Week 1 - Problem of the Week - Red blood cell, Micro and macrocytic anaemia, Haemolysis (Osmotic fragility test, G6PD enzyme screening activity

Question 1

This normal red blood cell has a smooth biconcave shape. a) What are the functional implications of this shape The nucleus is lost before leaving the bone marrow. This has consequences for the red cell. b) How does the red cell get into the blood?

Answer 1

A) – The biconcave shape of the red blood cell causes maximises the surface area to volume ratio This also allows the membrane to squeeze between capillaries as the membrane is flexible B) The red cell leaves the bone marrow as a reticulocyte and enters the blood

Question 2

d) What are the consequences of not having a nucleus? For a day or two the new red cells look a bit more blue than older ones on the blood film. e) Why?

Answer 2

D) Having no nucleus allows the red blood cell to contain more hemoglobin and, therefore, carry more oxygen molecules. It also allows the cell to have its distinctive bi-concave shape which aids diffusion and therefore maximises the surface area to volume ratio. E) The young red cells look a bit more blue because these are reticulocytes which a large than the erythrocytes and have some RNA which stains blue – these will become erythrocytes

Question 3

The cellular contents are packed full of haemoglobin molecules. These are best when they are bright red. f) Why do haemoglobin molecules change colour?

Answer 3

F) Haemoglobin molecules change colour depending on how much oxygen they are carrying- the more oxygen the redder the molecule. When the iron is oxygenated, it becomes red. When the iron is deoxygenated, it becomes darker red.

Question 4

Each haemoglobin molecule contains four ‘chains’, each made up of three parts: a globin chain, a protoporphyrin ring and iron. g) Explain how each part of haemoglobin contributes to the function of a red blood cell (think what would happen if there were a shortage of each one of the h) Why does the haemoglobin molecule need to be in a red blood cell?

Answer 4

G) The globin chains are necessary in the molecule- the haem group binds to the globin molecule to enable the carrying of oxygen within the iron. Iron is needed as well as propyhrin to make up the haem group – a shortage of any would result in hypochromic microcytic anaemia H) Haemgolbin needs to be in a red blood cell as oxygen binds to this, without haemoglobin oxygen would be unable to circulate in the blood and the person would become hypoxic

Question 5

Case 1 Think of a spherocytic red blood cell whose membrane has been damaged. Its membrane is tight and it has lost its biconcave shape. However, it can manage to struggle through most blood vessels and it has same content of haemoglobin as normal red blood cells. a) Explain the different mechanisms by which the red cell membrane can be damaged?

Answer 5

Red cell membrane can be damaged when there are abnormal antibodies on the membrane - the spleen macrophages attack the antibodies causing the red cell membrane to be damaged and the cells which survive try to repair resulting in spherecotyres - seen in delayed haemolytic transfusion reaction In hereditary spherocytosis - there are molecular defects in the genes that code for the red blood cell protein resulting in abnormal shape of the red blood cell As the spleen normally targets abnormally shaped red cells (which are typically older), it also destroys spherocytes.

Question 6

In examining a blood film , reticulocytes and spherocytes are found. b) What is a reticulocyte? c) What is the significance of their presence in the blood?

Answer 6

Reticulocytes are immature red blood cells (cells that aren’t yet fully developed). Their presence shows that the bone marrow is working to produce more reticulocytes - -usualy seen in states of hypoxia

Question 7

Case 2 Think of a spherocytic red blood cell whose membrane has been damaged. Its membrane is tight and the biconcave shape has been lost. However, it can manage to struggle through most blood vessels and it has the same content of haemoglobin as normal red blood cells. A body is not anaemic despite these problems with the cell membrane. d) Why should this be? e) When would anaemia occur?

Answer 7

The body is not anaemia as the spherocytes have the same haemoglobin content as normal red blood cells The anaemia occurs because the spherocytes is degraded in the spleen or becuase the cells are more likely to lyse resulting in decreased number of red blood cells

Question 8

. Every cell in the body is exposed to free radicals and oxidative damage every day. a) Why are red blood cells more vulnerable to oxidative damage? b) What are the sources of energy and reducing powers which protect the red blood cell against oxidative damage?

Answer 8

A) Detoxification of H2O2, all relies upon the one enzyme, glutathionine, without production of this enzyme, more likely to get oxidation damage Embden Myerhof mathway - produced ATP (energy) and NADH - needed to reduce Fe3+ to Fe2+ Hexose Monophospahte shunt - glucose-6-phosphate dehydroxygenase oxidesed G6P to hexose phosphates and reduces NADP+ to NADPH = the NADPH can now mae glutathionine to protect against oxidative damage

Question 9

Consider a red blood cell with markedly reduced protection from oxidative damage. c) Describe how this could have arisen?

Answer 9

This could have arisen due to a gluocse-6-phosphate dehydrogenase deficiency There therefore wouldnt be NADPH to reduce GSSG to glutathionine This would result in the build up of free oxidative species that can give free radicals to cause oxidative damage to red blood cells

Question 10

The body is very vulnerable taking drugs that can induce oxidative stress such as dapsone. d) What would be the potential clinical manifestation of this?

Answer 10

The most prominent side-effects of this drug are dose-related hemolysis (which may lead to hemolytic anemia) and methemoglobinemia. Person would be very hypoxic with likely jaundice

Question 11

Hypochormic microcytic anaemia Here is a common problem with red blood cells. They can be seen on a blood film as small and stiff. They do not manage through the capillaries very well. They all look pale and pasty and have so few haemoglobin molecules. a) Why are they smaller than a normal red blood cell?

Answer 11

They are smaller than a normal red blood cell because there is a reduced haemoglobin in the cell - therefore the cell is smaller

Question 12

They don’t have enough haemoglobin molecules. b) Why might this be? c) What is going to happen to the patient as a consequence of failure of haemoglobin synthesis?

Answer 12

RBCs may not have enough haemoglobin in this case (hypochromic microcytic anaemia) due to haemaglobinsation defects Usuaully due to cytoplasmic defects when there is a deficiency in the constituents of Hb (iron, porphyrin, globin) c) as a consequence the patient will become anaemic - fatigue, paleness, dizzy, leg cramps

Question 13

d) How might the red cell try & improve oxygen delivery?

Answer 13

d) to improve oxygen delivery, kidney will sense hypoxa and therefore EPO will production will increase stimualting the production of more red blood cells to meet the oxygen demand

Question 14

Case 3 Hypochromic Microcytic Red Blood Cell It is difficult to make haemoglobin if there is a deficiency of β-globin chains. Every red blood cell in this patient has this problem and it’s been that way since birth a) Why is there this difficulty? b) How does it affect the patient?

Answer 14

This seems like the condition beta-thalassemia The condition is due to a point mutation in the beta globulin genes on chromosome 11 therefore causing reduced(B+) or absent(B0) B globulin from the parents B-thal trait - only one parent affected (B+/B or B0/B) - usually asympomatic - increased HbA2 B-thal intermedia - both parents affected (B+/B+ or B+/B0) - may require trnasfusion, low HbA B-thal major - no B globulins - no HbA - regular transfusions

Question 15

Case 4 Hypochromic Microcytic Red Blood Cell The reason for inability to synthesise haemoglobin is that there isn’t enough iron. a) Why might this problem happen? These red cells are from a patient who, unlike in Case 1, can do something about this problem. b) What questions should you ask the patient?

Answer 15

Insufficient iron from the diet Achlorydia - absence of acid secretions from stomach - required for iron absorption Menorrhagia or GI Bleeding Malabsorption B) ask patient about their diet, whether they have any conditions affecting their GI, whether they have heavy periods (if female) or have bled a lot recently

Question 16

Station 4: The Macrocytic Red Blood Cell This macrocytic red blood cell is in a patient who has megaloblastic anaemia due to cobalamin (Vit.B12) deficiency. a) What is the effect of B12 deficiency on all the cells of the body?

Answer 16

B12 is required for DNA and RNA synthesis of all blood cells therefore a deficiency can potentially result in pancytopenia

Question 17

Red cells, as you know, are produced in the bone marrow along with some other cells. b) What are the other blood cells produced in the bone marrow? c) Why are blood cells often the first to be affected when there’s no Vitamin B12 around?

Answer 17

b) - Platelets and leucocytes c) - unsure lol

Question 18

This red cell is bigger than normal red blood cell and has got loads of haemoglobin on board. d) Why are the cells big? e) Why is there no shortage of haemoglobin in the cells? f) Why then is the patient so anaemic?

Answer 18

D) the cells are so big because there is a failure of the red blood cells to become smaller during erythropoeisis - therefore macrocytotic There is no shortage in Hb of these cells because all the Hb constituents are present - iron, prophyrin, globin - no cytoplasmic defects, only nuclear F) - the patient is anaemic because there are simply less of the red blood cells as there is increased apoptosis of the abnormal cells

Question 19

A patient is feeling the effects of his megloblastic anaemia. g) What symptoms might he present to his doctor with?

Answer 19

Glossitis, angular cheleitis, a pale yellow tinge to your skin Neurological symptoms (eg sensory loss or motor control loss - subacute combined degeneration of the spinal cord) Symptoms of anaemia - extreme tiredness (fatigue) 1. lack of energy (lethargy) 2. breathlessness 3. feeling faint 4. headaches 5. pale skin

Question 20

Anaemias can be classified functionally as: Hypoproliferative: bone marrow contains an inadequate number of red cell precursors (erythroblasts). Maturation abnormalities: despite normal or increased numbers of bone marrow erythroblasts, these produce a reduced number of normal red cells, i.e. erythropoiesis is ineffective. Haemolytic/haemorrhagic: shortened red cell survival What are the main causes of hyproliferation?

Answer 20

INflammation Iron deficiency anaemia Acute bleeding Bone disorders

Question 21

Sometimes a bone marrow test is needed for diagnosis of the cause of anaemia What is usually measured to determine whether this is necessary?

Answer 21

Measure the reticulocyte count When reticulocytes are high, this is unlikely to help in diagnosis, since the marrow is clearly capable of producing red cells, but these are not surviving normally in circulation. When reticuloytes are low, the bone marrow biopsy may be needed for identification of the cause of anaemia

Question 22

What are reticulocytes? Reticlocytes cotain RNA/ribosomes which are precipitated in a blue-staining reticular pattern on incubation with what type of dye?

Answer 22

These are young immature red blood cells having just been formed by extrusion of the eryrhtoblast nucleus They are seen to contain RNA/ribosomes precipitated in a blue-staining reticular pattern on incubation with brilliant cresyl blue dye

Question 23

Reticulocytes lose their residual RNA during the first 1-2 days after their release from the bone marrow into the circulating blood Therefore what percentage of circulating blood is reticulocytes?

Answer 23

Since normal red cell lifespan is around 120 days,approximately 1% of circulating red cells are normally reticulocytes.

Question 24

Reticulocytes are increased in the blood in proportion to the amount of erythropoiesis (red cell production) in the bone marrow Provide a convenient way to assess whether red cell production in the bone marrow is appropriate to the degree of anaemia (and the associated increase in erythropoietin ‘drive’). What is the stain used to count the number of reticulocytes present in the blood?

Answer 24

Brilliant cresyl blue staining

Question 25

Reticulocyte counts are useful in distinguishing the two basic causes of anaemia. When there is decreased production of haemoglobin or red cells - IDA, B12/folate, aplastic anaemias - this all means low retic count or When there is premature loss or destruction - haemorrhage, haemolysis - this means high retic count Symtpoms of IDA?

Answer 25

Koilonychia - spooning of nails Angular chelitis/stomatitis Anaemia symptoms

Question 26

When sickle cell anaemia is present, where is the pain most commonly? What haemaglobinopathy does this child have?

Answer 26

Bone and abdomen (Spleen) as these have very small arteries and therefore infarcts are prevalent This child has beta thalassemia major - can see the extramedullary haematopoesis due to increased maxillary size

Question 27

Causes of Haemolysis Hereditary * Enzymopathic * Membrane disorder * Globin disorder Acquired * Immune * Non-immune Name on enzyme disorder causing haemolysis? What would be seen on the blood film?

Answer 27

Enzymopathic Glucose-6-phosphate dehydrogenase deficiency Bite cells seen on blood film - removal of Heinz bodies from the spleen Membrane disorder Hereditary spherocytosis - small spherical cells with less haemoglobin -

Question 28

Name one membrane disorder cuasing haemolysis? What would be seen on blood screen? Are they coombs negative or posiive? What happens to the retic count in haemolytic disorders?

Answer 28

Hereditary spherocytosis - the deformed RBs are trapped in spllen leading to extravascular hamolysis Increased number of small spherical cells on blood film All membrane defects are Coombs negative as no antibodies present Retic count goes up in haemolytic disorders

Question 29

Diagnostic laboratory tests in haemolysis * Direct Coombs test -when is this used? * Osmotic fragility test - when is this used and what happens here? * G-6-PD enzyme activity screening test - when is this used? WHat is the expected enzyme activity in someone with G6PDD?

Answer 29

Direct Coombs Test - detects antibody coated red cells using antisera to immunoglobulins; end point is red cell agglutination - Used for immune mediated haemolysis Osmotic Fragility Test - incubate red cells in increasingly hypotonic solutions and measure release of haemoglobin from lysed cells. Increased fragility in hereditary spherocytosis. (spherocytes - very cytolytic) Quantitate fluorescence of NADPH generation by G-6-PD. Detects enzyme activity <20% in G6PD Deficiency

Question 30

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Answer 30

The red cells are large because he has megaloblasts - abnormally large red cell precurosr with an immature nucleus - the formation of these is due to abnromalities of the DNA synthesis of the RBC The man has macrocytic anaemia due to megalobast formation - this is due to abnormality of the DNA synthesis MAcocytosis causes - cytotoxic drugs, B12 and folate deficinecy , alcohol and bone marrow conditions Hypothyoridism can also cause macorcytic anaemia - but is also linked to pernicious anaemia (B12 deficiency)

Question 31

A detailed lifestyle history should be sought; why? (what is the commonest cause of macrocytosis) ● What is the relevance of the low serum folate concentration? ● Is this the reason for the macrocytosis?

Answer 31

Excess alcohol ingestion is the commonest cause of macrocytosis - find out the patients alcohol intake , any bleeding as well or weight loss - for cancer The low serum folate could be simply due to him eating little for several weeks - folate stores only last 4 months Liely the reason for his macrocytosis may be of vitB12 deficiency as penricious anaemia is linked to hypothyroidism or of something more sinister (GI bleed causes)

Question 32

Clinical Problem - Six months later he re-attends feeling tired after his GP notes a haemoglobin concentration of 80 g/L and Mean Cell Volume of 70 fl. Ferritin is 9 μg/l, transferrin saturation 1%. He is commenced on oral Ferrous Sulphate but fails to attend for an upper GI endoscopy as he has no other symptoms and his tiredness has improved.● Why are the red cells small? ● Are microcytic anaemias abnormalities of DNA synthesis or haemo-globin synthesis?

Answer 32

Red cells are small because it is likely that Mr Jones has iron deficinecy anaemia This is an abnormality of haemo-globin synthesis and therefore not enough Hb causes the cells to become microcytic Once the iron stores are depleted, the patient also becomes anaemic as the cells cannot further eyrhtopoeies - decreased RBC, decreased MCV and MCH

Question 33

● What are the best tests for diagnosing iron deficiency? ● What are the common causes of iron deficiency? ● How is iron deficiency investigated? (what is performed in symptomless indivduals with iron deficiency anaemia)

Answer 33

Measure serum ferritin - best test - measures the storage iron levels. Can measure transferrin (total iron binding capacity) - this measures the transported iron Common causes - menstrual bleeding and GI bleeding are the most common causes Can be caused by diet, malabsoprtion as well Investigations to identify the cause of iron deficiency are directed by symptoms, in symptomless individuals upper GI endoscopy followed by barium enema should be performed.

Question 34

Clinical Problem Twelve months later he is brought in as an emergency having collapsed in the pub. On admission he is pale with a blood pressure of 85/40 and tachycardia (150 bpm). Abdominal examination reveals : epigastric tenderness. Haemoglobin is 110 g/L, MCV 95fl. Reticulocyte count is 2 %. Intravenous access is established and synthetic colloid replacement commenced. ● What does the reticulocyte count tell you about red cell production?

Answer 34

Usually reticulocyte count is about 1% of the total RBC volume. As it is 2% in this patient, this implies that the patient is probably haemolytic or hameorrhic (more likely in this case) Reticulocytosis (associated with increased erythropoietin in iron replete (abundantly supplied) bleeding patients) becomes manifest only at 1-2 days after an acute bleed.

Question 35

● What are the signs of the shocked patient? What are the common causes of shock?

Answer 35

He is hypotenisve, tachycardic and anaemic Commonly caused by GI bleeding Also septic, cardiogenic, neurgoenic (this person has hypovalaemic) and anaphylactic

Question 36

Why might haemoglobin not be accurate in acute shock?

Answer 36

In acute bleeding the Hb concentration may underestimate the extent of blood loss until redistribution of body water has allowed dilution of the remaining red cells. This is because the loss of blood is proportional to loss of plasma - therefore Hb conc stays near about the same, but if total blood volume was full - the Hb would be a lot more decreased

Question 37

Clinical Problem Blood is sent for cross-match and red cell transfusion commenced later by which time his blood pressure has stabilised. Ten minutes into the blood transfusion, he is noted to be pyrexial (37.80C). The transfusion rate is slowed and the temperature settles. * How is blood “cross-matched”?

Answer 37

Blood is cross matched to test for ABO incompatibility, RhD protein and irregular antibodies To test for ABO - add anti-A, anti-B and anti-AB antibodies to the patients red cells - looks for agglutination - Direct Coombs test Prove the forward result by adding the patients plasma to red cells of a known group to see if the cells agglutinate Same direct coombs for rhesus disease To test for irregualr antibodies, indirect coombs testing

Question 38

Why is indirect coombs testing used to detect for irregular antibodies?

Answer 38

For ABO incompatibility and rhesus status - IgM is the immunoglobulin that binds here and this will crosslink the red blood cells, Therefore addition of the antibody will cause IgM to agglutinate all the cells For irregular antibodies- IgG is the immunoglobulin here and this is unable to cross-link cells, therefore after we have allowed the antibody to mix with the cells, we first must wash out any unbound antibody before adding anti-human immunoglobulina antibody (anti-human IgG antibody) which binds to the IgG and cross links to cause agglutination

Question 39

Clinical Problem Blood is sent for cross-match and red cell transfusion commenced later by which time his blood pressure has stabilised. Ten minutes into the blood transfusion, he is noted to be pyrexial (37.80C). The transfusion rate is slowed and the temperature settles. What are complications of the blood transfusion?

Answer 39

Febrile nonhemolytic transfusion reactions are due to white cell antibodies or hypersensitivity to donor plasma proteins. - seems this has occurred in the patient Can give paracetamol and continue slowly This transfusion reaction can often be confused with immediate haemolytic transfusion reactiojn _ STOP IMMEDIATELY - much more worrying