Pre-analytical factors that can affect - PowerPoint Presentation

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Pre-analytical factors that can affect coag

test results

Underfilled tube

High hematocrit

Hemolysis

Traumatic blood draw (tissue factor)

Delay in testing

Excessive agitation of blood in tube (platelet tests)

Slide3

Effect of high hematocrit on coag tests

Elevated citrate concentration may prolong clotting times

Slide4

Thromboplastin:

Tissue factor

Phospholipid

Calcium

The Prothrombin Time

Add thromboplastin (excess of tissue factor + phospholipid + calcium) to citrated plasma. Not sensitive to XI, IX, VIII levels

More sensitive than aPTT to warfarin effect

Usually expressed as International Normalized Ratio (INR)

Also contains a heparin-

neutralizing agent

Slide5

Why the INR?

Tissue factor in the thromboplastin may be recombinant, or derived from human or animal tissue

Phospholipid composition varies among thromboplastinsAs a result different thromboplastins have varying sensitivity to the effect of warfarin

The INR system makes PT results from different laboratories comparable to one another in patients receiving vitamin K antagonists (

not

in liver disease or other coagulopathies)

Slide6

ISI (International Sensitivity Index) is reagent- and method-specific; higher number indicates

lower

sensitivity to changes in clotting factor levels

INR =

Patient PT

Mean Normal PT

(

)

ISI

Slide7

INR =

(

)

1.24

22.0

12.6

= 2.0

INR =

(

)

2.46

16.2

12.2

= 2.0

Reagent A: ISI = 1.24, mean normal = 12.6 sec

PT = 22 sec

Reagent B: ISI = 2.46, mean normal = 12.2 sec

PT = 16.2 sec

Slide8

INR values with two different reagentsPatients on warfarin

PATIENT #

INR

INR

1

3.4

2.7

2

2.8

2.5

3

3.5

2.3

4

2.6

2

5

2.2

1.2

6

2.3

2.4

7

1.9

1.7

8

3

2.8

9

2.2

2.7

10

4

4

REAGENT E (ISI 2.98)

REAGENT B (ISI 0.96)

Slide9

Uses of the PT/INR

Best single test of the integrity of the fibrin clotting system

Detects most clinically significant acquired coagulopathiesDoes not detect the most common inherited clotting factor deficiencies (VIII, IX, XI)

Routinely used to monitor warfarin therapy

Insensitive to heparin at usual therapeutic concentrations

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Activated partial thromboplastin time (aPTT)

“Partial thromboplastin”

Phospholipid +

Activator (provides surface for generation of XIIa)

Incubate citrated plasma with phospholipid + activator (generates XIIa

→

XIa

→

IXa). Then add calcium to allow clotting to proceed to completion.

Not sensitive to VII level.

More sensitive to heparin than PT

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Uses of the PTT

Screen for inherited clotting factor deficiency (hemophilia, factor XI)

Monitor heparin therapyMany institutions switching to anti-

Xa

assay

Screen for acquired coagulation inhibitorsFactor VIII antibody

Lupus anticoagulant

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Limitations of the PTT

A long PTT does not always indicate a bleeding disorder

Factor XII deficiency

Lupus anticoagulant

Normal PTT does not rule out a common pathway defect

High factor VIII levels can mask defects lower in the pathway

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The aPTT

should be ordered selectively

Results of 1025 consecutive tests, excluding heparin monitoring

Robbins and Rose, Ann Intern Med 1979;90:796

# TESTS

# PATIENTS

Abnormal result

143

97

On anticoagulant

64

37

Liver disease

41

27

No cause found, no bleeding

15

14

Normal on repeat testing

9

9

Known hemophilia

5

4

History of intestinal bypass

5

4

Other malabsorption (CF)

2

1

Technical problem with test

1

1

Newly dx'd bleeding disorder

0

0

# abnormal: 143 (14%)

Slide14

What causes a long PT/INR and a normal PTT?

Factor VII deficiency

Mild deficiency of “common pathway” factors

Warfarin

Vitamin K deficiency

Liver disease

PTT

PT/INR

Slide15

What would cause a long PTT with a normal INR?

Deficiency of VIII, IX, XI

Deficiency of a contact factor (usually XII) (does not cause bleeding)

Heparin

Factor VIII inhibitor

Lupus-type inhibitor (antiphospholipid antibody)

PTT

PT/INR

Slide16

What if both PT/INR and PTT are long?

Liver disease

Vitamin K deficiency

Warfarin

DIC

High level of heparin

Other inhibitor affecting common pathway (eg, direct thrombin inhibitor)

Isolated deficiency of X, V, II, fibrinogen (rare)

PTT

PT/INR

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Thrombin time: thrombin + plasma. Very sensitive to heparin and other thrombin inhibitors. Prolonged by low fibrinogen, dysfibrinogenemia, high levels of fibrin degradation products.

Urea solubility: clot immersed in concentrated urea (breaks noncovalent bonds) clot dissolves unless crosslinked by factor XIIIa). For diagnosis of severe factor XIII deficiency (v. rare)

PT/INR

aPTT

Thrombin time

Other tests

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Mixing Study

Purpose: to determine whether long aPTT or PT is due to clotting factor deficiency or circulating inhibitor (

eg, factor VIII inhibitor, heparin, lupus-type inhibitor)

Mix patient plasma 1:1 with normal plasma, measure aPTT or PT

Incubate mixture for one hour, repeat aPTT or PT

Certain inhibitors (

eg

, factor VIII antibody) take time to work

Failure to correct prolonged clotting time by mixing with normal plasma implies presence of a circulating inhibitor

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Clotting factor assay

Serial dilutions of patient plasma in factor-deficient plasma

Serial dilutions of normal plasma in factor-deficient plasma (calibration curve)Measure aPTTs of both sets

Semi-log plot - % of normal factor vs aPTT

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3%

<1%

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Patient C

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Patient C

100/.5 = 200%

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100%

50%

10%

5%

1%

20

40

60

80

aPTT (sec)

% test plasma

Normal plasma

Patient

≥50%

Lupus inhibitor or other non-competitive clotting inhibitor → non-parallel plot

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Bethesda Assay for Inhibitors

Serial dilutions of patient plasma in normal plasma

Incubate 2 hoursAssay residual factor activity

1 Bethesda Unit neutralizes 50% of factor in an equivalent volume of normal plasma

Example: 1:100 dilution of patient plasma + normal plasma

→ 50% residual factor activity, so inhibitor titer is 100 BU

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Bethesda Assay

Residual factor activity

dilution pt plasma

50%

1:1

1:10

1:100

1:1000

100 BU

Slide27

The decline and fall of the bleeding time

Advantage: an in vivo test that theoretically measures both vascular and platelet function

Disadvantages

Poor standardization

Accuracy depends on experience of operator

Poor sensitivity, very poor specificity

Does not predict bleeding risk

Slide28

Rodgers and Levin, Semin Thromb Hemost 1990; 16:1

The bleeding time accurately detects aspirin use

Slide29

Rodgers and Levin, Semin Thromb Hemost 1990; 16:1

The bleeding time does not predict surgical bleeding

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Platelet function analysis

Whole blood passed through capillary tube coated with collagen plus either ADP or epinephrine (high shear)

Time to occlusion measured

Moderate sensitivity to platelet function defects, VWD

PFA-100

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Bleeding time

vs

PFA for detection of VWD

BT

C-Epi

C-ADP

Thromb Haemost 2003;90:483

Slide33

Platelet function analysis

Advantages vs bleeding time

In vitro test

Well-standardized

Somewhat better sensitivity and specificity

Disadvantages

Does not assess vascular function

Does not predict bleeding risk

Abnormal test result → test for specific defects in primary hemostasis

Test not useful if platelets <100K or if patient taking ASA, etc

PFA-100

Slide34

Platelet aggregometry

Various platelet agonists added to whole blood or platelet rich plasma

Thrombin, ADP, collagen (2 concentrations), arachidonic acid,

ristocetin

(2 concentrations)

Aggregation measured by changes in conductance (in whole blood) or turbidity (in PRP)

Release measured by

chemiluminesence

Significantly more sensitive than PFA

Many abnormal results nonspecificExpensive

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Saline agg

Thrombin rel

Risto low agg

Risto high agg

Collagen agg

Low

High

High

Low

Collagen rel

AA agg

ADP agg

AA rel

ADP rel

2 nM ATP

Ch 1

Ch 2

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Risto low

Risto high

Pt

Control

Type I VWD

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Collagen agg

Low

High

High

Low

Collagen rel

AA agg

ADP agg

AA rel

ADP rel

Pt

Normal

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Took Excedrin 5 days ago

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AA agg

ADP agg

AA rel

ADP rel

Pt

Normal

Taking ASA 81 mg/d and Plavix 75 mg/d

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AA agg

Coll agg

AA rel Coll rel

Coll agg

Low

High

Coll release

Low High

PFA: Coll/ADP 91 (nl 65-120)

Coll/Epi 139 (nl 85-175)

Slide42

Assessment of the fibrinolytic

system

Fibrinogen

(dilute thrombin time assay)

D-dimer

(immunoassay)

α

2-antiplasmin activity

(chromogenic substrate assay)Thromboelastography

Slide43

Global assessment of clotting: thromboelastography

Measures mechanical strength of clot vs time

Sensitive to most major defects in fibrin clot formation, platelet plug formation, excessive fibrinolysis

Can also detect hypercoagulability

Useful “point of care” test in OR, etc to guide blood product use

30 min

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World J Transplant 2012;2:1

Slide46

Effect of Coagulation Factor Deficiency on TEG

Normal

Factor

deficiency

Slide47

Effect of platelet abnormality on TEG

Normal

Thrombocytopenia

or

dysfunctional platelets

Slide48

Effect of hyperfibrinolysis on TEG

Normal

Hyperfibrinolysis

Slide49

Chromogenic substrate-based assay

Peptide containing target sequence of enzyme linked to chromophore

Colored cleavage product (in this case nitroaniline) detectable by spectrophotometry

Enzyme specificity determined by target sequence

Rate of color generation proportional to enzyme activity

Slide50

Examples of chromogenic assaysAnti-Xa assayPatient plasma added to mixture of Xa and chromogenic substrate (± antithrombin)

Residual Xa activity inversely proportional to inhibitor level

Protein C activityPatient plasma + venom enzyme that selectively activates protein C; activated protein C cleaves substrate

Slide51

Von Willebrand factor measurementsVWF antigen: via ELISAVWF activity

Ristocetin

cofactor assay: patient plasma + ristocetin + formalin fixed platelets

Alternative assay uses beads coated with monoclonal Ab against GP1b binding site in VWF rather than platelets

Multimer analysis: via gel electrophoresis

Ristocetin

-induced platelet aggregation

VWF

propeptide level

Slide52

VWF multimer analysis

Slide53

Enhanced ristocetin-induced platelet aggregation in type 2B VWD

Red = low dose

ristocetin

Black = high dose

ristocetin

Normal plasma Patient plasma

Patient platelets Normal platelets

Slide54

Von Willebrand propeptide

Propeptide

noncovalently bound to VWF multimers, released together with VWF into blood

Blood level normally proportional to VWF level

If

propeptide level significantly higher than VWF level, implies abnormally rapid clearance of VWF from blood

Some inherited VWD variants

Acquired VWD

Measurable propeptide level rules out type 3 VWD