W7CL1 - Blood Loss Anaemia & Approach to Disorders of Haemostasis

Question 1

Acute Blood Loss

Answer 1

Initially lose blood volume
- i.e. proportional loss of plasma & cells
Loss of up to ~20% of blood volume is tolerated
Hypovolaemic shock occurs with loss of ~40% of blood volume
Loss of ~ 50% of blood volume results in death
Blood volume can be estimated as approximately 70 mL/kg for adults (80 mL/kg in children and 100 mL/kg in neonates)

Question 2

Response to Acute Blood Loss

Answer 2

Plasma volume expansion to maintain blood volume
May take up to 48h
RBC fraction relatively & progressively diluted as plasma volume expands
↑ EPO due to hypoxia => release of reticulocytes stored in the marrow within hours

Question 3

Mechanisms of Haemorrhagic Diathesis

Answer 3

Overwhelm normal mechanisms of haemostasis
- e.g. trauma, surgery, giving birth (post-partum haemorrhage)
Defective mechanisms of haemostasis
- e.g. Haemophilia, Von Willebrand Disease, platelet disorders

Question 4

Post-Partum Haemorrhage

Answer 4

Primary post-partum haemorrhage is defined as a blood loss of >500ml within 24 hours of the birth
It is further classified into minor (500-1000ml) or major (> 1000 ml) with a further sub classification into moderate (1000-2000 ml) or severe (> 2000 ml or > 30% of blood volume)

Question 5

Post Partum Haemorrhage - History to Consider

Answer 5

If there is a known (previously diagnosed) history of a patient’s bleeding disorder it can be managed/prevented/reduced by targeted therapy especially when it is known a haemostatic challenge will occur
If there is no known coagulopathy with the patient or their relatives consider:
- type of bleeding e.g. rapid, diffuse, oozing
- onset, duration and severity
- drug taking (prescribed and non-prescribed)

Question 6

Deficiency of Several of Vitamin K Dependent Factors

Answer 6

Clinical
- both warfarin therapy and liver disease => multiple factor deficiency
- may exhibit purpura and petechiae => compound factor deficiency
Assays
- PT, aPTT, factor assays, TT, Fibrinogen
- e.g. Liver function tests

Question 7

Circulating Anticoagulant Present

Answer 7

Clinical 
- e.g. Heparin, factor antibodies (e.g. FVIII ‘inhibitors’), lupus anticoagulant
Assays
- aPTT with heparin neutralising agent
- aPTT with 1:1 mix with plasma
- aPTT with excess phospholipid
- Bethesda assay

Question 8

Consumptive Coagulopathy

Answer 8

Clinical
- e.g. Disseminated Intravascular Coagulation (DIC)
- inciting causes; sepsis, trauma, liver disease etc.
Assays
- DIC screen
- platelet count, PT, aPTT, FDP, D-dimer

Question 9

Laboratory Investigations of Bleeding Disorders

Answer 9

Quality assurance
Platelet disorders (& VWF)
Coagulation factor disorders
Fibrinolysis

Question 10

Quality Assurance Issues

Answer 10

Many haemostasis tests may be affected by medications
- e.g. aspirin affects platelet function
- must be known for meaningful interpretation of results
Haemolysis icterus, lipaemia may affect some assays & should not be analysed
Reference intervals should be established wherever possible

Question 11

Measurement of Platelets

Answer 11

Automated analysers
Must reliably distinguish platelets from RBC
Measure:
- platelets
- large platelets
- platelet distribution width
- immature platelets
- plateletcrit

Question 12

Platelet Function Analysers - PFA-100

Answer 12

Mimics platelet adhesion and aggregation at a site of vessel injury
- high shear rates
- collagen exposure
Membrane coating
- collagen
- agonist (e.g. Epinephrine, ADP)
Time to membrane occlusion recorded (closure time)
Sensitive to platelet adhesion, aggregation deficiencies

Question 13

Von Willebrand Factor

Answer 13

Used to determine the amount and functionality of VWF
Important in the diagnosis of Von Willebrand disease and ‘ruling out’ VWD in the investigation of platelet disorders
Assays include:
1. Von Willebrand factor antigen
- assesses the total amount of VWF
- commonly measured by ELISA
2. Ristocetin cofactor assay
- assesses functionality of VWF
- principle: washed platelets do not ‘agglutinate’ with ristocetin unless vWf present
3. Multimeric analysis
- assesses the components of VWF
- used in the classification of subtypes of vWD

Question 14

Laboratory Studies for Platelet Disorders

Answer 14

Platelet concentration
Platelet function analyser (PFA-100)
Platelet aggregometry
Von Willebrand factor

Question 15

Laboratory Studies of Coagulation Factor Disorders

Answer 15

Prothrombin time (PT)
International normalized ratio (INR) 
Activated partial thromboplastin time (APTT)
Thrombin time
Factor assays

Question 16

Formation and Detection of Fibrin Clot

Answer 16

Generation of ‘thrombin burst’
Thrombin cleaves fibrinopeptides A and B (highly negatively charged) from E domain
Changes charge of E domain from negative to positive
=> Positively charged E domains & Negatively charged D domains
Allows spontaneous fibrin polymer formation
Subsequently stabilized by FXIII

Question 17

Prothrombin Time (PT)

Answer 17

Also referred to as one stage prothrombin time (OSPT)
Measures the clotting time of re-calcified plasma in the presence of optimal concentration of tissue extract (‘thromboplastin’) => “tissue factor induced plasma coagulation time”
Assesses ‘extrinsic’ (& common) pathway
- => measures activity of plasma clotting factors: VII, (X, V, II, I)

Question 18

PT & INR

Answer 18

International Normalised Ratio (INR)
Require patient & control times
INR = Raw ratio adjusted to reflect sensitivity and/or reactivity of the thromboplastin reagent
INR = (test/normal)^ISI
ISI = International Sensitivity Index
- a derived figure assigned to the thromboplastin being used
- should preferably be close to 1.0

Question 19

Interpretation of PT/INR

Answer 19

Common pathological causes for prolonged PT/increased INR

• Oral Anticoagulant Therapy (specifically Warfarin)
• liver disease
• vitamin K deficiency
• DIC
• inherited deficiency/defect of factor VII

Question 20

Activated Partial Thromboplastin Time (aPTT)

Answer 20

Measures clotting time after contact activation with addition of phospholipid & CaCl2
FXIa and FXIIa must be activated using water wettable surface agents
Assesses intrinsic (& common) pathway
Measures activity of plasma coagulation factors
- XII, XI, IX, VIII ( X , V, II, I)

Question 21

aPTT Interpretation

Answer 21

Artefactual prolongation of the aPTT may be due to:
- the presence of heparin in the sample
- difficult or slow collection
- addition of an incorrect volume of blood to the tube
- delay in mixing blood with the citrate anticoagulant
- suboptimal specimen storage or a prolonged interval between collection and testing
Common pathological causes of a prolonged aPTT:
- DIC
- liver disease
- heparin treatment
- circulating anticoagulant (‘inhibitor’)
- inherited factor deficiency

Question 22

Thrombin Time

Answer 22

Thrombin is added to plasma and the clotting time measured
Affected by:
- the concentration of fibrinogen
- the presence of inhibitory substances e.g. heparin, fibrin/fibrinogen degradation products

Question 23

TT Interpretation

Answer 23

Causes of prolonged thrombin time include:

• congenital deficiency/defect, hypofibrinogenaemia
• DIC
• increased FDPs
• heparin treatment
• hypoalbuninaemia
• paraproteinaemia

Question 24

Fibrinolysis

Answer 24

Digestion of a fibrin clot
Plasminogen is activated to plasmin
Plasmin => digestion of fibrinogen & fibrin
- formation of fibrinogen/fibrin degradation products (FDPs)
Plasmin digestion of fibrinogen:
- early FDPs: fragments X and Y
- late FDPs: fragments D and E
Plasmin digestion of cross-linked fibrin:
- D-Dimers: D-D fragment
- other combinations
Inhibited by Plasminogen Activator Inhibitors (PAIs)
- e.g. α2 - antiplasmin

Question 25

Assessment of Fibrinolysis

Answer 25

Fibrin/Fibrinogen Degradation Products (FDPs)
- special tube with antifibrinolytic agent & thrombin
- latex agglutination method
- contain antibodies to fragments D & E
- normal values: < 10 mg/mL
D-dimers
- latex agglutination method
- detects cross-linked fibrin D-dimers
- normal values: < 200 mg/L