Bleeding and Clotting Disorders and

their Investigation

William Armour
Consequences of dysfunction
 Symptoms of bleeding disorders
• Easy bruising
• Purpuric rash
• Prolonged bleeding from minor cuts
• Bleeding from mucous membranes
• Bleeding into joints (haemarthrosis)
 Haemostasis
• Blood clotting and coagulation
• Vessel repair and clot dissolution
• Maintenance of the integrity of blood vessels
• Maintenance of the blood volume and
minimise blood loss
• Ensure delivery of oxygen to the tissues
• Ensure removal of waste from the tissues
 Haemostatic Disorders
• Bleeding and clotting abnormalities may be
due to HEREDITARY or ACQUIRED
disorders of any of these components.
Components of Haemostasis

Blood vessel wall

• Endothelium

Blood Cells – Primarily Platelets

• Leukocytes

Coagulation system

• Clot formation
• Fibrinolysis or clot breakdown
First Leg of Stool - Endothelium
Endothelium Coated with Glycocalyx

• AT - antithrombin
• GAG - glycosaminoglycan
• NF-jB - nuclear factor-jB
• PAR - protease-activated receptor
• T - thrombin
Endothelium Coated with Glycocalyx
• Glycocalyx is composed of membrane-binding
domain-containing
– proteoglycans
– glycoproteins
• Conjugated with oligosaccharides and plasma
proteins
– albumin
– antithrombin
Endothelium Coated with Glycocalyx
• Functions
– Antithrombogenicity of the vascular endothelium
• Regulates
– Thrombus formation
– Vascular permeability
– Inflammation
Endothelial Cells without Glycocalyx
• Endothelial cells synthesis adhesion molecules
– E-selectin
– Intercellular adhesion molecule 1
• Recruit leukocytes
• Increase vascular permeability when
glycocalyx between endothelial cell and basal
membrane is damaged
 Endothelium - Overview
• Blood vessel lined with endothelial cells
• If the blood vessel is damaged
– Blood vessel constriction slows blood loss
– Sub-endothelial collagen may be exposed
 Cellular Coagulation Pathway

• Necrotic neutrophils release procoagulant alarmins

• DNA
• high mobility group box 1 (HMGB1)
• FXIIa - activated factor XII
• PAMP - pathogen-associated molecular pattern
• PRR - pattern recognition receptor
 Blood Vessel Abnormalities
• Hereditary haemorrhagic telangiectasia
• Scurvy (Vitamin C deficiency)
• Infection, measles, rubella, scarlet fever
• Autoimmune vasculitis

• Bleeding tendency with normal platelet count and

function and normal clotting studies suggest a
vascular problem.
– Remember our stool has three legs
Second Leg of Stool - Platelets
 Platelets or Thrombocytes
• Platelets are formed by megakaryocytes
• Biconcave discs
– normally, 2 - 4 µm in diameter
– Volume increases in a thrombotic or inflammatory
environment
• 1011 produced daily
• Lifespan of 10 days in circulation
 Platelets or Thrombocytes
• Produced in response to thrombopoietin
• Thrombopoietin made by liver and kidneys
• Primary clot formation
– Consumptively forms bridges between
endothelium and leukocytes
– Forms aggregates with leukocytes
 Platelets or Thrombocytes
• Co-ordinators of Inflammation
• Activated via
– vWF binding to Glycoprotein Ib (GpIb) on platelet
– Toll-like receptor (TLR)-4
– Platelet-activating factor (PAF)
• Produced by
– Neutrophils
– Monocytes
– Endothelial cells

• Platelets release many active products

 Platelet Release Products
• Arachidonic acid precursor for Thromboxane
A2 (TXA2)
• Adenosine diphosphate - aggregation
• 5 hydroxytryptamine - aggregation
• Thromboxane A2 - increases aggregation,
vasoconstriction, platelet granule release
 Platelet Granules
Dense Granules Alpha Granules
• ADP • Fibrinogen
• Ca2+ • vWF
• Serotonin (5HT) • Factor V (FV)
• FVII
• FVIII
• Platelet-derived growth
factor (PDGF)
 Platelet Aggregation
Promotes Inhibits
• Vasoconstriction • Vasodilation
• TXA2 • Prostacyclin
• ADP • Nitric Oxide (NO)
• Serotonin (5HT)
• vWF (adhesion)
 von Willebrand’s Disease
• (Relative) lack of Von Willebrand’s
factor(vWF)
• Von Willebrand’s disease is a hereditary lack
of vWF where platelets can’t adhere
• Measuring vWF is one laboratory
investigation of bleeding disorders
• Range of disorders due to size which can be
differentiated using electrophoresis
• Ristocetin aggregation inhibited
 Bernard Soulier Syndrome
• Bernard Soulier Syndrome is a lack of
Glycoprotein Ib
• Lack of glycoproteins are very rare
• Use a fluorescent monoclonal antibody to the
specific glycoprotein
• Measure using flow cytometry and UV light
• Lack of GPIb gives no fluorescence
 Glanzman’s Thrombasthenia
• Glanzman’s thrombasthenia is a lack of
Glycoprotein GPIIb GPIIIa complex.
– GPIIb GPIIIa complex binds fibrin
– Promotes platelet aggregation
• This results in an abnormality in the ability of
the platelets to aggregate
• Detection by fluorescence flow cytometry
 Aspirin affects PLT function
• Aspirin inhibits cyclo-oxygenase (COX)
• Cox inhibitor prevents eicosanoid synthesis
• Thromboxane A2 not produced
• Reduced PLT aggregation
• Implications for the laboratory
• Patients must not take aspirin for 2 weeks before
blood sampled for PLT function tests
• Sample quality is very important.
 Thrombocytopenia
• Platelets are measured as part of a full blood
count (FBC)
• Normal range 150 - 400 x 109 per litre
 Thrombocytopenia
• Low platelets due to a number of causes
– Reduced production
– Increased destruction
• Consumption (acute disseminated intravascular
coagulation (DIC))
– Artefactual
• Inadequate mixing of blood
• Pseudothrombocytopenia (platelet clumps)
• No symptoms until < 20 x 109/l
 Pseudothrombocytopenia
• The presence of a spurious low platelet count
• No associated clinical symptoms of bleeding
• Caused by an in-vitro platelet aggregation
• Failure of the automated platelet analysis to provide a
count which reflects the in-vivo platelet count.
• Caused by the presence of cross-reacting
(aminoglycosides (kanamycin)) antiplatelet
autoantibodies to Glycoprotein GPIIb GPIIIa
complex.
 Actual Thrombocytopenia
Caused by:
 Thrombocytopenias
• Idiopathic Thrombocytopenic Purpura (ITP)
Immune-mediated, infection, HLA antibodies
• Thrombotic thrombocytopenic purpura (TTP) Due
to the vWF multimer not broken down
• Heparin-induced thrombocytopenia (HIT) Avoid
using low molecular weight heparin
• Disseminated intravascular coagulation (DIC)
Platelets used up- consumptive coagulopathy
Third leg of our Stool - Coagulation
 Citrate Sample Quality
Sample quality is critically important for accurate
results.
• Under-filled - over dilution by anticoagulant
• Overfilled - under dilution by anticoagulant
• Haemolysis - incorrect needle size, excessive
tube mixing, excessive suction, prolonged
tourniquet, and difficult collection (vein
stenosis).
• Inadequate mixing - clotted sample
 Citrate Sample Quality
• Optimally centrifuge at 18–25°C for 10 min at
1500–2000 g in a centrifuge that has a rotor
with swing out buckets.
– Generate platelet poor plasma (<10 × 109/L)
• After centrifugation samples should be
examined for haemolysis, icterus and lipaemia.
 Citrate Sample Quality
• Most tests should be performed within 4 hours
after draw.
– Except if no patient coagulation factor is actually
involved:
• D-dimer assay test within 24 hours of draw
• Heparin assay test within 24 hours of draw
• Out of date sample tubes are discarded
 Coagulation
• Series of plasma proteins reacting to form a fibrin
clot
• ‘Intrinsic’ and ‘extrinsic’ pathways
• ‘Intrinsic’ – Contact factor (Negative Surface)
• ‘Extrinsic’ – Thromboplastin (Tissue Factor)
• Serine proteases
– Coagulation amplification via a central loop
Coagulation
Coagulation Classic Cascade Model
 Limiting the spread of clotting
• Clot only required at the site of injury
• Do not want a clot to spread to patent vessels
• Endothelial cells release Prostacyclin and NO
• Prostacyclin inhibits platelet aggregation
• Prostacyclin and nitric oxide are vasodilators
• Tissue plasminogen activator converts
plasminogen to plasmin-fibrinolytic
Testing the Coagulation Pathway
 Prothrombin Time
• Measures ‘Extrinsic’ pathway
• Sample – Citrated plasma (removes Ca 2+)
• PT uses Innovin (or similar) which contains
– Human Tissue Factor
– Phospholipids
– Calcium Chloride
• Time measured from reagent addition to clot
formation
• Normal range 14 – 16 seconds
 International Normalised Ratio
(INR)

( )
𝐼𝑆𝐼
𝑃 𝑇 𝑝𝑎𝑡𝑖𝑒𝑛𝑡
𝐼𝑁𝑅=
𝑃 𝑇 𝑁𝑜𝑟𝑚𝑎𝑙
• In the lab ideally INR should be calibrated against
traceable standards of known INR values
– ISI and mean PT of the normal pool determined by
calibration
• International Sensitivity Index (ISI) should be close to
1.00
• Calibration minimizes system-related differences
 INR Calibration
• Using the known INR standards log
(INR) is known
• The gradient of the line is the ISI
• The intercept is ISI times the log PT
mean normal.
• Patients treated with vitamin K
antagonists (VKA) are used in the
calibration.
• 0 – 4.5 most accurate range
 Limitations of PT and INR
• Occasionally lupus anticoagulant interferes with the
‘external’ pathway complicating any warfarin
therapy.
• ISI studies suggest significant differences between
kits and methods
• Optical testing can be confounded by Icteric,
lipaemic and haemolysed samples.
Activated Partial Thromboplastin Time (APTT)

• Used to measure ‘intrinsic’

pathway
• 2 Reagents
• Reagent 1 contains:
– A negative surface (kaolin,
ellagic acid)
– Artificial phospholipid
• Reagent 2 contains:
– Calcium Chloride
• No Tissue factor in the reagents
• Normal range 30-50 seconds
 Limitations
• Lupus anticoagulant
may interfere with
results confounding
clinical interpretation.
• Optical testing can be
confounded by Icteric,
lipaemic and
haemolysed samples.
 Fibrinogen
• Guidelines recommend Clauss
fibrinogen assay
• Thrombin, Owren's Veronal
Buffer (OVB) are the reagents
used.
• The assay is calibrated against a
known traceable standard plasma.
• OVB is used to dilute the
standard human plasma known
concentrations
• Normal range 1.5-7.5g/l
 Limitations
• This is a functional assay
and may not correlate with
the antigen but should
correlate with the in-vivo
utility
• High levels do not correlate
to risk of thrombosis
 Fibrinolysis
• Breakdown of the fibrin clot
• Plasminogen converted to Plasmin
• Produces fibrin split products
• Fibrin(ogen) degredation products (FDP’s)
• Cross link degredation products (XDP’s)
• D-Dimers from stabilised fibrin (clot specific)
• Indicator of thrombosis or DIC
 D-Dimer

• Measures break down of

clots
• Effectively measures
plasmin
• Is a negative prognostic
factor
• Uses antibody coated
latex beads
 Limitations
• Cannot positively provide
diagnosis
• Raised in many conditions
• Very sensitive to sample
integrity issues
• Is extremely expensive
Comparison of All Methods
Coagulation Disorders
 Coagulation Disorders
• Hereditary Factor deficiencies: -
• VIII Haemophilia A. X linked. Mainly males.
• IX Haemophilia B (Christmas disease)
• XI Haemophilia C
• Acquired Factor deficiencies
• Factor inhibitors eg factor VIII inhibitor
• 50:50 dilution with normal plasma corrects
factor deficiency but not factor inhibitors.
 Treatment of Haemophilias
• Human factor VIII concentrates
– Can be used to treat vWF deficiency
– Infection risk
• Recombinant factor VIII (rFVIII)
– Von Willebrand factor (VWF) is critical for the in
vivo survival of factor VIII (FVIII)
– No viral infection risk
• Porcine factor VIII
– Unacceptable to some
– May develop antibodies (foreign protein)
 Chronic (Compensated) DIC
• Compensated DIC always precedes
uncompensated DIC as the livers ability to
keep up is overwhelmed
• Pathogenesis:
– Generation of a hyperthrombinemic state
– Alterations of physiologic anticoagulants
– Inhibition and impairment of the fibrinolytic
mechanism
– Activation of proinflammatory cytokines
 Chronic (Compensated) DIC
• There are no clinical signs of a well
compensated DIC.
• A possible cause is:
– Tissue Factor on the surface of:
• metastatic solid tumor cells in the blood system
• Viral envelop (herpes simplex virus 1 (HSV1))
– Activates the coagulation system forming small
mobile clots.
• The analytical results:
– D-dimer raised,
– fibrinogen may be low,
 Acute (Uncompensated) DIC
• Consumption coagulopathy
• Platelets and clotting factors used up
• Red cells are fragmented by fibrin strands
• Leads to:
– Bleeding including a purpuric rash (non-blanching)
– In systemic microthrombi
– End-organ failure
• Can be caused by:
– Neisseria meningitidis
– Meningococcal septicaemia
– Solid tumour metastases
 Acute DIC
• Analytical Results
– Raised D-dimer
– Low fibrinogen
– Low Platelets
– Schistocytes present on the film
Acute DIC caused by N. meningitidis?
• Gram negative diplococci
• Gram negative cell wall is an endotoxin
• Damages endothelial cells, platelet adherence
• Activates the clotting pathways
• Leads to blockage of the microcirculation
• In organs leads to organ failure
• Extremities leads to gangrene
 Possible cause of Acute DIC
Haemolytic Transfusion Reaction
• ABO incompatible transfusion
• IgM antibodies activate complement
• Inflammatory mediators, pain, hypotension
• Membrane attack complex (MAC) – Lysis
• Activation of clotting cascade
• DIC may result consuming platelets
 Venom-induced consumption
coagulopathy (VICC)
• VICC very similar to DIC
• Characterised by low or undetectable levels of
fibrinogen
• VICC does not usually result:
– In systemic microthrombi
– End-organ failure
• VICC presents with:
– A rapid onset
– A rapid resolution
Russell Viper
 Lupus Anticoagulant
(Antiphospholipid Syndrome)
• Dilute Russell Viper Venom Time (DRVVT)
• Venom activates factor X directly in the presence
of factor V and phospholipid
• Prolonged in antiphospholipid syndrome
• Shows Lupus anticoagulant. Functional assay
• Antiphospholipid antibodies by ELISA now
done. Quantitative assay
 Lupus Anticoagulant
• Lupus is normally an incidental laboratory finding
with no clinical signs. However, there may be:
– Bleeding (occasionally a cross-reactive antibody to FX)
– Thrombotic episodes
• It is caused by the autoimmune production of
antibodies to phospholipids
• The typical analytical results are:
– APTT prolonged,
– Mixing test shows an inhibitor (not fully correcting)
 Thrombophilias
• Increased risk of thrombosis
• Antithrombin (AT) deficiency
• Protein C and S deficiency
• Factor V Leiden
• Prothrombin G20210A
• Antiphospholipid syndrome (DRVVT)
 Antithrombin Deficiency
• Antithrombin is a natural anticoagulant
• Deficiency leads to a thrombotic tendency
• Functional assay looking at neutralization of
thrombin in the presence of added heparin
• Heparin enhances antithrombin so clotting
should prolong with additional heparin.
• Can’t treat with heparin
• Use Hirudin from Leech (Hirudo medicinalis)
Hirudio medicinalis
 Protein C and S deficiencies
• Protein C and S are natural anticoagulants
• They require vitamin K for synthesis
• Affected by warfarin therapy
• Warfarin reduces Protein C and S but also
warfarin dependent clotting factors so the
balance is maintained.
• Measure by ELISA
 Activated protein C resistance
• Naturally occurring anticoagulant
• Activated protein C cleaves factor Va
• Factor V Leiden mutation at site of cleavage
• Factor Va resistant to clevage by Protein C
• Factor V Leiden has 5% prevalence
• 20-25% will develop thrombosis
• Most common inherited thrombophilia
 Prothrombin G20210A
• Mutation of the prothrombin gene
• 2-3% of caucasians
• 4-8% develop thrombosis
• May have higher levels of prothrombin
• Genetic test to detect presence
Therapy
 Thrombolytic Therapy
• Streptokinase can be used to treat active
thrombosis
– Pulmonary Embolism (PE),
– Acute Myocardial Infarct (MI)
– Acute ischemic stroke (AIS)
– Deep vein thrombosis (DVT)
– Acute peripheral arterial occlusion
– Occlusion of indwelling catheters
– Intracardiac thrombus formation.
 Thrombolytic Therapy
• Streptokinase
– Serine protease that cleaves plasminogen into
active plasmin
• Tissue Plasminogen Activator (TPA)
– As it is produced from streptococcus
• Often exerts febrile reactions
• Other allergic reactions
• Dose dependant hypotension
 Thrombolytic Therapy
• Recombinant Tissue Plasminogen Activator (rTPA)
– Alteplase
• More fibrin ­specific with a plasma half-­life of 4­6 minutes
• Used to treat:
– acute cardiovascular events (STEMI)
– pulmonary embolism(PE)
– acute ischemic stroke(AIS)
• Not antigenic
– Reteplase
• Similar to Alteplase
Treatment for Thrombotic Tendency –
Warfarin
• Used to prolong blood clotting time reducing the
risk of thrombosis
• International Normalised Ratio (INR)
• Drs tell patients warfarin thins the blood
• No effect on blood viscosity
 Warafrin
• Vitamin K re-cycling inhibitor.
• Warfarin mimics both the reduced and
oxidized forms of Vitamin K stopping the
catalytic re-cycling of vitamin K.
• The vitamin K-dependent clotting factors are:
– Factors II, VII, IX and X (and proteins C, S, and
Z)
– All these factors are made functionally deficient by
warfarin.
 Heparin
• Heparin is a glycosaminoglycan loosely
composed of a mixture of chains of alternating
residues of D-glucosamine and a glucuronic
acid (or iduronic acid).
Heparin – Sulphonated Oligosaccharides

Unique pentasaccharide that binds to antithrombin in a two-step process

 Heparin
• Glycosaminoglycans are large complexes of
negatively charged hetero-polysaccharide chains.
The molecular weight of heparin ranges from 5 kD
to 30 KD with a mean molecular weight of 15 kD.
• Assuming an average of 2.5 sulphates per
disaccharide unit each unit will have a molecular
weight of 552.43. Thus, heparin will have a range
of 9 to 54 disaccharide units (with a length range
of 83 to 500 Angstroms).
 Unfractionated Heparin (UFH)
• Unfractionated Heparin is a full mixture of
chain lengths from 9 to 54 diasaccharide units.
• UFH is administered via intravenous line often
into the arm.
 Heparin Acts
• Heparin exerts its anticoagulant properties
indirectly by binding with anti-thrombin (AT) and
facilitating the subsequent inhibitory effect of AT
on thrombin and activated factor X (factor Xa).
• Unfractionated heparin (UFH) containing at least
18 saccharide sequences can influence the action
of AT on thrombin.
• However, UFH fragments of any length
containing a unique pentasaccharide sequence can
inhibit the action of factor Xa.
 Low Molecular Weight Heparin
(LMW Heparin)
• In LMW heparin more than 60% of the chains
contain less than 16 diasaccharide units
• So LMW Heparin only acts against Xa
• Thus, it is also called Anti-Xa
• It is administered subcutaneously
• Tinzaparin is the LMW Heparin of choose in
cancer centers:
– As it has the ability to prevent both metastatic
dissemination of cancer cells and tumor
angiogenesis
 Tinzaparin
• Tinzaparin is measured using the Anti-Xa
assay
• The test sample MUST be drawn 4 hours after
administration of Tinzaparin
– This allows repeatable assessment of dosage
 Non-Vitamin K Oral
Anticoagulants (NOACs)
• Direct competitive reversible antagonists of
activated factor X (Xa)
– Apixaban
– Edoxaban
– Rivaroxaban
• Direct competitive reversible antagonists of
thrombin (FII)
– Dabigatran
 References
• Lewis, Bain and Bates 2006
• Naish, J (2009) Medical Sciences. Saunders.
• Gould, B.E. (2002) Pathophysiology for
Health Professionals.
• Underwood, J.C.E. (2004) General and
Systematic Pathology