Examinations of coagulation

disturbances

Jan Živný
Department of Pathophysiology
[email protected]
 Outline
• Hemostasis:
– Definition, key players
• Thrombosis
– Examinations in DVT/PE
– Laboratory approach to hypercoagulation
• Bleeding
– Evaluation of platelets and vessel wall defects
– Plasma coagulation factors examination
• Case Report
 HEMOSTASIS = the arrest of bleeding
from an injured vessel
VESSEL WALL
ENDOTHEL

PLASMA
PLATELETS FACTORS
(procoagulation,
anti-coagulation)

Hemostatic abnormalities can result in procoagulation or/and

anti-coagulation conditions
Hemostasis is a integral part of inflammatory response
Thrombosis And Embolia
 Hemostatic And Coagulation Abnormalities:
Thrombosis And Embolia

THROMBOSIS

VENOUS ARTERIAL MICROCIRCULATORY

EMBOLIA
TISSUE ISCHEMIA

PULMONARY SYSTEMIC KARDIOVASCULAR

FAILURE

DVT/PE
 Pathogenesis of Thrombosis
Virchow’s postulate
Thrombi are formed as a result of the one or
more abnormalities in:

• blood vessels
• blood flow
• blood coagulability
 Abnormal Blood Vessel

a. Trauma
b. Vasculitis
c. Atherosclerosis
d. Others
 Abnormal Blood Flow

a. Decreased mobility
b. Vessel Obstruction
c. Eccomomy class
syndrome
d. Pregnancy
 Abnormal blood :
Congenital Thrombophilia
Factor General People With
Population Thrombosis

APCR: factor V Leiden mutation 3-8% of Caucasians 20-25%

   Prothrombin G20210A 2-3% of Caucasians 4-8%

Antithrombin deficiency 1 in 2-5000 1-1.8%%

  Protein C deficiency 1 in 300 2.5-5.0%

Protein S deficiency Unknown 2.8-5.0%

Hyperhomocysteinemia 11% 13.1-26.7%

 Abnormal Blood:
Acquired thrombophilia
Venous Thrombi
 VENOUS THROMBOSIS

TRIGGER

MIN.-HOURS

DVT distal DVT proximal

(infra) popliteal localization ileo-femoral localization

HOURS
YEARS DAYS

POSTTHROMBOTIC
SYNDROM LUNG EMBOLIA

DVT/PE
 CLINICAL MANIFESTATION OF DVT / PE
Deep Venous Thrombosis (DVT)

• A/ assymptomatic:
• cause of > 50% of PE
• B/ symptomatic:
• swelling,
• passive pain or palpation pain
• changes in color and temperature of skin
• Homan’s sign (increased resistance or calf
pain during dorsiflection of foot)
DVT/PE
 CLINICAL MANIFESTATION OF DVT / PE
Postthrombotic syndrome

• sign of chronic DVT

• usually develops 3-15 years after the
episode DVT
• varicose surface veins
• erythema, dermatitis
• skin ulcerations (lower extremities)
 CLINICAL MANIFESTATION OF DVT / PE
Pulmonary embolism (PE)
• complication of DVT
• Signs depend on the extend of embolism:
• dyspnea, tachypnea, tachycardia
• pleuritic chest pain
• increased jugular vein filling
• hemoptysis
• cardiovascular failure and sudden death
• important cause of morbidity and mortality of
hospitalized patients
 CLINICAL DIAGNOSIS OF DVT

• Real time ultrasound with Doppler

• 24-66% sensitivity
• Impedance pletysmography
• Magnetic resonance imaging (MRI)
• Contrast venography
• 5-10% complication (phlebitis)
• Accumulation of 125I-Fibrinogen
•low specificity and sensitivity
DVT/PE
 Clinical Diagnosis Of PE

• Ventilation / Perfusion lung scan

• in all clinically stabilized pts with suspicion
• Pulmonary angiography
• Computer tomography (CT)
• Right ventricular heart catetrization
• Echocardiography
• Blood gas analysis (ABG) ?
• ECG ?
• Chest X-ray ?
 Laboratory Approach to
Hypercoagulation states
Fibri(noge)n degradation products concentration (FDP)
<1000 g/L)
- haemocoagulation failure
- ELISA or semi quantitative agglutination methods
D-dimer: < 500 g/L
- cross-linked FDPs specific for stabile fibrin
- high sensitivity but low specificity for DVT/PE
 Laboratory Approach to
Hypercoagulation states
• Antithrombin activity (AT):
– normal 100-80 % of control plasma activity
– deficiency of AT increase the risk of thrombophilia
and DIC

• APCR (Activated Protein C Resistence)

– Factor V Leiden and Prothrombin 20210 by
Polymerase Chain Reaction
• Assays of specific anticoagulant proteins
 Laboratory Approach to
Hypercoagulation states
• Antiphospholipid antibodies:
• Immunoassays are available for several
phospholipids
• Evaluation of fibrinolytic system:
• plasma levels and activity of plasminogen
activators (e.g. tPA), inhibitor of plasminogen
activators (PAI-1), plasminogen, inhibitor of
plasminogen (alpha2-AP), lipoprotein (Lp)
Bleeding
Hemostatic And Coagulation Abnormalities:
Bleeding
BLEEDING

DIFUSE
SURGICAL FROM SMALL MICROVASCULAR:
INJURIES: purpura, petechiae, ecchymoses
s.c., i.m. needle (>3mm)
injections tissue apoplexia
mucosal erosions

Injury exceeds Failure of posttraumatic A) thrombocytopenias

the limits of hemostasis B) disintegration of
functional Dysfunction of: platelets, microvascular intima
hemostasis plasma coagulation
system, Endothelial cells
BLEEDING
 Blood Platelets

Platelet blood components may be stored for 5 days at room temperature

Platelets
 Idiopathic Thrombocytopenic Purpura

Large ecchymotic area over the thigh Bone marrow biopsy showing both
following minor trauma. The platelet megakaryocytic and erythroid
count at the time was 7000/uL hyperplasia
 Platelets and von Willebrand factor I.
Platelet count (PLT) - 150k - 400k /L
- for surgery optimal > 100k /L
- severe thrombocytopenia PLT < 20k /L
Mean platelet volume (MPV) - 6 - 8 fL
- some hereditary thrombocytopaties have large PLT
Aggregometry
- used for classification of congenital platelet disorders
- Aggregation is induced in PLT rich plasma or in WB
collected to sodium citrate by the addition of activator
(ADP, thrombin, collagen)
PLT LABORATORY
Tests for Platelet Agregation
 Platelets and von Willebrand factor II.
Flow cytometry
- immunological measurement of PLT receptors
Antibodies to platelets
- diagnosis of immune-mediated thrombocytopenias
vWF evaluation - various immunoassays;
- function test Ristocetin-induces PLT aggregation:
– decreased in most vWD
 Model of von Willebrand Factor–
Factor VIII Complex in Plasma

vW factor:
circulates as a complex with factor VIII (protecting factor VIII from
degradation )
involved in the interaction with collagen and platelet glycoproteins
(GpIb and GpIIb3
 Platelet aggregation test with Ristocetin
On fixed platelets using patients PPP
• normal (red) plasma
• patient (blue) plasma

Patient had:
• normal aPTT
• factor VIII activity 77%
• vWf antigen 61%
• Ristocetin co-factor activity
<12.5%
• vWf multimer pattern
showing absence of high
molecular weight multimers
Ristocetin can induce platelet aggregarion only in the presence of
large multimers of von Willebrand factor
von Willebrand factor protein multimer analysis
on agarose gel electrophoresis
• Lane 1
• normal control plasma:
• Lane 2
• Patient vWf disease Type 2B
• High molecular weight multimers are
absent
• Lane 3
• patient with acquired von Willebrand
syndrome (AVWS) secondary to ET.
• high and intermediate molecular
weight multimers are absent.
 Bleeding Time (Duke 1910, Ivy 1941)

• Functional test of primary hemostasis (platelets)

• Time required for spontaneous hemostasis after the
standard injury

• Duke: Standard incision of ear auricle 2-5 min.

• Ivy: Standard incision (6x1 mm) on the volar
aspect of the forearm, with the BP cuff inflated to
40 mm Hg (Ivy) 4-8 min

BLEEDING LABORATORY
 Bleeding Time - Ivy

Prolonged:
- Disorders of platelet
function
- von Willerand disease
- thrombocytopenia
(< 100k/L)
- aspirin (>5 days)
•Standard incision 6x1 mm
• BP cuff inflated to 40 mm Hg
•Normal value: 4-8 min
 Capillary resistance test (Rumpel-
Leede)
• Test of capillary vessel wall integrity
• diagnosis of hereditary purpuras (e.g. Weber-Rendu-Osler
pupura)

• Number of petechiae on the forearm area (4x4

cm) after the application of standard pressure of
10,5 kPa for 10 min using a BP cuff
• > 5 petechiae indicates increased fragility of
capillaries -
PLASMA COAGULATION FACTORS
Venepuncture

Blood is collected into

tubes with acid citrate
dextrose (ACD) and
centrifuged to obtain
platelet poor plasma
(PPP)
 Prothrombin Time = PT (Quick’s test):
Measures the activity of TF activation ptw. (extrinsic) and common ptw.

- Fibrin fibers appearance (i.e. Clotting time) is measured

after the addition of thromboplastin (TF and phospholipids)
to recalcified (CaCl2) plasma.
- Use:
- screening for disordered coagulation
- monitor therapy with coumarin (max. INR = 4.5)
- test of the liver biosynthesis

BASIC PCS LABORATORY

 Prothrombin Time = PT (Quick’s test):
Measures the activity of TF activation ptw. (extrinsic) and common ptw.

Longer PT:
• deficiency of FV or vit. K dependent FII, VII, IX, X
• severe FBG deficiency
• hi FDP
- In some settings is not influenced by heparin (to 1 U/mL)
- Normal values: 11-14 s (INR 0.9-1.1)
 Ratio of a patient's prothrombin time to a normal (control)
sample, raised to the power of the ISI value for the analytical
system used.
ISI usually between 1.0 and 1.4
The activated partial tromboplastin time = aPTT
diagnosis of contact (intrinsic) factor deficiency

- The clotting time is measured after the addition of

phospholipids (allow formation of FX- and FII complexes)
into the recalcified plasma that has been preincubated with
particulate material (e.g. micronized silica or kaolin) to
initiate activation of contact system.
-Use:
- screening for disordered coagulation
- diagnosis of contact (intrinsic) factor deficiency (less
then 30% of normal) F XII, PK, HMWK
- deficiency of FXI, FIX , FVIII (hemofilia C, B, A)
- Lupus anticoagulants
- monitoring of heparin therapy.
BASIC PCS LABORATORY
The activated partial tromboplastin time = aPTT
diagnosis of contact (intrinsic) factor deficiency

Typically 28-36 s
-Prolonged test time:
- Deficiency of FII, V, X;
- Deficiency of contact system (FXII, PK, HMWK); FXI,
FIX, FVIII (hemophilia C, B, A)
- lupus anticoagulants
- severe deficiency of FBG; high levels of FDP
- disordered conversion of FBG
- Shortened test time: Prothrombotic situations
- Results are expressed as a patients aPTT vs. normal aPTT
- Heparin therapy requires 1.5 - 2.5 times prolongation of
aPTT
BASIC PCS LABORATORY
 Other Plasma Coagulation System (PCS)
activity tests

Fibrinogen concentration (FBG): 2-4 g/L

- clotable fibrinogen (only functional) or total fibrinogen
(immunoassay)
- Hypofibrinogenemia: e.g. DIC
- Hyperfibrinogenemia: e.g. inflammation - acute phase protein
Thrombin time of plasma (TT): 17 - 24 s
- direct test of fibrinogen - fibrin conversion
- prolonged: dysfibrinogenemia, severe hypofibrinogenemia, hi
FDPs
(inhibits polymerization), heparin therapy (can be neutralized by
prothamin sulfate)

PCS LABORATORY
 aPTT or PT Mixing Studies
• aPTT with the mixture of 50:50 patients / normal
plasma
 remains prolonged when an inhibitor
(antibodies) is present

• aPTT or PT test using selectively deficient plasma

to be mixed with pts. plasma
 diagnosis of hemophilia A (FVIII), B (FIX), C
(FXI), and other PCS deficiencies

HEMOSTASIS LABORATORY
 Tests useful for the bedside examination
Lee-White test - coagulation time of whole blood w/o anticoagulant
- polystyrene or glass tube at 37oC.
- Normal time 4-10 min. (depends on setting)
- Function of PCS - fast orientation
Thrombin time of whole blood - coagulation time of whole blood
collected to the tube w/o anticoagulant and standard amount of
thrombin
- Presence of fibrinogen YES / NO
- DIC decompensation- fast screening method
Activated coagulation time (ACT) - blood drown w/o anticoagulant is
transferred to the tube with contact activator (silica or kaolin) and
mixed at 37oC until coagulum is present (normal about 150 s).
- Routine use for the heparinization control of extracorporeal
circulation or hemodialysis (required 180-300 s; heart surgery > 600 s)
BEDSIDE LABORATORY
 CASE REPORT: KOA 1
History:
F, 55 years
At 26
• 10 days after 3rd delivery - DVT of lower right
extremity and SVT of lower left extremity
• Therapy with heparin then OA of warfarin 6 month
At 41, 43
• 2 uncomplicated gyn. surgeries (ovarial cyst)
At 45
• Episodes of techycardia: ECHO showed mitral stenosis
At 46, 53, 54- SVT of lower extremities
 CASE REPORT: KOA 1

Q: What might be the cause of

thrombophilia of the patient?
What laboratory tests do you suggest?
KOA 1/II
protein S activita 52%
APC ratio = (APTT w APC / APTT w/o APC) = 2 - 5 normal
= 1.15 patient

DNA analysis of F V:
• The mutation (1691G→A substitution) removes a cleavage site of
the restriction endonuclease MnII (Arg506Gln Leiden)
• PCR treatment with MnII and then DNA electrophoresis will give
a quick diagnosis..
Q: What is the role of Activated protein C (APC)
in plasma coagulation?

Q: Explain the mechanism of APC -resistance ?

 KOA 1/III Regulation of Thrombin
Activity
HPS

T AT

TF PKS T
T
FVa AT
PS
FVIIIa
APC
HPS
PC T
TFPI PC

TM
ENDOTEL
KOA 1/IV

During hospitalization acute episodes of atrial

fibrilation: ECHO t=revealed thrombus in the LA plus
mitral stenosis
Cardiosurgery: Thrombectomy and mitral valve
replacement
Long term therapy with anticoagulants with INR = 3.5 –
4.5
KOA 1/V

• What is INR?
• What are the expected results of basic
coagulation examinations?
Vit.K def./OA
PLT BT APTT PT TT FBG

N N P P N N
 To Remember
• Hemostasis is part of inflammatory response
• Bleeding time
• aPTT
• PT (Quick)
– INR
• Thrombin time (DIC fast screen)
• D-dimer
• APC resistance
Thank you
 PT PTT Acquired Disorder Congenital Disorder
Long Normal Liver disease or vitamin K Factor VII deficiency
deficiency
Normal Long Acquired factor VIII inhibitor Factor VIII, IX, or XI deficiency
Long Long DIC, liver disease, Lupus Fibrinogen, prothrombin, factor
anticoagulant (LA) V or X deficiency
Normal Normal Thrombocytopenia, qualitative Mild factor deficiency, mild von
platelet disorder Willebrand disease, factor XIII
deficiency
 Expected Results Of Laboratory Tests (I)

Porucha PLT BT APTT PT TT FBG

Trombocytopenie L P N N N N
Hemofilie A N N P N N N
Hemofilie B N N P N N N
Hemofilie C N N P N N N
vW choroba N P N/P N N N
LA N N P N/P N N
 Expected Results Of Laboratory Tests (II)

Porucha PLT BT APT T PT TT FBG

FV-def. N N P P N N
N N P P N N
FII-def.
N N P P N N
FVII-def.
Vit.Kdef./OA N N P P N N

FBG-def. N N P P P L
Heparin N N/P P N/P P N
Dual Function of Native Factor V
3D overview of the protein C activation
and the FVa degradation
Schematic model of certain reactions of
the protein C anticoagulant system
Structures of the β-CCP1 of C4BP,
protein S and APC
Schematic models of APC-mediated activation of
PAR-1 and protein S binding to apoptotic cells.
Schematic representation of blood coagulation
and the protein C anticoagulant system
Simplified Model of von Willebrand Factor
Functions in Platelet-Plug Formation.