HIS28 Inherited And Acquired Bleeding Disorder I

Question 1

Haemostasis

Answer 1

Body mechanism to
—> prevent excessive blood loss
—> stop excessive clot formation (i.e. prevent thrombosis)

Must be activated at appropriate TIME and appropriate SITE

Components:

1. Blood vessel
2. Platelets
3. Coagulation factors
4. von Willebrand factor

Question 2

Blood vessel

Answer 2

• Keep blood fluid in normal state
  —> inhibit haemostasis unless activated
• Initiate haemostasis when endothelium damaged
  —> Vasoconstriction
  —> Collagen underneath exposed
  —> Tissue factor released

Question 3

von Willebrand factor

Answer 3

Produced by endothelium, circulating in plasma

1. Bridge between platelet and collagen (sub-endothelial matrix) in platelet adhesion (via GPIb/V/IX complex)
   —> bind to exposed collagen first
   —> guide platelets to adhere to collagen (like an angel)
2. Carry and protect Factor 8 in circulation
   —> without vWF
   —> Factor 8 t1/2 very short (rapid degradation in plasma)

Question 4

Functions of Platelets

Answer 4

Primary haemostasis

Structure:

• Membrane: **GP with specific functions + **Phospholipids
• Cytoplasmic granules: Alpha granules + Dense granules —> ***ADP, TXA2, 5-HT

Function:
1. Form primary haemostatic plug to close vascular defect
- adhesion to injured vessel
—> aggregation of platelets to each other
—> release
—> fusion
—> provide surface for coagulation cascade

2. Release 5-HT and TXA2 to stimulate vasoconstriction
   —> reduce bleeding + concentrate platelets/clotting factors at injury site
3. Clot formed by platelet is less stable (temporary)
   —> provide surface for coagulation cascade
   —> generate a stable fibrin clot

Question 5

Platelet adhesion

Answer 5

1. Primary adhesion
   - vWF adhere to collagen
   —> platelet adhere to vWF via GPIb/V/IX complex
   —> activated platelet
   —> Ca influx + signalling
   —> platelet degranulation (ADP, TXA2, 5-HT)
   —> trigger platelet degranulation + aggregation
2. Platelet-platelet tethering
   —> release more platelet granules (+ve feedback)
   —> platelet attach to each other via GPIIb/IIIa
3. Aggregation

Question 6

Blood coagulation factors

Answer 6

Features:

1. Enzymes and co-factors (Proteins mainly produced by liver)
   - Factor 7 (shortest t1/2) and 8
   - Factor 1 (fibrinogen) and 2 (prothrombin) (longest t1/2)
   - other coagulation factors

2. Cascade
—> Platelet
—> TF (from damaged site)
—> TF-7a complex
—> Factor 10a
—> Thrombin
—> Fibrin + Factor 13a (cross link fibrin monomer —> stable fibrin polymer) + +ve feedback
—> stabilised, cross-linked fibrin clot

3. ***Amplification
4. ***Positive feedback
5. ***Activate platelets

End result: form ***stable (vs primary) haemostatic plug within short time

Question 7

Current concept of coagulation

Answer 7

Initiated by 7a-TF complex
—> activate Factor 10
—> generate some Thrombin
—> activate Factor 5, 8 (positive loop) + Factor 9 (reinforce formation of tenase + prothrombinase) + Factor 11 (further activate Factor 9)

Question 8

Endogenous anticoagulants (x rmb)

Answer 8

1. Activated Protein C (APC)
   - inhibit Factor 5a, 8a
2. αTHR

—> Clot can only be formed at injury site + injury time

Question 9

***Evaluation of bleeding disorders

Answer 9

1. History
   - Personal (age of onset, severity, progression)
   —> Inherited vs Acquired (much more common)
   - Family history
   - Trauma history? nature of trauma triggering bleeding?
   —> no trauma history (spontaneous bleeding): bleeding disorder
   —> profuse bleeding / bruising with minor injury: bleeding disorder
   - Drug history
2. Pattern of bleeding
   - **Mucocutaneous / Continuous oozing —> **Platelet / Vessel / vWF defect
   - **Deep-seated (joint, soft tissue) / Delayed (re-bleeding after initial cessation) —> **Coagulation factors, Severe vWF deficiency (~ Factor VIII deficiency: Haemophilia A)
   - Drugs (e.g. Warfarin)
3. Past surgical history (e.g. tooth extraction)
   - any excessive bleeding
4. Menstrual history
5. Associated systemic illness (e.g. liver / renal disease, autoimmune disease)
6. Other symptoms of cytopenia
   - anaemic symptoms
   - fever (suggesting neutropenia / leukopenia as well —> Pancytopenia)
   —> suggest BM disorder

Question 10

***Mucocutaneous bleeding vs Deep-seated bleeding

Answer 10

Mucocutaneous bleeding (***Platelet / Vessel / vWF defect):
1. Bruising
2. Petechiae
3. Retinal bleeding
4. Menorrhagia
5. Oral mucosa / Gum bleeding
6. GI bleeding

Deep-seated bleeding (***Coagulation factors, Severe vWF deficiency):
1. Muscle haematoma
2. Haemarthrosis
3. Intracranial bleeding

Question 11

***Tests for bleeding disorders

Answer 11

1. Platelet
   - count
   - morphology
   - function (platelet function test)
   —> by aggregometry that measures aggregation response of platelets to different agonists
   - **Numerical disorder (Thrombocytopenia)&raquo_space;> Platelet dysfunction (except drug-induced platelet dysfunction)
   - **Thrombocytopenia:
   —> ↓ Production from BM
   —> ↑ Consumption (immune / non-immune)
   —> Abnormal sequestration in spleen (hypersplenism)
   —> Dilutional (e.g. massive blood transfusion)
2. Coagulation factors
   - functional assay for Factor level
   - ***Coagulation screening test (in-vitro)
   —> Prothrombin time (PT) —> test Extrinsic + Common pathway
   —> Activated partial thromboplastin time (APTT) —> test Intrinsic + Common pathway
   —> Thrombin time (TT)
   —> Fibrinogen level
3. von Willebrand factor
   - function
   - amount

Question 12

Coagulation screening test

Answer 12

Test:
- Prothrombin time (PT)
- Activated partial thromboplastin time (APTT)
- Thrombin time (TT)
- Fibrinogen level

Procedure:
- Exact volume of blood collected into bottle containing sodium citrate —> binds (remove) Ca —> anticoagulated blood
—> centrifugation
—> all coagulation factor + vWF are in plasma
—> add back necessary components (e.g. PT / APTT / TT reagent with appropriate amount of Ca + Phospholipid) to initiate clotting

Question 13

Warfarin

Answer 13

MOA:
Antagonise Vit K action
—> reduce Factor 2, 7, 9, 10 (Vit K dependent factors)
—> ∵ Factor 7 short t1/2
—> changes in Factor 7 level well reflect therapeutic effect of Warfarin
—> ∴ use PT

Why need to monitor:
1. Narrow therapeutic index
2. Variable individual response

Question 14

Prothrombin Time (PT)

Answer 14

Test for Extrinsic + Common pathway:

Extrinsic:
- Factor 7 (only factor in extrinsic pathway)

Common:
- Factor 10
- Factor 5
- Prothrombin (Factor 2)
- Fibrinogen (Factor 1)

International Normalised Ratio (INR):
- PT will vary with type of thromboplastin (PT reagent) used
- Individual thromboplastin calibrated against international WHO reference thromboplastin —> assign an International sensitivity index (ISI) (WHO standard: 1)
- ISI of test thromboplastin = ISI of WHO standard x slope of graph
- calibration with 20 normal people, 60 patients on Warfarin
- ***Use for Warfarin titration only!!!
- NOT use to monitor new oral anticoagulants
- NOT use to monitor DIC

INR = [PT of patient sample / mean PT of 20 normal people]^ISI of test thromboplastin

Question 15

Activated Partial Thromboplastin Time (APTT)

Answer 15

Test for Intrinsic + Common pathway:

Intrinsic:
- Factor 12 (contact factor —> initiate coagulation)
- Factor 11
- Factor 9
- Factor 8
- Prekallikrein, Kallikrein, HMWK (contact factor —> initiate coagulation; level cannot be measured directly)

Common:
- Factor 10
- Factor 5
- Prothrombin (Factor 2)
- Fibrinogen (Factor 1)

Question 16

***Interpretation of PT, APTT

Answer 16

1. Isolated prolong PT
   —> Factor 7 deficiency (most likely)
2. Prolong PT associated with Prolong APTT
   —> Factor(s) deficiency / inhibitor in Extrinsic + Intrinsic / Common pathway
   —> e.g. Vit K deficiency, Vit K antagonist (Warfarin), Liver disease
3. Isolated prolong APTT
   —> Factor 12, 11, 9, 8 deficiencies
   —> Inhibitors (AutoAb) of Factor 8 / other factors (rare) / Lupus anticoagulant (Anti-phospholipid syndrome: AutoAb target phospholipid —> APTT depend most heavily on phospholipid availability)
   —> Contact factor deficiency (Factor 12, Prekallikrein, Kallikrein, HMWK)
   —> Unfractionated Heparin

Question 17

Thrombin Time (TT)

Answer 17

Test for Fibrinogen —> Fibrin:

Prolonged TT:
- Hypofibrinogenaemia (quantity)
- Dysfibrinogenaemia (quality, abnormal structure)
- Thrombin inhibition
- Inhibitor of Fibrinogen —> Fibrin

e.g.
1. Liver disease e.g. Cirrhosis (↓ synthesis / abnormal fibrinogen)
2. DIC (consumption of clotting factors / contain inhibitors of fibrinogen —> fibrin)
3. Heparin therapy (thrombin inhibition)

Question 18

Interpretation of Fibrinogen level

Answer 18

Detected by functional assay (i.e. quantitative + qualitative):

↓ Fibrinogen level:
1. Liver disease e.g. Cirrhosis (↓ synthesis / abnormal fibrinogen)
2. DIC (consumption of clotting factors)
3. Inherited defects e.g. Hypofibrinogenaemia, Afibrinogenaemia, Dysfibrinogenaemia

↑ Fibrinogen level:
1. ***Acute phase reaction (acute phase protein) e.g. acute inflammation, sepsis, acute infection
2. Female sex, pregnancy, oral contraception (hormonal changes)

Question 19

***Summary of Haemostatic test

Answer 19

1. PT (extrinsic + common pathway):
   - Factor 7
   - Factor 5, 10, prothrombin, fibrinogen
2. APTT (intrinsic + common pathway):
   - Factor 8, 9, 11, 12
   - Factor 5, 10, prothrombin, fibrinogen
3. TT:
   - Quantitative / qualitative defect of fibrinogen (Factor 1)
   - inhibitors of fibrin formation
   - inhibitors of thrombin
4. Fibrinogen level (by functional assay):
   - Quantitative / qualitative defect of fibrinogen

Question 20

In-vivo vs In-vitro coagulation

Answer 20

There is NO intrinsic / extrinsic pathway in in-vivo coagulation!!!

Different from in-vitro coagulation:
- Factor XII, Kallikrein, Prekallikrein, HMWK —> only required in in-vitro coagulation!!!
- In-vivo has anticoagulants e.g. Activated Protein C

Question 21

***Workup of patient with Prolong APTT

Answer 21

Prolong APTT (most common problem for coagulation tests)

• Isolated APTT
  —> Factor 8, 9, 11, 12, Contact factor deficiencies/inhibitors (most common factor 8 inhibitor)
  —> Presence of Lupus anticoagulant
• Associated with prolong PT —> then do TT / Fibrinogen assay
  —> Normal TT (i.e. NOT involve fibrinogen to fibrin)
  1. Vit K deficiency / antagonists (Factor 2, 7, 9, 10)
  2. Common pathway factor deficiency / inhibitors (Factor 2, 5, 10)
  3. Multiple factor deficiencies involving multiple pathway

—> Prolonged TT (+/- ↓ Fibrinogen level) (i.e. Involve fibrinogen to fibrin)
1. ***Liver disease (∵ quantitative / qualitative defect of fibrinogen)
2. Unfractionated Heparin (mostly TT + APTT prolong only)
3. Direct thrombin inhibitors (TT ↑↑ + APTT + PT prolong)
4. Afibrinogenaemia / Dysfibrinogenaemia / Hypofibrinogenaemia (TT + APTT + PT prolong)
5. DIC (all coagulation factors consumed)

Question 22

Autoimmune thrombocytopenia (AITP) - Diagnosis of exclusion (排除曬其他野先consider)

Answer 22

Platelet sensitisation with **AutoAb (usually IgG)
—> premature removal from circulation by **Reticuloendothelial system (autoimmune haemolysis)
—> target antigens for AutoAb: ***GPIIb-IIIa / GPIb-IX

May occur:
1. Without identifiable antecedent / associated illness (typically in young / middle-aged adults, female predominant)
2. Secondary to other autoimmune diseases
3. Acute post-viral event (esp. young children)

Investigations:
- **Low platelet
- Exclusion of platelet consumption by other causes (e.g. DIC)
- BM exam show **adequate / even ↑ megakaryocytes
(- AutoAb on platelets/serum
- characterisation of antigenic specificity of AutoAb)

Question 23

Alloimmune thrombocytopenia (very rare)

Answer 23

1. Neonatal Alloimmune thrombocytopenia
   - Fetomaternal incompatibility for human platelet antigen (HPA)
   —> sensitisation of mother
   —> Maternal AlloAb cross placenta
   —> Thrombocytopenia in foetus (c.f. Haemolytic disease of newborn)
2. Post-transfusion purpura
   - severe thrombocytopenia 1 week after transfusion (contains HPA-1a positive platelets) in HPA-1a negative patients previously sensitised to HPA-1a (Alloimmunised)
   —> contain Ab against HPA-1a
   —> **immune complex formed between released **HPA-1a antigen and Ab
   —> adsorbed onto patient’s HPA-1a negative platelets
   —> destruction of platelets
3. Platelet refractoriness
   - Repeated failure to obtain satisfactory platelet count increment after transfusion of platelets
   - AlloAb against donor platelets (mainly HLA Ab / Platelet specific Ab (less common))

Question 24

Inherited Platelet disorders

Answer 24

Functional disorders (功能上差):
1. Defects of **Adhesion —> Bernard-Soulier syndrome —> Defect of GP Ib-IX, Thrombocytopenia, Giant platelets
2. Defects of **Release reaction —> Deficiency of α-granules (Grey platelet syndrome) / δ-granules (Storage pool disease)
3. Defects of ***Aggregation —> Glanzmann’s thrombasthenia due to defect of GP IIb-IIIa

Numerical disorder / Hereditary thrombocytopenia (數字上少):
1. Bernard-Soulier syndrome
2. Wiskott-Aldrich syndrome
3. May-Hegglin anomaly (giant platelets with Dohle body-like inclusions in neutrophils)

Question 25

Inherited Coagulation disorders

Answer 25

1. Haemophilia A
   - deficiency of Factor 8
   - genetic defect in Factor 8 gene on long arm of X chromosome (Xq28)
   - X-linked
   - positive family history of bleeding tendency on maternal side
   - 1/3 new cases from spontaneous mutation
2. Haemophilia B
   - less common than Haemophilia A
   - deficiency of Factor 9
   - genetic defect in Factor 9 gene on long arm of X chromosome (Xq26)
   - X-linked

BOTH Clinical feature: **Haemarthrosis, Soft tissue bleeding, **Isolated prolong APTT

Differentiation by
***Clotting factor assay
- Haemophilia A —> low Factor 8 activity
- Haemophilia B —> low Factor 9 activity

Problems with Haemophilia A and B:
1. Recurrent bleeding in major joints —> progressive joint deformity
2. Inhibitor (AlloAb) to infused factor concentrate —> replacement therapy less effective
3. High cost of treatment
4. Transfusion transmitted infection e.g. HIV, HBV, HCV
5. Detection of carrier female: Phenotypic diagnosis (not always accurate) vs Molecular diagnostics (Factor 8 gene is a big gene, technically demanding)

3. **Von Willebrand disease (VWD)
   - defect in vWF (qualitative / quantitative)
   —> **Defect in platelet adhesion + ***↓ Factor 8 level
   - gene for vWF on short arm chromosome 12 (12p)
   - Autosomal inheritance

Clinical feature: ***Mucocutaneous bleeding, Isolated prolong APTT (∵ ↓ Factor 8 level)

Further tests for VWD:
- vWF antigen assay
- Ristocetin cofactor (RiCof) assay
- Collagen-binding assay
- Factor 8 assay
- Ristocetin-induced platelet aggregation test
- Factor 8 binding assay
- Multimeric study

Diagnosis of mild VWD can be difficult:
- Significant bleeding history
- Positive family history
- Compatible lab results (only on repeated testing)
(- Molecular diagnosis by next generation sequencing)

4. Other
   - very rare
   - mostly ***Autosomal recessive
   - Factor 12 deficiency NOT associated with bleeding
   - Factor 7 deficiency: Isolated prolong PT
   - Factor 10 deficiency: Prolong PT + APTT
   - Fibrinogen deficiency / Dysfibrinogenaemia: Prolong PT + APTT + TT

Question 26

Acquired bleeding disorders

Answer 26

1. Vit K deficiency (common)
2. Vit K antagonism (very common)
3. Haemorrhagic disease of new born (very uncommon)
4. Liver disease (very common)
5. Uraemia (very common)
6. ***DIC (common and clinically important)

Question 27

Acquired bleeding disorder in details

Answer 27

1. Vit K deficiency (common)
   - Obstructive jaundice (i.e. no bile), Malabsoprtion state, Killing of intestinal flora by oral broad-spectrum antibiotics

• Vit K: fat-soluble, cannot be synthesised by human, must be absorbed from food in upper small intestine in presence of bile, some flora also produce Vit K
• required in γ-carboxylation of glutamic acid residues (post-translational event in hepatocytes)at amino-terminal domain of Factor 2, 7, 9, 10 (Vit K dependent clotting factor) + Protein C, Protein S (Naturally occurring anticoagulants)
  —> Gla domain
  —> able to bind Ca to adhere to platelet (biologically active)
• Coagulation screening: Prolong PT, APTT, Normal TT

2. Vit K antagonism (very common)
   - oral anticoagulants (coumarin, phenindione derivatives) for primary / secondary prevention of thromboembolic disorders (competitive Vit K epoxide reductase inhibitor)
   —> creates functional Vit K deficiency state
   —> Prolong PT, APTT, Normal TT
3. Haemorrhagic disease of new born (very uncommon)
   - immature liver to produce bile, sterile gut, low Vit K content in breast milk
   —> prone to develop Vit K deficiency (if only breast fed and not given Vit K supplement)
4. Liver disease (very common)
   - ALL coagulation factors (except vWF) (also Antithrombin, Protein C, S, Plasminogen, α2-antiplasmin) synthesised in hepatocytes
   - Reticuloendothelial cells lining sinusoids clear activated intermediates of coagulation and fibrinolysis from bloodstream
   - Acute / Chronic liver disease
   —> **↓ Hepatic synthetic function —> ↓ clotting factors, inhibitors
   —> **Thrombocytopenia (∵ Portal hypertension —> increased splenic sequestration i.e. hypersplenism)
   —> **Cholestasis —> ↓ Vit K absorption —> ↓ active Vit K dependent factor + inhibitors
   —> **Acquired dysfibrinogenaemia
   —> ***Failure to clear activated intermediates of coagulation and fibrinolysis from bloodstream

• Coagulation screening: Prolong PT + APTT + TT

Treatment:
- Parenteral Vit K replacement (improve bleeding tendency)
- Clotting factor replacement by Fresh Frozen Plasma (FFP)

5. Uraemia (very common)
   - defects in platelet function + platelet-vessel wall interaction
   - aggravated by low Hct
   - **Mucocutaneous bleeding
   - Coagulation screening: **Normal

Treatment:
- ↑ Hct (via RBC transfusion / erythropoietin therapy)
- Peritoneal / Haemodialysis
- DDAVP (Desmopressin) administration / Cryoprecipitate transfusion

6. **DIC (common and clinically important)
   - **Inappropriate + Excessive activation of haemostasis
   - Microthrombotic lesions (rare)
   - Chronic + Compensated (if trigger is weak and persistent, usually no bleeding)
   - Acute + Uncompensated (trigger strong enough)
   —> **Widespread thrombosis + **Generalised haemorrhage
   —> Tissue ischaemia
   —> Multi-organ dysfunction

Causes:
1. Infections: septicaemia, viral infections
2. Malignancies: APL, metastatic carcinoma
3. Obstetric conditions: septic abortion, amniotic fluid embolism, abruptio placentae (early detachment of placenta from uterus)
4. Massive tissue trauma: extensive trauma / burns
5. Extensive IV haemolysis: ABO incompatibility

Pathogenesis:
1. Entry of tissue thromboplastin into blood
2. Direct activation of coagulation
3. Severe vascular endothelial injury
4. Direct activation of platelets
—> ALL leads to **Thrombin generation
—> **Fibrin generation (Widespread IV coagulation) + Secondary fibrinolysis
—> **Consumption of clotting factors, natural anticoagulants, platelets (*結血, 溶血都無曬)

Lab results:
- Prolong PT + APTT + TT + ↓ Fibrinogen (∵ consumed)
- ↑ Fibrin degradation products (***D-dimer) from Secondary fibrinolysis
- ↓ Platelet (Thrombocytopenia)

(- APTT may be normal due to elevated Factor 8 (acute phase protein)
- Fibrinogen may be normal ∵ acute phase protein)

Treatment:
- Remove precipitating trigger
- Control bleeding
- Frequent monitoring of coagulation screening
- Transfusion of platelet, fresh frozen plasma (supportive for bleeding)