: hPT,

hexagonal PL tests, PNP

ELISAELISA forfor detectiondetection ofof thethe autoauto--antibodyantibody


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Reaction

ELISAELISA forfor detectiondetection ofofthethe autoauto antibodyantibody

againstagainst 2-glycoprotein I

2-glycoprotein IOn plate

Reaction mix

Anti- 2-glikoprotein I

antibody(patient serum)

Peroxidase-labelledanti-human

immunglobulin

2-glycoprotein I

(serum)


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When should be thrombophilia

investigation done?

When should we take sample for


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When should we take sample for

thrombophilia tests?

Genetic test: any time but tests should be done once in alifetime

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


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Which

tests toorder?

Thrombophilia markers with strong


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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


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Where the thrombophilia

investigations should be done?


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In such laboratories where all listed factorscan be tested and the results are also

interpreted

laboratory diagnosis of coagulation disorders

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