Red Blood Cells Clinical Biomedicine

Review of Red blood cells, haemoglobin and anemia

What is the size of RBCs

–Mean diameter 7μ,

–mean volume 78 – 101fl

What percentage of rbc is Hb

25% by volume, but 33% by weight

What is useful about the biconcave shape of red blood cells

Allows pliability as some rbc is bigger then capillaries allows it to squeeze, also increases surface area for gaseous exchange

How far will a rbc travel and how long is it’s lifespan

300miles roughly 170,000 circuits through the heart and they last about 120 days in a healthy individual

What are the % breakdown of a rbc membrane

40% lipids, 52% protein and 8% carb

Roughly what is the red cell cytoskeleton

Describe the metabolic activity of a red blood cell

Anaerobic glycolysis to avoid oxidation of iron which creates an inoperative oxygen carrier. The ATP produced maintain membrane deformability and ion/water exchange. Glycolysis results in production of 2,3 DPG

In a healthy individual where are rbc made and how many are destroyed and replaced daily

In an adult, is usually confined to marrow spaces of sternum, pelvis and long bones

Approx 10¹¹ RBCs are destroyed and replaced per day (nearly 1 million every second)

RBC production can be uprated by 10X

1mm³ blood normally contains 3.5 - 5.5 x10⁶ RBCs

What are two types of immature rbcs

Nucleated RBCs or normoblasts

Reticulocytes, which aren’t nucleated but do have some organelles still present usually 1-2 days into ciruclation

What drives RBC production

EPO(erythropoietin), a hormone produced primarily by the kidneys in response to low oxygen levels.

How are RBC nuclei removed

They are extruded and phagocytosed by macrophages,

Which receptor allows a developing rbc get iron

CD71

What is haemolysis

A shortening of the RBC lifespan =  haemolysis. (May be compensated by increased production, so not necessarily anaemic – “compensated haemolysis”)

Whats the Spleen job

Discriminating filter that pluck out granular debris and defective membrane, and destroy senescent cells. Macrophages line the cords which filter into the splenic sinuses.

What is removed by macrophages in the spleen AND what feature makes this process more effective

nuclear material (Howell-Jolly bodies)

cytoplasmic organelles,

Siderotic (= iron containing) granules  (Pappenheimer bodies),

oxidised Hb (methaemoglobin, i.e. Heinz bodies)

Reticulocytes are sticky which possibly slow the passage to allow greater time for the macrophages to work

How is oxygen transported from the lungs to respiring tissues

O₂ is reversibly bound to a haem group (= an Fe atom in a porphyrin ring) – a physical, not chemical interaction

Fe atom remains in reduced form. (Fe³⁺ = non-functional methaemoglobin, MetHb).

How is CO2 transported

RBCs associated with 95% of total CO2 transport

25% bind directly to Hb while 70% is converted to bicarbonate ions (HCO3-) via carbonic anhydrase. The remaining 5% is dissolved in plasma.

How does Hb act as a buffer

Hydrogen ions left from dissociation of carbonic acid to bicarbonate bind to globin chains of Hb, preventing acidosis

What is the difference between oxygenation and oxidation

Oxygenation is the PHSYICAL binding of oxygen to haemoglobin whereas oxidation is the CHEMICAL binding which is incompatible for oxygen transport

What is the structure of Haemoglobin

Tetrameric molecule about 68kDa, 4 globin chains of two types in each Hb molecules.

3 different variations of Hb

Adult Hb (HbA) = α₂ß₂            Hb A₂ = α₂δ₂ Fetal Hb (HbF) =  α₂γ₂

Each globin chain surrounds a Haem group which comprises of 1 atom Fe & porphyrin ring C₃₄H₃₂FeN₄O₄ so 4 haem groups in each Hb molecule

Describe oxygen binding with reference to the oxygen dissociation curve

Binding of first oxygen is relatively difficult but after binding there’s a conformational change making the 2nd and 3rd binding a lot easier.

Relatively high affinity of fully oxygenated Hb prevents arterial deoxygenation but after first oxygen molecule is given up the next two are more easily released

What factors affect oxygen affinity

Confirmational change due to binding.

2,3 diphosphoglycerate or 2,3DPG promoting deoxygenation

Acidosis induces Bohr effect

Fetal Hb has increased O2 affinity due to less acitve binding to 2,3 DPG therefore reducing deoxygenation

Potential advantages of artificial RBCs

Storage temperature and shelf-life

Immediate, universal administration

No lag in effectiveness as with natural blood (due to 2,3-DPG and nitric oxide depletion during storage)

No risk of disease transmission

Not dependant on donors

Avoid religious / cultural issues, e.g. Jehovah’s Witnesses

Why can’t Hb be transfusedin solution

Free globin chains are toxic to kidneys, scavenge nitrous oxide leading to vasoconstriction and hypertension, short half life

What are some examples of artificial red blood cells

Re-manufactured” Hb (human or animal) – polymerised bovine globin chains, e.g. Hemopure (licensed in S. Africa in 2001).

Conjugated Hb (e.g. to polyethylene glycol) (Hemospan)

Artificial / bioengineered cells (e.g. Yeasts, E.coli, lipososmes - “neohemocytes”) containing Hb

Blood “pharming” – in vitro differentiation and growth of  stem cells into RBCs. First clinical trial Nov 2022 (UK)

What is Anaemia

Reduced Hb in blood

What are the symptoms of anaemia and when does it typically display symptoms

In general appear when [Hb] falls below 90 – 100 g/l

• Slow-onset anaemia - much better tolerated than rapid

  (as low as 60 g/l in an otherwise well, young patient)

• Shortness of breath

• Weakness

• Pallor

• Lethargy

• Palpitations

• Headaches

• Heart failure and confusion (older patients)

The clinical signs are pallor of mucous membranes and nails bed, depending on the anaemia, concave nails, jaundice, leg ulcers, bone deformities and recurrent infection could occur

How is anaemia classified

2 Parameters: Size (Microcytic, normocytic and macrocytic) and Hb concentration (Hypochromic, normochromic, hyperchromic)

What are the regular parameters for MCHC, MCH and MCV

mcv - 80-101fl

MCHC 300-350 g/l

MCV 27 - 34pg

What is normocytic, normochromic anaemia

MCV normal, MCHC normal, MCH normal but RBC count is reduced. This could be due to an acute bleed, marrow failure, haemolysis or renal failure

What is microcytic, hypochromic anaemia

RBCs reduced in volume and have less Hb. MCV, MCH and MCHC all reduced

Iron deficiency, thalassemia, anaemia of chronic disorder

Macrocytic, normochromic anaemia

RBCs increased in volume Hb at normal conc, RBC count low

MCV increased

MCHC is normal

MCH increased

B12 or folate deficiency

What levels or blood loss cause issues

1L-1.5L ok if lying or sitting but standing may be hard

1.5L - 2L loss, variable loss of consciousness, SOB and sweating

>2L severe shock possibly irreversible and potentially death

Does anaemia set in straight away after an acute bleed

No, as blood cell and fluid are both lost. It may take 2-3 days post-bleed before it becomes anaemia. Reticulocytes takes 3-5 days to respond

What does iron deficiency anaemia look like

Small, pale Red blood cells with some being miss-shapen

How do you treat IDA

Oral administration e.g. ferrous sulphate tablets or parenteral (injected)

Should rise about 10g/l per week and blood transfusion in severe cases

How does B12 or folate deficiency display in blood cells

Macrocytic, normochromic with oval macrocytes and hype segmented neutrophils, where the nucleus is split into clusters

What causes hereditary elliptocytosis

(Spectrin, Glycophorin C or protein 4.1 disorder)

Incidence 1in 3 - 4,000

A splenectomy may be required

This is normocytic and normochromic

What is hereditary spherocytosis

Incidence 1 in 2,000 in North Europeans (but much less common elsewhere)

Spectrin, Ankyrin, band 3 or protein 4.1  disorder

Many are clinically silent

Considerable genetic

   heterogeneity

Increased osmotic fragility

Splenectomy may be required

How can a patient get acquired impairment of erythropoiesis

Bone marrow infiltration - Replacement of erythropoietic tissue by tumour usually from prostate, breast, leukemia or fibrotic tissues

Transient failure from parvovirus or drugs like cytotoxic drugs

How does anaemia due to bone marrow infiltration present

Tear-drop poikilocytes

If NRBCs and immature WBCs are also present, is suggestive of marrow infiltration leading to extra-medullary haemopoesis

What is extra-medullary haemopoesis

Usually associated with severe anaemia due to bone marrow infiltration or fibrosis

haemopoesis outside the bone marrow.

Indicated by:

–Tear-drop poikilocytes

–NRBCs

–Immature WBCs

What are two inherited impairment of erythropoiesis disorders

Fanconi Anaemia. 1 in 160,000 but strong racial association. Progressive BM failure causing death from haemorrhage or infection. Increased risk of solid tumours

Diamond Blackfan anaemia 7 in 1,000,000. Presents in early infancy decrease in erythroid precursors

What are some causes of AIHA

Causes of AIHA:

50% are idiopathic

Lymphoproliferative disorders

Mycoplasma and EBV infection

Drug-induced, e.g. Penicillin

Other autoimmune diseases

What is microangiopathic anaemia

RBC fragmentation within the circulation commonly associated with mechanical heart valves. DIC, HUS, TTP

You can see RBC fragments (schistocytes)

What does a pyruvate kinase deficiency cause

Haemolysis through failure of glycolytic pathway and inadequate ATP you can see Burr Cells produced by cellular dehydration

What does a G6PD deficiency mean

Hb oxidises to MetHb

Challenge by oxidising agent (fava beans, some malaria medication) results in significant haemolysis

Spleen then removes chunks of cells creating keratocytes or bite cells

What are the 2 categories of haemoglobinopathy

Structural variations (>800), where Hb is made in essentially normal amounts but its structure is abnormal

The thalassemia syndromes, in which there is a variable loss of ability to produce a particular type of globin chain.

What are some structural variations of Hb

1.HbS (Sickle cell)   α₂ß₂ ^{6 Glu → Val}

2.HbD Punjab   α₂ß₂ ^{121 Glu→Gln}

3.HbE   α₂ß₂ ^{26 Glu→Lys}

4.HbC   α₂ß₂ ^{6 Glu → Lys}

Alpha

HbG Philadelphia  α₂^68Asp→Lysß₂  

Hb Reading  α₁ or α₂ ^48Leu→Pro ß₂

What are the clinical significance of heterozygote and homozygote expression of Beta chain variation

What is sickle cell disease

HbS is insoluble in deoxy state and forms tactoids distorting RBCs

This can trigger violent haemolytic episodes which causes clotting

How is the frequency of HbS gene maintained in malarial areas

In a balanced polymorphism:

–Heterozygote is more fit than either of the homozygotes

–Hb AA → P. Falciparum (malaria).

–Hb SS → life-limiting haematological disorder

Benefit to heterozygotes is at the expense of homozygotes

–HbS heterozygote has some resistance to Plasmodium falciparum whilst not usually sickling

What are some causes of unstable Hbs

1) abnormality of heme pocket, so heme is not   firmly bound, and water can enter > metHb

2) Interference in binding of α & β chains

3) Interference with α chain structure

Most cases are new mutations with normal parents

What does unstable Hbs cause

Oxidation of heme precipitates and damages the cell membrane the precipitates are Heinz bodies and these are removed by macrophages

What is Thalassemia

A group of inherited RBC disorders characterised by reduced globin chain synthesis (α or β)

Generally prevalent in populations that evolved in warm, humid areas where malaria was endemic, but now affects all races. (Thalassemias provide varying resistance to malaria)

What is α-thalassemia

α-thalassemia – impaired ability to synthesise

  α globin chains

 → excess β chains → β tetramers = Hb H 

  (in fetus, excess γ chains, = Hb Barts )

β tetramers (HbH bodies) aggregate, can be stained by e.g. brilliant cresyl blue

usually a mild, microcytic hypochromic anaemia.

What is β -thalassemia

β -thalassemia – impaired ability to synthesise

   β globin chains.

Mutations in HBB gene on chromosome 11.

Many mutations identified

β^o mutations allow no β chain production, β⁺ allow some

Excess α-chains bind to RBC membrane, damaging it → ineffective erythropoesis & reduced RBC survival

Reduction in available β-chains causes an increase in production of γ and δ chains, so get increased production of HbF (α₂γ₂)  and HbA₂ (α₂δ₂)

What is Casgevy

Casgevy^TM is a gene therapy in which the regulator gene that normally stops HbF production at birth (BCL11A) is knocked out using CRISPR from the patient’s (harvested) stem cells.

The modified stem cells are re-infused and re-colonise in the (prepared) bone marrow, producing HbF-containing RBCs. (As HbF has γ, not β chains it provides viable RBC function and lifespan).

Approved by UK Medicines Regulator. Cost US$2million per patient.