Week 1 - C - Intro to anaemia & microcytic anaemia - Iron deficiency, Sideroblastic anaemia, Thalassemia, , Automated Analyser results

Question 1

What is the definition of anaemia?

Answer 1

This is a reduced total red blood cell count so that there is insufficient haemoglobin to deliver oxygen to the cells Anaemia is due to a ‘spurious or relative’ low blood count when there is too much plasma

Question 2

Anaemia can be defined as a haemoglobin (Hb) level of less than: what? Reference ranges for normal Hb can vary from lab to lab Anaemia can also be defined in terms of a reduction in red blood cell count (RBCs) and haematocrit (Hct). What is anaemia based on the haemtocrit?

Answer 2

Anaemia is defined as a Hb level of less than * 130g/l in men or * 115g/l in women Anaemia is also based on the haematocrit * Men

Question 3

Where does red blood cell production take place?

Answer 3

Red blood cell production - eryhtropoiesis takes place in the bone marrow

Question 4

The haemaglobin levels and the haemtocrit levels are measured in different ways Spectrophotometry is a tool that hinges on the quantitative analysis of molecules depending on how much light is absorbed by colored compounds. Haemoglobin is measured using a spectrophotomeric method How is haemoglobin concentration measured?

Answer 4

The red blood cell is burst (lyse) to create haemoglobin solution which is stabilised The optical density of the Hb is then measured d at 540nm and the optical density is proportional to the Hb coencentration using a certain law The Hb concentration is then calculated against known reference standard cyan-metHb concentration solution

Question 5

What is the law that allows for the calculation of the haemoglobin concentration from measuring the optical density of the haemoglobin?

Answer 5

This is Beer’s law

• Burst (lyse) the red cells to create Hb solution
• Stabilise the Hb molecules (cyan-metHb)‏
• Measure the optical density (OD) at 540nm

OD Proportional to the concentration (Beer’s Law)

Question 6

What is the method known as that measures Hb cocnentration?

Answer 6

Spectrophotometry - uses how much light is absorbed by the compound to measure its concentration Have to first lyse the red blood cell to get Hb solution and then stabilise the solution before measuring its optical density and using Beer’s law to work out its concentration

Question 7

Hametocrit is a Ratio (or commonly expressed as the percentage) of the whole blood that is red cells if the sample was left to settle How do modern machines measure haemtocrit?

Answer 7

The calculate this value by adding the calculated volume of the red cells it counts compared to the total volume of blood

Question 8

In rare situations Hb/hct are not a good marker of anaemia What are 2 cases where this is true?

Answer 8

* 1st example - When somebody becomes acutely unwell due to an arterial bleed - results in the total blood volume decreasing but the Hb & Hct concentration stay the same because they are relative to the total blood volume. Cant tell they are anaemic until given fluids (saline) * 2nd example - haemodiltuion - when the plasma volume increases therefore increasing the total blood volume but therefore decreasing the Hb and Hct concentration - example is during pregnancy

Question 9

1st example - When somebody becomes acutely unwell due to an arterial bleed - results in the total blood volume decreasing but the Hb & Hct concentration stay the same because they are relative to the total blood volume. Cant tell they are anaemic until given fluids (saline) 2nd example - haemodiltuion - when the plasma volume increases therefore increasing the total blood volume but therefore decreasing the Hb and Hct concentration - example is during pregnancy Basically, which is a false negative / positive for anaemia?

Answer 9

* 1st example where the blood is acutely lost and the Hb/Hct measurements remain normal would be a false negative for anaemia * 2nd example where there is haemodilution due to plasma expansion for example in pregnancy would be a false positive for anaemia

Question 10

What is the bodies response to anaemia? (acutely)

Answer 10

There is an icnreased red cell production and therefore the reticulocyte count will increase acutely to try and restore normla blood levels

Question 11

Describe reticulocytes? How would the blood film be described if seeing reticulocytes?

Answer 11

Reticulocytes are red cells that have just left the bone marrow They are anucleated but have a smalll amount of RNA and therefore stain purple/deeper red - They are larger than average red cells (erythrocytes) Blood film would be described as polychromatic (multiple colours)

Question 12

When there is upregulation of reticulocytes by the bone marrow (usually in response to anaemia), how long does this acute upregulation take? Do reticulocytes have the biconcave appearance of erythrocytes? Describe the diameters of the erythrocyte?

Answer 12

This usually takes a few days They do not have the biconcave appearances Diameters of erythrcoyte - 8 nanometers in width 2 nanometers thickness at edge 1 nanometer thickness at centre

Question 13

These are far quicker than carrying out spectrophotometry to measure Hb and is also used to calculate Hct What are the three things that automated analysers measure?

Answer 13

Automated analysers measure * The haemoglobin concetration * The number of red cells (giving a concentration) * The size of the red cells (noted as mean cell volume or MCV)

Question 14

What is it that automated analysers measure again?

Answer 14

Automated analysers measure * Haemoglobin concentration * The number of red blood cells (concentration) * The size of the red blood cells (Mean cell volume)

Question 15

Measured: Haemoglobin concentration The number of red blood cells (concentration) The size of the red blood cells (Mean cell volume) How can the automated analysers be used to identify the : haemtocrit, Mean cell haemoglobin (MCH) and Mean cell haemoglobin concentration (MCC)?

Answer 15

* Haemotcrit - have the number of red cells and the mean cell volume - therefore can work out the total volume of red cells in the total blood volume * Mean Cell Haemoglobin from the number of red cells (concentration) and the Hb concentration * Mean Cell haemoglobin Concentration - can use the haemoglobin concentration, the number of cells to get MCH, then if you know the haemoglobin in the cell, can get concentration from dividing this by the size (MCV)

Question 16

To assess anaemia We can also look at the: * Blood film * look at cellular morphology Reticulocyte count * assess marrow response To classify the cause of anaemia we can look at the pathophysiology of anaemia or the morphological characterisitcs Broadly, what are the two causes of anaemia?

Answer 16

Decreased production of the red blood cells or Increased destruction of the cells

Question 17

What is the difference in the reticular cell count in decreased production and increased destruction of the cells?

Answer 17

In decreased production of the cells - the reticulocyte cell count is decreased also In increased destruction - the reticulocyte cell count is increased acutely as an attempt to defend against the reducing red cell count

Question 18

Decreased production can either be due to hypoproliferation where there is a reduced amount of erythropoeisis or Maturation abnormality where there erythropoiesis is abnomal What are the two causes of maturation abnormalities?

Answer 18

Can have this due to cytoplasmic defects - impaired haemaglobinisation - where the formation of haemaglobin is impaired or Nuclear defects - where there is impaired cell division

Question 19

Does cytoplasmic maturation defects typically occur in micro or macrocytic anaemia? Does nuclear defect causing impaired cell division typically occur in micro or macrocytic anaemia?

Answer 19

Cytoplasmic defects - impaired haemaglobinisation typically occurs in microcytic anaemia - the there is a reduced red blood cell size but decreased number Nuclear defects - impaired nuclear cell division results in abnomrally large but low numbers of red blood cells - there is an increased red blood cell size but decreased number

Question 20

Increased blood loss or destruction of red cells (high reticulocyte count)‏ Caused by : Bleeding or haemolysis How do these cause an increased reticulocyte cell count? Name a condition that causes excessive haemolysis?

Answer 20

Bleeding or haemolysis - can cause the bone marrow to have erythroid hyperplasia where there are more red blood cells produced to combat the hypoxia caused by the anaemia resulting in an increased reticulocyte cell count * A condition that causes excessive haemolysis - glucose 6 phosphate dehydrogenase deficienxy * This leads to a decreased NADPH & therefore reduced glutathionine which results in an increased reactive oxygen species and free radicals which damage cells

Question 21

What type of anaemia would increased blood loss o destruction of red blood cells result in?

Answer 21

This would result in something known as normocytic anaemia - the red cells are a normal size, their just isn’t enough

Question 22

A useful way for understanding anaemia but limitations for establishing a cause for anaemia in an unknown patient

Practical classification based on cell size and Hb count is more useful

What is the useful way for telling nuclear from cytpolasmic defects?

Answer 22

This would be to measure the mean cell volume (size of the red blood cells in the automated analyser)

Question 23

What type of anaemia do low and high mean cell volumes relate to?

Answer 23

Low mean cell volume - this relates to mcirocytic anaemias - consider problems with haemaglobinisation High mean cell volumes - this relates to macrocytic anaemias - consider problems with maturation due to nuclear defects

Question 24

Haemoglobin synthesis occurs in the cytoplasm - defects result in small cells What makes up the haem group? What enzyme catalyses this?

Answer 24

Fe2+ and protoporphyrin IX are converted to haem by the enzyme ferrocheletase Haem is made up from iron and a prophyrin ring and then bind with one globin per haem molecule to form haemoglobin

Question 25

Hb is synthesised in the cytoplasm To make Hb need * Globins * Haem * Porphyrin ring * Iron (Fe 2+)‏ Shortage in these results in small red cells with a low hb content How are the small red cells with the low Hb count described under the microscope?

Answer 25

The small red cells would be described as microcytic (small cells) and Hypochromic - reduced colour as they do not have as much haemoglobin

Question 26

How is hypochromia able to be measured from the automated analysers?

Answer 26

Hypochromia can be measured form the mean cell haemoglobin - low haemoglobin will mean less coloured cell

Question 27

Hypochromic, microcytic anaemias = Deficient haemoglobin synthesis: cytoplasmic defect Hypochromic microcytic anaemias can be down to haem deficiencies or globin deficiens What is the most common cause of micocytic anaemias?

Answer 27

The most common cause is due to iron deficiency anaemia

Question 28

Name other causes of microcytic anaemias? Give at least one globin cause

Answer 28

Microcytic anaemias - Iron deficiency anaemia as said is the most common Can be caused due to some cases of chronic disease Problems with porphyrin synthesis Globin defieincy - thalassemia (no production of one or more globin chains)

Question 29

Congenital sideroblastic anaemia is a very very rare cause of anaemia in which the bone marrow produces ringed sideroblasts rather than healthy red blood cells (erythrocytes) This is where the body has a microcytic anaemia with a normal or raised iron Sideroblastic anaemia is a type of porphryia in which the red cells and haemgolobin is decreased WHat causes sideroblastic anaemia?

Answer 29

In sideroblastic anemia, the body has iron available but cannot incorporate it into hemoglobin, which red blood cells need in order to transport oxygen efficiently. and therefore the body is not iron deficient but the red cells are microcytic It is usually x-linked and x-linked sideroblastic anaemia is caused by a mutation in ALA synthase in the x chromosome

Question 30

Sideroblastic anaemia should be suspected whenever a microcytic anaemia is not responding to iron Look at the feritin to see iron availability and look at a fillm and a marrow * What should be seen on the film? * WHat should be seen in the marrow? What stain is used? Sideroblasts are atypical, abnormal nucleated erythroblasts (precursors to mature red blood cells) with granules of iron accumulated in the mitochondria surrounding the nucleus.

Answer 30

• In the film - can see hypochromasia due to there not being haemoglobin pigment
• Ringed sideroblasts are seen in the bone marrow
• Ring sideroblasts are named so because iron-laden mitochondria form a ring around the nucleus.
• Diagnosed using a Prussian blue stain

Question 31

What is the treatment options for sideroblastic anaemia? (remember iron has already failed as the cells cannot incorporate iron into the haem molecule) What is the enzyme if sideroblastic anaemia?

Answer 31

The enzyme involved in sideroblastic anaemia is ALA synthase - this enzyme is mutation in this X-linked sideroblastic anaemia resulting in anaemia The enzyme ALA synthase is involved in the making up pophryin and therefore this is why the pophryin cannot bind to Iron Treatment option - would be to give pyridoxine (vitamin b6) or repeated transfusion if severe anaemia

Question 32

WHat are the microcytic anaemias again? Find The Small Cell What are the macrocytic? Cant Be Fucked

Answer 32

Microcytic- proprhyrin defects can also cause it * Fe2+ deficiency * Thalasemmia * Sideroblastic anaemia - this is a porphyrin defect * anaemia of Chronic disease Macrocytic * Cytotoxic drugs eg methotrexate * B12 deficiency * Folate deficiency

Question 33

Can exist in Fe2+ or Fe3+ state Iron is essential * Oxygen transport * Hb, myoglobin * Electron transport * Mitochondrial production of ATP What is the pathway in which iron is prevented from turning into its Fe3+ state? Is Fe2+ ferric or ferrous?

Answer 33

This is the Embden-Myerhof pathway which produces ATP and NADH from anaerobic respiration (glycolysis) The NADH reduces iron from Fe3+ to Fe2= Fe2+ = ferrous - transported iron (transferrin bound) Fe3+ = ferric - stored iron (ferritin bound)

Question 34

Where is the bulk of iron in the body present?

Answer 34

The bulk of iron in the body is present in the haemoglobin

Question 35

When fully saturated, one gram of haemoglobin will bind to what volume of oxygen? We don’t generally lose much iron so therefore dont absorb vast amounts (only really women who lose iron) How much iron do we absorb per day?

Answer 35

When fully saturated, 1g of Hb will carry 1.34mls O2 We absorb roughly 1mg of iron per day

Question 36

An average human holds 5 litres of blood in the body What is the level of iron on average in the blood?

Answer 36

On average there is 2500mg iron in the blood continuously A rule of thumb, there is half the iron to blood - eg 2500 mg of iron in 5000mls (humans have bout 5 litres of blood) So say you lose, 100ml of blood, that is the equivalent of losing 50mg iron

Question 37

Where is most of the iron stored and what is it bound to when stored? What does iron have to bind to when transported?

Answer 37

Most of the iron is in Hb Most of it is stored in the liver and is bound to ferritin when stored Iron has to be bound to transferrin when transported

Question 38

Haemoglobin is a good way of assessing the concentration of iron in the blood - this is the functional iron How is the serum and storage iron measured?

Answer 38

Serum iron is measured by measuring transferrin (also known as the total iron binding capacity (TIBC) ) Storage iron is measured by measuring the serum ferritin

Question 39

Protein with two binding sites for iron atoms Transports iron from donor tissues (macrophages, intestinal cells and hepatocytes) to tissues expressing transferrin receptors (especially erythroid marrow) Why is serum transferrin raised in iron deficiency anaemia? (ie why is the total iron binding capacity raised in IDA) What happens to the %saturation of transferrin with iron measures?

Answer 39

There will be increased transferrin in IDA as this will be an attempt to transfer more iron and as there is deficient iron, the transferrin saturation will therefore be very low

Question 40

% saturation of transferrin with iron measures iron supply reduced in iron deficiency reduced in anaemia of chronic disease When will the saturation be increased above normal?

Answer 40

In conditions where there is too much iron eg haemachromatosis

Question 41

Large intracellular protein Spherical protein stores up to 4000 ferric ions Tiny amount of ferritin present in serum reflects intracellular ferritin synthesis in response to iron status of the host What does serum ferritin tell us?

Answer 41

Serum ferritin tells us the measure of iron storage A low serum ferritin means there is an iron deficiency

Question 42

When an iron deficiency anaemia is suspected from a blood test revealing a low Hb, low mean cell volume (microcytic anaemia) in eg a female with menorrhagia What would be carried out to confirm the diagnosis?

Answer 42

Measure the serum ferritin to measure the iron stores (could also measure total iron binding capacity (TIBC = transferrin measurment) or the % transferrin saturation)

Question 43

Sometime there can be a high ferritin, any examples of why?

Answer 43

If we get an infection and want to get rid of it, we can get rid of iron as the infection requires this making our bodies an unfavourable environment – therefore ferritin is an acute phase protein as it will increase in concentration to mop up more iron (ferritin might still be high but might still be iron deficient)

Question 44

What are causes of iron deficiency anaemia?

Answer 44

Women of child bearing age and children are going to require more iron Not eating enough iron - eg vegetarians or vegans Losing too much eg menses or GI loss Not absorbing enough iron - eg malabsorptive conditions

Question 45

If male or female greater than 55 with iron deficiency anaemia, what would be at the top of the differentials? What are conditions that can cause malabsorption of iron? Where in the bowel is iron absorbed?

Answer 45

If greater than 55 with IDA - then think carcinoma - bowel cancer Conditions that can cause malabsorption eg crohn’s, ceoliacs, achlrhydia iron is absorbed in the proximal small bowel

Question 46

What is achlorydia?

Answer 46

Achloryida is the absence of hydrochloric acid in the gastric secretions. Iron requires acidic secretions to be absorbed If the MCV volume is low and there is a good history of menorrhagia in a women, further tests may not be required and oral iron supplements can be started

Question 47

Identifying iron deficiency is not sufficient Why are they iron deficient? * Diet? Malabsorption? Blood loss? Causes of chronic blood loss * Menorrhagia or Haematuria * Gastrointestinal * Tumours * Ulcers * Non-steroidal anti-inflammatory agents What is the average menstrual blood loss &what is heavy?

Answer 47

Averga emenstrual blood loss is 30-40mls Heavy menstruation would be losing roughly 60mls of blood

Question 48

How much iron would be lost if 30mls of blood was lost during menstruation? (roughly) And if 60mls if heavy?

Answer 48

Due to iron being about half of the blood content (ie 2500mg iron in blood, total blood volume is 5000ml) Then losing 30ml blood would equate roughly to 15mg iron Losing 60ml blood would equate to roughly 30mg iron As we only absorb 1mg iron per day (30mg./month) then losing 30mg during menses can cause iron deficiency anaemia

Question 49

What are symptoms of iron deficiency anaemia?

Answer 49

Angular chelieits Glossits Koilonychia Fatigue Paleness

Question 50

Iron deficiency anaemia Symptom not diagnosis Iron replacement therapy may relieve symptom without treating the underlying problem Investigations essential to identify diagnosis Early surgery of GI tumours may be curative What is the treatment for IDA? (name the drugs)

Answer 50

1st line - oral iron supplements (ferric fumarate, ferric sulphate, ferric gluconate) 2nd line - IV iron replacement (ferric carboxymaltose) 3rd line if severe -red cell transfusion

Question 51

What are side effects of iron replacement? What can be taken with iron supplements to help absorption? How long is therapy with iron continued for?

Answer 51

Side effects - constipation, black stools, nausea , abdo discomfort Can take abscorbic acid (vit C - eg orange juice) with supplements to help absorption Continue therapy until Hb returns to normal and the for 3 months after to replete stores

Question 52

How fast should the haemoglobin levels rise if on iron?

Answer 52

They should rise by 10g/l per week with a modest reticulocytosis

Question 53

If suspecting a GI cause of IDA Iron deficiency anaemia is a symptom and requires investigation for an underlying cause Can do gastroscopy, upper or lower GI endoscopy, H.pyori test (stool antigen and then urea breath test) What is treatment for H.pylori infection? What are the ALARM symtpoms for an upper GI malignancy in patients >55?

Answer 53

Hpylori infectin - lanzoprazole, amxocillin, clarithromycin ALARM * Anaemia * Loss of weight * Anorexia * Recent onset * Malaena * Swallowing difficulty