Haemoglobin and rbcs COPY Flashcards
Electrophoresis
Method of analysing molecules by measuring migration in electric field. Support used: agarose for DNA, polyacrylamide for proteins, which acts as sieve thru which molecules migrate.
Spectrophotometry
Analysing molecules based on spectral properties. Spectrophotometer measures absorbance
Haem component
Synthesised in mitochondria . ls contained in other proteins eg myoglobin cytochromes peroxydases catalases. Combo of protoporphyrin ring with central ferrous iron atom. Iron usefully in FE 2+ form. Able to combine reversibly with oxygen. Bound within pockets of globin proteins.
Globin component
Synthesised in rough endoplasmic reticulum. Eight functional globin chains arranged in two clusters - alpha cluster and beta cluster.
Where is Hb found and conc in body and conc of Fe in g of Hb
Found only in RBCs. Normal conc in adults 120-165 g. Each g of hb contains 3.4 mg Fe.
2ndary struc and quaternary struc
2 - 75% of alpha and beta chains in form or a-helices. 4 - approx sphere, hydrophilic surface, hydrophobic core, haem groups found within pockets.
Absorbance
Fraction of incident light absorbed by solution. A = log10
How many subunits in Hb
4 : 2 alpha and 2 beta
Binding of O2
Cooperative, meaning binding of 1 O molecule to haem grp results in change in conformation of other subunits, affecting their ability to bind to O. In Hb this is a positive cooperative effect so as O binds to 1 haem the affinity of others increase. Twice as much O can be delivered. Seen in sigmoidal nature of O association curve.
Myoglobin
O binding protein in muscle. 1 haem grp per molecule and greater affinity for O than haemoglobin saturating at lower pO2 values. Lack of cooperativity means its poor at releasing O under same conditions.
Pulse oximeter
A pulse oximeter is a non-invasive way of measuring oxygen saturation levels. It relies on the difference in absorbance of oxyhaemoglobin and deoxyhaemoglobin. Oxygenated haemoglobin absorbs more infrared light but less red light than deoxygenated haemoglobin.
Carboxyhaemoglobin (CoHb)
Generated by the binding of carbon monoxide to ferrous iron (Fe2+) in Hb. Since hb has a 200-fold greater affinity for CO than O, it can readily outcompete O for binding to the four haem groups of Hb.
Methaemoglobin (MetHb)
Generated when the Fe2+ ion is oxidised to the Fe3+ (ferric) state which results in greatly impaired oxygen binding. Relatively low levels of MetHb can cause the oxygen dissociation curves to shift leftwards, which can result in tissue anoxia, as oxygen is not readily released by MetHb.
What does enzyme methemoglobin reductase do
Reduces methaemoglobin back to haemoglobin. Disorder methaemaglobinaemia can be hereditary eg as a deficiency in methemoglobin reductase or production of a mutant form of haemoglobin known as haemoglobin M, which is resistant to reduction.
how can Methaemaglobinaemia be acquired
following exposure to chemicals including aniline dues such as p-chloroaniline, nitrates, and local anaesthetics such as benzocaine.
Normal Hb runs further towards positive electrode. Why is normal Hb (A) more negatively charged than Hbs (Sickle hb)
Difference in charge due to point mutation in one aa of B chain. Glutamate in normal protein (hydrophilic, - charged) replaced by valine (hydrophobic, uncharged)
What are rbcs (erythrocytes) wbcs (leukocytes) and platelets derived from
Pluripotent Haemopoietic stem cells (HSCs). The cells above are produced throughout life in bone marrow. Blood cells all originate in bone marrow.
HSCs distributed in …
Ordered fashion in bone marrow amongst mesenchymal cells, endothelial cells and the vasculature with which HSCs interact.
Haemolysis is regulated by …
Genes, transcription factors, growth factors such as micro environment
What is haemopoiesis
Production and differentiation of blood cells
2 essential characteristics of HSCs
Self renewal (so some remain HSCs so pool not depleted). Differentiation to mature progeny (other daughter cells follow this path). These cannot renew themselves.
Origins of blood cells

Sites of haemopoiesis in fetus
Derive from mesoderm. Initially formed in vasculature of Yolk sac.
Disruption of regulation of hp can disturb …
Balance between proliferation and differentiation and may lead to leukaemia or bone marrow failure.
Haemopoietic growth factors are…. and they…
glycoprotein hormones which bind to cell surface receptors.
Rbcs are produced under influence of
erythropoietin
Granulocytes (neutrophils, cinephils and base officials) and monocytes production is under influence of
cytokines such as interleukins and granulacyte and granulacyte macrophage colony stimulating factors known as G-CSF and G-M CSF
Production of platelets is under influence of
Thrombopoietin.
Which growth factor is not produced by cells of bone marrow
Erythropoietin, produced in kidney
Lymphoid differentiation

Myeloid differentiation

Development of rbcs
Cell division until the cells reached the late erythroblast stage, when cell extrudes its nucleus.
Why is there a Blue tinge in early rbcs
Due to RNA content, add methylene blue to test for RNA.
WHat is required for erythropoiesis
Iron, vitamin B12, folate, erythopoietin. low amounts of these = anaemia; microcytic (smaller rbcs)(iron deficiency), macrocytic (bigger)(folate/B12 deficiency)
Erythropoietin
is a glycoprotein synthesised mainly in kidney in response to hypoxia (body not getting enough O). Ep interacts with ep receptor on red cell progenitor membranes and so stimulates bone marrow to produce more rbcs.
Iron function
Synthesis of Oxygen transport proteins haemoglobin and myoglobin. Also cofactor for protein and enzymes involved in energy metabolism, respiration, dna synthesis and apoptosis.
Mitochondrial proteins
cytochromes a, b and c: for production of ATP
cytochrome P450 for hydroxylation reactions
(e.g. drug metabolism)
Iron absorbtion
Iron is absorbed in the duodenum.
Haem iron (animal derived) is in ferrous (Fe2 +) form: this is the best absorbed form.
Non-haem iron is present mainly in ferric (Fe3 +) form in food and requires action of reducing substances (e.g. ascorbic acid, vitamin C) for absorption
Sources of non-haem iron such as soya beans often contain phytates,bind to iron, reducing absorption
Iron homeostasis
Excess iron is potentially toxic to organs eg heart and liver
There is no physiological mechanism by which iron is excreted
Therefore, iron absorption is tightly controlled: only 1-2 mg per day is absorbed from diet
Iron in plasma is bound to transport protein transferrin, delivers iron to bone barrow for ep and for use in enzymes and muscles.
Most iron recycled.

How do duodenal enterocytes alter iron absorbtion
Iron usually reduced to fe2+ form in duodenum before being taken up into cell, stored as ferrutin. Alternately oxidised to Fe3+ and transported to plasma via ferroportin.
Hepcidin synthesis suppressed by erythropoietic activity: ensures iron supply by increasing ferroportin in the duodenum enterocyte, which increases iron absorption.
When storage iron is high, hepcidin synthesis is increased, which binds and degrades ferroportin.
This prevents the efflux of iron from the enterocyte, so it is lost when the cell is shed into the gut lumen.
In inflammatory states hepcidin production is increased, this causes anaemia, because reduction in iron , resultant anaemia called anaemia of chronic disease.
Erythropoiesis regulation
Pro-inflammatory cytokines (IL-1 TNFα IL-6 IFNγ), reduction in ep and reduce production of epo.
B12 and folate
B12 and folate needed to synthesise dTTP, needed for synthesis of thymidine. Deficiency inhibits DNA synthesis, and affects all rapidly dividing cells, especially in bone marrow; cells can grow but unable to divide normally, process called megoloplastic-erythropoiesis, rapidly dividing cells lining epithelial surfaces of mouth and gut and gonads also affected.
sources of B12 and folate
Vitamin B12:
Meat, Liver & kidney, Fish, Oysters & clams, Eggs, Milk & cheese, Fortified cereals
Folic Acid:
Green leafy vegetables, Cauliflower, Brussels sprouts, Liver & kidney
Whole grain cereals
Yeast
Fruit
ABsorbtion of B12
B12 Cleaved from food proteins by HCl and then binds to diff proteins. passes to duodenum, cleaved from proteins and binds to glycoprotein intrinsic factor (resistant to digestion by gut enzymes and transports B12 to ileum
- Stomach:
B12 combines with Intrinsic factor (IF) made in the gastric parietal cells.
- Small intestine:
B12-IF binds to receptors in the ileum.
- Vitamin B12 deficiency may result from:
- inadequate intake e.g. veganism
- lack of acid in stomach (achlorhydria)
inadequate secretion of IF: pernicious anaemia (an autoimmune disorder)
Malabsorption e.g. coeliac disease
Folic acid
Takes place in small intestine mainly in duodenum. Total body stores: 10 mg (3 months). Requirements of folate increase during pregnancy and increased rbc production eg sickle cell anaemia. Folate supplementation required here.
Red cell destruction
At end of lifespan rbcs phagocytosed by macropahges in spleen. iron from Haem returns to bone marrow where it is recycled. Catabolysm of haem produces bilirubin, which is excreted in bile.
Red cell membrane
Biconcave in shape: helps manoeuvrability thru small blood vessels to deliver O. Membrane made up of liquid bilayer supported by protein cytoskeleton and contains transmembrane proteins: maintain integrity, shape and elasticity/deformability of rbc.
Disruption of vertical linkages in membrane (usually ankyrin/spectrin) causes
Hereditary Spherocytosis, (autosomal dominant)
Spherocytes are cells that are approximately spherical in shape, a round, regular outline and lack central pallor, result from the loss of cell membrane without the loss of an equivalent amount of cytoplasm so the cell is forced to round up
Red cells become less flexible and are removed prematurely by the spleen –haemolysis
Disruption of horizontal linkages in membrane causes
produces Hereditary Elliptocytosis,
Elliptocytes may also occur in iron deficiency
Hb
Haemoglobin A in adults is a tetramer: it is made up of 4 subunits, each composed of a globin chain (2 α, 2 β) bound to a haem group.
Each haem group consists of a ferrous iron ion (Fe2+) held in a ring known as a porphyrin.
- each Fe2+ can bind to 1 oxygen molecule.
Oxygen Hb dissociation curve
The normal position of the curve depends on: H+ ion concentration (pH), CO2 in red blood cells, Structure of Hb, Concentration of 2,3-DPG.
Left shift (gives up oxygen less readily), HbF, CO
Right shift (easy oxygen delivery): High CO2 - low pH – ‘Bohr effect, High 2,3-DPG, HbS
Bohr effect - lower affinity for Hb, more likely to release, enhances O delivery proportionally to metabolic activity.
rbcs metabolism
metabolic pathways -Highly adapted:
- Generation of ATP to meet energy requirements
- Maintenance of:
– haemoglobin function
– membrane integrity and deformability
– RBC volume
Relevance of metabolsim of rbcs - G6PD AND 2,3 DPG
G6PD
- Important enzyme in the hexose monophosphate (HMP) shunt
- HMP shunt coupled to Glutathione metabolism, protects red cell from oxidant damage
- Oxidants generated in the blood stream, e.g. during infection, or may be exogenous e.g. drugs, broad beans
- Deficiency of G6PD causes red cells to be vulnerable to oxidant damage
2,3-Diphosphoglycerate (2,3-DPG)
- Produced by Rapaport-Luebering shuttle
- Allosteric effector - modulates haemoglobin oxygen affinity
- Binds to β-globin chain in central cavity of haemoglobin molecule
- Role in adaptive response to anaemia, hypoxia and high altitude
Glucose-6-phosphate dehydrogenase (G6PD) deficiency
Most prevalent enzyme disorder.
X-linked inheritance so affected individuals are usually hemizygous males (but occasionally homozygous females)
G6PD deficiency usually causes intermittent, severe intravascular haemolysis (breakdown of unstable blood cells) as a result of infection or exposure to an exogenous oxidant
Extrinsic oxidants may be foodstuffs (e.g. broad beans), chemicals or drugs
Distribution parallels malaria: selective advantage, resistance to falciparum malaria
Meaning of : microcytic, normocytic, macrocytic
Microcytic – describes red cells that are smaller than normal or an anaemia with small red cells
Normocytic – describes red cells that are of normal size or an anaemia with normal sized red cells
Macrocytic – describes red cells that are larger than normal or an anaemia with large red cells
Causes of microcytosis
- Defect in haem synthesis
- Iron deficiency
- Anaemia of chronic disease
- Defect in globin synthesis (thalassaemia)
- Defect in α chain synthesis (α thalassaemia)
- Defect in β chain synthesis (β thalassaemia)
Macrocytes
Rbcs larger than normal. Can be of diff types: round, oval, polychromatic (young immature rbc)
CAuses of macrocytosis
Lack of vitamin B12 or folic acid (megaloblastic anaemia)
Liver disease and ethanol toxicity
Haemolysis (polychromasia)
Pregnancy
Colour of rbcs
Normal red cells have about a third of the diameter that is pale
This is a result of the disc shape of the red cell; the centre has less haemoglobin and is therefore paler
Hypochromia
Means cells have larger area of central pallor than normal. Results from lower Hb content and conc and flatter cell. Rbcs that show hypochromia: hypochromic. Hypochromia and microcytosis often go together, with iron deficiency being common cause.

Polychromasia
Polychromasia (‘many colours’) describes an increased blue tinge to the cytoplasm of a red cell
It indicates that the red cell is young
Polychromatic cells are larger than normal red cells i.e. polychromasia is one of the causes of macrocytosis

Young red cells: reticulocytes and reticulocytosis
- To detect young rbcs (reticulocytes) : new methylene blue stain. THis stains for higher RNA content.
- Reticulocytosis : presence of increased numbers of reticulocytes. May occur when bone marrow pumps out young rbcs, as response to bleeding of rbc destruction (haemolysis).
- Detecting polychromasia or increased numbers of reticulocytes gives you similar information
- However identification of reticulocytes is less subjective so they can be counted reliably

Anisocytosis
Describing differences in red blood cell size. red cells show more variation in size than is normal

poikilocytosis
Describing differences in red blood cell shape

Polikilocytes come in variety of shapes
Spherocytes
Elliptocytes
Irregularly contracted cells
Sickle cells
FRagments
TArget cells
TArget cells
Rbcs with accumulation of Hb in centre of area with central pallor. May occur in number of diff conditions : obstructive jaundice, liver disease, haemoglobinpathies, hyposplenism (spleen doesnt function properly or has been removed)
Sickle cells
Sickle cells are ‘sickle’ or crescent shaped
They result from the polymerisation of haemoglobin S, which in the deoxygenated form is much less soluble than haemoglobin A
Haemoglobin S occurs when one or two copies of an abnormal β globin gene (βS) are inherited.
The mutation in the beta haemoglobin gene is the charged glutamic acid residue in position 6 is replaced by an uncharged valine molecule.
Red cell fragments
Fragments or schistocytes are small pieces of rbcs.
They indicate that a red cell has fragmented
Red cell fragmentation may result from a shearing process caused by the platelet-rich blood clots in the small blood vessels e.g. disseminated intravascular coagulopathy*
REference range determination
A reference range derived from carefully defined reference population
Samples are collected from healthy volunteers with defined characteristics
They are analysed using the instrument and techniques that will be used for patient samples
The data are analysed by an appropriate statistical technique
Not all results outside the reference range are abnormal
Not all results within the normal range are normal
Interpreting red cells on blood count and film
Is there anaemia?
If so, are there any clues in the blood count?
Are there any clues in the clinical history?
The correct interpretation of a blood count may also require you to examine a blood film
You should examine and describe red cell:
Size ,Shape, Age (polychromasia), Poikilocytes
Where do rbcs originate from
Common myeloid progenitor
In sickle hb ..(HbS)
- 2 normal a chains and 2 variant beta chains. These are almost identical to normal beta globin but aa valine replaces glutamic acid at positon 6 of chain (mutation in codon 6 of beta globin chain)
- Glu Valine
- Polar Non polar
- Soluble Insoluble
- Deoxyhaemoglobin S is insoluble
- HbS polymerises to form tactoids that distort the red blood cell to result in a sickled shape
- Autosomal recessive inheritance pattern:
- HbSS: sickle cell anaemia
- HbAS: sickle trait (usually asymptomatic)
Sickle cell disease incorporates
- Sickle cell anaemia (HbSS)
- A number of compound heterozygous states that lead to a disease syndrome due to sickling
e.g. HbSC
HbS b thalassaemia
Haemolysis (breakdown) of sickled red blood cells :
- Shortened red cell lifespan: 20 days
- Anaemia: baseline Hb concentration in HbSS is ~60-80 g/l (compared to 125-160 for HbA)
- Process of haemolysis leads to Gall Stones (increased red cell breakdown products)
- And leads to Aplastic Crisis (Parvovirus B19 where bone marrow erythropoiesis has been shut down)
Blockage to microvascular circulation (vaso-occlusion)
- Refers to blockage of venules that capillaries drain into
- Occurs as consequence to polymerisation of abnormal hb in low O conditiosn - when they form the rigid sickled shape.
- Rigid sickle cells can blog vessels leading to…
- Tissue damage and necrosis (Infarction)
- Pain
- Dysfunction
Effect of Hbs on oxygen hb dissociation curve
- HbS shows right shift in oxygen dissociation curve compared to HbA
- HbS is a low affinity haemoglobin and so gives off O more readily to tissues.
- Individ with sickle cell anaemia (HbSS) who have lower concs of Hb (around 60-80 g/l) do not suffer from the symptoms of anaemia that somebody with HbA of that
- The baseline Hb concentration in individuals with HbSS is lower than for HbA of that conc level would feel.
- Because of readiness of HbS to give off oxygen to tissues, individ with this level ( 60-80) do not suffer normal symtoms of anaemia.
- Anaemia results from reduced rbc survival. The greater oxygen delivery to tissues results from a reduced etythropoietic drive (synthensised in response to hypoxia) and this contributes to anaemia.
- It would be expected in HbA to therefore have feelings of tiredness, breathlessness, palpitations with the heart beating faster to improve oxygen delivery
- However this does not result in symptoms of anaemia
- This can be partly explained by the Oxygen-Hb dissociation curve
Pathophysiology of SCD
- Hypoxia
- Polymerisation HbS
- Rigid ‘sickling’ of rbcs so
- Haemolysis and Reduced rbc survival OR Vaso-occlusion
- Anaemia, Jaundice, Gallstones OR Bone, Kidney, Cerebral, Retina, Lung, Spleen
Early presentation of sickle cell disorders
- Symptoms rare before 4-6 months of age
- Onset coincides with switch from fetal to adult Hbsynthesis
- Early manifestations
- -Dactylitis (inflammation of digits)
- -Pooling of rbcs in spleen (splenic sequestration)*
- -Infection
SCD and the spleen
- The spleen is involved in:
- –Immune defence
- –Breakdown and removal of old, malformed or damaged red blood cells: ‘quality control’
- Repeated splenic vaso-occlusion (usually with no symptoms) in HbSS leads to the spleen no longer working by age of 5 years: ‘functional hyposplenism’
- Hyposplenism increases susceptibility to encapsulated bacterial infection so need to receive :
- –Immunisations
- –Prophylactic (preventative) antibiotics
Complications associated with sickle cell disease
Acute chest syndrome in SCD (emergency could be fatal)
Stroke in Sickle Cell Disease
Avascular Necrosis of the Femoral Head
Osteomyelitis due to Salmonella Infection
Gallstones
Laboratory features of sickle cell disease
- Hb low (typically 60 - 80 g/l)
- Reticulocytes usually high (high cell turnover because premature destruction in haemolysis)
- Blood film
- Sickled cells (crescent shape)
- Boat cells (look like boats)
- Target cells (dougnuts)
- Howell Jolly bodies (contain purple dots - DNA remnants from nuclei which have not been removed)

Diagnosis of sickle cell disease
- Principle: In the presence of a reducing agent oxyHb converted to deoxy Hb
- Solubility decreases
- Solution becomes turbid
- Does not differentiate AS from SS, it will only detect presence of HbS due to reduction of solubility
- Definitive diagnosis requires Electrophoresis or High Performance Liquid Chromatography (HPLC) to separate proteins according to charge
Sickle cell anaemia includes :
just HbSS not HbSC. sickle cell DISEASE involves both
The molecular alteration of the beta chains is a …. which protects against
missense mutation, malaria
Clinical manifestations may start in utero because b-globin is part of fetal Hb true or false
False - fetal hb does not include beta chains its just alpha and gamma
Oxygen delivery in lower PH
like in metabolically active tissues
lowers O affinity of Hb and facilitates release of O to tissues. Right shift on sigmoid oxygen dissociation curve
Other funtions of Hb
Transport of CO2 and of nitric acid , most CO2 carrried in plasma as bicarcbonate ions
Effects of 2,3 -DPG, hypoxia and pH on oxygen dissociation curve
2,3-DPG binds to the haemoglobin molecule to increase its liberation of oxygen. This chemical is more present during high levels of metabolism.
HbF, low 2,3-DPG, hypoxia and high pH can cause the ODC to shift to the left. HbS, high 2,3-DPG and low pH can cause the ODC to shift to the right.
Multipotent haemopoietic stem cells ( HSCs) give rise to
lymphoid stem cells - from which
myeloid stem cells - from which red cells (erythrocytes), granulocytes, monocytes and platelets are derived.
HSCs have two major characterstics
Have ability to self-renew
Some daughter cells remain as HSCs so pool of HSCs is not depleted
Differentiate to mature progeny
The other daughter cells follow a differentiation pathway.
Normal Hb is called
Haemoglobin A
Foetal HbF
is made of 2 alpha and 2 gamma subunits. It has a higher affinity for oxygen than HbA which means that the foetus will get oxygen from the mother’s blood.
DUring inflammation what happens to hepcidin
hepcidin production is stimulated and iron entry into plasma is inhibited, causing the hypoferremia and anaemia of inflammation.
DNA synthesis needs 4 immediate precursors:
dATP, dTTP, dCTP, dGTP
Normal rbcs blood film

Microcytosis blood film

Macrocytosis blood film
