laboratory diagnosis of coagulation disorders
TRANSCRIPT
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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