Haemophilia

Question 1

Types of Bleeding

Answer 1

Joint bleeds - usually large weight bearing joints - knees, ankles and elbows - pain, swelling, warmth and restricted movement. Not always associated with significant trauma
Muscle bleeds - less common than joint bleeds, but can cause damage by compression of local blood vessels and nerves
Cerebral bleeds - potentially life threatening
Mouth and mucosal membranes - a little blood goes a long way!
Prolonged bleeding - after surgery and invasive procedures
Excessive bruising - usually to be found on arms and legs, “Thumbprint bruising on chest

Question 2

Haemophilia A

Answer 2

X Chromosome disorder, 1 in 10,000 live male births
Deficiency or defect in the procoagulant activity of factor VIII
Normally FVIII acts as a cofactor in the activation of Factor X
FVIII circulates attached to vWF
The Haemophilia A Mutation, Structure, Test and Resource Site – HAMSTERS

Question 3

Haemophilia B

Answer 3

X Chromosome disorder, 1 in 35,000 live male births
Deficiency or defect in the procoagulant activity of factor IX
Normally FIX acts as an activator of Factor X in the presence of FVIII and Phospholipid
FIX Leiden
Haemophilia B mutation database (KCL)‏

Question 4

classification of haemophilia

Answer 4

mild, moderate and severe

Question 5

Acquired Haemophilia

Answer 5

Acquired Haemophilia is due to the development of an autoimmune inhibitor against FVIII (or rarely FIX or VWF) in a previously normal individual
It is rare and affects both males and females
It is sometimes associated with drug therapy, auto immune conditions and pregnancy but most cases arise spontaneously
Approx 75% of acquired inhibitors destroy the FVIII molecule, 25% inhibit the FVIII molecule, but do not destroy it

Question 6

vWD causes

Answer 6

von Willebrand factor (vWF) is an adhesive glycoprotein that circulates in large multimers 800 – 20,000kDA
Synthesized by endothelial cells and megakaryocytes
Initiates platelet adhesion to exposed collagen at sites of vascular damage and between platelets themselves
Transports and protects FVIII from in-vivo proteolysis
Abnormalities of VWF gives a heterogeneous set of VWD results
Incidence - approx 1% of the population world wide affecting both males and females.

Question 7

vWD disease tests

Answer 7

The vWF normal range exhibits high degree of variation within the population (40 –200 iu/dl), with a link between the vWF level and ABO blood group of the patient
Significant variation in vWF in patients with the same mutation
vWD are sub-typed using clinical and laboratory criteria
vWF antigen – ELISA, Latex
vWF activity – CBA ELISA, Ristocetin cofactor
vWF:FVIII Binding study
vWF Multimeric structure

Question 8

vWD disease subtype

Answer 8

Type 1 – moderate quantitative deficiency
Type 3 – severe quantitative deficiency
Type 2a – Functional defect – small multimers
Type 2b – Spontaneous binding of large multimers to platelets
Type 2M – reduced plt gp1b platelet binding
Type 2N – reduced binding to FVIII

Question 9

Acquired vWD

Answer 9

Autoantibody to vWF – binds to circulating vWF and the Ag:Ab complex is removed and destroyed
‘Mechanical’ removal - associated with aortic valve stenosis and para-valvular leakage of replacement heart valves (Heyde’s Syndrome)
A mild form of vWD associated with hypothyroidism

May present as a vWD type 1, 2 or 3

Question 10

Laboratory Diagnosis

Answer 10

Consistently Prolonged PT or APTT
repeat sample, poorly collected blood samples can lead to erroneous results
Are they anticoagulated ?
Warfarin interferes with VITK metabolism - low FII,VII,IX,X
Heparin binds with ATIII to increase anti IIa / Xa potency of ATIII / NOAC
Clinical Details
Liver Disease - low Fib,FII,VII,IX,X
Malignant conditions with paraproteins (interference with optical detection systems)
DIC (general consumption of all factors and Fib)
Family History / Clinical Presentation
Some bleeding disorders have a characteristic appearance

Question 11

Factor Assays

Answer 11

One stage factor assays (APTT – FVIII, FIX, FXI, FXII)
100ul Dilution of patient / standard in buffer
100ul Deficient plasma (e.g. FVIII or FIX)
Phospholipid (platelet subst.)
Kaolin / Cephalin / Silica (-ve surface)
CaCl – time clot formation after addition of calcium excess
Two stage / Chromogenic factor assay
FXa generation based assay – measures the ability of the FVIII to activate FX to Fxa (qualitative not quantitative)

Question 12

vWD assays

Answer 12

Latex Immunoassays (VWF Antigen)
Latex particles coated with IgG monoclonal antibody specific for human VWF, latex particles will aggregate around VWF molecules in patient plasma, analyser measures light transmission
Ristocetin Co-Factor
Testing the ability of the VWF molecule to bind to GP1b platelet receptors
Collagen Binding
Testing the ability of the VWF molecule to bind Collagen (GP2a/3a)
vWF Multimers
Electrophoresis of vWF and staining to determine the physical size of vWF molecules

Question 13

Fibrinogen defects

Answer 13

Defined as fibrinogen activity <1.5g/ltr
Chromosome 4
Dysfibrinogenemia – production of a 'normal' amount of a defective protein detected by a discrepancy between the functional (Clauss) and quantitative assays
Common in Liver Disease
May be prothrombotic or haemophilic
Afibrinogenemia - absent production (approx 1 in 1,000,000)
Hypofibrinogenemia – reduced production
Haemophilic (very rarely thrombotic

Question 14

Factor II Deficiency

Answer 14

Chromosome 11
Produced in the Liver
Genetic mutation leading to reduced FII is very rare (approx 1 in 2,000,000)‏
Autosomal Recessive inheritance
Acquired FII deficiency associated with lupus anticoagulant more common (Acquired Hypo-prothrombinemia ~ ?15 cases per year worldwide)

Question 15

Factor V Deficiency

Answer 15

Chromosome 1
FV deficiency (Owrens Parahaemophilia) is very rare approx 1 in 1,000,000
Autosomal Recessive inheritance
Leads to a strong bleeding tendency – Excessive bruising, Epistaxis, Gum Bleeds and muscle bleeds after trauma

Question 16

Factor VII Deficiency

Answer 16

Chromosome 13
Produced in the Liver
FVII deficiency (Proconvertin Deficiency) is rare approx 1 in 400,000
Autosomal Recessive inheritance
Strong bleeding history – Epistaxis, Gum Bleeds, Menorrhagia, 50% of patients with severe FVII deficiency (<1% FVII) will experience a serious CNS bleed with high morbidity and mortality

Question 17

Factor X Deficiency

Answer 17

Chromosome 13
Produced in the Liver
Rare disorder – Stuart-Prower disease 1 in 1,000,000
Autosomal Recessive Inheritance
Strong bleeding history - Joint Bleeds, Epistaxis, GI Bleeding
Typically diagnosed neonatally due to an umbilical stump that bleeds excessively

Question 18

Factor XI Deficiency

Answer 18

Chromosome 4
Rare deficiency 1 in 1,000,000 in most populations, however 1 in 8 Ashkenazi Jews are carriers of a FXI mutation
Autosomal recessive
Prolonged bleeding from minor wounds
Milder bleeding history – mild epistaxis and menorrhagia

Question 19

Other Defects

Answer 19

Factor XII, Pre-Kallikrein, High Molecular Weight Kininogen deficiencies cause prolongation of the APTT but do not lead to a bleeding tendency
Liver disease causes decreased production of factors II, VII, IX, X, Fib
Vitamin K deficiency (dietary or anticoagulant) causes decreased production of the functional forms of factors II, VII, IX, X - however non-functioning variants are also produced which can be detected using echis carinatus snake venom
Combined FV/FVIII deficiency – 100 cases worldwide, Mediterranean localisation (intracellular protein trafficking mutation - ERGIC-53)
FXIII / α2Antiplasmin – delayed onset bleeding

Question 20

Replacement Product

Answer 20

Prevention of damage and deterioration within major joints – knees, hips, elbows, ankles
can be treated for bleeding either prophylactically or “on demand”. Each treatment modality has advantages and disadvantages from both a medical and economic point of view
In a trial of 133 haemophilia patients (R. Khoriaty et al 2005) the average amount of clotting factor used per 1kg body weight per year was 2181.7 units (£829) for prophylaxis vs. 711 units (£270) for on demand treatment
The total number of bleeds per year was significantly less for prophylactic dosing (7.4) vs. on-demand treated patients (11.4).

Question 21

Complications of replacement products:

Answer 21

Development antibodies due to exposure to ‘foreign protein’
Viruses – Hep B, Hep C, HIV
vCJD (Creutzfeldt–Jakob Disease)

Question 22

FVIII Concentrates

Answer 22

Recombinant Ultra–high-purity products:
Helixate NexGen (CSL Behring) - Cultured BHK cells
Kogenate (Bayer) - Cultured BHK cells
Advate (Baxter) – Cultured CHO cells
Xyntha (Pfizer) – Cultured CHO cells, β domain deleted

Recombinant products typically require the addition of human albumin as a stabilising protein, or utilise human albumin during the cell culture step

Question 23

Development of Inhibitors

Answer 23

Approx. 25% of severe and moderate haemophilia A patients develop an inhibitor – usually pre-teenage
Treatment with bypassing agents (FEIBA) or activated recombinant factor VII (rFVIIa)
Inter-patient variability complicates dosing and prevents optimal response
Develop treatment strategies for both the dose and choice in individual patients
Treatment
Remove stimulus (switch product)
Immunosuppression

Question 24

Fresh Frozen Plasma

Answer 24

Defined as the fluid portion of one unit of human blood that has been centrifuged, separated, and stored frozen at −18°C (or colder) within 6 hours of collection.
Post massive blood transfusion – replace clotting factors after red cells have been replaced
Cannot be used for replacement of isolated factor deficiencies – all clotting factors are contained in FFP

Question 25

Cryoprecipitate

Answer 25

Cryoprecipitate is prepared by slow thawing of fresh frozen plasma (FFP) at 4°C for 10–24 hours. When cryoprecipitate appears as an insoluble precipitate and is separated by centrifugation, it contains significant quantities of FVIII (about 5 IU/ml), von Willebrand factor (VWF), fibrinogen, and FXIII (but not FIX or XI).
The resultant supernatant is called cryo-poor plasma and contains other coagulation factors such as factors VII, IX, X and XI

Question 26

DDAVP

Answer 26

Used in Mild Haem A or Type I vWD – no use in Haem B
Desmopressin (1-deamino-8-D-arginine vasopressin, also known as DDAVP) is a synthetic analogue of antidiuretic hormone (ADH). The compound boosts the plasma levels of FVIII and VWF after administration
works by triggering release of VWF stored in the Weibel-Palade bodies in the endothelial cells raising the patient’s own vWF (and FVIII)

Question 27

DDAVP advantages

Answer 27

A single intravenous infusion at a dose of 0.3 mg/kg body weight can be expected to boost the level of FVIII approx 3 fold
Advantages of DDAVP over plasma products are the much lower cost and the absence of any risk of transmission of virus
A concentrated nasal spray available and a spray dosage in an adult of 300 mg is equivalent to the standard intravenous dose of 0.3 mg/kg

Question 28

Tranexamic Acid

Answer 28

an antifibrinolytic agent that competitively inhibits the activation of plasminogen to plasmin
use in FXI deficiency, to cover dental, gynae, or urological surgery and may obviate the need for replacement therapy with concentrates or plasma
mucosal bleeding