Microcytic and Macrocytic Anaemia

Question 1

Describe anaemia and give the normal reference ranges for RBC’s.

Answer 1

○ Caused by decrease production or increased destruction of RBC’s, or by blood loss.

○ Reference ranges:
Male - 13.3-16.7g/dL
Female - 11.8-14.8g/dL

Question 2

Describe symptoms of anaemia.

Answer 2

○Yellowing of skin - due to breakdown of RBC’s.
○Shortness of breath - due to inability of blood to supply adequate oxygen to tissues.
○Fatigue - decreased oxygen carrying capacity.
○Pallor - oxygenated blood appears red, but due to decreased Hb blood is less oxygenated.
○Splenomegaly - due to excess breakdown of RBC’s in haemolytic anaemia, and breakdown of Hb into bilirubin.

Question 3

What are the normal ranges for FBC?

Answer 3

○MCV - 80-95fL
○MCH - 27-34pg
○Erythrocyte count - 4.5-6.5 x10^12/L (males), 115-155 x10^12/L (females)

Question 4

Describe the classification of anaemia.

Answer 4

Microcytic, hypochromic anaemia:
○MCV <80fl
○MCH <27pg
○Caused by iron deficiency or thalassaemia.

Normocytic, normochromic anaemia:
○MCV 80-95fl
○MCH >=27pg
○Caused by haemolytic anaemia.

Macrocytic anaemia:
○MCV >95fl
○Caused by vitamin B12 or folate deficiency.

Question 5

Describe the FBC for anaemia.

Answer 5

○Leucocyte and platelet count to differentiate between anaemia and pancytopenia.

○High reticulocyte count - compensatory mechanism, where bone marrow releases immature erythrocytes to compensate for low oxygen levels.
○Healthy individuals - 0.5-1.5%
○Anaemia - >2.5%

Question 6

What can be seen in the blood film of patients with anaemia?

Answer 6

○Anisocytosis - abnormal RBC size.
○Poikilocytosis - abnormal RBC shape.
○Target cell
○Pencil cell - RBC cannot carry oxygen due to size and shape.
○Spherocyte - RBC becomes too round which causes vessel blockage.
○Basket cell
○Normoblast - nucleated RBC.
○Basophilic stippling - presence of many basophilic granules.

Question 7

Describe iron cycling.

Answer 7

○ Iron is ingested by food and absorbed by intestines, but most iron is reabsorbed from jejunum and duodenum.
○ Iron is transported by transferrin, and delivered to erythroblasts in bone marrow, which incorporate iron into Hb.
○ Breakdown of RBC’s after 120 days by macrophages of reticuloendothelial system, releasing iron from Hb into plasma.
○ Macrophages store some iron as ferritin & haemocydorin, depending on overall body iron level.

Question 8

Describe iron uptake, storage, and utilisation.

Answer 8

○ Iron can be absorbed as haem iron or non-haem iron.
○ Haem iron is absorbed by duodenal enterocyte, where it’s degraded to release iron.
○ Inorganic iron is converted from ferric state (Fe3+) to ferrous state (Fe2+), facilitated by ferrireductase.
○ Divalum metal transporter (DMT1) readily absorbs ferrous iron into enterocyte, where it’s combined with apoferritin to form ferritin.
○ Ferroportin delivers iron to circulation, which is regulated by hepcidin.

Question 9

Describe some lab tests performed to determine microcytic anaemia.

Answer 9

○FBC
○Blood film
○Serum ferritin
○Serum iron

○Total iron binding capacity (TIBC):
○How much iron is bound.
○A high TIBC suggests iron levels are low as there is a lot of iron still left that can be bound.
○The capacity to bind iron is high (as there is a lot of unbound iron).

○Perl’s stain can be used on a bone marrow biopsy to detect presence of iron.

Question 10

What would be seen in the FBC in microcytic anaemia?

Answer 10

○ <80fl MCV
○Low MCH - less Hb, less haem, so less iron.
○Low ferritin
○Low RBCC

Question 11

What would be seen in the blood film in iron deficiency anaemia?

Answer 11

○Microcytosis - small RBC’s.
○Hypochromia - pale RBC’s, with large centre of pallor.
○Elliptocytosis - long thin cells.
○Tear drop poikilocytes - tear shaped cells.
○Anisocytosis - unequal size of RBC’s

Question 12

Describe some treatments for iron deficiency anaemia.

Answer 12

○Iron supplements - ferrous sulphate.
○Blood transfusion and iron therapy in severe cases.
○Vitamin C - help to absorb iron.
○Birth control pills - decrease amount of blood loss during menstruation, if menstrual cycle is causing anaemia.

Question 13

Describe how macrocytic anaemia is classified.

Answer 13

○Classified into megaloblastic and non-megaloblastic anaemia, depending on the appearance of developing erythroblasts in the bone marrow.
○Megaloblasts are large cells containing large abnormal nuclei and finely dispersed chromatin, arising due to defective DNA synthesis.
○Vitamin B12 and folate act as coenzymes in DNA synthesis pathway.
○Thus deficiency in either of these can cause impaired DNA synthesis, resulting in appearance of megaloblasts.
○Megaloblasts are identified as abnormal cells and removed from circulation, resulting in ineffective erythropoiesis.

Question 14

Describe vitamin B12.

Answer 14

○Synthesised by microorganisms.
○Found in foods of animal origin (ex. Liver, meat, fish etc) bound to methycobalamin.
○Vitamin B12 is extracted by pepsin and acidic environment of stomach, after which it combines with intrinsic factor (IF) and is absorbed.

Question 15

Describe how vitamin B12 is extracted and absorbed from foods.

Answer 15

○ Vitamin B12 is synthesised by microorganisms and is found in foods of animal origin, bound to methycobalamin.
○ It’s extracted by pepsin and the acidic environment of the stomach.
○ Vitamin B12 then binds to intrinsic factor (IF), which is synthesised by gastric parietal cells.
○ The IF-B12 complex binds to cubam in the ileum, a specific surface receptor for IF, which is composed of cubilin and amnionless.
○ The cubulin IF-B12 complex is endocytosed by amnionless into ileal cells.
○ IF is destroyed, while vitamin B12 is absorbed into the blood, where it attaches to transcobalamin and haptocorrin.
○ Transcobalamin transports vitamin B12 to bone marrow and other tissues.

Question 16

What biochemical reactions is vitamin B12 involved in?

Answer 16

○ Vitamin B12 acts as a cofactor for methionine synthase, which is responsible for methylation of homocysteine to methionine.
○ Vitamin B12 delivers a methyl group.
○ Vitamin B12 deprivation and mutation in methyl tetrahydrofolate reductase (MTHFR) inhibits synthesis of thymidine, impairing DNA synthesis.
○ It’s also involved in converting methylmalonyl CoA to succinyl CoA, which is an important early step in the synthesis of haem.
○ Vitamin B12 deprivation causes absence of succinyl CoA and methionine and results in a build-up of substrates.

Question 17

What are causes of B12 deficiency?

Answer 17

○Inadequate dietary intake - malabsorption, due to lack of IF - pernicious anaemia.
○GI disease
○Surgical removal on site of IF synthesis
○Drug induced
○Increased requirement of vitamin (pregnancy).

Question 18

Describe pernicious anaemia, associated with macrocytic anaemia.

Answer 18

○Gastric mucosa are attacked by immune cells, so cannot absorb B12.
○ Causes B12 deficiency.
○Usual onset 60 years.
○Helicobacter pylori infection can initiate an autoimmune gastritis, causing iron deficiency in younger individuals and pernicious anaemia in elderly.

Question 19

What are some lab diagnosis for macrocytic anaemia?

Answer 19

○Measure amount of B12, serum folate and anti-IF antibodies.

Question 20

Why is folate important?

Answer 20

○Important for thymidine synthesis and for development of a healthy foetus.
○Intestinal absorption is a lot less than B12.

Question 21

Describe some causes of folate deficiency.

Answer 21

Inadequate dietary intake:
○Folate is lost easily as it is heat labile - 90% is destroyed by cooking.
○Body stores only last for three months.

Intestinal malabsorption:
○Coeliac disease, causing intestinal villi destruction and malabsorption of folate.

Increased requirement:
○Pregnancy - required by foetus for spinal cord.
○Women deficient in folate may have babies with spinobifoda.

Drug induced, which interferes with absorption or metabolism:
○Alcohol - affects body’s ability to absorb folate, causing increase loss in urine.
○Methotrexate:
○Inhibits dihydrofolate reductase, resulting in decreased supply of folates.
○Patients treated with long-term, low-dose methotrexate for rheumatoid arthritis or psoriasis can be aided with folate.

Question 22

Describe the biochemical basis for megaloblastic anaemia.

Answer 22

○The polymerisation of four deoxyribonucleoside monophosphates forms DNA.
○Folate deficiency inhibits the rate limiting step by limiting thymidine monophosphate (dTMP) synthesis from deoxyuridine monophosphate.
○5,10-methylene THF polyglutamate is needed as a coenzyme for this reaction.
○B12 is needed to convert methylTHF to THF.
○Homocysteine is converted to methionine in this reaction.
○In folate deficiency, B12 has no methyl group to bind to, so homocysteine cannot be converted.
○THF is a substrate for folate polyglutamate synthesis, which are intracellular folate coenzymes.
○B12 deficiency decreases folate coenzyme 5,10-methylene THF polyglutamate, which is needed for dTMP synthesis.

Question 23

What would FBC result be for macrocytic anaemia?

Answer 23

○Reduced Hb.
○Increased MCV >100.

Question 24

What would you see on the blood film of someone with macrocytic anaemia?

Answer 24

○Macrocytes
○Megaloblasts
○Poikilocytes
○Anisocytes
○Hypersegmented neutrophils - 7-8 lobes instead of 2-5, due to defective DNA synthesis and structural abnormalities in nuclear chromatin.

Question 25

What other findings would you get for macrocytic anaemia?

Answer 25

○Low levels of serum B12.
○Low levels of serum and RBC folate.
○In pernicious anaemia, may find autoantibodies to IF or gastric parietal cells.

Question 26

Describe hepcidin.

Answer 26

Produced by liver.
Regulates iron homeostasis.
Inhibits export of iron from ferroportin by using lysosomes to degrade ferroportin.