Laboratory Evaluation of Haemostasis &
Introduction to Bleeding Disorders

Blood Module
Dr Jim Faed

1
 recap

Haemostasis – the big picture









 

  

  
  






  
 

 
 




Haemostasis achieved
2
 Initiation of Coagulation by the Tissue Factor pathway
recap
Slow activation of FX to Xa and Prothrombin (II) to Thrombin (IIa)
results in activation - FVa, FVIIIa, FXIa and more platelets activated

Ca++
FVIIa `
FVII

Platelet
Tissue
Tissue Injured
Phospholipid surfaces Factor
factor cell
expressed
Ca++ Phospholipid more
IX IXa platelets activated

` FVIII FVIIIa
Ca++ + Phospholipid
FV FVa
X Xa
II IIa FXI FXIa
Prothrombin Thrombin

3
Advanced level diagram of cell membrane based dynamics for coagulation
 recap

Rapid Phase of activation of Coagulation

cell membrane theory of activation of coagulation
Factor XIa
IX IXa Ca++
VIIIa
Ca++ platelet phospholipid

Fibrinopeptides A & B
X Xa Ca++
Va
Ca++ platelet phospholipid

II IIa Fibrin monomer

Thrombin burst
Fibrin polymer
Fibrinogen
Stable Fibrin
Factor XIII FXIIIa (cross-linked 4
meshwork of fibres) 4
 Basic – key info
Basic tests of blood coagulation
Intrinsic coagulation pathway
All factors present in blood
Blood sample: citrated plasma
Contact activation on Kaolin
Phospholipid added (platelet substitute)
(calcium chelated)
Slow activation – 5-10 min
Factor XII F XIIa Extrinsic Coagulation Pathway
Requires tissue factor
F XI F XIa
Tissue Factor & Ca++
Ca++ + F IX F IXa
F VIIIa
F VIIa Factor VII
FX F Xa Prothrombin
F Va Time (PT)
Activated F II FIIa 8–11s
Partial Prothrombin activated to Thrombin
Thromboplastin Fibrinogen assay &
Time (APTT) Thrombin clotting time
22-34s (TCT)
Fibrinogen converted to Fibrin
12-18s 5
 The Basic Tests and their
sensitivity to factor deficiencies
Activated Partial Prothrombin Thrombin
Thromboplastin Time Clotting Fibrinogen
Time (APTT) (PT) Time (TCT)

Clinically XI, IX, VIII VII I I

important
factor X, V,
deficiencies
(II, I – not very sensitive to
detected
these deficiencies)

Deficiency
detected but
not clinically
XII
important
6
 Basic – key info

Coagulation Factor assays

  Coagulation Factor assays are usually
performed as coagulation bioassays
  Activity
is measured as a percentage of
biological clotting activity - except fibrinogen
  100% is the Factor conc. in a pool of ‘normal’ plasma
  1% of any factor is equivalent to the activity present if
normal plasma is diluted 1:100 in plasma that completely
lacks that factor
  APTT & PT are prolonged if factor conc. is <40-50%
  Fibrinogenis assayed in mass concentration,
reference range 1.8-4.0 g/L

  Other factors have a much lower concentrations: mg/L
7
 Coagulation factor assays
  Usually based on a mixing test –
  Determine the ability of patient’s plasma to correct
the coagulation time in a plasma deficient in one
factor Coagulation time is evaluated
against a standard graph for
the assay
Patient’s 100%
plasma + Ca++

%
Factor
Plasma deficient
in factor assayed present
0%
Phospholipid +
Contact activator Clotting Time to clot - seconds8
detected
 Basic – key info

Causes of bleeding
  Defective vessel wall
  Only a small number of conditions are common and, or important
  Platelet disorders
  Thrombocytopenia
  Ref. range 150-400x10 /L
9

9
  Bleeding increases if platelet count <100x10 /L

  Defective platelet function

  Drugs that affect platelet function

  Inherited conditions

  Von Willebrand disease

  Low levels or abnormal vWF molecule (& FVIII - minor)
  Reduced platelet aggregation & prolonged bleeding
  Defective coagulation
9
  Large number of conditions: acquired or inherited
 Basic – key info

Assessing bleeding disorders

  History

  Location of bleeding

  Mucosal: nose (epistaxis), endometrial – heavy periods

  Joints/muscles

  Skin

  Other

  Pattern of bleeding

  Frequency

  Bleeding with surgery/trauma, menstruation, spontaneous

  Drug history & diet – anti-platelet agents

  Family history (for possible inherited disorders)
10
 Basic – key info

Assessing bleeding disorders

  Tests

  Blood screen – platelet count & morphology

  Coagulation – basic tests:

  APTT, PT, fibrinogen, (TCT)

  If
appropriate, second stage tests – Specialist
evaluation

  von Willebrand factor assays

  Platelet function studies

  Coagulation factor assays

11
Bleeding disorders to be discussed in
this lecture

  Disseminated intravascular coagulation (DIC)

(consumption coagulopathy)

  Vitamin K and coagulation

  Vitamin K deficiency conditions

  INR – standardised Prothrombin Time test result

  Warfarin – an anticoagulant that acts by
blocking vitamin K cycle
12
 Basic – key info
Bleeding disorders
Disseminated Intravascular Coagulation
  What is it –
  Widespread activation of platelets and coagulation
  Coagulopathy arises from ‘consumption’ of coagulation

factors and platelets

  Causes include –
  Release of procoagulant material into the circulation

  Widespread focal or diffuse damage to endothelial cells

  Potential results are –

  Mild DIC - no adverse effects

  Moderate or severe DIC –

  Bleeding

  Thromboses and ischaemia 13
 Basic – key info
Disseminated Intravascular Artery

Coagulation
Endothelial damage

  Pathogenesis -

  Tissue factor / other cell products
released in blood vessels

Microvasculature
Tissues and

  Any cause of extensive
endothelial damage

  Some snake venoms

  Resulting in –

  Platelet activation & aggregation

  Activation of coagulation

  Formation of micro thrombi

  Secondary activation of fibrinolysis
14

Vein
 Fibrinolysis is activated in DIC

  Activators and Inhibitors – concept


















  


































Activator



  



 tPA

 

 






































Fibrinolysis is always activated in DIC































  Fibrin is










cleared
from most

small
blood














vessels: high levels of FDP’s produced


  Inadequate activation results in
microvascular thromboses 15
 Basic – key info

DIC: Clinical picture & diagnosis

  Clinical features
  Variable – those of the underlying condition
  Bleeding and, or microvascular thromboses

  Laboratory findings: consumptive coagulopathy

  Falling fibrinogen conc., prolonged TCT

  Falling platelet count

  High/rising level of fibrin degradation products (FDPs)

  Reduced coagulation factors

  prolonged APTT & PT – only in severe cases

  Protein C and Antithrombin may fall significantly
  RBC fragmentation seen in blood film

  minor feature in most cases
16
 Basic – key info
Diseases causing Disseminated
Intravascular Coagulation - 1
  Infections
  Gram negative septicaemia
  Endotoxin activates monocytes - Toll 4 receptors

  Tissue factor expressed by monocytes (in blood)

  Occurs in all cases of gram negative septicaemia

  Especially severe in meningococcal septicaemia as
very high levels of endotoxin are released
  Control mechanisms

  Protein C, Antithrombin, Fibrinolysis

  May be overcome by prothrombotic factors resulting in
thromboses becoming clinically apparent
  Platelets, Fibrinogen & other coag. factors used up17
 advanced level concepts
Protein C, aPC, Thrombin and PAR-1
signalling
FVi Anticoagulant
FVIIIi PS
role
aPC
FVa
FVIIIa

IIa IIa
PC aPC
Thrombin Thrombin

Thrombo
PAR-1
EPCR EPCR EPCR (Thrombin
modulin receptor)

Can anti-inflammatory effects S1P-1 S1P-3

of aPC be enhanced?
New modified aPC molecules Anti-apoptotic, Apoptotic,
with anti-inflammatory effects anti-inflammatory Pro-inflammatory signal
but no anticoagulant effects signalling inducing vascular 18
are being studied. leakage
 advanced level concepts

Activated Protein C (aPC) &

Plasminogen Activator Inhibitor I (PAI-I)
Plasminogen tPA
(tissue
activates plasminogen
inhibits activator)

Plasmin inhibits aPC

proteolysis PAI-I from

monocytes /
macrophages
Fibrin FDP’s

In meningococcal septicaemia – very low aPC 19

and very high PAI-1 blocks fibrinolysis

 21 yr old with meningococcal
septicaemia
Adult Female
Parameter 16:15 18:22 03:10
Reference range
Haemoglobin (Hb.) 137 116 67 115-165 g/L
9
Platelet Count 269 50 27 150-400x10 /L
9
Total Leucocyte Count 2.05 14.9 4.00-11.00x10 /L
9
Neutrophils 1.55 12.43 2.00-7.50x10 /L
APTT >100s 24-35 s
Fibrinogen <0.7 1.8-4.0 g/L
Protein C 22% 70-140%
Antithrombin 67% 70-150%

20
 Basic – key info

Treatment of Acute DIC - basics

  Treat the cause (if possible) and the DIC will stop

  Infection – antibiotics

  Trauma / shock – treat the shock

  Secondary bleeding caused by DIC

  Replacement – blood component therapy

  Thrombocytopenia & bleeding – Platelet Concentrate

  Low fibrinogen & bleeding – Fibrinogen-rich blood

component (Cryoprecipitate)

  Severe cases with a prolonged APTT not controlled by

Cryoprecipitate - Fresh Frozen Plasma

  Verysevere cases: Intensive Care Unit management
of multi-organ failure (Specialist care) 21
 Basic – key info

Other Disorders Causing DIC - 2

  Any severe or widespread tissue injury

  Prolonged shock with secondary hypoxic tissue injury

  Extensive tissue trauma – release of tissue products

  Severe burns

  Severe viral infections causing endothelial injury, eg

dengue,


  Traumatic brain injury with brain myelin (lipid) released
into blood

  Platelets and coagulation activated

22
 Less common causes of DIC –
covered later in ELM & ALM

  Obstetric complications

  Amniotic fluid embolism – Fetal hair and waxy skin secretions

  Eclampsia – a hypersensitivity state

  Severe allergic hypersensitivity reactions

  Causing severe vasculitis or anaphylaxis

  Incompatible blood transfusion reaction

  Malignancy – may occasionally cause a chronic DIC

  Promyelocytic leukaemia – DIC is common

  Mucin-secreting adenocarcinomas

  Others: cell products released from necrotic cells

  Liver failure

  Acute severe liver failure - severe hepatitis: viral, alcoholic

  Chronic liver failure: occasional cases of cirrhosis

  Snake venoms

  Some venoms activate Coagulation, Fibrinolytic, or sometimes Complement
23
cascades
New topics -

  Vitamin K deficiency causing bleeding

  Liver disease

  Anticoagulants warfarin

  The INR: standardisation of the PT test for
monitoring Warfarin anticoagulation

  And to finish –

  Some minicases for diagnosis and

  A differential diagnosis chart

24

Vitamin K and post-translational modification
of coagulation factors: II, VII, IX & X and the
coagulation inhibitors Proteins C & S

epoxide Nature 2004 427: 493-494

reductase
OH (VKOR) O
R R

O
CH3 CH3
HO O

vit K reduced CO2 vit K epoxide

O2 H 2O
carboxylase
COO- COO-
COO-

glutamic acid γ-carboxyglutamic acid 25

 Basic – key info

Bleeding and Vitamin K deficiency

  Vitamin K is needed to permit addition of carboxyl groups to

Factors II, VII, IX & X, and Proteins C & S
  Lack of Vitamin K -
  Factors II, VII, IX & X do not function normally if sufficient
γ-carboxyl groups are not present
  Unable to bind to activated platelet phospholipid
  Risk of bleeding

  Vitamin K deficiency occurs in 3 groups of clinical conditions

  Small bowel malabsorption disorders
  Liver disease
  Neonate (newborn infant) & premature infant
26
 Basic – key info

Adults and Vitamin K deficiency

Vitamin K3, Menadione
  Vitamin K - a fat soluble vitamin
  Vegetable sources, and
  Bacterial synthesis in colon
  Causes of Deficiency
  Low intake – uncommon issue
  Malabsorption disorders

  Bile salts (biological detergents) required to emulsify fats and
fat soluble vitamins in food during small bowel digestion and
absorption

  Liver stores of Vitamin K will usually last 1-3+ weeks

  Deficiency results in prolonged PT & APTT & a bleeding tendency

  Beware: the abdominal surgery patient with a wound infection
and antibiotic therapy who has not eaten for 1-2 weeks! 27
 Basic – key info

Vitamin K and Liver disease

  Most coagulation factors are synthesised in the liver

  Severe liver disease results in lower factor levels

  Vitamin K-dependent factors (II, VII, IX & X) fall more
than others (fibrinogen, V, VIII)

  Injection of pharmacological doses of Vit K may improve
coagulation status enough for liver biopsy or surgery

Day 2 (24h Reference

Example Day 1 after Vit K) Range
APTT 48 40 22-34 s
PT 15.6 13.4 8.0-11.0 s
Fibrinogen 1.9 1.9 1.8.4.0 g/L 28
 Basic – key info

Vitamin K and the Neonate

  Neonate
  Limited Vitamin K transported across placenta
  Low levels of Vitamin K-dependent factors at birth
  Falling levels after birth (no oral intake in first 24 hrs)

  Normal feeding: trough Factor levels at 48 hrs are ~10-30%

  Risk of bleeding into the brain ~1:1000
  Vitamin K always given after birth
  0.5mg IM at birth, or
  3 oral doses (poorly absorbed even in full term normal infant)
  Premature infants
  Increased risk of intracerebral bleeding after birth
  Physical moulding of head during birth processes
  Intramuscular dose of Vit K must be given – not able to
absorb sufficient Vit K 29
 Nature 2004 427: 493-494

Warfarin anticoagulation: inhibiting Vitamin K

WARFARIN

epoxide
OH O
reductase
R (VKOR) R

O
CH3 CH3
HO O

vit K reduced CO2 vit K epoxide

O2 H 2O
carboxylase
COO- COO-
COO-

glutamic acid γ-carboxyglutamic acid 30

 Basic – key info

Warfarin anticoagulation
(Wisconsin Alumnus Research Foundation)

  Benefits

  Useful for treating patients with pathologically
increased risk for clotting

  Oral medicine – injection not needed

  Potential problems

  Risk of bleeding

  A narrow therapeutic window exists for warfarin

  Careful clinical control needed – regular lab testing

  INR (Standardised Prothrombin Time (PT)) is best test

31
 Basic – key info
Standardising the Prothrombin time: the
INR – International Normalised Ratio

  Standardisation of Commercial PT test results

  The International Normalised Ratio is the –

  Ratio of the patient’s PT to a PT on normal plasma

  Corrected to show the test response expected with the
International Reference Thromboplastin

PT of Patient’s plasma Sensitivity Index for Thromboplastin

PT of normal plasma

  The INR corrects for differences between test brands

32
 Basic – key info
Common causes for warfarin
treatment to go out of control

  Changed intake of vitamin K in food

  Vomiting, binge eating

  Sudden changes in dietary patterns, eg when travelling

  Decreased absorption of vitamin K

  Gastroenteritis and other causes for malabsorption

  Change of medications (and some foods)

  Increased drug clearance

  Reduced warfarin effect) by displacement from albumin or by
induction of hepatic cytochrome

  Inhibition of warfarin metabolism by hepatic cytochromes

  Any cause for liver cell injury, including
gastroenteritis, heart failure, hepatitis, et al 33
 Case 1
This 22 year old woman has several large bruises on her
legs and forearms. She bleeds from cuts for longer than
other people and has heavy periods lasting 10 days.

Platelets TCT Fibrinogen APTT PT

Test
225 11 2.2 36 9
result
Ref. 150-400
10-14s 1.8-4.0g/L 22-36s 8-10.5s
Range x109/L

Differential diagnosis

Additional tests needed

34
 Case 2
An 8 year old girl with numerous bruises and petechiae
has the following test results. (Hb 125, MCV 85, MCH 28)

Platelets TCT Fibrinogen APTT PT

Test 5 12 2.0 30 9.5
150-400
Ref Range 10-14s 1.8-4.0g/L 22-36s 8-10.5s
x109/L

Differential diagnosis

Further tests
35
 Interpreting test results: Case 3
Patient 24 hours post surgery; has had a rigor –
temp. now 38.6°C; BP has fallen: 145/95 to 100/60
APTT 36 22-34 s
PT 15 8.0-11.0 s
INR 1.7 0.8 – 1.2
TCT 22 10-18 s
Fibrinogen 0.9 1.8-4.0 g/L
FDP’s (D-dimers) 7 < 0.5 FEU/L
Platelet count (now) 80 150-400 x109/L
Platelet count (24h ago) 225

  Differential diagnosis

  Treatment
36
 Case 4
A 1 year old boy has several large bruises and is being
investigated for child battering. (Hb 115, MCV 79, MCH 27)

  

















 

 
 





! 

Differential diagnosis

Further tests

37
 Case 5
A 45 year old man requires a liver biopsy to assess his
liver due to the presence of chronic hepatitis C. (Hb 122,
MCV 90, MCH 28)

  











 




 

 
 





! 

Differential diagnosis

Further tests
38
 Further tests – mostly
Case Differential diagnosis
Specialist level
Platelet capillary defect (from clinical history): •  PFA-100 test or bleeding
1. •  Von Willebrand disease or time
•  A platelet function defect •  vWF studies, FVIII

Severe thrombocytopenia: •  None needed

•  Acute immune thrombocytopenia (acute
2. leukaemia excluded as blood screen is
otherwise normal)
Sudden fall in fibrinogen and platelet count, •  None needed to
raised FDPs (& mild increase in APTT & PT) diagnose DIC.
3. •  Disseminated Intravascular Coagulation •  Identify & treat cause of
(DIC) DIC
Prolonged APTT , other screening tests •  Haemophilia A or B:
4. normal: FVIII or FIX defic.
•  A haemophilia •  Rarely FXI or FXII defic

Mild thrombocytopenia : not clinically important •  Liver function tests if not

and assessed recently
5. Mildly prolonged APTT & PT: •  Renal function tests
•  Moderately severe liver disease 39
MICN2: Blood Module - Haemostasis 3 - Coagulation tests &
Introduction to bleeding disorders

recap

Haemostasis – the big picture


 


& 

Laboratory Evaluation of Haemostasis &
Introduction to Bleeding Disorders

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Blood Module  


Dr Jim Faed
Haemostasis achieved
1 2

Initiation of Coagulation by the Tissue Factor pathway recap

recap
Slow activation of FX to Xa and Prothrombin (II) to Thrombin (IIa) Rapid Phase of activation of Coagulation
results in activation - FVa, FVIIIa, FXIa and more platelets activated cell membrane theory of activation of coagulation
Ca++ Factor XIa
FVIIa `
FVII
IX IXa Ca++
Platelet VIIIa
Tissue
Tissue Injured
Phospholipid surfaces Factor Ca++ platelet phospholipid
factor cell
expressed
Ca++ Phospholipid more
IX IXa platelets activated
Fibrinopeptides A & B
X Xa Ca++
` FVIII FVIIIa Va
Ca++ + Phospholipid Ca++ platelet phospholipid
FV FVa
X Xa II IIa Fibrin monomer
II IIa FXI FXIa Thrombin burst
Prothrombin Thrombin Fibrin polymer
Fibrinogen
Stable Fibrin
3
Factor XIII FXIIIa (cross-linked 4
Advanced level diagram of cell membrane based dynamics for coagulation meshwork of fibres) 4

Basic – key info

Basic tests of blood coagulation The Basic Tests and their
Intrinsic coagulation pathway
All factors present in blood sensitivity to factor deficiencies
Blood sample: citrated plasma
Contact activation on Kaolin
(calcium chelated) Activated Partial Prothrombin Thrombin
Phospholipid added (platelet substitute)
Thromboplastin Time Clotting Fibrinogen
Slow activation – 5-10 min
Time (APTT) (PT) Time (TCT)
Sta

Factor XII F XIIa Extrinsic Coagulation Pathway

ge

Requires tissue factor

1

F XI F XIa Clinically XI, IX, VIII VII I I

Tissue Factor & Ca++
Ca++ + F IX F IXa important
F VIIIa factor X, V,
Sta

F VIIa Factor VII deficiencies

(II, I – not very sensitive to
g

FX F Xa detected
e2

Prothrombin these deficiencies)

F Va Time (PT)
Activated F II FIIa 8–11s
Partial Deficiency
Prothrombin activated to Thrombin
Thromboplastin Fibrinogen assay & detected but
Thrombin clotting time not clinically
XII
Time (APTT)
22-34s (TCT) important
Fibrinogen converted to Fibrin
12-18s 5 6

Dr Jim Faed, Pathology Dept 1

MICN2: Blood Module - Haemostasis 3 - Coagulation tests &
Introduction to bleeding disorders

Basic – key info

Coagulation Factor assays Coagulation factor assays

  Coagulation Factor assays are usually   Usually based on a mixing test –
performed as coagulation bioassays   Determine the ability of patient’s plasma to correct
  Activity is measured as a percentage of the coagulation time in a plasma deficient in one
biological clotting activity - except fibrinogen factor Coagulation time is evaluated
  100% is the Factor conc. in a pool of ‘normal’ plasma against a standard graph for
  1% of any factor is equivalent to the activity present if the assay
normal plasma is diluted 1:100 in plasma that completely Patient’s 100%
lacks that factor plasma + Ca++
  APTT & PT are prolonged if factor conc. is <40-50%
%
Factor
Plasma deficient
  Fibrinogen is assayed in mass concentration, in factor assayed present

reference range 1.8-4.0 g/L Phospholipid +

0%
Clotting
  Other factors have a much lower concentrations: mg/L
7 Contact activator Time to clot - seconds8
detected

Basic – key info Basic – key info

 Causes of bleeding Assessing bleeding disorders

  Defective vessel wall   History
  Only a small number of conditions are common and, or important
  Location of bleeding
  Platelet disorders
  Mucosal: nose (epistaxis), endometrial – heavy periods
  Thrombocytopenia
9   Joints/muscles
  Ref. range 150-400x10 /L
9
  Bleeding increases if platelet count <100x10 /L
  Skin
  Defective platelet function   Other
  Drugs that affect platelet function
  Pattern of bleeding
  Inherited conditions
  Frequency
  Von Willebrand disease   Bleeding with surgery/trauma, menstruation, spontaneous
  Low levels or abnormal vWF molecule (& FVIII - minor)
  Reduced platelet aggregation & prolonged bleeding   Drug history & diet – anti-platelet agents
  Defective coagulation   Family history (for possible inherited disorders)
9 10
  Large number of conditions: acquired or inherited

Basic – key info

Assessing bleeding disorders Bleeding disorders to be discussed in

this lecture
  Tests
  Blood screen – platelet count & morphology   Disseminated intravascular coagulation (DIC)
  Coagulation – basic tests: (consumption coagulopathy)
  APTT, PT, fibrinogen, (TCT)
  If appropriate, second stage tests – Specialist   Vitamin K and coagulation
evaluation   Vitamin K deficiency conditions
  von Willebrand factor assays
  INR – standardised Prothrombin Time test result
  Platelet function studies
  Warfarin – an anticoagulant that acts by
  Coagulation factor assays blocking vitamin K cycle
11 12

Dr Jim Faed, Pathology Dept 2

MICN2: Blood Module - Haemostasis 3 - Coagulation tests &
Introduction to bleeding disorders

Basic – key info Basic – key info

Bleeding disorders Disseminated Intravascular Artery

Disseminated Intravascular Coagulation Coagulation

Endothelial damage
  What is it –
  Pathogenesis -
  Widespread activation of platelets and coagulation
  Tissue factor / other cell products
  Coagulopathy arises from ‘consumption’ of coagulation
released in blood vessels

Microvasculature
factors and platelets
  Any cause of extensive

Tissues and
  Causes include – endothelial damage
  Release of procoagulant material into the circulation   Some snake venoms
  Widespread focal or diffuse damage to endothelial cells
  Resulting in –
  Potential results are –   Platelet activation & aggregation
  Mild DIC - no adverse effects
  Activation of coagulation
  Moderate or severe DIC –
  Formation of micro thrombi

  Bleeding
  Secondary activation of fibrinolysis

  Thromboses and ischaemia 13 14

Vein

Basic – key info

Fibrinolysis is activated in DIC DIC: Clinical picture & diagnosis

  Activators and Inhibitors – concept   Clinical features

  




  Variable – those of the underlying condition




























  Bleeding and, or microvascular thromboses
Activator



  



 tPA





  Laboratory findings: consumptive coagulopathy


  Falling fibrinogen conc., prolonged TCT












 

 


  Falling platelet count


























  High/rising level of fibrin degradation products (FDPs)










  Reduced coagulation factors
Fibrinolysis is always activated in DIC
  prolonged APTT & PT – only in severe cases































  Fibrin is










cleared
from most


small
blood












  Protein C and Antithrombin may fall significantly

vessels: high levels of FDP’s produced   RBC fragmentation seen in blood film


  Inadequate activation results in
  minor feature in most cases
microvascular thromboses 15 16

Basic – key info advanced level concepts

Diseases causing Disseminated Protein C, aPC, Thrombin and PAR-1
Intravascular Coagulation - 1 signalling
  Infections FVi Anticoagulant
FVIIIi PS
role
  Gram negative septicaemia
aPC
FVa
  Endotoxin activates monocytes - Toll 4 receptors FVIIIa

  Tissue factor expressed by monocytes (in blood)

  Occurs in all cases of gram negative septicaemia IIa PC aPC
IIa
Thrombin

  Especially severe in meningococcal septicaemia as Thrombin
PAR-1
very high levels of endotoxin are released Thrombo
EPCR EPCR EPCR (Thrombin
modulin receptor)
  Control mechanisms

  Protein C, Antithrombin, Fibrinolysis Can anti-inflammatory effects S1P-1 S1P-3
of aPC be enhanced?

  May be overcome by prothrombotic factors resulting in
New modified aPC molecules Anti-apoptotic, Apoptotic,
thromboses becoming clinically apparent anti-inflammatory
with anti-inflammatory effects Pro-inflammatory signal
  Platelets, Fibrinogen & other coag. factors used up17 but no anticoagulant effects signalling inducing vascular 18
are being studied. leakage

Dr Jim Faed, Pathology Dept 3

MICN2: Blood Module - Haemostasis 3 - Coagulation tests &
Introduction to bleeding disorders

advanced level concepts

21 yr old with meningococcal
Activated Protein C (aPC) & septicaemia
Plasminogen Activator Inhibitor I (PAI-I) Parameter 16:15 18:22 03:10
Adult Female
Reference range
Haemoglobin (Hb.) 137 116 67 115-165 g/L
Plasminogen tPA Platelet Count 269 50 27 150-400x10 /L
9

(tissue Total Leucocyte Count 2.05 14.9 4.00-11.00x10 /L

9

activates plasminogen Neutrophils 1.55 12.43 2.00-7.50x10 /L

9

inhibits activator) APTT >100s 24-35 s

Fibrinogen <0.7 1.8-4.0 g/L
Plasmin inhibits aPC Protein C 22% 70-140%
Antithrombin 67% 70-150%
proteolysis PAI-I from
monocytes /
macrophages
Fibrin FDP’s

In meningococcal septicaemia – very low aPC 19 20

and very high PAI-1 blocks fibrinolysis

Basic – key info Basic – key info

Treatment of Acute DIC - basics Other Disorders Causing DIC - 2

  Treat the cause (if possible) and the DIC will stop
  Any severe or widespread tissue injury

  Infection – antibiotics
  Prolonged shock with secondary hypoxic tissue injury

  Trauma / shock – treat the shock

  Extensive tissue trauma – release of tissue products

  Secondary bleeding caused by DIC
  Severe burns

  Replacement – blood component therapy
  Severe viral infections causing endothelial injury, eg

  Thrombocytopenia & bleeding – Platelet Concentrate dengue,

  Low fibrinogen & bleeding – Fibrinogen-rich blood
  Traumatic brain injury with brain myelin (lipid) released
component (Cryoprecipitate) into blood

  Severe cases with a prolonged APTT not controlled by

  Platelets and coagulation activated
Cryoprecipitate - Fresh Frozen Plasma

  Very severe cases: Intensive Care Unit management
of multi-organ failure (Specialist care) 21 22

Less common causes of DIC –

covered later in ELM & ALM New topics -

  Obstetric complications

  Amniotic fluid embolism – Fetal hair and waxy skin secretions

  Vitamin K deficiency causing bleeding

  Eclampsia – a hypersensitivity state
  Liver disease

  Severe allergic hypersensitivity reactions

  Causing severe vasculitis or anaphylaxis

  Anticoagulants warfarin

  Incompatible blood transfusion reaction
  The INR: standardisation of the PT test for

  Malignancy – may occasionally cause a chronic DIC

  Promyelocytic leukaemia – DIC is common
monitoring Warfarin anticoagulation

  Mucin-secreting adenocarcinomas

  Others: cell products released from necrotic cells
  And to finish –

  Liver failure
  Some minicases for diagnosis and

  Acute severe liver failure - severe hepatitis: viral, alcoholic

  A differential diagnosis chart

  Chronic liver failure: occasional cases of cirrhosis

  Snake venoms

  Some venoms activate Coagulation, Fibrinolytic, or sometimes Complement
23 24
cascades

Dr Jim Faed, Pathology Dept 4

MICN2: Blood Module - Haemostasis 3 - Coagulation tests &
Introduction to bleeding disorders

Vitamin K and post-translational modification Basic – key info

of coagulation factors: II, VII, IX & X and the
Bleeding and Vitamin K deficiency
coagulation inhibitors Proteins C & S
  Vitamin K is needed to permit addition of carboxyl groups to
epoxide Factors II, VII, IX & X, and Proteins C & S
Nature 2004 427: 493-494
reductase
OH (VKOR) O   Lack of Vitamin K -
R R   Factors II, VII, IX & X do not function normally if sufficient
O γ-carboxyl groups are not present
CH3 CH3   Unable to bind to activated platelet phospholipid
HO O
CO2   Risk of bleeding
vit K reduced vit K epoxide
O2 H 2O
carboxylase   Vitamin K deficiency occurs in 3 groups of clinical conditions
COO- COO-   Small bowel malabsorption disorders
COO-
  Liver disease
  Neonate (newborn infant) & premature infant
glutamic acid γ-carboxyglutamic acid 25 26

Basic – key info Basic – key info

Adults and Vitamin K deficiency Vitamin K and Liver disease

Vitamin K3, Menadione
  Vitamin K - a fat soluble vitamin   Most coagulation factors are synthesised in the liver
  Vegetable sources, and   Severe liver disease results in lower factor levels
  Bacterial synthesis in colon   Vitamin K-dependent factors (II, VII, IX & X) fall more
than others (fibrinogen, V, VIII)
  Causes of Deficiency
  Injection of pharmacological doses of Vit K may improve
  Low intake – uncommon issue
  Malabsorption disorders coagulation status enough for liver biopsy or surgery
  Bile salts (biological detergents) required to emulsify fats and
fat soluble vitamins in food during small bowel digestion and Day 2 (24h Reference
absorption Example Day 1
after Vit K) Range
  Liver stores of Vitamin K will usually last 1-3+ weeks APTT 48 40 22-34 s
  Deficiency results in prolonged PT & APTT & a bleeding tendency
PT 15.6 13.4 8.0-11.0 s
  Beware: the abdominal surgery patient with a wound infection
and antibiotic therapy who has not eaten for 1-2 weeks! 27 Fibrinogen 1.9 1.9 1.8.4.0 g/L 28

Basic – key info Nature 2004 427: 493-494

Vitamin K and the Neonate
Warfarin anticoagulation: inhibiting Vitamin K

  Neonate WARFARIN
  Limited Vitamin K transported across placenta
  Low levels of Vitamin K-dependent factors at birth epoxide
  Falling levels after birth (no oral intake in first 24 hrs) OH O
reductase
  Normal feeding: trough Factor levels at 48 hrs are ~10-30% R (VKOR) R

  Risk of bleeding into the brain ~1:1000 O

CH3 CH3
  Vitamin K always given after birth HO O
  0.5mg IM at birth, or CO2
vit K reduced vit K epoxide
  3 oral doses (poorly absorbed even in full term normal infant) O2 H2O
carboxylase
  Premature infants
COO- COO-
  Increased risk of intracerebral bleeding after birth COO-
  Physical moulding of head during birth processes
  Intramuscular dose of Vit K must be given – not able to
absorb sufficient Vit K 29 glutamic acid γ-carboxyglutamic acid 30

Dr Jim Faed, Pathology Dept 5

MICN2: Blood Module - Haemostasis 3 - Coagulation tests &
Introduction to bleeding disorders

Basic – key info Basic – key info

Warfarin anticoagulation Standardising the Prothrombin time: the

(Wisconsin Alumnus Research Foundation) INR – International Normalised Ratio

  Benefits

  Standardisation of Commercial PT test results

  Useful for treating patients with pathologically
increased risk for clotting
  The International Normalised Ratio is the –

  Oral medicine – injection not needed

  Ratio of the patient’s PT to a PT on normal plasma

  Corrected to show the test response expected with the

  Potential problems International Reference Thromboplastin

  Risk of bleeding

  A narrow therapeutic window exists for warfarin PT of Patient’s plasma Sensitivity Index for Thromboplastin

  Careful clinical control needed – regular lab testing
PT of normal plasma

  INR (Standardised Prothrombin Time (PT)) is best test

  The INR corrects for differences between test brands

31 32

Basic – key info

Common causes for warfarin
Case 1
treatment to go out of control

  Changed intake of vitamin K in food This 22 year old woman has several large bruises on her

  Vomiting, binge eating
legs and forearms. She bleeds from cuts for longer than
other people and has heavy periods lasting 10 days.

  Sudden changes in dietary patterns, eg when travelling

  Decreased absorption of vitamin K Platelets TCT Fibrinogen APTT PT

  Gastroenteritis and other causes for malabsorption Test
225 11 2.2 36 9

  Change of medications (and some foods) result

  Increased drug clearance Ref. 150-400
10-14s 1.8-4.0g/L 22-36s 8-10.5s

  Reduced warfarin effect) by displacement from albumin or by Range x109/L
induction of hepatic cytochrome

  Inhibition of warfarin metabolism by hepatic cytochromes Differential diagnosis

  Any cause for liver cell injury, including
Additional tests needed
gastroenteritis, heart failure, hepatitis, et al 33 34

Interpreting test results: Case 3

Case 2 Patient 24 hours post surgery; has had a rigor –
temp. now 38.6°C; BP has fallen: 145/95 to 100/60
An 8 year old girl with numerous bruises and petechiae APTT 36 22-34 s
has the following test results. (Hb 125, MCV 85, MCH 28) PT 15 8.0-11.0 s
INR 1.7 0.8 – 1.2
Platelets TCT Fibrinogen APTT PT
TCT 22 10-18 s
Test 5 12 2.0 30 9.5 Fibrinogen 0.9 1.8-4.0 g/L
150-400 FDP’s (D-dimers) 7 < 0.5 FEU/L
Ref Range 10-14s 1.8-4.0g/L 22-36s 8-10.5s
x109/L Platelet count (now) 80 150-400 x109/L
Platelet count (24h ago) 225
Differential diagnosis

  Differential diagnosis
Further tests
  Treatment
35 36

Dr Jim Faed, Pathology Dept 6

MICN2: Blood Module - Haemostasis 3 - Coagulation tests &
Introduction to bleeding disorders

Case 4 Case 5
A 1 year old boy has several large bruises and is being A 45 year old man requires a liver biopsy to assess his
investigated for child battering. (Hb 115, MCV 79, MCH 27) liver due to the presence of chronic hepatitis C. (Hb 122,
MCV 90, MCH 28)
  














  







 






 


 
 





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Differential diagnosis
Differential diagnosis
Further tests
Further tests
37 38

Further tests – mostly

Case Differential diagnosis
Specialist level
Platelet capillary defect (from clinical history): •  PFA-100 test or bleeding
1. •  Von Willebrand disease or time
•  A platelet function defect •  vWF studies, FVIII

Severe thrombocytopenia: •  None needed

•  Acute immune thrombocytopenia (acute
2. leukaemia excluded as blood screen is
otherwise normal)
Sudden fall in fibrinogen and platelet count, •  None needed to
raised FDPs (& mild increase in APTT & PT) diagnose DIC.
3. •  Disseminated Intravascular Coagulation •  Identify & treat cause of
(DIC) DIC
Prolonged APTT , other screening tests •  Haemophilia A or B:
4. normal: FVIII or FIX defic.
•  A haemophilia •  Rarely FXI or FXII defic

Mild thrombocytopenia : not clinically important •  Liver function tests if not

and assessed recently
5. Mildly prolonged APTT & PT: •  Renal function tests
•  Moderately severe liver disease 39

Dr Jim Faed, Pathology Dept 7