Week 1 - G - Haemolysis - Erythrocytosis, Extra/intra vascular - Premature destruction, Abnromal cell membrane/RBC metabolsim/haemgolobin

Question 1

Hameolysis is the premature destruction of red cells What is the breakdown product of haemoglobin that lets you know that haemolysis has taken place? How long do red blood cells typically live for? What percentage of RBCs are accounted for by reticulocytes normally?

Answer 1

This would be the increase in bilirubin RBCs normally live for approximately 120 days Reticulocytes live for 1-2 days and therefore 1% of the total circulating RBC concentration is reticulocytes

Question 2

Why are red blood cells particualry susceptible to damage?

Answer 2

* They need to have a biconcave shape to travel in circulation successfully * They have limited metabolic reserve and rely exclusively on glucose metabolism for ATP (as they have no mitochondria) * They cannot generate new proteins once in circulation as they have no nucleus therefore if any proteins are damaged they are unable to repair themselves

Question 3

What does the biconcave shape allow for?

Answer 3

The biconcave shape enhances oxygen transport - primary responsibility of RBC Also allows the RBC to withstand high pressure in arteries and squeeze through small blood vessels in tissues - flexible membrane

Question 4

What is the difference between compensated and decompensated haemolysis?

Answer 4

Compensated haemolysis - the increased rate of red cell destruction is compensated for by the increased red cell production in the bone marrow - therefore Hb is maintained Decompensated haemolysis - the increased rate of red cell destruction exceeds the bone marrow capability for red cell production - they become anaemic and Hb falls

Question 5

What are the consequences of haemolysis? What would a bone marrow biopsy actually show? What would the reticulocyte count in the blood show? Ie to the bone and breakdown products

Answer 5

The consequences include: Erythroid hyperplasia - the kidneys detect hypoxia and produce EPO to try an stimulate the bone marrow to produce more RBCs There is an increase in the Hb breadown products eg bilirubin * Bone marrow biopsy would show an increase in red cell precursor * Reticulocyte count would show reticulocytosis

Question 6

It is not possible to directly measure red cell survival routinely - technically there are ways but they are very complicated and not done routinely We rely on detecting the consequences by measuring the breakdown prodcuts or reticulcoytes for example to understand the cause of the haemolysis What are the two bone marrow responses to haemolysis?

Answer 6

Erythroid hyperplasia Reticulocytosis

Question 7

Are reticulocytes nucleated red cells? WHat is the difference between a reticulocyte and erythrcoyte?

Answer 7

Reticulocytes are not nucleated - the late eyrythroblast enucleates before becoming a reticulocyte and leaving the bone marrow A reticulocyte is slightly bigger than a RBC and has some RNA hence why is partly blue -polychromatic on blood film

Question 8

Apart from haemolysis, what else can cause a reticulocytosis?

Answer 8

Haemorrhage - will have raised reticulocytes here Also Reticulocyte count raised in response to treatment for iron deficiency anaemia

Question 9

What stains can be used to detect reticulocytes? The stains bind to the RNA in the reticulocyte - these are known as supravital staining (ie the cells have been removed from the body)

Answer 9

New methylene blue or brilliant cresyl blue staining can be used - this stain addition then counting the proportion of reticulocytes to erythrocytes used to be the way of counting the number of reticulocytes before automated analysers

Question 10

Automated analysers now count reticuloytes instead of doing it manually What must be added to enable the flourescent cells to be counted?

Answer 10

Fluorochrome is added which binds to ribosomal DNA and the fluorescent cells are counted

Question 11

Bone marrow aspiration and trephine biopsy can be used to see the lots of red cells precursors to show the erythroid hyperplasia if wanted WHat are the two different types of haemolysis?

Answer 11

Extravascular and intravascular haemolysis

Question 12

Where does extravascular and intravascular haemolysis take place?

Answer 12

Extravascular haemolysis takes place in the reticuloedothelial system - predominantly in the liver but also in the spleen Intravascular haemolysis takes place in the circulation

Question 13

Intravascular and extravascular haemolysis have: Different mechanisms therefore different breakdown products detected Useful classification as knowing intra/ extravascular helps determine the cause of haemolysis Which type of haemolysis is more common? If it is chronic haemolysis, what happens to the organ?

Answer 13

Extravascular haemolysis is more common If chronic haemolysis or over a long duration, the organ will become hyperplastic - splenomegaly +/- hepatomegaly

Question 14

When the RBCs are broken down in haemolysis, what is the haem converted to? Is there excess unconjugated or conjugated bilirubin? What can the excess bilirubin lead to the formation of? Is bilirubin a normal product of red cell breakdown?

Answer 14

The iron is recycled The haem is converted to biliverdin by heme oxygenase and then to bilirubin by biliverdin reductase The increase in bilirubin is unconjugated biilirubin The excess biliruin can lead to the formation of gall stones Yes, it is normal - just present in excess in haemolysis

Question 15

Where are red cells broken down in intravascular haemolysis? What happens to the contents?

Answer 15

Red cells are destroyed in the circulation spilling their contents into the circulation also

Question 16

The contents of the RBC breakdown is not held safely in the spleen in intravascular haemolysis and these contents spill into the circulation These contents can be very toxic When the contents are spilled into the circulation what are the contents, just name them for now?

Answer 16

Haemaglobinaemia Methaemalbuminaemia Haemaglobinuria Haemosiderinuria

Question 17

What is haemaglobinaemia and methaemalbuminaemia?

Answer 17

Haemaglobinaemia - this is where there is free Hb in circulation Methaaemalbuminaemia - this is where the haemoglobin binds to albumin int he circulation

Question 18

What is haemaglobinuria? WHat may people think if they are passing this?

Answer 18

This is where the haemaglobin is excreted directly into the urine and is pink - the urine then turns black if left to stand (initially pink then oxidises to become black) They may think they are passing old blood instead of Hb Excess iron that is released may be processed in the kidneys and eventually released into the urine as a compound known as hemosiderinuria

Question 19

The products of extravascular haemolysis are normal breakdown products (just present in excess) What is seen in extravascular that is not seen in intravascular? The products of intravascular are abnormal,w hat are the 4 products again?

Answer 19

Will see splenomegaly +/- hepatomegaly in extravascular haemolysis Haemaglobinaemia - free Hb in circulation Methaemalbuminaemia - Hb bound to albumin Haemaglobinuria - Hb in urine, turn urine pink then urine turns black if left to sit due to oxidation Haemosiderinuria - excess iron processed in the kidneys and then excreted as haemosiderin

Question 20

Severe INTRAVASCULAR HAEMOLYSIS MAY BE LIFE THREATENING as the abnormal products released are toxic WHat are some of the causes of intravascular haemolysis? This is much rarer than extravascular remember (extravascular basically accounts for the causes not mentioned below)

Answer 20

ABO incompatibility (acute immediate haemolytic transfusion reaction) Glucose-6-phosphate dehydrogenase deficiency Severe falciparm malaria Rarer still Paraoxysmal nocturnal haemaoglobinuria, paroxysmal cold haemoglobinuria

Question 21

What is blackwater fever? What parasite causes it?

Answer 21

Blackwater fever (BWF) is a severe clinical syndrome, characterized by intravascular hemolysis, hemoglobinuria, and acute renal failure Blackwater fever is a complication of malaria infection in which red blood cells burst in the bloodstream (hemolysis), releasing hemoglobin directly into the blood vessels and into the urine, frequently leading to kidney failure - it is caused by the parasite plasmodium falciparum

Question 22

Is delayed haemolytic transfusion reactions extra or intra vascular? How is this different from ABO?

Answer 22

In delayed haemolytic transfusion reactions, the cells are phagocytosed by the spleen becuase of the presence of irregualr antibodies (all-antibodies) leading to extravascular hameolysis. ABO incompatibility - activation of complement by IgM antibodies leads to haemolysis in circulation

Question 23

What binds haemoglobin in circulation? If the levels of this are low, what is indicated?

Answer 23

Haptoglobin binds haemgolobin that is in circulation If haptoglobin is low - this is sensitive that intravascular haemolysis is taking place as there is less plasma haptoglobin present because Hb has bound to it

Question 24

Investigations for haemolysis Confirm the haemolytic state How is this done? (ie what results are expected for both intra and extravascular haemolysis)

Answer 24

Full blood count and film Reticulocyte count - useful to see reticulocytosis - seen in both Measure serum unconjugated bilirubin - tests to see if there is haemolysis - would be raised Measure serum haptoglobins - low in intravascular Can measure urine urinobilogen to see if there is increased bilirubin breakdown in urine

Question 25

How can you differ between extravascular and intravascular haemolysis?

Answer 25

* In extravascular haemolysis - there is splenomegaly and hepatomegaly * In extravascular hameolysis - there is increased plasma haemoglobin (normal levels of plasma haptoglobin) * There is methaemalbuminaemia Also there is haemoglobinuria and haemsoidenuria In both, get increased bilirubin, urinary urobiliogen and serum LDH

Question 26

Important to take a history for haemolysis - cause could be genetic or acquired haemolysis Name a cause of each?

Answer 26

Genetic * Hereditary shperocytosis * G6PDD Acquired Transfusion reaction - * Acute immediate (intravascular) * Delayed (extravascular)

Question 27

What could be seen on the blood film in haemolysis?

Answer 27

Blood film Maybe might see spherocytes - evidence of membrane damage Red cell fragments - mechanical damage Heinz bodies may be present - due to oxidative damage Others such as sickle cells, or due to other disorders

Question 28

What may be causes of spherocytes present?

Answer 28

Spherecoytes may be present due to autoimmune haemolytic anaemia where antibodies attack the cell causing extravascular haemolysis or Genetic - eg hereditary spherocytosis (spherical RBCs trapped in spleen and haemolysed)

Question 29

What may cause the presence of heinz bodies? When the heinz body containing red cells are processed through the spleen what do they become?

Answer 29

Heinz bodies are formed by damage to the Hb molecules, usually through oxidant damage Heinz bodies are usually present in the condition G6PDD where the enzyme to oxidise glutathionine isnt present resulting in oxidative damage to RBCs Bite cells are formed once the damaged RBCs go through the spleen

Question 30

If suspecting an underlying autoimmune haemolytic anaemia, what test would we want to do?

Answer 30

If suspecting this would want to carry out a Coomb’s Test (direct anti-globulin test)

Question 31

As well as talking about the site of rec cell destruction - intra or extra vascular - we can talk about the site of the red cell defect 4 main reasons: Normal red cells can be destroyed either due to immune or mechanical reasons And then there are three reasons why abnromal red cells may be haemolysed, name them?

Answer 31

Abnormal red cell membrane Abnormal red cell metabolism Abnormal haemaglobin These mechanisms can also be congenital or acquired

Question 32

Lets talk about normal red cells that are destroyed prematurely due to immune means What is the difference between autoimmune and alloimmune haemolysis?

Answer 32

Autoimmune haemolysis - the patient has their own antibodies destroying their red cells Or Allo-immune - Alloantibody. In blood banking, an antibody formed in response to pregnancy, transfusion, or transplantation targeted against a blood group antigen that is not present on the person’s red blood cells. (antibody formed in response to a blood group antigen not present on the persons own red blood cells)

Question 33

Autoimmune haemolysis of normal cells can be classified as warm or cold autoantibody reations What immunoglobulin mediates warm and cold? Is warm or cold more common?

Answer 33

Warm autoimmune haemolytic anaemia (warm AIHA) - mediated by IgG and is by far the most common Cold AIHA - mediated by IgM

Question 34

Most types of IgG haemolytic aneamia are idiopathic There are secondary causes however such as? At what temp do the warm AIHA bind at? What is the treatment?

Answer 34

Autoimmune disorders such as SLE Lymphomas Drugs sometimes such as penicillins Infection The IgG mediated AIHA binds at 37 degrees -nomal body temp and is treated with steroids/immunosuppression

Question 35

Cold AIHA are mediated by IgM How do these activate the haemolysis? What temp do they bind at? Most cases are again idiopathic but what conditions can they be associated with?

Answer 35

These bind at temp less than 4degrees OFten made worse by Raynauds The IgM causes a cell-surface complement activation leading to the haemolysis (by the membrane attack complex - C5b-C9) Associated with EBV, mycoplasma and some lympomas

Question 36

What is the treatment of IgM mediated AIHA?

Answer 36

Keep the patient warm

Question 37

What test can be used to identify the antibody in autoimmune haemolytic anaemia? (warm (IgG) or cold (IgM)) This test will detect the presence of IgG for the warm AIHA and the presence of the complement protein C3d.which is suggestive of cold AIHA

Answer 37

Can use Direct Coomb’s test - should positive There will be antibody/complement present on the red cell and addition of anti-human immunoglobulin will cause agglutination of the cells confirming their presence

Question 38

Alloimmune haemolysis What are conditions when this occurs? (this is when there are pre-formed antibodies in the persons blood which will attack foreign red blood cell antigens) Transfusion and passive mother to child Extra or intra vascular? (someones elses antibodies attack your own)

Answer 38

Immune response due to transfusion reactions Acute immediate haemolytic transfusion reaction - ABO incompatibility and IgM mediated - this is intravascular Delayed haemolytic transfusion reaction - irregular antibodies - this is IgG mediated - extravascular and occurs 7-10 days after transfusion Passive transfer of antibody Haemolytic disease of the newborn * - Rh D - ABO incompatibility - Others eg anti-Kell

Question 39

Normal red cells can be destroyed by immune means as discussed (autoimmune and alloimmune) They can also be destroyed by mechanical means Mechanical hemolytic anemia is a form of hemolytic anemia due to mechanically induced damage to red blood cells. Red blood cells, while flexible, may in some circumstances succumb to physical shear and compression What are types of acquired mechanical red cell destruction?

Answer 39

Disseminated intravascular coagulation Haemolytic uraemic syndrome and thrombotic thrombocytopenic purpura Leaking heart valves Malaria

Question 40

Disseminated intravascular coagulation is widespread activation of coagualtion from release of procagulants into the circulation with consumption of clotting factors and platelets -lots of small clots form in small vessels meaning no resources if a bleed occurs Causes - maliganncy, sepsis, trauma What are signs of the disease? What causes the passing rbcs to haemolyses?

Answer 40

Get severe bruising and bleeding Fibrin strands lyse the passing red blood cells due to this disease

Question 41

When treating DIC, what is given to replace the fibrinogen, and the coaguulation factors? What platelet level merits platelet replacement? (usually when active bleeding) What is seen on blood tests? Name what the substance is that would be increased in the blood? - it is a fibrin degradation product

Answer 41

Usually try and treat the cause of the DIC firstly if low bleeding risk If high bleeding risk or active bleeding 1st - Fresh frozen plasma - replaces the coagulation factors 2nd line - would cryoprecipitate which would replace the fibrinogen If plasma levels <50x10^9 - then platelet transfusion Decreased platelets and fibrinogen, increased fibrin degradation products - D-dimers

Question 42

What is the normal plasma platelet level?

Answer 42

Normally the platelet count is 150-450 x10^9 /l

Question 43

What are both haemolytic uraemic syndrome and thrombotic thrombycytopenic purpura due to?

Answer 43

E.coli 0157 - most common cause of renal failure in chidlren under 5 years of age

Question 44

What is a mechanical destruction of RBCs in circulation known as? - this mechanical destruction causes intravascular haemolysis (usually due to HUS/TTP - E.coli 0157, DIC, Leaking heart valves and malaria) Why do the red cells fragment (haemolyse) in these conditions?

Answer 44

This is referred to microangiopathic haemolytic anaemia (MAHA) - Red cell fragmentation as a result of mechanical (extrinsic) damage making them more likely to shear or compress

Question 45

Microangiopathic results in numerous red cell fragments known as what on blood film?

Answer 45

These red cell fragments are seen on blood film and are known as schistiocytes A schistocyte is a fragmented part of a red blood cell. Schistocytes are typically irregularly shaped, jagged, and have two pointed ends.

Question 46

Name the different types of immune mediated and mechanical acquired haemolysis of normal red cells

Answer 46

Immune mediated * Autoimmune - Cold (IgM) and warm (IgG) autoimmune haemolytic anaemia * Allo-immune - Transfusion reactions and passage of blood from foetus to mother Mechanical (microangiopathic haemolytic anaemia) * Haemolytic uraemic syndrome * Thrombotic thrombocytic purpura * Disseminated intravascular coagulation * Leaky heart valves - Infections and Burns

Question 47

Lets talk about acquired causes of abnormal cell membranes Acquired membrane defects are very rare What are some examples?

Answer 47

Liver Disease (Zieve’s Syndrome) Vitamin E deficiency - never been seen by the lecturer Paroxysmal Nocturnal Haemoglobinuria

Question 48

Zieve’s syndrome is an acute metabolic condition that can occur during withdrawal from prolonged alcohol abuse. It is defined by hemolytic anemia hyperlipoproteinaemia (excessive blood lipoprotein), jaundice (elevation of unconjugated bilirubin), and abdominal pain. What is seen on the blood film?

Answer 48

Polcyhcromatic macrocytes Anaemia

Question 49

Paroxysmal nocturnal haemaglobinuria RARE - but can be asked about in 4th year life-threatening disease of the blood characterized by destruction of red blood cells by the complement system, a part of the body’s innate immune system. This destructive process occurs due to the presence of defective surface proteins on the red blood cell, which normally function to inhibit such immune reactions. What is seen in the urine and when is this most pronounced?

Answer 49

In the urine, can see the presence of haemoglobinuria - turns pee pink and black if left to stand and Haemosiderinuria - turns pee brownish The classic sign of PNH is red discoloration of the urine due to the presence of hemoglobin and hemosiderin from the breakdown of red blood cells.[8] As the urine is more concentrated in the morning, this is when the color is most pronounced

Question 50

All cells have proteins attached to their membranes, often serving as a mode of communication or signaling between the cell and the surrounding environment. These signaling proteins are physically attached to the cell membrane in various ways, commonly anchored by glycolipids such as glycosyl phosphatidylinositols (GPI). PNH occurs as a result of a defect in the assembling of these glycolipid-protein structures on the surface of blood cells. What is the most common defective enzyme in PNH?

Answer 50

The most common defective enzyme in PNH is phosphatidylinositol glycan A (PIGA), one of several enzymes needed to make GPI.

Question 51

WHat happens when the defect in the enzyme PIGA occurs in a haematopoeitc stem cell?

Answer 51

A mutation in the PIGA gene can lead to the absence of GPI anchors expressed on the cell membrane. When this mutation occurs in a hematopoietic stem cell in the bone marrow, all of the cells it produces will also have the defect. - marrow failure (pancytopenia)

Question 52

What is ZIeve’s syndrome due to? What is paroxsymal nocturnal haemaglobuminuria due to?

Answer 52

Zieves syndrome - due to withdrawal form prolonged alcohol abuse - It is defined by hemolytic anemia hyperlipoproteinaemia (excessive blood lipoprotein), jaundice (elevation of unconjugated bilirubin), and abdominal pain. PNH - haemglobin in the urine - due to a defect in the GPI The most common defective enzyme in PNH is phosphatidylinositol glycan A (PIGA), one of several enzymes needed to make GPI. (glycosyl phosphatidylinositols)

Question 53

Abnormal cell membrane can occur due to acquired conditions (Zieve’s syndrome, vitamin E deficiency, Paroxysmal nocturnal haemaglobinura) There can also be congenital causes What is the most common example?

Answer 53

This would be Hereditary spherocytosis

Question 54

What is the most common mode of inheritance of hereditary spherocytosis?

Answer 54

This is an autosomal dominant condition mainly (can be autosomal recessive rarely)

Question 55

How does an abnormal red cell membrane cause hereditary spherocytosis?

Answer 55

This is because if the red cell membrane is abnormal Then the cell loses its biconcave shape The cell is less able to squeeze through small vessels and withstand pressures in circulation - both make it more susceptible to destruction Also oxidative environment in spleen will lead to its destruction

Question 56

As hereditary spherocytosis is present from birth, what will be seen on bloods? What organ will be enlarged? What is the main inheritance of the disease?

Answer 56

Spleen will be very enlarged here In the bloods, will see Increased unconjugated bilirubin - could cause formation of gallstones The condition is mainly autosomal dominant

Question 57

In hereditary spherocytosis -will see the presence of spherocytes on blood film Also will have a anaemia with a normal or elevated MCV (due to reticulocytosis) Elevated bilirubin Also will be Direct Coomb’s negative - rules out immune mediated defects What test can be carried out to diagnose this condition?

Answer 57

Osmotic fragility testing - place cells in increasingly hypotonic solutions and measure the degree of cell lysis by the haemoglobin produced

Question 58

What is important to supplement in HS? What may the treatment be?

Answer 58

Remember to provide folic acid supplementation - bone marrow will be working overtime using this May require RBC transfusion for symptomatic anaemia or Splenectomy if gets very bad May also require cholecystectomy if gallstones affect the gall bladder

Question 59

Covered premature destruction of normal cells (immune mediated or mechanical) Abnormal cell membrane - main cause hereditary spherocytosis Now abnormal cell metabolism Red cells are dependent on glycolysis to generate ATP What enzyme is key for protection against oxidative damage.?

Answer 59

This would be glucose-6-phosphate dehydrogenase Its deficiency would result in build up of reactive oxidative species

Question 60

What is the other most common enzyme defect for abnormal cell metabolism? What is the inheritance? What is the inheritance in G6PDD?

Answer 60

Pyruvate kinase deficiency - autosomal recessive G6PDD - x-linked recessive

Question 61

Some patients with G6PDD will only show symptoms if exposed to certain drugs which cause oxidative damage When may this occur? What are the cells known as in G6PDD on blood film?

Answer 61

Malaria prophylaxis Dapzone for leprosy Henna use They are known as bite cells - the damaged Hb as precipitated in the red cell and become heinz bodies - the fucntioning spleen bites this Heni body out

Question 62

What does pyruvate kinase enzyme catalyse?

Answer 62

Catalyses the final step of glycolysis - PEP to pyruvate

Question 63

Name two conditions for abnromal ahemoglobin

Answer 63

Thalassemia * Alpha thal trait * Haemoglobin H disease * Haemoglobin Barts Hydrops Foetalis * Beta thal trait * Beta thal intermedia * Beta thal major Sickle cell disease * Sickle cell trait (HbS) (only one beta mutated) * Sickle cell anaemia (HbSS both beta mutated)

Question 64

What does this boy have?

Answer 64

Can see extramedullary haematopoeisis Can see hair on end sign on Xray Also has Target cells on xray - seen in thalassemia Probably has hepatosplenomegaly Would have majorly increased HbF Beta thalassaemia major

Question 65

Blood film of a RhD negative man who had iron deficiency anaemia and received a RhD positive unit of blood. Ten days later he became jaundiced and anaemic again. What does he have? What antibodies will be raised?

Answer 65

He developed an alloantibody to RhD and destroyed the transfused blood. Extravascular haemolysis - delayed haemolytic transfusion reaction There will be increased IgG immunoglobulin

Question 66

22 yrs female. Splenomegaly Intermittent mild jaundice Gallstones Father had cholesystectomy in young adulthood for gallstones What does she have? (would obviously perform tests)

Answer 66

Has hereditary spherocytosis Removal of all gall bladder due to excess bilirubin causing gall stones formation

Question 67

Anaemic Hb80-100 supravital stain Thai girl Intermittant jaundice when has fever/unwell and oxidant drugs What are the cells the arrows are pointing to here? What does she have?

Answer 67

Haemoglobin H disease A form of alpha thalassaemia. 3 of 4 alpha genes are not working so there is an excess of beta chains which can precipitate. HbH prep.is a modified retic stain. HbH(β4) containing golf balls(blue) are seen as well as retics (red) The golf ball cells are also known as inclusion bodies