IMED1001 Blood and Drugs Lecture Notes

Lecture 1 Bone Marrow Structure & Function

Learning Outcomes:
1. Explain process of haemopoiesis.
2. Known about cytokines that influence haemopoiesis.
3. Understand the components of blood and their function.
4. Know that cells are present in normal blood
5. Understand the blood count.

Notes:
● Haematopoiesis (haem is blood, poiesis is making) is the process of
blood cell production in bone marrow.
○ Cells proliferate and differentiate within the marrow.
○ They come from progenitor cells and mature.
● The progenitor cell is the pluripotent hematopoietic stem cells; the
most primitive cell.
○ We need cell hormones; known as cytokines.
● The HSC can differentiate to form all forms of blood cell.
○ Red blood cells (erythrocytes-brown),
○ Leucocytes (purple),
○ Platelets (cell fragment),
● Bone marrow is body’s most active tissue- starts from
fetal.
○ Blood cells are deciduous and mature one’s
do not proliferate.
■ 24 x 10​12​ lifespan of a erythrocyte.
■ 2 x 10​11​ erythrocytes die a day.
■ Platelets survive 10 days.
■ Granulocytes only spend hours in the
blood (WBC, used only to fight
infection).
● We begin making in uterus; from the yolk sack
(3 weeks), then liver (3 month), then bone
marrow (fully active month 7)
○ At birth the entire skeleton has fully
active bone marrow.
● As we age no more from all bones; begins to be
mostly vertebra.
○ Tibia and rib dip off earlier.
● Bone marrow is within the hard bony cavity.
 ○ We require cytokines and regulators, both growth factors along with vitamins and metals.
● Bone marrow is structured: haemopoietic cells are within bone matrix.
○ Immature cells adhere to matrix and mature cells lose adhesive proteins and can leave.
● Trabecular bone is the thin pieces of bone that give bone marrow structure.
○ We produce blood cells from next to this bone, as it gains nutrients from it.
○ Most immature next to the trabecular bone,
and further away more mature.
○ All the white is fat.
○ When we are born we have 100% cells, no
fat. As we age 100-age.
○ There must be good variance between cells
size and colour.
● Normal bone marrow:
○ Erythropoiesis: 20%, as it only takes 3-4 days
to make them.
○ Granulopoiesis: 60%
○ Lymphocytes: 15%
○ Megakaryocytes <0.1% (they are big cells)
○ Other: plasma cells and macrophages.
● Cytokines are cell hormone growth factors that
regulate hematopoiesis.
○ They control differentiation, control number of cells made, and affect function of cell.
● Different types of growth factors include:
○ Acting on primitive cells,
○ Some act on later cells committed to a particular cell lineage.
● Erythropoietin, a growth factor, increases red blood cell production.
○ EPO is abused by sports competitors.
○ Cyclists will due to dehydration increase blood cell number.
○ Anaemic, low hemoglobin (O​2​), kidney gets signal, and EPO made in kidney (can’t fail)
■ Renal failure: EPO injections of
blood transfusions.
● G-CSF, GM-CSF: control leukocyte production.
● IL3 and GM-CSF are examples of broad specificity
cytokines which act on pluripotent stem cells.
○ Cytokine binds to cell receptor and signal is
transmitted to the cell nucleus.
● Stem cell growth factors include:
○ IL-3
○ Stem cell factor
● Committed progenitor cell growth factors include:
○ Erythropoietic
○ G-CSF
○ GM-CSF
 ○ M-CSF
○ IL-5
○ Thrombopoietin.
● How to assess haemopoiesis:
○ Blood count first, then...
○ Bone marrow examination:
■ How many cells?
■ Are they in the right place?
■ Which cells are present?
■ Do they look normal?
■ Measure growth factors (EPO serum)

Lecture 2 What is Blood?

Learning Outcomes:
1. Explain process of haemopoiesis.
2. Known about cytokines that influence haemopoiesis.
3. Understand the components of blood and their function.
4. Know that cells are present in normal blood
5. Understand the blood count.

Notes:
● Cells in water, proteins and electrolytes.
● Average is 5 liters.
○ 20-30ml/kg red cells.
○ Plasma 40-50ml/kg.
● Plasma is 55%, 45% erythrocytes and buffy coat of other stuff is less than 1%
● The blood count is a count of number of blood cells (CBC, FBC are all blood count)
● It will count number RBC, WBC and platelets.
○ Also all 5 types of WBC.
○ Amount of RBC haemtocrit (% blood RBC), RBC size/number and haemoglobin.
● Machine takes less than a minute to process 1 sample of blood.
○ Shakes and wobbles, takes aloquit and does blood count.
● 3 letter acronym:
○ RBC: rbc count x10​12​/L
○ HGB: g/L
○ HCT: %blood RBC
○ MCV: mean singe RBC volume fL (femtoliter)
○ MCH:mean cell haemoglobin
○ MCHC: mean cell haemoglobin concentration.
○ RDW:RBC distribution width.
○ RET%: % RBC that are reticulocytes.
○ PLT: platelet x10​9​/L
 ○ MPV: mean platelet volume
● Based on standard caucasian range →
● MCV, is mean size of RBC.
○ We look at this to figure out why a person is anemic.
○ Microcytic: less than 80fL:
■ Iron deficiency, thalassemia, haemoglobinopathies, anemia chronic disease.
○ Normocytic:
■ Blood loss, haemolytic RBC membrane, enzyme deficiency, extrinsic, stem cell
defect.
○ Macrocytic: greater than 100 fL:
■ Megaloblastic, excess alcohol, liver disease, reticulocytosis, drug therapy,
marrow failure.
● Reticulocyte count reduced: failure of erythropoiesis.
● Reticulocyte count increased: approach BM erythroid response.
● WBC count acronym:
○ WBC: x10​9​/L
○ NE%: % WBC neutrophils
○ NE#: # neutrophils. (more import)
○ LY: lymphocytes
○ MO: monocytes
○ EO: eosinophils
○ BA: basophils
○ NRBC: nucleated rbc
● Abnormality in blood count, then we look at
blood film to see morphological approach of
blood.
○ Stained with romanowsky stain.
○ RBC goes brown, WBC purple pink
orange and platelets are small dots.
● Odd sizes and different colours is a problem in
RBC blood film.
○ Severe burns can do it.
● Tear drop poikilocytes: weird shape RBC due
to bone marrow problems.
○ Fibrous tissues in their bone marrow
from scar tissues.

○ Likely to
be anemic, when less
RBC present.
● Thalassaemia:
inherited red cell
disorder.
 ○ They look like bullseye with haemoglobin in the centre.
● Neutrophil, eosinophil and basophil are all granulocytes.
● Monocytes are garbage removal and lymphocyte is the fighting cell.
○ Lymphocytes when activated by infection increase in size and cytoplasm.
● Leukaemia is normally in play when all the blood cells look the same.
● Below is an example of how she does figures out in her brain.

● Coagulation, Virchow’s triad:

○ Clotting factors: circulating proteins
○ Platelets: derived megakaryocytes in
the BM
○ Endothelium: blood vessel lining.
■ Any 3 not functioning
properly then clotting
disorders.
● Coagulation cascade:
○ A cascade sets off fibrin to then fill in
the breach.
● Thrombocytopenia (not enough platelets)
○ Petechiae in acute immune
thrombocytopenia.
○ Platelet count is 10 x 10​9​/L. Normal is
150-400.
○ Result is petchiae- lots of horrible rash.
● Blood transfusion: donation and products.
○ Whole blood is given.
 ○ We divide it into separate components, we separate into plasma and cells.
○ We keep plasma into separate portions.
■ Packed red blood cells, with anticoagulate.
■ Packed platelets, for those with bad count.
■ Fresh frozen plasma, so the clotting factors does not coagulate.
■ Immunoglobulins can be purified into specific factors A, B and albumin.
● We give blood for those with acute loss, anaemia, marrow failure, transplantation,
thrombocytopenia, and immune deficiencies.
● Risks for transfusion include:
○ Wrong blood, volume overload (heart failure), allergy, fever, immune reaction, infection.
● Blood grouping is super important to understand.

Lecture 3 How to Clot But Not Too Much

Learning Outcomes:
1. Haemostasis:
a. Primary haemostasis: platelets and Von Willebrand Factor
b. Clotting cascade: generation of fibrin clot
c. Fibrinolysis: breakdown of the fibrin clot
2. Tests of haemostasis:
a. Bleeding history
b. Platelet count
c. PT/APTT/Fibrinogen
d. D Dimers.

Notes:
● Blood homeostasis is to stop blood loss at site of infection.
○ As well as to switch off clotting so that blood remains in a liquid state.
● Haemostasis: 4 elements:
○ Vascular endothelium,
○ Platelets,
○ Procoagulant and anticoagulant proteins
○ Fibrinolytic system.
● Quick localised and well regulated response.
○ Clot formation low and clot lysis high then BLEEDING issues
○ Clot formation high and clot lysis low then THROMBOSIS issues.
● Vasoconstriction is the primary action from injury:
○ Constriction of blood vessels reduced blood flow

1. Uninjured: Intact endothelium prevents platelet adherence by production of nitrous oxide (NO)
and prostacyclin.
a. Upon vascular injury: reflex vasospasm of smooth muscle.
b. Exposes proteins in the subendothelium that activate coagulation:
 i. VWF
ii. Tissue factor
2. Platelet adhesion and activation
a. Circulating platelets bind to the subendothelial matrix through platelet surface receptor,
via VWF (glycoprotein 1b/IX/V complex)
b. Other interactions contribute to platelet adhesion, eg GP1a/lla binding to collagen
c. Platelets activated by collagen, VWF, thrombin and ADO.
d. Activated platelets change shape and release granules which contain ADP, TXA2,
fibrinogen and VWF.
i. They express glycoprotein llb/lla fibrinogen receptor.
e. ADP and TXA2 help recruit other platelets to form
the platelet clot.
3. Formation of the fibrin clot (Secondary haemostasis)
a. Tissue factor combines with factor VIIa to form
FVIIa-TF complex.
b. This complex activates other clotting proteins leading
to thrombin production.
c. Large scale production of thrombin (factor II) takes
place on platelet surface.
d. Thrombin converts fibrinogen to fibrin (makes the
mesh of clot)
i. Also activates factor 13; helps to crosslink the clot.
4. Fibrinolysis, the process of clot breakdown:
a. Tissue plasminogen activator (tPA) activates plasminogen and converts it to plasmin.
b. Plasmin enzymatically attacks fibrin molecule producing fibrin degradation products
(FDP), cleared by macrophages.
i. FDP sometimes called D Dimers
5.

● 4 processes in platelet response:

○ Adhesion,
○ Aggregation,
○ Secretion,
○ Pro-coagulant activity.
● Central to haemostasis, initial closure of
vessel wall.
● Vasoactive substances, platelet agonists
and coagulation proteins from platelet
granules are released at the site of injury.
● In propagation phase activated clotting
factors bind to platelet surface and
increase thrombin generation.
● Deficiency in platelet number is
idiopathic/immune thrombocytopenic
purpua (ITP)
○ Purpua is the rash and easily
bruised skin from reduced platelet
count.
● Thrombin activates coagulation factors, platelets,
anticoagulant factors and determines the balance of
fibrinolysis.
● Intrinsic pathway or extrinsic pathway can cause
coagulation.
○ Extrinsic pathway is vascular injury
○ Intrinsic is blood touching something not
self.
○ → phospholipid is PL.
○ Biological amplification takes place: 1mole
XIa generates 10​7​ moles IIa.
○ Clotting factors number I-XIII.
○ Factors II, VII, IX and X require post
translational gamma carboxylation.
○ Most clotting factors are serine proteases.
○ Phospholipid derived from activated platelet membrane.
● The coagulation cascade explained simply to memorise:
○ Binding of factor VII to tissue factor (TF) initiates coagulation.
○ Generates trace amounts of factor IIa (thrombin)
○ Activates factor XI to XIa, factor V to Va and factor VIII to VIIIa.
○ Massive highly focused thrombin burst.
○ Rapidly converts soluble fibrinogen to insoluble fibrin.
○ Fibrin reinforces and stabilises the platelet plug.
● Tests of coagulation:
○ APTT and PT are the two main tests (two different tests have two different activators, run
different pathways)
○ APTT: measures contact factor activating factor VII to end on factor X.
○ PT: measures factor VIIa to fibrin.
● Principles of tested:
 ○ Citrate-decalcified blood (can’t clot) centrifuged to generate plasma.
○ Platelets are removed from centrifuge.
○ Clotting proteins are left in plasma and we add an activator, platelet substitute and
calcium.
■ Then we measure time to form a clot.
● Most important screening test is the clinical history:
○ Drugs, family history and personal bleeding history.
○ In vitro tests are not the whole story.
○ Lab screening:
■ Platelet count (needed to clot normally)
■ Prothrombin time (PT)
■ Activated partial thromboplastin time (APTT)
● Plasma contains: water, clotting proteins, antibodies, hormones, salts, vitamins, proteins.
● PT, is prothrombin time: extrinsic and common pathways:
○ Sensitive to abnormalities in VII, X, V, II and fibrinogen.
○ Time for clot to form after addition of TF, phospholipid and calcium to decalcified
plasma.
○ Reference range usually 13-15 seconds.
○ Basis to monitor oral vitamin K antagonists.
○ Relationship between PT and factor deficiency is not linear.
■ Prolongs exponentially lower at factor concentrations.
● International normalised ratio, INR: ratio of PT test compared to normal lab PT (corrected for
tissue factor used)
○ Its to create a standardised result if you move
○ So we can monitor drugs like warfarin.
● APTT, is activated partial thromboplastin time, intrinsic and common pathways.
○ Measures intrinsic and common pathways
○ We use contact activator Kaolin.
○ Deficient factor less than 40 percent we can pick up.
● Fibrinogen or factor 1, normal range 2-4 g/L
○ Functional method to measure, called Clauss assay:
■ Clot diluted with plasma w/high thrombin.
■ Use diluted plasma to remove effects of inhibitory substances eg FDPs heparin.
■ High thrombin concentration so clotting time independent of thrombin
concentration for wide range of fibrinogen levels.
○ Immunological methods (antibody mediated testing)
■ Measure concentration not functional activity.
○ Then you can test to see if you’ve not go enough or if its not working effectively.
●

Lecture 4 Disorders of Homeostasis

Learning Outcomes:
 1. Know the pathophysiology of haemostasis – if you understand this you can work out
what goes wrong with disease
2. Major bleeding disorders
a. Platelet and coagulation factor problems
b. Congenital and acquired
3. Presentation and major risk factors for VTE

Notes:
● Patients with diagnosis of haemophilia they should have a card.
○ Usual product replacement on it, and
quantity to administer in emergency.
● Von-Willebrand's disease:
○ VW protein has role in primary
haemostasis.
○ Carrier protein for FVIII
○ Autosomal dominant inheritance, effects
males and females equally.
○ Most patients have a mild bleeding
tendency.
■ Women with heavy bleeding,
epistaxis, mucosal bleeding,
easy bruising.
○ Types of VWD:
■ Type 1: decrease quantity of
VW factor.
● More mild, nose bleeds n heavy periods.
■ Type 2: decreased function of the antigen.
● Severity depends on abnormality.
■ Type 3: rare and severe deficiency from VWF from inheritance of 2 mutated
genes.
○ Must specifically test for VWD as APPT may or may not be prolonged.
■ Check for VWF antigen, and its function.
○ Normal is greater than 50% and abnormal is less than 30%, but between 30-50% is a gray
zone.
○ Treatment:
■ DDAVP (desmopressin)
● They will release their own stores of VWF and it will normally cover
those with mild VWF (4-6 hours)
● IV infusion that increases release of VWF from storage granules in
platelets and endothelium.
■ Tranexamic acid tablets, mouthwash.
● Stabilize fibrin clot may be take for first 4-5 days after procedure.
 ■ Biostate:
● FOr severe deficiency type 3, or non responders to DDAVP.
○ Plasma derived product prepared from donors that contains
FVIII and VWF.

○ Given same day as procedure.

● Vitamin K deficiency:
○ Several carboxylase enzymes within hepatic cells.
○ Necessary activation for factors II, VII, IX and X.
○ Vitamin K1 from dietary greens.
○ Vitamin K2 made in gut flora.
○ Deficiency:
■ Neonatal hemorrhagic disease:
● All neonates vitamin K deficient.
● All babies must receive as not much vit K in milk.
■ Adults:
● Any cause of malnutrition or malabsorption.
○ Diagnosis:
■ Prolonged PT
■ Normal fibrinogen and normal/prolonged APTT.
● Why patients with liver disease bleed:
○ Clotting factors creation decreased
○ Decreased absorption of vitamin K
○ Decreased clearance of activated clotting factors
○ Hyperfibrinolysis
○ Thrombocytopenia
○ Acquired platelet dysfunction.
● Massive transfusion as a cause of bleeding disorders.
○ Defined as replacement of more than 50% blood volume in 12-24 hours.
 ○ In adults usually more than 10 units of packed cells
■ Packed cells don't have clotting factors and platelets.
■ Test will show prolonged INR, prolonged APTT, low fibrinogen and low
platelets.
■ Coagulopathy potentiated by acidosis or hypothermia.
○ Many institutions now have massive transfusion protocol.
● Disseminated intravascular coagulation, DIC, death is coming.
○ Systemic process blood exposed to procoagulant factors like tissue factors.
○ Massive thrombin generation, widespread coagulation, and fibrinolysis and depletion of
factors.
■ Acute causes:
● Sepsis,
● Severe trauma
● Pregnancy
complications
● Snake bite
● Acute leukaemia.
■ Chronic causes:
● Cancer
○ Abnormal RBCs, breaking into
fragments and absence of purple
platelets.
● Thrombosis (clot formation> clot lysis):
○ Risks for venous thromboembolism
(VTE)
■ Endothelial injury,
■ Hypercoagulability,
■ Abnormal blood flow.
● Deep vein thrombosis affecting the leg:
○ Swelling,
○ Redness,
○ Tenderness,
○ Pitting oedema (swelling of the leg and finger away-pitt)
○ Above knee DVT and above knee DVT
○ Diagnosis of DVT:
■ Only ⅕ will have DVT.
■ Cannot be diagnoses or excluded on exam/history alone.
■ Assessment of pre-test probability of DVT, as prediction guide.
■ D-dimer test
■ Then ultrasonography of leg.
● Fibrinolysis: the breakdown of blood clots yields D-dimer.
● Plasmin breaks down cross linked fibrin to d-dimers.
● Occurs after a thrombus has found.
 ○ The D-dimer doesn’t mean you have a DVT.
○ If the D-dimer is negative you CAN EXCLUDE DVT from diagnosis.
● Pulmonary embolism:
○ Clot in lung
○ Chest pain, palpitations, shortness breath
○ Could be chest infection, pneumonia, cardiac failure.
○ Diagnosis:
■ History xam.
■ CXR/ECG/FBP.
■ Calculate pre-test probability.
■ D-dimer negative can exclude.
■ Imaging studies: Ventilation perfusion lung scan or CT pulmonary angiogram.
● VTE:
○ Usually starts in the calf veins.
○ Majority of DVT are proximal.
○ Pulmonary embolism arises from proximal DVT.
○ 50% untreated DVT are expected to cause PE.
○ 10% PE are rapidly fatal.
○ 30% untreated symptomatic non-fatal PE will have fatal recurrence.
● Treatment Of VTE aims:
○ Relieve symptoms.
○ Prevent PE,
○ Prevent death,
■ UK: 20-30% medical patients at PM have PE as contributor or cause of death.
○ Prevent recurrence
○ Prevent complications.
○ ANTIcoagulation is mainstay of
treatment.
● Treatment duration:
○ →
● Risk factors for VTE:
○ Acquired conditions:
■ Previous VTE
■ Major trauma/surgery
■ Cancer
■ Drugs, the pill,
pregnancy,
■ Overweight, smoker, old.
○ Inherited conditions:
■ Antithrombin deficiency
■ Protein C deficiency
■ Protein S deficiency
■ Factor V leiden mutation
 ■ Prothrombin gene mutation
■ Dysfibrinogenemia
○ Other:
■ Hyperhomocysteinemia
■ Elevated factor VIII level.
● Natural inhibitors of coagulation:
○ Antithrombin, Active protein C and Protein S.
● Antithrombin:
○ Inhibitor of thrombin, XA and serine proteases in coagulation cascade.
○ Heparin cofactor:
■ Slowly inactivates thrombin in absence of heparin.
■ Rapidly inactivates thrombin in presence of heparin due to conformational
change.
○ Active sites: reactive centre, heparin binding site.
○ Inherited autosomal dominant
○ Acquired causes: liver disease, warfarin therapy, protein loss, DIC.
● Protein C:
○ Vitamin K dependent protein made in liver.
○ Autosomal dominant inheritance.
○ Anticoag. Effect only active activated protein C.
○ APC inactivates a and VIIIa.
○ Effect markedly enhanced by protein S.
○ aPC also has anti-inflammatory action, protects endothelial barrier and enhances
fibrinolysis
● Protein S:
○ Autosomal dominant inheritance
○ Vitamin K dependent
○ Cofactor for protein C.
○ Acquired deficiency in pregnancy OCP, liver
disease.
● Factor V Leiden:
○ Point mutation
○ Arginine to glutamine at position 506.
○ Protein C unable to inactivate protein V
○ Activate protein C resistance
● Prothrombin Gene mutation:
○ Prothrombin precursor for thrombin.
○ G20210A transition 3’ untranslated
region of gene is risk factor for
thrombosis.
○ Heterozygous carriers have 30%
plasma prothrombin levels.
 ○ Mechanisms: altered mRNA processing or decay rate?

Lecture 5 Anticoagulant, Antiplatelets and Thrombolytic drugs

Learning Outcomes:
1. Describe the pharmacology, use and monitoring of anticoagulant, antiplatelet and fibrinolytic
agents

Notes:
● We are concerned with preventing thrombosis
○ CVD results from imbalanced homeostasis towards excessive thrombosis.
● Drugs to treat or prevent this imbalance attempt to tip balance away from clot formation.
○ Anticoagulant,
○ Profibrinolytic
○ Antiplatelet.
● Drugs may affect:
○ Coagulation (formation of fibrin)
○ Fibrinolysis (fibrin breakdown)
○ Platelets (adhesion and activation)
● Anticoagulants:
○ Classical anticoagulants:
■ Warfarin, oral.
■ Heparin, SC/IV
○ New/direct oral anticoagulants (NOACs, DOACs)
■ Direct Anti-Xa (rivaroxaban, apixaban, edoxaban)
■ Direct Anti-IIa (dabigatran)
● Warfarin:
○ Derived from coumarin, toxin from
plants.
○ Active component in rat poisons.
○ Inhibits vitamin K epoxide reductase:
■ Inhibits formation of vitamin K.
■ Vitamin K antagonist
■ Leads to reduced formation of
vit K dependant coagulation
proteins
● Factor II, VII, IX, and
X.
○ Only active in vivo (doesn’t work on samples)
○ Delayed onset of activity.
■ Doesn’t affect formed coagulant factors (therefore only works in vivo)
■ Effects formation of new anticoagulants.
 ○ Orally active
○ Rapidly absorbed, long half life (dosage 1/day)
○ Strongly binds to plasma protein.
○ Interacts with different drugs and is affected by diet (vit K dietary intake)
■ Dosage is difficult and must be monitored.
○ Warfarin dose:
■ Increased activity in
● Vitamin K deficiency,
● Hepatic disease (impaired synthesis coagulation)
● Hypermetabolic states
● Drug interactions
○ Antihemostatic drugs (aspirin)
○ Liver enzyme medication (cimetidine)
■ Decreased activity in
● Pregnancy
● High vitamin K diet
● Drug interactions
○ Barbiturates and alcohol stimulate liver to increase clearance.
○ Vitamin K supplements.
○ Monitoring Warfarin:
■ Prothrombin time:
● Time for coagulation of plasma after stimulation of extrinsic factor
pathway by addition of tissue factor/calcium.
■ International normalised ratio, INR:
● The PT of patient divided by a normal PT adjusted for batch of reagents.
○ Warfarin adverse effects:
■ Haemorrhage:
● Difficult to reverse.
● Oral vitamin K, no immediate effect, fixes long term.
● Fresh frozen plasma (FFP) must be used immediately.
■ Dosage must be carefully titrated based on INR.
○ Warfarin summary:
■ Chronic use, not for acute thrombosis.
■ Delayed onset
■ Dosing problematic
■ Difficult to reverse.
● Heparin
○ Intravenous or subcutaneous adminstration.
○ Highly sulfated glycosaminoglycan derived from pig, or
cow mucosa.
○ Strong negative charge.
○ HIghly variable molecular weight, 60 - 100 kDa)
○ Mimics effect of human heparan sulphate.
 ○ Active part of the molecule is a repeated pentasaccharide unit.
○ Binds to and increases activity of endogenous antithrombin (AT)
○ Increases ability of antithrombin to bind to activated factor II (thrombin) by 1000-fold.
■ Also binds other coagulation proteins.
○ Once heparin AT complex binds to a thrombin molecular, heparin is released leaving
inactive T-AT complex behind; heparatin can go bind another antithrombin molecule
circulating.
■ Potential use of heparin to desensitise patient to subsequent heparin injection by
consuming antithrombin.
○ As you run out of antithrombin; the effect of heparin will lower… works worse over time
○ Heparin pharmacodynamics and monitoring:
■ Not orally available.
■ Effect is immediate
■ Monitored by activated partial thromboplastin time, APTT.
● Time for clot formation after stimulation of intrinsic coagulation by
addition of calcium and phospholipid.
■ Effect reversibile by administration of protamine sulphate.
○ Heparin adverse effects:
■ Haemorrhage, administer protamine.
■ Thrombocytopenia
■ Osteoporosis through inhibition of vitamin D.
○ Low molecular weight heparin:
■ Heparin which has been treated to remove many of the non-active parts of the
protein.
■ Molecular weight is more consistent
■ Pharmacokinetics are more predictable.
■ Molecule is far more active and potent
■ Cannot be reversed.
■ Pentasaccharides now on the market.
● Summary of classical anticoagulants:
○ Warfarin for chronic thromboembolic risk
○ Heparin for acute anticoagulation:
■ Percutaneous coronary intervention, cardiopulmonary bypass
■ Bridge to warfarin
○ Relatively ineffective alone for cardiovascular disease.
● New anticoagulants:
○ Direct thrombin inhibitors:
■ Ximelagatran withdrawn dabigatran approved.
■ Does not require antithrombin, does not require monitoring.
■ Overcomes dietary and drug interactions of warfarin
○ Oral factor Xa inhibitor:
■ Rivaroxaban
■ Alternative to heparin for certain procedures and in AF.
 ■ Irreversible.
● Pro Fibrinolytics:
○ Streptokinase/Urokinase:
■ Derived from bacteria
■ Inexpensive and effective
■ Antigenic, single use (streptokinase)
○ Alteplase (hrtPA)
■ Human recombinant protein
■ Extremely expensive
■ Clot selective; more active on fibrin bound
plasminogen.
○ Can be used for after the event to rapidly break down
a thrombus or embolism causing the infarct.
■ Effective treatment for myocardial
infarction, stroke.
○ Adverse effects:
■ Inflammation
■ Overdose leading to haemorrhage.
● May treat with aminocaproic acid.
● Platelets:
○ Critical role in haemostasis
○ Now maladaptive:
■ We live longer
■ We bleed less often
■ We predispose ourselves to atherosclerosis
■ 10% of normal platelet number is all that is required to prevent spontaenous
bleeding.
○ Atherothrombosis:
■ Sudden, unpredictable atherosclerotic plaque disruption (rupture/erosion) leading
to platelet activation and thrombus formation.
■ Underlying conditions that results in myocardial infarction, ischemic stroke and
vascular death.
○ Endothelial cells when health produce nitrous
oxide and prostaglandin: inhibit platelets, ADP,
cAMP,
○ P2y1 and P2Y12.
○ CRUCIAL process for exam: watch lecture
on atherothrombosis formation.
■ 7/3/18 1:30 mins through lecture.
● Aspirin:
○ Inhibits cyclo-oxygenase, irreversible.
○ Reduces production of thromboxane.
■ COX-1 in platelets.
 ■ Platelets permanently affect, no nuclear material to produce more.
○ Also inhibits PGI2 production in endothelial cells.
■ COX-2 in endothelial cells.
■ Endotheliales rapidly restore PGI2 by synthesising new COX-2.
○ Thus aspirin prevents one of the mechanisms of platelet amplification following initial
activation.
■ Tips balance away from thrombosis.
○ Short half life, irreversible, 9 days for new platelets.
○ Adverse effects bleeding and GI bleeds.
○ Interacts with other NSAIDS affecting COX.
○ Monitoring resistance:
■ Monitored with arachidonic acid stimulated platelet aggregometry
■ Resistance is an issue especially for diabetes
● Fibrinogen Receptor Inhibitors:
○ Antibodies or small molecules which bind to and block GPIIb-IIIa integrin
○ Prevents binding of fibrinogen to the platelets
■ Therefore prevents platelet aggregation
○ Platelet activation and degranulation occurs normally
○ Only effective in infected acutely
○ Long term receptor blockade causes more problems than it prevents
○ Used in PCI
● Thienopyridines:
○ P​2​Y​12​ adp receptor antagonists
○ Inhibits amplification of platelet response by ADP released degranulation.
■ Does not affect pathways platelet activation (other ones like collagen, thrombin)
○ Metabolised in the liver:
■ Interacts with other liver drugs
■ Response variable
■ Irreversible
○ Monitoring:
■ Functional monitoring ADP stimulated platelet aggregometry
■ Resistance associated with CYP450 mutations
■ Effect of aspirin and
thienopyridine is most
effective for cardiovascular
disease.
● Some bleeding risk,
especially 3 and 4
generation
thienopyridines
○
Lecture 6 Thrombosis, Embolism and Tissue Infarction
Learning Outcomes:
3. n

Notes:
● CVD most common cause of death in Australia.
● Abnormal clotting is major contributor to CVD.
● Blood has RBC, WBC, platelets, fibrin and clotting factors.
● Anatomy:
○ Arteries,
○ Arterioles,
○ Capillaries,
○ Veins,
○ Venules,
○ Lymphatics.
○ Vessels have interna/endothelia surface.
■ Media, muscle.
■ Externa/Adventita, connective tissue.
● Clotting can be physiological or pathological.
○ Physiological: response to injury
○ Pathological: cause of tissue damage/death.
● Most vascular disease: narrowing or blockage of the lumen of hollow tube.
○ Means build up of toxic metabolites and deprivation of nutrients/oxygen/glucose.
● Clotting factors:
○ Procoagulant: platelets, clotting factors, vWF, fibrinogen.
○ Anticoagulant: protein S, protein C, ATIII, fibrinolysis.
● Initiation event: platelet plug → coagulation…. Fibrinolysis.
○ Fibrinolysis: plasminogen from liver in circulation.
■ Activated to form plasmin.
■ Plasmin cleaves fibrin.
■ Fragments entre circulation and cleared.
● Oxygen deprivation:
○ Ischaemia: reduced blood flow.
■ May have normal oxygen content.
■ Usually due to obstruction.
■ Tissue damage combo:
● Decreased ATP generation,
● Failure of sodium pump,
● Cascade of events including cell swelling, calcium influx, rupture of
lysosomes and membrane rupture, production of toxic metabolites and
free radicals.
■ Acute limb ischaemia:
 ● Thrombus or embolus in peripheral artery.
● Blocks downstream blood flow.
○ Hypoxia:
■ Deficiency of oxygen in blood available for tissues
■ Reduced oxidative respiration.
■ Can be due to low oxygenation:
● Cardiorespiratory failure
● Asphyxia
■ Can be due to low carrying capacity:
● Anaemia
● Carbon monoxide poisoning
■ Can be due to low blood.
○ Leads to irreversible cell injury: necrosis.
■ Cells breakdown past a certain point.
■ Tissue will die.
○ Infarction is the process that leads to infarct: an area of ischemic necrosis caused by the
occlusion of the vascular supply to the affected tissue.
■ Usually due to arterial occlusion:
● Thrombus, embolus, vasospasm, expansion of
artheroclerotic plaque, compression of vessels, vasculitis.
○ Myocardial infarction:
■ Local tissue death of heart muscle.
■ Rupture plaque, embolism travel or something else blocking
coronary artery.
■ Infarction is irreversible and necrotic.
● Thrombosis: formation of blood clot within a vessel.
○ Impact depends on site, size and type of vessel.
● Virchow’s Triad:
○ Endothelial injury:
■ Direct physical injury to vessel.
■ Chemical or metabolic abnormality.
● Drugs, tobacco, fish oil.
■ Atherosclerosis
■ Infection.
■ Injury activates endothelial cells.
● Changes gene expression towards
pro-coagulant state.
● Downregulation in thrombomodulin and overactivity of thrombin.
● Inflammation of endothelium downregulates protein C and other
anticoagulant.
● Antifibrinolytic effects: plasminogen inhibitors, decreased production of
t-PA.
● Activation of platelets form plug which can clot.
 ○ Abnormal blood flow.
■ Normal blood flow is laminar.
■ Cells tend to flow in center of lumen,
plasma at the periphery moving slower than
blood.
■ Turbulence: disrupts of laminar flow, brings platelets into contact with vessel.
● More stress on walls.
● Pockets of relative stasis: where some blood barely moves.
● Allows clogging cascade activation, protein aggregation/activation and
fibrin aggregation.
○ Keeps them in wall contact and prevents dilution of activated
factors by flowing fresh blood/clotting inhibitors.
○ Hypercoagulable states:
■ Hypercoagulability, also called thrombophilia: any disorder of the blood which
predisposes thrombosis.
■ Can be from primary genetic or secondary acquired disorders.
■ Primary:
● Factor V:
○ Variation up to 15% caucasians called factor V leiden.
○ Loss of antithrombotic counter pathway.
○ Heterozygotes: 5X increased v. thrombus, homozygotes: 50X
increase.
● Prothrombin:
○ 2% carry variation in gene
○ Leads to elevated prothrombin levels and 3X increased risk of
venous thrombosis.
■ Secondary:
● Immobilisation: not moving.
● Major trauma
● Malignancy:
● DIC: breakdown of cell membrane of some bacteria in septicaemia leads
to clotting factor consumption.
○ Many capillaries forming thrombus.
○ Running out of clotting factors and blood starts clotting.
● Characteristics of thrombi:
○ They are attached to vessel wall and propagate towards heart, can detach.
○ Laminations from lines of Zahn:
■ Platelet and fibrin layers alternating with darker red cell rich layers.
■ Only forms in flowing blood, forms more likely in faster flowing blood.
■ Mural thrombi: large thrombi attached to wall of heart or aorta.
○ Arterial or cardiac thrombus: typically arise at sites of endothelial injury/turbulence.
○ Venous thrombi: typically at sites of stasis.
■ More enmeshed red cells, fewer platelets, less often have lines of Zahn.
 ■ Deep veins of lower extremities most commonly affected.
■ Upper extremities sometimes too.
○ When you die: your blood clots. So must determine if post death.
● Possible sequelae:
○ Propagation: extension of clot or build up of layers.
○ Dissolution: activation of fibrinolysis.
○ Occlusion: thrombus blocks lumen and
blocks blood supply or drainage.
○ Organisation and recanalisation:
■ Organisation: fibroblasts and
then endothelial cells grow into
blood clot, securing it.
■ Recanalisation: capillary
channels form within the
fibrotic clot, partially
res-establishing lumen.
○ Embolisation: bit of the clot breaks off.
● Embolus: detached intravascular solid, liquid
gaseous mass carried by blood from origin
point to distant site- where it usually causes
tissue dysfunction or infarction.
○ Can travel long or short distances.
○ Classify via: consistency (gas/liquid), circulatory compartment (artery/vein) or type of
embolic material:
■ Thromboemboli: a thrombus that has embolized.
● Pulmonary:
○ Tend to ends up in the lungs.
○ Due to Virchows triad.
○ Normally stasis in deep veins travel.
○ Stops blood reaching lung.
○ Leads to VQ mismatch, increased right ventricular pressure.
○ Larger embolism: acute right heart failure and sudden death.
○ Smaller emboli: pulmonary haemorrage, infarct, hypoxia.
○ Multiple small embolic: chronic pulmonary hypertension,
chronic RHF
■ Sequelae:
● Lysis
● Superimposed thrombus
● Organisation and recanalisation.
● Systemic:
○ Often arterial cardiac origin.
○ Lodge in vessels at bifurcations or narrowing.
○ Localised infarcts.
 ■ Air/gas emboli:
● Air surgical, trauma.
● Gas: nitrogen, diver.
○ High pressure more nitrogen dissolves into blood.
○ Rapid return to low pressure results in bubbles forming in
microvasculature.
■ Fat emboli:
● Trauma of long bones
● Marrow gets into circulation.
● Fat is irritant to endothelial so will trigger profuse clotting
● Intravascular coagulation.
■ Amniotic fluid emboli:
● Rare by complicated.
● Happens in labour.
● Tear in placental membrane or uterine veins.
● Amniotic fluid into maternal circulation.
● Can trigger disseminated intravascular coagulation.
■ Septic emboli: colony of bacteria of fungi
● Infectious colonies of bacteria detach and lodge elsewhere.
■ Foreign body emboli:
● Intravenous drug users.
●

Lecture 7 Red Blood Cells Production and Function

Learning Outcomes:
1. Describe the process by which RBC are produced.
2. Understand components required for erythropoiesis.
3. Describe process of RBC destruction.

Notes:
● RBC:
○ Made in bone marrow, small flexible biconcave disc shape
○ RC membrane: lipids proteins
○ Carry haemoglobin.
■ Must be small to travel small capillaries.
○ Deformable: thus no nucleus
○ Require iron, folate, vitamin B12, EPO.
○ Normal lifespan 120.
● What is haemoglobin:
○ Haem: made in mitochondria contains iron
○ Globin: 2 alpha and 2 beta globin polypeptide chains
■ Defects in global chain: thalassaemia and sickle cell disease.
● Erythropoiesis: process of making RBC under EPO influence.
○ EPO made by kidney
○ Process occurs in kidney.
○ Erythroid islands in marrow
○ Stages of development red cell precursors.
○ Radial organisation: central
proerythroblast near macrophage,
peripheral mature cells.
○ Nuclear extrusion to produce RBC.
○ Macrophage feeds developing RBC
with iron.
● RBC:
○ Phospholipid membrane with proteins
○ Contains Hb
○ Normal Hb:
■ Infant: 110-140g/L
■ Adult male: 130-180g/L
■ Adult female: 115-155g/L
○ Normal RBC count: 3.8-6.0 X 10​12​/L
● Normal haemoglobin structure:
○ Hb two chains of each type.
○ HbA 98%
○ HbA​2​ 2%
○ HBF 1% (foetal haemoglobin combination of
both)
● We have different forms of haemoglobin.
● EPO: regulates red blood cell production.
○ Stimulus to EPO production is renal O​2​ tension.
■ Anemia, low O​2 pressure,
​ defective cardiac or pulmonary function.
● Requirements:
○ Metals like iron and cobalt
○ Vitamins like B12, folate and thiamine
○ Amino acids and other cytokines
● Iron:
○ Integral part of haemoglobin
○ From our diet.
○ Transported to marrow bound to transferrin
○ Can only absorb 5-10% of iron in diet.
○ Require 1-2 mg a day.
○ Hard to absorb iron in acidic environment.
○ Absorbed in upper part of small intestine, and transferred in transferrin.
○ Stored as ferritin in macrophages.
○ Lose iron in perspiration, feces, and blood loss.
● Cobalamin, B12:
○ Required from diet.
○ Animal products.
○ Large stores.
● Folate:
○ Fruits, veggies.
○ Absorbed in upper small bowel.
○ Small stores, 3 months.
○ Deficiencies result in anaemia.
● Hb and globin chains:
○ Alpha
○ Non-alpha:
● RBC cell membrane:
○ Deformable, stabile.
○ Embedded proteins.
○ Peripheral membrane elasticity proteins
○ Integral: embedded with lipids.
● Red Cell metabolism:
○ RBC need ATP for energy.
○ Anaerobic glycolysis.
○ Embden-Meyerhof pathway: makes ATP.
■ 1 mol glucose yields 2 mol ATP by
glycolytic pathway.
■ ATP to maintain RBC shape and
energy for deformability
■ 2 mol of lactate produced
■ Lactate transported to liver to
convert to glucose.
● RBC: no nucleus, no reproduce
○ No cell organelles, no synthetic
activities.
○ RBC can squeeze through tight
spots in microcirculation
○ Can do, reversibly deformable.
● Normal shape is biconcave shape with
central pallor; pale staining region where
nucleus is.
● Removed in reticuloendothelial system.
● Iron recycled.
● Haem broken down to bilirubin.
● Globin degraded to amino acids and
returned to pool
 ● Haemolysis is process of breaking down RBC.
● Reduced haemoglobin is more important that reduced RBC.
● Causes: not enough bone marrow production, increased loss, premature destruction.
○ Symptoms depend on speed and patient age.
● Reduced bone marrow production:
○ Primary: bone marrow failure, red cell aplasia.
○ Secondary: infections, drugs, infiltration, absence of ingredients, BM hypoxia, ineffective
erythropoiesis
● Increased red blood cell loss:
○ Bleeding, haemorrhage
○ Haemolysis: premature red blood cell breakdown.
●
Lecture 8 Anemia General Concepts and Iron
Learning Outcomes:
4. n

Notes:
● Anaemia reduction in haemoglobin concentration below the normal range for age and gender of
individual.
● Anemia is usually accompanied by reduction in RBC count and haematocrit (% blood
haemoglobin)
● Anaemia is not a disease but rather a manifestation.
○ Symptoms:
■ Tiredness
■ Dizziness
■ Short of breath
■ Headache
■ Tachycardia (fast heart beat)
■ Older people: heart failure and chest pain (angina)
■ Infants: irritable, failure to thrive
○ Signs:
■ Pallor: mucous membranes in eyes eg are pale.
■ Increased cardiac output: tachycardia, heart murmur.
■ Specific types or causes of anemia
● Jaundice: haemolytic anaemia.
● Koilonychia (spoon fingernails): iron deficiency aenima.
● Splenomegaly: haemolytic.
● Stool colour change: haemolytic.
● Anaemia not diagnosis
● Need to establish the cause
● Can’t treat without the cause.
● May need to do blood transfusion to abate symptoms.
● Mean cell volume MCV: can guide cause of anemia.
● MCV and blood film are important.
● Stained blood film:
○ Romanovsky stain
○ Assess RBC morphology
○ Assess other cell types for abnormality.
○ Test by results for iron, folate, B12, Hb electrophoresis
● Causes of anaemia:
○ Bone marrow production reduced:
■ Primary:
■ Bone marrow failure aplastic anaemia
● Red cell aplasia (aplasia means not making)
● Bone marrow dysfunction, myelodysplasia (older ppl)
■ Secondary:
● Insufficient nutrients, iron folate B12 EPO.
● Infection
● Drugs
● Marrow infiltration
○ Leukaemia
○ Cancer
● Increased loss of RBC:
○ Blood loss:
■ Bleeding, acute.
■ Chronic: slow bleeding usually gastrointestinal, menorrhagia.
○ Haemolysis:
■ Premature red blood cell breakdown
● Inherited
● Acquired
● Defect of the cell or the environment.
● Clinical clues to cause of anemia:
○ Blood loss:
■ Menorrhagia
■ Melaena
○ Splenomegaly:
■ Chronic haemolysis
■ Extramedullary haemopoiesis
○ Bone marrow failure: bruising bleeding infection
○ Jaundice.
● MCV less than 80, microcytic anemia:
○ Almost always iron related.
● MCV more than 120, macrocytic anaemia:
○ Folic acid and B12 deficiency.
● MCV is normal range 80-100 femtoliters.
 ○ Haemolytic failure, haemolysis, renal failure.
● Iron: important mineral in human body.
○ Exists all cells in various amounts
○ Important for: red blood, myoglobin, enzyme
(cytochrome system in mit) immune system.
● Normal body content is 4g.
○ Normal RBC 2.5 g
○ Myoglobin 300mg
○ Enzyme 200mg
○ Storage iron 1g.
● Absorbed transported to marrow bound by transferrin.
○ Recycled by macrophages.
○ Excess stored in macrophages and liver as
ferritin.
● Need a 1mg a day.
● Causes of iron deficiency:
○ Reduced intake: poor intake, absorption
○ Increased usage: blood loss, increased usage.
○ Poor intake:
■ Developing world.
■ Anaemia in paediatrics.
○ Poor iron absorption/malabsorption.
■ Stomach or bowel gastrectomy, coeliac disease.
○ Chronic blood loss:
■ GI: ulcers, carcinoma, varices, diverticulosis,
hemorrhoids.
■ Uterine bleeding, menorrhagia.
○ Increased iron utilisation:
■ Growth, neonates, puberty,
pregnancy.
● Negative iron balance is first stage
● Iron deficient erythropoiesis: reduced iron
stores, mildly reduced erythron iron
● Iron deficiency anemia: reduced iron stores and
reduced erythron iron.
● Clinical features:
○ Atrophic glossitis: smooth tongue
○ Angular cheilitis: redness and fissures
○ Koilonychia: fingernails spoon.
● Haematology of iron deficiency anaemia:
○ Hypochromic microcytic anemia
○ Blood film: hypochromic microcytic cells pencil cells and elliptocytes.
● Lab features of iron deficiency:
 ○ Mild thrombocytosis
○ Reduced reticulocyte count
○ Bone marrow:
■ Reduced erythropoiesis
■ Reduced iron stores.
● Lab measures:
○ Serum iron.
○ Serum transferrin
○ Transferrin saturation: % iron transporter occupied.
○ Serum ferritin: reflects body iron stores, best method.
● Other investigations in iron deficiency:
○ malabsorption , coeliac serology
○ Blood loss in Gi and uterine:
■ Gastroscopy
■ Colonoscopy
■ Capsule endoscopy
■ Pelvic scopic
● Iron replacement therapy:
○ Orals
■ Response in a week.
■ Poor absorption or poor compliance is
problem
○ Intramuscular or intravenous if severe
■ Oral iron side effects.‹
○ Duration: to normalise Hb and ferritin
○ Reponse: increase Hb; reticulocytosis.
● Blood transfusion: rarely required.
● Treat the underlying cause.
●

Lecture 9 Folate, Vitamin B12 and Anaemia

Learning Outcomes:
5. B12 metabolism
6. Folate metabolism
7. Causes and consequences of deficiencies
8. Megaloblastic anemaia

Notes:
● Vitamin B12, Cobalamin:
○ Nuclear maturation required
○ From diet: animal products.
○ Absorbed in ileum requires intrinsic factor
 ○ Large stores, years.
● Folate:
○ Fruit, vegetables
○ Absorbed in upper small bowl
○ Small stores, 3 months.
○ Deficiencies result in macrocytic anaemia.
● Vitamin B12: essential cofactor in DNA and cell metabolism.
○ Converts homocysteine to methionine.
■ Important in methylation of DNA RNA and proteins
○ Converts methylmalonyl CoA to Succinyl CoA (L-methylmalonyl CoA Mutase)
■ Important in fatty acid breakdown and energy production.
● Vitamin B12:
○ Solely from diet.
○ Derived from animal products.
○ Absent from vegetables or fruits
○ Need 1micrograms a day
○ We can store for years.
○ We normally get over 10-15 micrograms.
● Acidic stomach:
○ Dietary B12 will detach from protein and bind to R-binder.
○ R-B​12​ complex pass into duodenum.
■ Less acidic duodenum, B12 is released by binder.
○ B12 will bind to intrinsic factor, IF.
■ Made by parietal cells in gastric mucosal.
○ IF-B​12 complex
​ travel to terminal ileum: cublinin or amnion receptor will take up the
complex.
■ The B​12​ is then slice from the complex.
○ Liver lets it leave as TcI-B​12​ or TcII-B​12 into
​ blood (2 is actively absorbed by cells via
endocytosis)
● Causes of B12 deficiency:
○ Diet: inadequate intake
○ Vegans: no animal products.
○ Infants born to B12-deficient mum.
○ Malnutrition, famine, poverty.
● Most common cause:
○ Malabsorption in stomach or terminal ileum.
○ Gastric causes: pernicious anemia; gastrectomy.
○ Intestinal causes: defects of the ileum; surgical, Crohn's, bacterial overgrowth.
● Pernicious anemia:
○ Autoimmune gastritis: recognise gastric layer of body as foreign.
○ Reduce secretion of IF, hence less B12 absorbed.
○ Manifested by antibodies: act on parietal cells or on the IF itself.
○ We test for antibodies in clinical world.
 ○ Normally older females are the ones with it, w/family history.
○ Blood group A, blue eyes, white eye.
● Pernicious Anemia is a macrocytic anemia: larger than 100.
○ We replace the B12.
○ Bypass the gastric absorption; thus via intramuscular injections.
● Jaundice and anemia will take place as a byproduct.
○ Can have neuron and psychiatric implications
○ Neurons need B​12​ to fire.
● Can expect to find:
○ Macrocytic anemia
○ Leukopenia
○ Thrombocytopenia
○ Low B​12​ level, especially active B​12
○ Classical changes under microscope
○ Bilirubin will be raised.
● 1000 micrograms hydroxocobalamin IM injection.
○ 3 times a week for 2 weeks, 6 doses.
● Large doses of oral B12 can be given too.
○ Less reliable than IM.
● B9, Folate.
○ Water soluble B vitamin
○ Need 100 micrograms
○ We get 200-250 a day
○ We store enough for 2-3 months.
○ Folate is building block for DNA synthesis.
○ B​12​ is a cofactor in converting folate to tetrahydrofolate, building block for DNA.
● Cause of deficiency
○ Diet:
○ Poor absorption
○ Not enough greens
○ Increased requirements:
■ Physiological: pregnancy, lactation, prematurity.
■ Pathological: haemolytic aenemia, inflammatory conditions, exfoliative
dermatitis, Crohn disease.
○ Excess folate lsos, dialysis (protein bound)
○ Drugs: anticonvulsants.
○ Other: alcoholism.
● Investigate deficiency:
○ Diet drugs alcohol
○ Jaundice
○ Anaemia
○ Folate in serum low
○ And red cell level low
 ○ Normal or low B​12.
● Replace:
○ Folic acid 5mg per day for 4 months
○ Corrects anaemia.
● Neural tubes defects:
○ Fetal growth and development characterised by
many cell divisions
○ Critical requirement
○ Neural tube will not close in developing infants.
○ Women in pregnancy require adequate B12.
● Megaloblastic anaemia:
○ Hematology: macrocytic
■ Hypersegmented neutrophils
■ Mild haemolysis
■ Increased bilirubin
■ Hypercellular bone marrow
○ Biochemistry:
■ Reduced serum B​12​ or RBC folate
■ IF or parietal cell antibodies detected.
● Abnormal appearance of RBC in megaloblastic anaemia.
○ Cytoplasm of red cell concentrated with haemoglobin
○ Should mature from dark blue to light pink.
○ Nucleus in red cell will become more condensed as it matures till its excreted.
● BM:
○ Red cells nucleus maturation will lag behind the cytoplasm maturation which should be
up to date.
○ Nucleus is staying large.
○ These changes are megaloblastic.
○ Nuclear has lacy appearance.
● Liver disease shown by:
○ Target cells cholestasis
○ Acanthocytes, spur cells in end stage.

Lecture 10 Haemolytic Anaemia and

Haemoglobinopathies
Learning Outcomes:
9. n

Notes:
● Anaemia is a low haemoglobin.
● Haemolytic anaemia: RBC life span lower than 120 days.
 ○ Usually bone marrow response and increases erythropoiesis (EPO)
○ Red cell production can increase 6-8 fold.
● Anemia occurs when:
○ Red cell survival is less than 15 days.
○ Haematinic deficiency especially folate, B12.
○ Bone marrow disease.
● Physiological RBC destruction:
○ RBC broken down by macrophages.
○ Globin to amino acids
○ Haem binds to haptoglobin and degraded to
protoporphyrin → made to bilirubin.
○ Iron released and recycled.
○ Normally they die outside blood vessels.
● If they die intravascular: hemoglobin in plasma, different
coloured plasma/urine.
● Haemolytic anaemia: when RBC not living long enough,
and not enough bone marrow made new RBC.
○ Due to RBC destruction:
■ Increased bilirubin
■ Increased Lactate Dehydrogenase (LDH)
■ Reduced haptoglobins (Hb-haptoglobin complex made from free haemoglobins
liberated)
○ Evidence of damaged RBC:
■ Spherocytes
■ Fragmented red cells, known as schistocytes.
○ Evidence of increased RBC production:
■ Reticulocytosis (purple-y), polychromasia
■ Nucleated red blood cells (should be in bone marrow, its overworked)
● Extravascular:
○ Red cell lysis occurs outside blood vessels
○ Blood film: polychromasia and altered RBC shape.
○ LDH elevated
○ Bilirubin elevated.
● Intravascular:
○ Red cell lysis occurs inside blood vessels
○ Blood film: fragmented red cells.
○ Haptoglobin low
○ Plasma haemoglobin present; hemosiderin in urine.
● Clinical features of haemolytic anaemia:
○ Anaemia
○ Jaundice (yellow eyes, skin)
○ Pigment gallstones may occur
○ Splenomegaly (worked to try clear RBC)
 ○ Ankle ulcers (sickle cell anaemia)
○ Expanded bone marrow (overworked)
○ Aplastic crisis: parvovirus (switching off erythrocyte activity in bone marrow virus)
○ Megaloblastic anemia: folate deficiency.
● Causes of haemolytic anaemia:
○ Hereditary:
■ Membrane defect: hereditary spherocytosis
● RBC membrane must be deformable and stable with bilayer and proteins.
● Membrane proteins (52%):
○ Peripheral membrane proteins for elasticity like spectrin,
ankyrin.
○ Integral membrane proteins embedded with lipids like Band 3
and glycophorin.
● Hereditary spherocytosis: common autosomal dominant defect in
structural RBC membrane proteins: spectrin, ankyrin and band 3.
○ RBC less deformable and loses membrane when passing through
spleen.
○ Red cells become spherical and rigid then destroyed.
● Erythropoiesis is normal.
● These patients aren’t anaemic all the time: fluctuating anaemia and
jaundice.
● Splenomegaly and gallstones.
○ Laboratory features:
■ Spherocytes on film
■ Polychromasia
■ Negative DAT (direct-antiglobulin-test)
■ Positive EMA, band 3
○ Treatment:
■ Splenectomy (remove the site of damage occurring to
RBC)
■ Folic acid (give them more so they can make more RBC)
■ Cholecystectomy (take out the gallbladder if too much
pain)
■ Membrane defect: Hereditary Elliptocytosis:
● Autosomal dominant milder than spherocytosis.
● Many asymptomatic.
● Mutations in spectrin, 10% have haemolysis
● Elliptical shaped red cells.
● Variants:
○ Hereditary pyropoikilocytosis (both with it)
○ Southeast asian Ovalocytosis.
■ Enzyme defect: G6PD deficiency.
● Glucose-6-phosphate dehydrogenase deficiency.
 ● Enzyme in hexose monophosphate shunt which generates reducing
power as NADPH.
● Most common RBC enzyme disorder worldwide
● Gene on X-chromosome, X linked, more males affected.
● Susceptible to oxidant stress.
○ Triggers include drugs, fava (broad beans), infection, hypoxia.
● Intravascular haemolysis, self-limiting
● Oxidised haemoglobin removed.
● Blood film: bite or blister cells missing parts.
● Treatment:
○ Remove, stop, treat offending agent.
○ Treat infection
○ Transfuse blood if necessary.
● Blood count between crisis will be normal.
■ Enzyme defect: pyruvate kinase deficiency:
● Glucose metabolism in RBC anaerobically.
● Embden-Meyerhof pathway.
● PK is final step.
● Enzyme lack means no ATP and RBC die.
● They can’t deform properly with no energy.
● Treatment:
○ Take out the spleen (which removes the RBCs)
○ Prickled shape RBC will take place when spleen removed.
○ Mild compensated haemolysis.
● Diagnosis: Pyruvate kinase assay.
■ Globin chain defect: haemoglobinopathies
● Alpha genes on chromosome 16
● Beta genes chromosome 11
● Haemoglobinopathies: abnormalities of globin chain synthesis.
○ Either total loss of gene due to mutation: structural amino acid
substitution, structural haemoglobinopathy is the name.
○ Or mutation caused reduced rate of globin chain production:
thalassaemia.
■ Most common monogenic disorder in world.
● Thalassaemia reduced production:
○ Has microcytic
● Structural haemoglobin:
○ Structural change like sickle cell.
● Thalassemia classification:
○ Clinical severity: trait, intermedia, major
○ Globin chain defect.
○ Large number of molecular defects.
● Clinical features:
 ○ Some will die.
○ Variable anaemia
○ Some with stunted growth
○ Some carriers
● Beta-thalassaemia is the reduction in number of beta globin chains
produced.
○ Those with major present at 3-6 months of age.
○ By 12 months little gamma from childhood and no new beta
made so low haemoglobin
○ Transfusion dependent for their entire life.
○ They will get too much iron.
○ We remove the iron from their body.
○ Spleen enlarged, liver enlarged, hair on brain x ray.
● Alpha-thalassaemia:
○ Alpha we have two genes on 16, therefore can have missing 1-4
different gene deletions.
○ Severity dependent on how many are leaving.
● Hb Barts Hydrops Fetalis:
○ Super anaemic, Hb 6.1g/dL
○ MC 110 fL
○ Erythroblastosis fetalis.
○ Kid days from liver and spleen squashing heart.
● HbS, sickle cell:
○ Beta globin chain weird crystals.
○ Make sickle cell shapes.
○ Can’t get around microcirculation.
○ They get stuck.
○ Tissue downstream cannot receive oxygen, tissue will die.
○ Tissue infarction, tissue death.
○ Spleen can die; autosplenectomy.
○
○ Acquired:
■ Immune haemolytic anaemia (antibodies from body attack body cell)
● Auto-antibody directed at RBC
● Causes are idiopathic (IDK why happened) or B cell lymphoma (cancer)
● Microscope: spherocytes and polychromasia on PB.
○ Looks like heredity spherocytosis.
● Positive direct antiglobulin test.
● Treatment:
○ Immunosuppression
(corticosteroids),
○ Treat the cause
○ Splenectomy
 ○ Transfusion of blood
● Alloimmune haemolytic anaemia:
○ Rhesus disease of newborn when mums antibodies affect the
baby.
● Drugs:
○ Drugs sometimes can do same thing.
● Appearance:
○ Identical to inherited spherocytosis:
■ Polychromasia,
■ Spherocytes
■ Dying leukemia
■ Nucleated RBC.
■ Fragmentation haemolysis:
● Microangiopathic haemolytic anaemia
● Red cells have exposed to an abnormal surface.
○ Heart valve: pinhole lesion in heart.
○ Fibrin strands in vasculature damage small blood vessels.
○ Mechanical damage to RBC
● Leads to anaemia.
○ Schistocytes
● Haemolysis is intravascular.
○ Can pee haemoglobin.
■ Oxidative haemolysis
■ Liver disease (spur cell anaemia)
● Acanthocytes are present in severe liver
disease.
● Weird shaped burr red cells.
■ Renal dysfunction:
● Severely messed up RBC: echinocytes that
are shaped like burrs.
■ Infections:
● Severe bacterial sepsis:
● Malaria
● Clostridium welchii.
● .
○
Lecture 11
Learning Outcomes:
10. n

Notes:
Lecture 12
Learning Outcomes:
11. n

Notes:

Lecture 13
Learning Outcomes:
12. n

Notes:

Lecture 14
Learning Outcomes:
13. n

Notes:

Lecture 15
Learning Outcomes:
14. n

Notes:

Lecture 16
Learning Outcomes:
15. n

Notes: