Haematology Flashcards
1
Q
Explain the synthesis of a red blood cells, including the synthesis of erythropoietin.
A
From bone marrow -> multipotent haemopoietic stem cells -> lymphoid stem cells / myeloid stem cells
Lymphoid stem cells -> T cell/ B cell/ NK cell
Myeloid stem cells -> granulocyte-monocyte/ erythroid/ megakaryocyte
Normal erythroid maturation
Myeloid -> proerythroblast -> erythroblast (squeeze cytoplasm out into sinusoid leaving nucleus behind which is then ingested by macrophage) -> erythrocytes
Process of production red cells is called erythropoiesis
Hypoxia/ anaemia -> kidneys -> erythropoietin increase -> increased bone marrow activity -> increased red cell production ->
Juxtatubular interstitial cells in kidneys synthesis 90% erythropoietin
Hepatocytes and interstitial cells synthesis 10%
2
Q
What are the essential characteristics of a stem cell?
A
Ability to self-renew and produce mature progeny
Ability to divide into two cells with different characteristics, one is another stem cell and the other a cell capable of differentiating to mature progeny.
3
Q
Describe the basic physiology of the red blood cells.
A
The erythrocytes survives around 120 days in the blood stream.
Main function is oxygen transport. Also transports some CO2.
Ultimately destroyed by phagocytise cells of the spleen; also liver (less)
4
Q
Explain the synthesis of white blood cells.
A
The multipotent haemopoietic stem cell can also give ride to a myeloblast and a mono blast which in turn give rise to granulocytes and monocytes.
Cytokines such as G-CSF (granulocyte colony-stimulating factor), M-CSF (macrophage colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor) and various interleukins are needed.
No need to remember:
Normal granulocyte maturation
Myeloblast -> promyelocyte -> myelocyte -> metamyelocyte -> band form -> neutrophil
5
Q
Describe the basic physiology of neutrophils.
A
The neutrophil granulocyte survives 7-10 hours in the circulation before migrating to tissues.
It’s main function is defence against infection; it phagocytosis and then kills micro-organisms
Diagram
Circulating neutrophils - flow through blood vessels (in middle)
Marginated neutrophils - are adherent to endothelium
6
Q
Describe the physiology of the eosinophil.
A
Spends less time in circulation than neutrophil.
Main function is defence against parasitic infection.
7
Q
Describe the physiology of the basophils.
A
Have a role in allergic responses
8
Q
Describe the physiology of monocytes.
A
Spend several days in circulation.
Migrate to tissues where they develop into macrophages and other specialised cells that have a phagocytic and scavenging function.
Also store and release iron.
9
Q
Describe the physiology of platelets.
A
Have a role in primary haemostasis.
Platelets contribute phospholipid which promotes blood coagulation.
10
Q
Describe the physiology of lymphocytes.
A
Gives rise to T cells, B cells and natural killer (NK) cells.
Lymphocytes recirculate to lymph nodes and other tissues and then back to the blood stream.
Intravascular life span is very variable
Small lymphocyte = high nuclei: cytoplasmic ratio
Large granular lymphocyte = contain cytotoxic granules, e.g. NK cell, cytotoxic T cells
11
Q
Explain the following terms:
- anisocytosis
- poikilocytosis
- microcytosis
- macrocytosis
- microcyte
- macrocyte
- microcytic
- normocytic
- macrocytic
A
Anisocytosis: red cells show more variation in size than normal
Poikilocytosis: red cells show more variation in shape than normal
Microcytosis: red cells are smaller than normal (e.g. smaller than nucleus of lymphocyte)
Macrocytosis: red cells are larger than normal
Microcyte: a red cell that is smaller than normal
Macrocyte: a red cell that is larger than normal
Can be of specific types:
-round macrocytes
-oval macrocytes - deficiency of Vit. B12/ folic acid
-polychromatic macrocytes - lots of young cells .e.g. recent haemorrhage
Microcytic: describes red cells that are smaller than normal or an anaemia with small red cells.
Normocytic: describes red cells that are of normal size or an anaemia with normal sized red cells
Macrocytic: describes red cells that are larger than normal or an anaemia with large red cells
12
Q
Explain hypochromia.
A
Normal red cells that have about a third of the diameter pale.
This is a result of the disk shape of the red cell; the centre has less haemoglobin and is therefore paler
Hypochromia means that the cells have a larger area of central pallor than normal
This results from a lower haemoglobin content and concentration and a flatter cell
Red cells that show hypochromia are described as hypochromic
Hypochromia and microcytosis often go together.
13
Q
Explain hyperchromia.
A
Hyperchromia means that cells lack central pallor.
This can occur because they are thicker than normal or because their shape is abnormal.
Cells showing hyperchromia can be described as hyperchromatic or hyperchromic.
Has many causes since many abnormal shaped cells lack the central thinner area. However there are only two important types: sphere types (round outline, shape of sphere) and irregularly contracted cells (dense, irregular, clumped)
Spherocytes
Cells that are approximately spherical in shape
Have a round, regular outline and lack central pallor
They result from the loss of cell membrane without the loss of equivalent amount of cytoplasm so cell is forced to round up.
Spherocytes are seen in hereditary spherocytosis but not all the cells are spherical
Irregularly contracted cells
Are irregular in outline but are smaller than normal cells and have lost their central pallor
They usually result from oxidant damage to the cell membrane and to the haemoglobin
14
Q
Describe polychromatic, reticulocyes and reticulocytosis.
A
An increased blue tinge to the cytoplasm of a red cell. Indicates that the red cell is young.
Another way to detect young cells is to do reticulocyte stain
This exposed living red cells to new methylene blue, which precipitates as a network or reticulum
Detecting polychromatic or increased numbers of reticulocytes gives you similar information however identification of reticulocytes is more reliable so they can be counted.
15
Q
Describe the different types of poikilocytes.
A
Spherocytes
Irregularly contracted cells
Sickle cells
Are sickle or crescent shaped/ boat shaped
They result from the polymerisation of haemoglobin S when it is present in a high concentration.
Target cells
Are cells with an accumulation of haemoglobin in the centre of the area of central pallor.
They occur in obstructive jaundice, liver disease, haemoglobinopathies and hyposplenism
Elliptocytes
Elliptical in shape
They occur in hereditary elliptocytosis and in iron deficiency
Fragments
Or schistocytes are small pieces of red cells
They indicate that a red cell has fragmented
16
Q
Describe rouleaux, agglutinate and Howell-Jolly bodies.
A
Rouleaux
Are stacks of red cells
They’re resemble a pile of coins
They result from alterations in plasma proteins
Agglutinates
Agglutinates differ from rouleaux as they are irregular clumps rather than tidy stacks
They usually result from antibody on the surface of the cells
Howell-Jolly body
Nuclear remnant in a red cell
The commonest cause is lack of splenic function
17
Q
Define the following terms:
- leucocytosis
- leukopenia
- neutrophilia
- neutropenia
- lymphocytosis
- eosinophilia
- thrombocytosis
- thrombocytopenia
- erythrocytosis
- reticulocytosis
- lymphopenia
- anaemia/polycythemia
- pancytopenia
A
Leucocytosis: too many white cells Leukopenia: too few white cells Neutrophilia: too many neutrophils Neutropenia: too few neutrophils Lymphocytosis: too many lymphocytes Eosinophilia: too many eosinophils Thrombocytosis: too many platelets Thrombocytopenia: too few platelets Erythrocytosis: too many red blood cells Reticylocytosis: too many reticulocytes Lymphopenia: too few lymphocytes Polycythaemia (slow growing blood cancer in which your bone marrow makes too many red blood cells) Pancytopenia: reduction of all lineages (RBCs, WBCs, platelets)
18
Q
Describe atypical lymphocyte, left shift, toxic granulation and hypersegmented neutrophil.
A
Atypical lymphocyte
An abnormal lymphocyte
Often the term is used to describe abnormal cells present in infectious mononucleosis (glandular fever)
Atypical mononuclear cell is an alternative term
Left shift
There is an increase in non-segmented neutrophils or there are neutrophil precursors in the blood (infection/ inflammation)
Right shift = increase in segmented
Toxic granulation
Heavy granulation of neutrophils
Results from infection, inflammation and tissue necrosis (but is also a normal feature of pregnancy)
Hypersegmented neutrophil
There is an increase in the average number of neutrophil lobes or segments
Usually results from a lack of vitamin B12 or folic acid.
> or equal to 6 lobes
19
Q
What is meant by a reference and a normal range?
What is normal affected by?
How is a reference range determined?
A
Reference range: range derived from a carefully defined reference population Normal range is more vague (usually 95% of healthy population will have test results falling within a normal range.
Affected by: Age Gender Ethnic origin Physiological status e..g pregnancy Altitude - pO2 decreases = hypoxia, kidney increases erythropoetin, haemoglobin increases Nutritional status Cigarette smoking, alcohol intake - WBC count and haemoglobin effected
Reference range determined by:
Samples are collected from healthy volunteers with defined characteristics
They are analysed using the same instrument and techniques that will be used for patient samples
The data are analysed by an appropriate statistical technique:
-data with a normal (Gaussian) distribution can be analysed by deterring the mean and standard deviation and taking mean +/- 2SD as the 95% range
-data with a different distribution must be analysed by an alternative method
Not all results outside the reference range are abnormal
Not all results within the normal range are normal
Want to know if normal for the individual/ patient
A health-related range may be more meaningful than a 95% range
Ideal and non-ideal tests
The less the overlap the more ideal, no overlap is best but little overlap is expected so best you can hope for in practice
20
Q
State what those abbreviations are in a full blood count (FBC): WBC RBC Hb Hct PCV MCV MCH MCHC Platelet count
A
WBC - white blood cell count in a given volume of blood (x10^9/l)
RBC - red blood cell count in a given volume of blood (x10^12/l)
Hb - haemoglobin concentration (g/l)
Hct - haematocrit (l/l)
PCV - packed cell volume (% or l/l) (an older name for the Hct)
MCV - mean cell volume (fl)
MCH - mean cell haemoglobin (pg)
MCHC - mean cell haemoglobin concentration (g/) sometimes (g/dl)
Platelet count - the number of platelets in a given volume of blood (x10^9/l)
21
Q
How are the WBC, RBC and platelet count measured?
How is Hb measured?
How is PCV/ Hct measured?
A
WBC, RBC, platelet count
Initially counted visually, using a microscope and a diluted sample of blood
Now counted in large automated instruments, by enumerating electronic instruments generated when cells flow between a light source and a sensor or when cells flow through an electrical field.
Hb
Initially measured in a spectrometer by converting haemoglobin to a stable form (cyanmethaemoglobin) and measuring light absorption at a specific wavelength
Now measured by an automated instrument but principle is the same.
PCV/ Hct
Initially measured by centrifuging a blood sample (hence PCV)
How much of the column is packed with RBC’s, RBC’s at bottom, WBC’s lighter so at top, platelets (buffy coat)
22
Q
Describe MCV, MCH and MCHC.
A
MCV
Initially calculated by dividing the total volume of red cells in a sample by the number of red cells in a sample .i.e. dividing PCV by the RBC
Now determined in directing by light scattering or by interruption of an electrical field
Larger red blood cells = higher MCV
MCH
The amount of haemoglobin in a given volume of blood divided by the number of red cells in the same volume .i.e. the Hb divided by the RBC
MCHC
The amount of haemoglobin in a given volume of blood divided by the proportion of the sample represented by the red cells .i.e. the Hb divided by the Hct
MCHC is now measured electronically, most accurately on the basis of light scattering
23
Q
Describe the difference between MCH and MCHC.
A
MCHC can be seen as the density of red blood cells
The MCH is the absolute amount of haemoglobin in an individual red cell
In microcytic and macrocytic anaemias, the MCH tends to parallel the MCV
The MCHC is the concentration of haemoglobin in a red cell
The MCHC is related to the shape of the cell
24
Q
How do you interpret a blood count?
A
Is there leucocytosis or leukopenia? If so why?
Which cell line is abnormal?
Are there any clues in the clinical history? E.g. pneumonia
Is there thrombocytosis or thrombocytopenia? If so are there any clues in the blood count?
Are there any clues in the clinical history?
Interpret:
-WBC and differential (percentage of different types of cell)
-Hb
-MCV
-Platelet count
25
Describe polycythaemia, including its causes.
How do you evaluate polycythaemia.
Treatment?
Too many red cells in the circulation
The Hb, RBC and Hct/ PVC are all increased compared with normal subjects of the same age and gender (men>women, adults>children)
Pseudopolycythaemia = reduced plasma volume due to blood doping or overtransfusion (illegal blood transfusion by athletes)
True polycythaemia = increase in total volume of red cells in the circulation due to;
- appropriately increased erythropoietin - response to hypoxia
- inappropriate erythropoietin synthesis or use
- independent of erythropoietin
Evaluation:
Start with a clinical history and physical examination (splenomegaly, abdominal mass (sign of kidney) or cyanosis)
Next compare with an appropriate normal range
Is it genuine or apparent?
-a high Hb, RBC and PCV/Hct can result from a decrease in plasma volume, referred to as pseudopolycythaemia or apparent polycythaemia
-when the abnormalities result from an increase in the number of circulating red cells there is a true polycythaemia
Causes:
Blood doping in professional cyclists
Medical negligence (blood transfusion) -weight of patient needs too be considered
Appropriately raise levels of erythropoietin:
-altitude
-hypoxia (can be seen by cyanosis and clubbing of fingers)
Erythropoeitin inappropriately administered to haematologically normal subjects (cyclists)
Renal/ other tumour inappropriately secretes erythropoietin
Abnormal function of the bone marrow -inappropriately increased erythropoiesis that is independent of erythropoietin. This is an intrinsic bone marrow disorder called polycythaemia vera and is classified as a myeloproliferative neoplasm. Can lead to thick blood - hyperviscosity which can lead to vascular obstruction
Treatment
If there is no physiological need for a high haemoglobin, or if hyperviscosity is extreme, blood can be removed to thin the blood.
If there is intrinsic bone marrow disease, drugs can be used to reduce bone marrow production of red cells
26
How does clinical context help us with interpreting an FBC that shows polycythaemia?
A young healthy athlete - her very suspicious
A breathless cyanosis patient - probably due to hypoxia
An abdominal mass - it could be carcinoma of the kidney
Splenomegaly - a pointer to polycythaemia vera
27
Describe the term anaemia.
Anaemia is a reduction in the amount of haemoglobin in a given volume of blood below what would be expected in comparison with a healthy subject of same age and gender
The RBC and Hct/PCV are usually also reduced
Occasionally it results from an increase in the volume of plasma rather than a decrease in the amount of haemoglobin. In a healthy person, anaemia resulting from this cannot persist because excess fluid in circulation is excreted so anaemia can be regarded as a decrease of absolute amount of haemoglobin in the circulation.
28
What are the mechanisms of anaemia and distinguish this from the term cause?
Mechanisms
- reduced production of red cells/ haemoglobin in the bone marrow
- loss of blood from the body
- reduced survival of red cells in the circulation
- pooling of red cells in a very large spleen so less in circulation
The mechanism of anaemia might be reduced synthesis of haemoglobin in the bone marrow
The cause of this could be either a condition causing reduced synthesis of haem (lack of iron) or one causing reduced synthesis of globin (inherited mutation - thalassemia)
29
How can we classify anaemia to figure out the cause?
By cell size
Microcytic (usually also hypochromic - cell smaller, reduced haemoglobin)
Normocytic (usually also normochromic - defect not in syntheiss of haemoglobin)
Macrocytic (usually also normochromic)
30
Explain microcytic anaemia.
Common causes:
Defect in haem synthesis
-iron deficiency e.g. iron trapped in macrophages in the stored form haemosiderin and is not mobilised for synthesis of haem
-anaemia of chronic disease - chronic infection e.g. rheumatoid arthritis, TB
Defect in globin synthesis (thalassaemia)
-defect in alpha chain synthesis (alpha thalassaemia)
-defect in B chain synthesis (B thalassameia)
31
Explain macrocytic anaemia.
Macrocytic anaemia usually results from abnormal haemopoiesis so that the red cell precursors continue to synthesise haemoglobin and other cellular proteins but fail to divide normally so red cells end up larger than normal
An alternative mechanism is premature release of cells from the bone marrow. Reticulocytes are larger so MCV increases
Common causes:
- Megaloblastic anaemia as a result of lack of vitamin B12 or folic acid
- Use of drugs interfering with DNA synthesis e.g. methotrexate used to treat leukemia
- Liver disease and ethanol toxicity
- Recent major blood loss with adequate iron stores (reticulocytes increased)
- Haemolytic anaemia (chronic macrocytosis) (reticulocytes increased)
32
What is the difference between macrocytic and megaloblastic?
A macrocytic anaemia is one in which average cell size is increased.
One cause of this is megaloblastic erythropoiesis. This refers specifically to the delay in maturation of the nucleus (defect in synthesis of DNA) while the cytoplasm continues to mature and the cell continues to grow.
A megaloblast is an abnormal bone marrow erythroblast.
It is larger than normal and shows nuclei-cytoplasmic dissociation (immature nucleus, not condensed)
It is possible to suspect megaloblastic anaemia from the peripheral blood features:
-oval macrocytes
-teardrop
-macrocytosis - high MCV
-anaemia
-neutrophils - hypersegmentation
But to be sure requires bone marrow examination
33
Explain normocytic anaemia.
Mechanisms:
- recent blood loss
- failure of production of red cells
- pooling of red cells in the spleen
Causes:
-peptic ulcer, oesophageal varices (enlarged/ swollen veins), trauma
-failure of production of red cells:
Early stages of iron deficiency or anaemia of chronic disease
Renal failure
Bone marrow failure (reduction of stem cells) or suppression
Bone marrow infiltration (cancer)
-hypersplenism e.g. portal cirrhosis, splenic sequestration (sickle cell anaemia)
34
Explain haemolytic anaemia.
Anaemia resulting from shortened survival of red cells in the circulation
Haemolysis can result from an intrinsic abnormality of the red cells.
Haemolysis can result from extrinsic factors acting on normal red cells.
Haemolytic anaemia can be classified as inherited or acquired:
Inherited haemolytic anaemia can result from abnormalities in the cell membrane, the haemoglobin or the enzymes in the red cell
Acquired haemolytic anaemia usually results from extrinsic factors such as microorganisms, chemicals or drugs that damage the red cell. Extrinsic factors can interact with red cells that have an intrinsic abnormality
Can also be classified as intravascular or extravascular:
-intravascular haemolysis occurs if there is very acute damage to the red cell
-extravascular haemolysis occurs when defective red cells are removed by the spleen
Often haemolysis is partly intravascular and partly extravascular
When to suspect haemolytic anaemia:
Otherwise unexplained anaemia, which is normochromic and usually either normocytic or macrocytic
Evidence of morphologically abnormal red cells
Evidence of increased red cell breakdown e.g. lactate dehydrogenase high, increased bilirubin - if liver doesn’t cope - high unconjugated bilirubin (gallstones, jaundice)
Evidence of increased bone marrow activity - high reticulocyte count
35
Explain hereditary spherocytosis.
Haemolytic anaemia or chronic compensated haemolysis resulting from an inherited intrinsic defect of the red cell membrane
After entering the circulation the cells lose membrane in the spleen and thus become spherocytic due to tethering of cytoskeleton
Red cells become less flexible and are removed prematurely by the spleen - extravascular haemolysis
The bone marrow responds to haemolysis by an increased output of red cells leading to polychromatic and reticulocytosis
Haemolysis leads to increased bilirubin production, jaundice and gallstones
Spherocytes are also more prone to haemolysis when osmotic pressure is reduced
Treatment:
Only effective treatment is splenectomy, but this has its own risks so is only done in severe cases (spleen protects against malaria and bacteria)
A good diet is important so that secondary folic acid deficiency does not occur
Alternatively, one folic acid tablet can be taken daily
36
Explain glucose-6-phosphate dehydrogenase (G6PD) deficiency.
G6PD is an important enzyme in the pentose phosphate shunt
It is essential for the protection of the red cell from oxidant damage
Oxidants may be generated in the blood stream e.g. during infection, or may be exogenous causing haemolysis:
-extrinsic oxidants may be foodstuffs (e.g. broad beans), chemicals (e.g. naphthalene) or drugs (e.g. dapsone, primaquine)
The gene for G6PD is on the X chromosome so affected individuals are usually hemizygous males (but occasionally homozygous females)
G6PD deficiency usually causes intermittent, sever intravascular haemolysis as a result of infection or exposure to an exogenous oxidant
These episodes of intravascular haemolysis are associated with the appearance of considerable numbers of irregularly contacted cells
Haemoglobin is denatured and forms round inclusions known as Heinz bodies, which can be detected by a specific test
Heinz bodies are removed by the spleen, leaving a defect in the cell
Acute haemolysis sometimes requires blood transfusion
Thereafter prevention is important - diet and drugs
37
Explain autoimmune haemolytic anaemia.
Autoimmune haemolytic anaemia results from production of autoantibodies directed at red cell antigens
The immunoglobulin bound to the red cell membrane is recognised by splenic macrophages which removes parts of the red cell membrane, leading to spherocytosis
Complement components can also be bound to the immunoglobulin molecule, and they are also recognised by receptors on splenic macrophages
The spherocytes are less flexible than normal red cells
The combination of cell rigidity and recognition of antibody and complement on the red cell surface by splenic macrophages leads to removal of cells from the circulation by the spleen (rigid so gets trapped in spleen)
Diagnosis is by:
Finding spherocytes and an increased reticulocyte count
Detecting immunoglobulin plus/minus complement on the red cell surface
Detecting antibodies (direct antiglobulin test - Coombs test) to red cell antigens or other autoantibodies in the plasma
Treatment is by:
Use of corticosteroids and other immunosuppressive agents
Splenectomy for severe cases
38
What is the treatment for microangiopathic anaemia?
Removing the cause e.g. treating severe hypertension or stopping a causative drug
Plasma exchange when it is caused by an antibody in the plasma that is leading indirectly to fibrin deposition (thrombus, thrombocytopenia)
39
Describe haemoglobin.
Synthesis occurs during development of RBC and begins in pro-erythroblast
-65% erythroblast stage
-35% reticulocyte stage
Haem synthesised before nucleus removed
Structure of haemoglobin made up on haem (synthesised in mitochondria) and globin (synthesised in ribosomes)
1) Fe enters bound to transferrin (iron-binding blood plasma glycoproteins)
2) Transferrin is endocysed through vesicles
3) Iron enters the mitochondria to form haem
Haem synthesis is regulated by delta-ALA (excess haem —> negative feedback
40
Explain the structure and synthesis of haem.
Contained in proteins e.g. haemoglobin, myoglobin, cytochromes, peroxidases, catalase, tryptophan
Same in all types of Hb - globin differs
Combination of protoporphyrin ring with central iron atom (ferroprotoporphyrin)
Iron usually in ferrous form (Fe2+)
Able to combine reversible with oxygen
Synthesised mainly in mitochondria which contains the enzyme ALAS
Regulation
41
Explain the synthesis and structure of globin.
Various types which combine with haem to form different haemoglobin molecules.
Eight functional globin chains, arranged in two clusters:
- b-cluster (b, g, f and e globin genes) on the short arm of chromosome 11
- a-cluster (a and z globin genes) on the short arm of chromosome 16
Globin gene expression and switching
Normal adult haemoglobin:
HbA>HbA2>HbF
Structure of globin
Primary
- a —> 141 aa
- non-a —> 146 aa
Secondary
-75% a and b chains - helical arrangement
Tertiary
- approximate sphere
- hydrophilic surface (charged polar side chains), hydrophobic core
- haem pocket
42
Explain the oxygen-haemoglobin dissociation curve.
O2 carrying capacity of Hb at different pO2
Sigmoid shape:
-binding on one molecule facilitates the second molecule binding (cooperativity)
-p50 = partial pressure of O2 at which Hb is half saturated with O2 (26.6 mmHg - HbA)
```
The normal position of curve depends on:
Concentration of 2,3-DPG
H+ ion concentration (pH)
CO2 in red blood cells
Structure of Hb
```
Right shift (easy oxygen delivery)
- high 2,3-DPG
- high H+
- high CO2
- HbS
Left shift (give up oxygen less readily)
- Low 2,3-DPG
- HbF
43
Define haemoglobinopathies.
Structural variants of haemoglobin or defects in globin chain synthesis (thalassaemia)
44
Define thalassaemia and describe its classification.
Genetic disorders characterised by a defect of globin chain synthesis
Most common inherited single gene disorder worldwide
(Worldwide distribution: malaria and thalassaemia overlap - protect malaria)
Classification
Globin type affected
-alpha
-beta
Clinical severity
-minor (trait) - asymptomatic but can pass gene
-intermedia - (in)dependent of transfusions (intermittent transfusions) non-dependent thalassaemia
-major - individuals need regular blood transfusions, transfusion dependent thalassaemia
45
Explain The general principle of beta (b) thalassaemia and inheritance.
Describe the laboratory diagnosis of Beta thalassaemia.
Deletion or mutation in b globin gene(s)
Reduced or absent production of b globin chains
Prevalence - mainly Mediterranean countries (Greece), Arabian Peninsula, Iran, Indian Subcontinent, Africa, South-east Asia
Autosomal inheritance
Laboratory diagnosis:
FBC- microcytic hypochromic indices, increased RBCs relative to Hb
Film - target ells, poikilocytosis but no anisocytosis
Hb EPS (electrophoresis)/ HPLC
-alpha - normal HbA2 and HbF, +/- HbH (harder to diagnose)
-beta - raised HbA2 and raise HbF
Globin chain synthesis/ DNA studies
-genetic analysis for B-thalassaemia mutations and Xmnl polymorphism (in B-thalassaemias) and a-thalassaemia genotype (in all cases)
46
Describe beta thalassaemia trait.
Describe beta thalassaemia major.
Also known as thalassaemia carrier
Carry a single abnormal copy of the beta globin gene
Usually asymptomatic
Mild anaemia
RBC’s are hypochromic microcytic
Have both normal HbA and abnormal beta thalassaemia haemoglobin
Thalassaemia major
Carry 2 abnormal copies of the beta globin gene
Severe anaemia, incompatible with life without regular blood transfusions
Clinical presentation usually after 4-6 months of life
Hypochromic sphere RBCs
Alpha chain precipitates
Pappenheimer bodies (iron deposits)
Clinical presentation:
Severe anaemia usually presenting after 4 months
Hepatosplenomegaly - RBCs enlarge
Blood film shows gross hypochromia, poikilocytosis and many NRBCs
Bone marrow - erythroid hyperplasia
Extra-medullary haematopoiesis
47
List the clinical features of beta thalassaemia.
```
Chronic fatigue
Failure to thrive
Jaundice - chronic haemolysis
Delay in growth and puberty
Skeletal deformity e.g. expansion of maxillary, frontal bones is a sign for hepatosplenomegaly
Splenomegaly
Iron overload
```
```
Other complications:
Cholelithiasis and biliary sepsis
Cardiac failure
Endocrinopathies
Liver failure
(Last three due to iron overload deposits)
```
48
What is the treatment for thalassaemia major?
Regular blood transfusions
-phenotypes red cells (match closely as possible)
-aim for pre-transfusion Hb 95-100g/L
-regular transfusion 2-4 weekly
-if high requirement, consider splenectomy
Iron chelation therapy
-start after 10-2 transfusions or when serum ferritin >1000mcg/l
-audiology and ophthalmology screening prior to starting
-Deferasirox (Exjade)
Oral
Dose 20-40mg/kg
SE: rash, GI symptoms, hepatitis, renal impairment
-Desferrioxamine (Desferal)
Long-established
Sc infusion 8-12 hours 5-7 days per week (or IV in cardiac iron overload)
Dose 20-50 mg/kg/day
SE: vertebral dyspepsia, pseudo-rickets, genuvalgum, retinopathy, high tone sensorineural loss, increased risk of Klebsiella and Yersinia infection
Compliance
Vitamin C
-Deferiprone (Ferriprox)
Oral
Dose 5-100 mg/kg/day
Effective in reducing myocardial iron
SE: GI disturbance, hepatic impairment, neutropenia, agranulolcytosis, Arturo-ashy
-Combination (reduce dosage of each, reducing toxicity)
Splenectomy - can cause thromboembolic disease
Supportive medical care
Hormone therapy
Hydroxyurea to boost HbF
Bone marrow transplant
Management of infection
-Yersinia
-other Gram negative sepsis
(These strive on iron, so iron overload helps them grow)
-prophylaxis in splenectomised patients - immunisation and antibiotics
49
How is iron overload monitored?
-Serum ferritin
>2500 associated with significantly increased complications
Acute phase protein
Check 3 monthly if transfused otherwise annually
-Liver biopsy
Rarely performed because not representative
-T2 cardiac and hepatic MRI
<20ms - increased risk of impaired LF function
Check annually or 3-6 monthly if cardiac dysfunction
-Ferriscan - R2 MRI
Non-invasive quantitation of LIC
Not affected by inflammation or cirrhosis
<3mg.g normal
>15mg/g associated with cardiac disease
Check annually to 6 monthly if result >20
50
Describe alpha thalassaemia.
Deletion or mutation in a globin gene(s)
Reduced or absent production of a globin chains
Affects both foetus and adult
Excess ? And ? Chains form tetramers of HbH and Hb Barts respectively
Severity depends on number of ? Globin genes affected
51
Describe sickle b thalassaemia, HbE beta thalassaemia, HbH disease.
Diagrams
52
What are the problems associated with treatment in developing countries?
Describe screening and prevention.
Lack of awareness of the problems
Lack of experience of healthcare providers
Availability of blood
Cost and compliance with iron chelation therapy
Availability of and very high cost of bone marrow transplant
Screening and prevention:
Counselling and health education for thalassameics, family members and general public
Extended family screening
Pre-marital screening? Discourage marriage between relatives?
Antenatal testing
Pre-natal diagnosis (CVS)
53
Describe the sickle gene and its effect on RBCs.
Missense mutation at codon 6 of the gene for b globin chain
Glutamic acid replaced by valine
Valine is non-polar, glutamine is polar
Valine is insoluble, glutamine is soluble
Deoxyhaemoglobin S is insoluble
HbS polymerises to form fibres - “factoids”
Intertetrameric contacts stabilise structure
Red cell effects
Stages in sickling of red cells:
-Distortion
Polymerisation initially reversible with formation of oxyHbS (sickle shaped)
Subsequently irreversible
-Dehydration - further concentrates haemoglobin
-Increased adherence to vascular endothelium —> ischaemia
Rigid - can’t travel across microvasculature as effectively
Adherent
Dehydrated
54
List the sickle cell disorders.
Sickle cell anaemia and compound heterozygous states e.g. SC, Sb thalassaemia
-genetically simple - autosomal recessive
-clinically heterozygous
Sickle cell disease (SCD) incorporates sickle cell anaemia and all other conditions that can lead to a disease syndrome due to sickling
55
What are the pathogenesis involved in sickle cell disorders.
-Shortened red cell lifespan - haemolysis
Anaemia
Gall stones
Aplastic crisis (parvovirus B19 - arrests maturation of RBCs)
-Anaemia partly due to a reduced erythropoietin drive as haemoglobin S is a low affinity haemoglobin (release O2 more readily)
-Blockage to microvascualar circulation (vaso-occlusion)
Tissue damage and necrosis (infarction)
Pain - acute pain crisis
Dysfunction
Consequences of vaso-occlusion in sickle cell disease:
HbS polymers —> irreversible shape —> trapped in microvasculature, attract other cells, exacerbates this
56
Explain hypertension in sickle cell disease.
Cell-free haemoglobin limits nitric oxide bioavailability in sickle cell disease
Pulmonary hypertension correlates with the severity of haemolysis
Likely mechanism is that the free plasma haemoglobin resulting from intravascular haemolysis scavenges NO and causes vasoconstriction
Associated with increased mortality
```
Pathogenesis
Lungs:
Acute chest syndrome
Chronic damage
Pulmonary hypertension
```
```
Urinary tract:
Haematuria (papillary necrosis)
Impaired concentration of urine (hyposthenuria)
Renal failure
Priapism
```
Brain:
Stroke
Cognitive impairment
Eyes:
Proliferative retinopathy
57
Describe the early presentation of sickle cell disorders.
How has this improved.
Symptoms rare between 3-6 months
Onset coincides with switch from feral to adult Hb synthesis
Early manifestations:
-dactylitis = inflammation of digit
-splenic sequestration = spleen enlargement, pooling of blood and anaemia
-infection-S pneumoniae
Educate parents to examine abdomen and palpate spleen
58
Explain sickle emergencies.
```
Septic shock (BP < 90/60)
Neurological signs or symptoms
SpO2 < 92% on air
Symptoms/signs of anaemia with Hb<5 or fall >3G/dl from baseline
Priapism > 4 hours
```
Acute chest syndrome
New pulmonary infiltrate on chest X-ray WITH fever
Develops in context of vaso-occlusive crisis surgery pregnancy
Diagnosis often delayed
Mechanical ventilation 15%
Mortality > 18 yr 9%
Avascular necrosis of the femoral head - due to ischaemic damage
Osteomyelitis due to Salmonella infection
Stroke in sickle cell disease
Affects 8% SS
Most common 2-9 years
Involves major cerebral vessels - middle cerebral artery, intercranial carotid artery
By 25 years prevalence of gallstones 50% in SS
Coinheritance of Gilbert syndrome (UGT 1A1 TA7/TA7 genotype) further increases risk - reduce bilirubin conjugation
59
What are the laboratory features of sickle cell disease.
Explain the diagnosis of sickle cell disease.
```
Hb low (typically 6-8 g/dl)
Reticulocytes high (except in aplastic crisis - decrease in Hb with reticulocytopenia without evidence of haemolysis)
Film
-sickled cells
-boat cells
-target cells
-Howell Jolly bodies
```
Diagnosis
Solubility test:
In presence of a reducing agent oxyHb converted to deoxyHb
Solubility decreases
Solution becomes turbid (cloudy/ opaque/ thick)
Does not differentiate AS from SS (detects there are sickle cells but can’t differentiate sickle cell trait)
Definitive diagnosis:
Electrophoresis or high performance liquid
Chromatography (HPLC) separates proteins according to charge
60
Explain the management of sickle cell disease.
General measures:
- folic acid - DNA synthesis
- penicillin
- vaccination - against capsule bacteria, influenza
- monitor spleen size
- blood transfusion for acute anaemic events, chest syndrome and stroke
- pregnancy care
```
Painful crisis:
-pain relief (opioids) - analgesia
-hydration
-keep warm
-oxygen if hypoxic
-exclude infection:
Blood and urine cultures
CXR
```
```
Painful crises triggered by:
Infection
Exertion
Dehydration
Hypoxia
Psychological stress
```
```
Pain management:
Opioids
-marked individual variation in response (intolerance, increase dosage)
-diamorphine most widely used
-most children receive oral opioid
Individual analgesia protocols
Patient controlled analgesia
Adjuvants - paracetamol, NSAIDs, Pregabalin/Gabapentin
```
```
Exchange transfusion
-stroke
-acute chest syndrome
Haemopoietic stem cell transplantation
-<16 yr with severe disease
-survival 90-95%, cure 85-90%
Induction of HbF
-hydroxyurea
-butyrate
```
61
What are the current disease-modifying therapies for SCD?
Transfusion
Hydroxycarbamide (hydroxyurea)
- HbF inhibits polymerisation of HbS
- infants with SCD do not usually develop symptoms until >3 months
- patients with higher HbF levels have fewer complications and improved survival
- increases production of foetal haemoglobin (HbF)
- decreases stickiness of sickle red blood cells
- reduces white blood cell production by the bone marrow
- improves hydration and size of red blood cells
- generates nitric oxide which improves blood flow
Haematopoietic stem cell transplantation
CNS disease:
-stroke
-abnormal TCD and silent infarct
-silent infarcts with cognitive deficiency
-abnormal MRA despite transfusions
-abnormal TCD and RBC alloantibodies
-CNS disease requiring transfusions with iron overload despite optimal care
(If hydroxyurea fails)
```
Limitations:
-donor availability
Long term effects:
-infertility
-pubertal failure
-chronic GvHD
-organ toxicity
-secondary malignancies
```
62
Describe sickle cell trait.
```
HbAS
Normal life expectancy
Normal blood count
Usually asymptomatic
Rarely painless haematuria
Caution: anaesthetic, high altitude, extreme exertion
```
63
What is haemostasis and what is it for?
The cellular and biochemical processes that enable both the specific and regulated cessation of bleeding in response to vascular insult.
To prevent blood loss from intact and injured vessels, enable tissue repair.
64
Describe the balance in haemostasis.
```
Too little = bleeding
Increase in fibrinolytic factors
Increase in anticoagulant proteins
Decrease in coagulant factors
Decrease in platelets
```
```
Too much = thrombosis (pulmonary embolism, DVT, HA, stroke)
Decrease in fibrinolytic factors
Decrease in anticoagulant proteins
Increase in coagulant factors
Increase in platelets
```
65
Summarise the haemostatic plug formation.
Response to injury to endothelial cell lining
Vessel constriction
Vascular smooth muscle cells contract locally
Limits blood flow to injured vessel —>
(Mainly important in small blood vessels; local contractile response to injury)
Formation of an unstable platelet plug
Platelet adhesion
Platelet aggregation
Limits blood loss and provides surface for coagulation —>
Stabilisation of the plug with fibrin
Blood coagulation
Stops blood loss —>
Vessel repair and dissolution of clot
Cell migration/ proliferation and fibrinolytic
Restores vessel integrity
66
Describe a normal artery wall.
Endothelial cells - anticoagulant barrier
TM, EPCR, TFPI, GAG
Subendothelium - procoagulant
Basement membrane
Elastin, collagen
VSMC - tissue factor
Fibroblasts -TF
Generally latent when endothelium intact. Platelet is in resting form.
67
Describe platelets and their role in haemostasis.
Small (2-4 um)
Anuclear
Life span around 10 days
Count: 150-350 x 10^9/L
Megakaryocytes (Nuclear lobes and granulated cytoplasm) primarily found within the bone marrow
Haematopoietic stem cell —> promegakaryocyte —> megakaryocyte —> platelet
Proplatelet protrusions into blood vessels which then bleb off into circulation
Ultrastructure features of the platelet:
A2B1 - collagen
Phospholipid membrane - coagualtion: highly dynamic, flip flop mechanism, negative end of phospholipid found in centre flipped to outside - highly attractive to clotting factor
AIIbB3 - fibrinogen
Micrfotubules and actomyosin - cytoskeleton, platelet rapidly change shape
GPVI - collagen receptor
A-granules: growth factors, fibrinogen, FV, VWF - released when platelets activated
GPIIb/V/IX - platelet capture by VWF
P2Y - ADP
Dense granules - ADP, ATP, serotonin, Ca2+
Platelet cytoskeleton:
Important for platelet morphology, shape change, pseudopods, contraction and clot retraction
Platelet activation - conversion from a passive to an interactive cell
Platelets have roles in haemostasis and thrombosis, cancer, atherosclerosis, infection and inflammation
In normal blood vessel: multimeric VWF circulates in plasma in globular conformation. Binding sites are hidden from platelet GpIb
Platelet adhesion
1) vascular injury damages endothelium and exposes sub-endothelial collagen
2) exposed sub-endothelial collagen binds to globular VWF
3) Tethered VWF unravelled by rheological shear forces of flowing blood, exposing binding sites
4) VWF unravelling exposes platelet binding sites (GpIb) - platelets get tethered
5) binding of VWF to platelet GpIb recruits platelets to site of vessel damage
6) platelets can also bind directly to collagen via GPVI and a2B1 (only at low shear (low flow) I.e. not in arteries/ capillaries
Activation
7) platelets become activated and will further recruit platelets
8) collagen and thrombin also activate platelets (change shape)
9) platelets bound to collagen/ VWF release ADP and thromboxane - activate platelets
Aggregation
10) Activated platelets (aIIbB3) recruit additional platelets
1) aIIB2 also binds fibrinogen - platelet plug develops. Helps slow bleeding and provides surface for coagulation (change membrane composition)
Platelet changes shape upon adhesion, activation and aggregation
Flowing disc-shaped platelet —>rolling ball-shaped platelet (touch collagen, VWF) —> hemisphere-shaped platelet (stick to collagen release factors, firm but reversible adhesion) —> spreading platelet (irreversible adhesion)
68
Describe platelet disorders.
Not enough platelets/ platelets that don’t work
Thrombocytopenia
< 100 x 10^9/L = no spontaneous bleeding but bleeding with trauma .e.g. childbirth
< 40 x 10^9/L = spontaneous bleeding common
Immune thrombocytopenia (ITP): purpura, multiple bruises, ecchymoses
< 10 x 10^9/L severe spontaneous bleeding
69
Describe coagulation factors.
The liver - most plasma haemostatic proteins, clotting factors
Endothelial cells - VWF, TM, TFPI
Megakaryocytes - VWF, FV
Clotting factors circulate as inactive precursors
Either serine protease zymogens or cofactors
Activated by specific proteolysis
Serine protease domain-containing proteins
FVII
FX
Prothrombin
FIX
FXI
Protein C
Once activated, serine protease domain catalysed proteolysis of target substrate.
Serine protease contain a catalytic triad His/Asp/Ser
These serine protease cleave substrates after specific Arg (and Lys residues)
70
Explain the initiation of coagulation.
When endothelial cells damaged, exposure of TF on surface of extravascular cells to clotting factors
1) FVII/ FVIIa bind cell surfaces via GIa domain (gives ability of clotting factors to interact with phospholipid surfaces)
All domains of FVII/FVIIa interact with TF
TF makes FVIIa 2x10^6 times more active
2) TF-FVIIa proteolytically activates FX and TIX
Removes activation peptide to yield active enzyme (cleave small bit)
3) FXa can activate prothrombin to generate thrombin
Activation is inefficient - only small quantities of thrombin are generated.
71
Describe tissue factors.
cellular receptor and cofactors for FVII/VIIa
only procoagulant factor that does not require proteolytic activation
Primary initiator of coagulation
47kDa integral membrane
Normally located at extravascular sites I.e. usually not exposed to the blood (VSMC, fibroblasts .etc.)
TF expressed higher in certain organs (I.e. lungs, brain, heart, testis, uterus and placenta)
TF in these locations provide further haemostatic protection in these organs.
```
Factor VII (FVII)
Serine protease zymogen
48kDa plasma glycoprotein
Expressed/ secreted by the liver
Domain structure:
-Gla domain
-2x EGF-like domains
-Serine protease domain
Circulates in plasma at around 10nM
Around 1% of plasma FVII circulates in its activated from (FVIIa)
```
Coagulation serine protease protein structure
FVII, FIX, FX and PC share:
-a homologous modular structure (4 domains)
-Gla domain - binding to phospholipid surfaces
-EGF domain is involved in protein-protein interactions
-all circulate in plasma in zymogen form
-activated by proteolysis
Gla domain containing proteins:
FVII
FX
Prothrombin
FIX
Protein C
Protein S
Gla domain - binding to phospholipid surfaces
Defines vitamin K-dependent proteins
Gla domains contain 9-11 y-carboxyglutamic acid residues
Gla domains bind 6/7x Ca2+ ions which causes a structural transition
Glutamic acid —> (via Vit K carboxylase) additional -COOH binded —> gives affinity to Ca2+ —> Ca2+ gives conformation to binding to -ve charged phospholipids
(Warfarin is Vit K antagonist so that clotting factors don’t have Gla domain)
72
Explain the propagation of coagulation.
1) Thrombin feedbacks to FVIII and FV.
2) FVIIIa acts as cofactors for FIXa —> FXa (more FXa produced)
3) FXa joins up with its cofactors FVa to activate prothrombin and increase thrombin production (x300)
Small amount of thrombin can activate coagulation system to greatly enhance the amount of thrombin
73
What happens to patient deficient in procoagulant factors?
```
Haemophilia A (FVIII deficiency)
Haemophilia B (FIX deficiency)
(X linked - primarily affects boys)
```
74
Explain the regulation of coagulation.
Clotting only occurs at site of injury, deposited fibrin shuts system down to prevent getting thrombotic event.
Inhibitory coagulation mechanisms:
(I) TFPI
(II) The protein C anticoagulant pathway (APC and protein S)
(III) antithrombin
Deficiencies of antithrombin, protein C and protein S are important risk factors for thrombosis (TFPI deficiency not too important)
TFPI (tissue factor pathway inhibitor), TFPI-FXa can bind/inactivate TF-FVIIa active site via Kunitz domain 1 (K1)
Protein C pathway - protein C is activated by thrombin-TM complex on EC
Activated protein C (APC) inhibits thrombin generation by proteolytically inactivating procoagulant cofactors FVa and FVIIIa by cleaving
(Factor V Leiden mutation arginine substituted for glutamine. Arginine is cleavage site for protein c so not inactivated —> increased risk of thrombosis.
Protein C activation
FIIa - serine protease, cleaves fibrinogen to form fibrin
1) thrombin binds to TM with high affinity
2) thrombomodulin converts thrombin to anti-coagulant, protein C localised to endothelial surface (where thrombin/ TM are)
3) Thrombin cleaves protein C to release activation peptide, activates protein C zymogen to APC (serine protease)
4) Protein S acts as a cofactors for APC activation at edge of clot, this stops fibrin from spreading beyond site if damage (haemostatic plug prevented from spreading)
TFPI regulates the initiation of coagulation
Protein C pathway regulates the propagation phase of coagulation by down-regulating thrombin generation - it does not inhibit thrombin
Antithrombin is a serine protease inhibitor (serpin)
AT inactivated many activated coagulation serine proteases (FXa, thrombin, FIXa, FXIa)
AT mops up free serine proteases that escape the site of vessel damage
(Heparin binds to antithrombin and enhances efficiency of antithrombin inhibiting thrombin and FXa. Heparin is cofactor for antithrombin)
75
Explain the process of fibrinolysis.
Plasminogen —> plasminogen via tissue plasminogen activator (tPA), tPA binds to fibrinogen and activates plasminogen to plasmin
Forms fibrin degradation products, FDP - elevated in DIC
Disseminated intravascular coagulation (DIC) is a condition in which blood clots form throughout the body, blocking small blood vessels.
TPA can be used in therapeutic thrombolysis for MI, ischameic stroke .etc. (Clot busters)
76
State the drugs and tests used in haemostasis.
Drugs
Anticoagulants (treat/protect against venous thrombosis): heparin, warfarin, DOACs (direct oral anticoagulants)
Antiplatelet agents (treat/ protect against arterial thrombosis) - aspirin, P2Y12 blockers
Tests
Coagulation (PT, APTT)- haemostatic potential
Platelet function tests
D-dimer - fibrin degradation product
77
Describe minor bleeding symptoms.
Describe a significant bleeding history.
Easy bruising, gum bleeding, frequent nosebleeds, post-operative bleeding, menorrhagia, post partum bleeding
Epistaxis not stopped by 10 mins compression or requiring medical attention/ transfusion
Cutaneous haemorrhage or bruising without apparent trauma
Prolonged (>15 mins) bleeding from trivial wounds or in oral cavity or recurring spontaneously in 7 days after wound. Spontaneous GI bleeding leading to anaemia
Menorrhagia requiring treatment or leading to anaemia, not due to structural lesions e.g. fibroids of the uterus
Heavy, prolonged or recurrent bleeding after surgery or dental extractions
78
Why do people have abnormal haemostasis?
Lack of a specific factor
- failure of production: congenital and acquired
- increased consumption/clearance
Defective function of a specific factor
- genetic defect
- acquired defect - drugs (antiplatelet drugs), synthetic defect, inhibition
79
Describe the disorders of primary haemostasis.
Platelets
Low numbers: thrombocytopenia
-bone marrow failure eg: leukaemia, B12 deficiency (megaloblastic anaemia - large immature cells, can’t synthesise DNA to divide)
-accelerated clearance eg: immune (ITP), DIC
Impaired function
-hereditary absence of glycoproteins or storage granules eg. ADP, GPIIb/IIIa
Auto-immune thrombocytopenic purpura(auto-ITP)
Antiplatelet autoantibodies coat platelet —> sensitised platelet —> macrophage of spleen in retinoendothelial system
Mechanisms (and causes) of thrombocytopenia
1) failure of platelet production by megakaryocytes
2) shortened half life of platelets
3) increased pooling of platelets in an enlarged spleen (hypersplenism) and shortened half life
Von Willebrand Factor
Von Willebrand disease
-hereditary decrease of quantity +/- function (common) - autosomal
-acquired due to antibody (rare)
VWF has two functions in haemostasis
Binding to collagen and capturing platelets
Stabilising Factor VIII
-Factor VIII may be low if VWF is very low
VWD is usually hereditary
-Deficiency of VWF (Type 1 - make little, not recessive or 3 - make none, recessive)
-VWF with abnormal function (Type 2)
The vessel wall
- Inherited (rare) Hereditary haemorrhagic telangiectasia Ehlers-Danlos
syndrome (collagen, elasticated skin) and other connective tissue disorders
-Acquired: Scurvy, Steroid therapy (thinning of connective tissue holding vessels - bleeding), Ageing (senile purpura), Vasculitis
```
In summary:
Platelets
-Thrombocytopenia
-Drugs
Von Willebrand Factor
-Von Willebrand disease
The vessel wall
-Hereditary vascular disorders
-Scurvy, steroids, age
```
80
Describe bleeding in disorders of primary haemostasis.
```
Immediate
Prolonged bleeding from cut
Epistaxes
Gum bleeding
Menorrhagia
Easy bruising
Prolonged bleeding after trauma or surgery
```
Thrombocytopenia – Petechiae
Severe VWD – haemophilia-like bleeding
81
What are the tests for disorders of primary haemostasis.
Platelet count, platelet morphology Bleeding time (PFA100 in lab)
Assays of von Willebrand Factor
Clinical observation
82
Describe disorders of coagulation.
The role of the coagulation cascade is to generate a burst of thrombin which will convert fibrinogen to fibrin
Deficiency of any coagulation factor results in a failure of thrombin generation and hence fibrin formation
The primary platelet plug is sufficient for small vessel injury
In larger vessels it will fall apart
Fibrin formation stabilises the platelet plug
Haemostasis: fibrin clot stabilising platelet plug
Haemophilia: failure to generate fibrin to stabilise platelet plug
Deficiency of coagulation factor production
Hereditary
-Factor VIII/IX: haemophilia A/B
Acquired (more common)
-Liver disease - Most coagulation factors are synthesised in the liver
-Dilution - Red cell transfusions no longer contain plasma (coagulation factors), Major transfusions require plasma as well as rbc and platelets
-Anticoagulant drugs – warfarin
Increased consumption
Acquired
-Disseminated intravascular coagulation (DIC)
-Immune - autoantibodies
DIC
- Generalised activation of coagulation – Tissue factor
- Associated with sepsis, major tissue damage, inflammation
- Consumes and depletes coagulation factors -Platelets consumed
- Activation of fibrinolysis depletes fibrinogen
- Deposition of fibrin in vessels causes organ failure
83
Describe bleeding in coagulation disorders.
superficial cuts do not bleed (platelets) bruising is common
nosebleeds are rare
spontaneous bleeding is deep, into muscles and joints
bleeding after trauma may be delayed and is prolonged
frequently restarts after stopping
Haemarthrosis – hallmark of haemophilia (swollen elbow)
Intramuscular injections should be avoided (bruising all over body)
84
Distinguish between bleeding due to platelet and coagulation defects I.e. primary vs secondary.
Platelet/Vascular
- Superficial bleeding into skin, mucosal membranes
- Bleeding immediate after injury
Coagulation
- Bleeding into deep tissues, muscles, joints
- Delayed, but severe bleeding after injury. -Bleeding often prolonged
Both can be life threatening
85
What are the tests for coagulation disorders.
Screening tests (‘clotting screen’)
- Prothrombin time (PT)
- Activated partial thromboplastin time (APTT)
- Full blood count (platelets)
```
Factor assays (for Factor VIII etc)
Tests for inhibitors
```
86
Describe disorders of fribrinolysis.
Disorders of fibrinolysis can cause abnormal bleeding but are rare
Hereditary
-antiplasmin deficiency
Acquired
-drugs such as tPA
-Disseminated intravascular coagulation - lots of fibrin therefore lots of fibrinolysis
87
What are the genetics of common bleeding disorders.
```
Haemophilia
-Sex linked recessive (SLR)
Von Willebrand disease
-Autosomal
-Type 2, (Type 1) AD
-Type 3 AR
All the rest (V, X etc.)
-Autosomal recessive (AR)
-And therefore much less common
```
88
Explain the treatment of abnormal haemostasis.
```
Failure of production/function
Replace missing factor/platelets
-Prophylactic
-Therapeutic
Stop drugs
```
Immune destruction
Immunosuppression (eg prednisolone)
Splenectomy for ITP
Increased consumption
Treat cause
Replace as necessary
Details of above: (No need to memorise)
Factor replacement therapy
Plasma
-Contains all coagulation factors Cryoprecipitate
-Rich in Fibrinogen, FVIII, VWF, Factor XIII ◻ Factor concentrates
-Concentrates available for all factors except factor V.
-Prothrombin complex concentrates (PCCs) Factors II, VII, IX, X
Novel approaches
Gene therapy
-Haemophilia B, (Haemophiia A)
Platelet replacement therapy
Pooled platelet concentrates available
In development
Other treatments:
DDAVP - vasopressin, causes endothelial to release VWF (help in type 1)
Tranexamic acid - antifibrolytic, competes with fibrin for tPA, crosses placenta low conc. in breast milk
Fibrin glue/ spray
89
Describe ABO blood groups.
A and B antigens on red cells formed by adding one
or other sugar residue onto a common glycoprotein and fucose stem on red cell membrane
Group O has neither A or B sugars - stem only
Antigens determined by corresponding genes A gene codes for enzyme which adds N-acetyl
galactosamine to common glycoprotein and fucose stem
B gene codes for enzyme which adds galactose
A and B genes are co-dominant
O gene is ‘recessive’ eg: person is blood group A - genes could be AA or OA
```
Person has antibodies against any antigen NOT present on own red cells, antigen-antibody interaction can be fatal
Naturally occurring (nearly from birth) - IgM: it is a ‘complete’ antibody, so:- fully activates complement cascade to cause haemolysis of red cells - lysis - release haemoglobin, release bilirubin, jaundice
In labs, IgM Abs interact with corresponding antigen to cause agglutination - can see incompatibility —> X-match: patient’s serum mixed with donor red cells - should not react: if reacts (agglutinates) = incompatible (anti-A, anti-B)
```
90
Describe RhD.
Blood groups: RhD positive (if have D antigen) or RhD
negative (if not)
Genes for RhD groups:
D - codes for D antigen on red cell membrane
d - codes for no antigen and is recessive
Therefore dd = no D antigen = RhD negative
DD or Dd = D antigen present = RhD positive
Small percentage are RhD negative
People who lack the RhD antigen (ie: RhD negative) CAN make anti-D antibodies AFTER they are exposed to the RhD antigen - either by transfusion of RhD positive blood or in women, if they are pregnant with an RhD positive fetus Anti-D antibodies are IgG antibodies (delayed RBC haemolysis, all symptoms slower, delayed haemolytic transfusion reaction - anaemia, high bilirubin, jaundice)
1) Future transfusions - patient must, in future, have RhD neg blood
2) HDN = haemolytic disease of the newborn
- if RhD neg mother has anti-D - and in next
pregnancy, fetus is RhD pos - mother’s IgG anti-D antibodies can cross placenta - causes
haemolysis of fetal red cells, anaemia- if severe: hydrops details; death - severe anaemia; hyperextension of arm, legs, neck due to brain damage (cross BBB)
BEFORE each transfusion episode, test patient’s blood sample for red cell antibodies
Therefore, before transfusing patient, as well as testing their ABO and RhD group, must do an ‘antibody screen’ of their plasma
91
Describe the blood components.
Split one unit of blood by centrifuging whole bag (red cells bottom, platelets middle, plasma top) then squeeze each layer into satellite bags and cut free (closed system) - no longer give whole blood, only parts needed as components wont be wasted - only need some and some components degenerate quickly
Red cells
1 unit from 1 donor - ‘packed cells’ (fluid plasma removed)
Shelf life 5 weeks; stored at 4oC (fridge)
Give through a ‘blood giving set’ - has filter to remove clumps/debris
Rarely need frozen red cells
(National Frozen Bank) - for rare groups/ antibodies - poor recovery on thawing - lose RBCs
FFP - fresh frozen plasma
1 unit from 1 donor
Stored at -30oC (frozen within 6h of donation to preserve coag factors) Shelf life 2 years Must thaw approx 20-30 mins before use (if too hot, proteins cook)
Give ASAP – ideally within 1h or else coag factors degenerate at room temp
Dose 12-15ml/kg = usually 3 units Need to know blood group - no x- match, just choose same group (as contains ABO antibodies, which could cause a bit of haemolysis
Indications:
1. If bleeding + abnormal coag test results (PT, APTT) - Monitor response - clinically and by coag tests
2. Reversal of warfarin (anticoagulant) eg for urgent
surgery (if PCC not available)
3. Other conditions occasionally
NB not just to replace volume/ fluid loss
Cryoprecipitate
From frozen plasma thawed at 4-8oC overnight
residue remains
Contains fibrinogen and factor VIII
Same as FFP - store at -30oC for 2 yrs Standard dose = from 10 donors
(5 in a pack)
Indications:
If massive bleeding and fibrinogen very low Rarely hypofibrinogenaemia
Platelets
1 pool from 4 donors
Store at 22oC (Room temp) - constantly agitated
Shelf life 7 days only - (risk of bacterial infection)
Need to know blood group - No cross-match, just choose same group (as platelets have low levels of ABO antigens on, so wrong group platelets would be destroyed quickly) - and can cause RhD sensitisation, as some red cell contamination
Indications:
Mostly haematology patients with bone marrow failure (if platelets <10 x 109/L)
Massive bleeding or acute DIC
If very low platelets and patient needs surgery
If for cardiac bypass and patient on anti-platelet drugs
1 pool is usually enough - rarely need more
92
Describe fractionated products (large pool).
Factor VIII and IX
- For haemophilia A and B respectively (males)
- Factor VIII for von Willebrand’s disease
- Heat treated - viral inactivation
- Recombinant factor VIII or IX alternatives increasingly used, but expensive
Immunoglobulins:
- IM: Specific - tetanus; anti-D; rabies
- IM: Normal globulin - broad mix in population (eg: HAV)
- IVIg – pre-op in patients with ITP or AIHA
Albumin
4.5%
→ Useful in burns, plasma exchanges, etc
→ Probably overused (not indicated in malnutrition)
20% (salt poor)
→For certain severe liver and kidney conditions only
93
Describe donors
Aim to keep blood safe for patient (eg transmitting infections; drugs; disease)
By testing for infections (some) - not failsafe And by questioning for risk behaviour - to exclude them e.g. Hep B/C, HIV
Also aim to prevent harm to donors - by
questioning them, to exclude risky ones (eg: people who have heart problems, etc)
Exclude high risk donors Use voluntary, unpaid donors
94
Describe haemopoiesis.
When can you see immature neutrophils?
Production of blood cells in bone marrow
Normal haemopoiesis (polyclonal healthy/ reactive)
-normal marrow
-reactive marrow
Malignant haemopoiesis (abnormal/ colonial)
-leukaemia (lymphoid, myeloid), myelodysplasia, myeloproliferative
Differentiation and maturation
Myeloblast —> promyelocyte —> myelocyte —> metamyelocyte —> neutrophil
Cytokines influence differentiation and proliferation:
Erythroid - erythropoietin
Lymphoid -IL2
Myeloid - G-CSF, M-CSF
Immature WBCs and nucleated RBCs in blood film = leukoerythroblastic feature.
Situations with immature WBCs:
-after chemotherapy - growth factor GCSF given to recover neutrophils
-sepsis - bone marrow compensates
95
Describe the leukocytes.
```
Bone marrow:
Lymphoblasts
-myeloblasts
-promyelocytes
-myelocytes
-metamyelocytes
```
```
Peripheral blood:
Immunocytes:
-T lymphocytes
-B lymphocytes
-NK cells
phagocytes:
-granulocytes: neutrophils, eosinophils, basophils
-monocytes
```
Bone marrow = immature cells, proliferating and differentiating from stem cells
Peripheral = mature cells respond to infection
In haemopoietic cancers both immature and mature cells found in peripheral
96
Describe causes of abnormal white blood cell count.
```
Cell production
Increase:
Reactive
-infection
-inflammation
Malignant
-leukaemia
-myeloproliferative
```
```
Decrease:
Impaired BM function
-B12 or folate deficiency
-BM failure
-Aplastic anaemia
-Post chemotherapy
-Metastatic cancer
-Haematological cancer
```
Cell survival
Increase:
Failure of apoptosis (e.g. acquired cancer causing mutations in some lymphomas)
Decrease:
Immune breakdown - auto-antibodies
97
Give an example of increased white cell number.
Eosinophilia
Two causes: reactive; primary (malignant)
Normal haemopoiesis; abnormal haemopoiesis
```
Normal
(stimulated by)
• Inflammation
• Infection
• Increased cytokine production
– Distant tumour
–Haemopoietic or non haemopoietic
```
```
Abnormal haemopoiesis
(autonomous cell growth)
• Cancers of haemopoietic cells
• Leukaemia
-Myeloid or lymphoid
–Chronic or acute
• Myeloproliferative disorders
```
Reactive causes:
Parasitic infestation
Allergic diseases e.g. asthma, rheumatoid, polyarteritis,pulmonary eosinophilia. Neoplasms, esp. Hodgkin’s, T-cell NHL Hypereosinophilic syndrome
Malignant Chronic Eosinophilic
Leukaemia (PDGFR fusion gene)
98
How do you investigate a raised WCC?
* History and examination
* Haemoglobin and platelet count
* Automated differential - when no differential, machine cant recognise different cell types. Only happens in certain cases e.g. malignancy
* Examine blood film
* Abnormality White cells only, or all 3 lineages (red cells/platelets/white cells) ?
* White cells 1 cell type only, or all lineages? (e.g. neuts/eos/monocytes/lymphocytes) a;; types = reactive but one exception is chronic myeloleukaemia
* Mature cells only or mature and immature cells?
99
What are the common causes of abnormal white cell counts?
Phagocytes
Neutrophils Eosinophils Monocytes
Immune cells
Lymphocytes
100
Describe neutrophilia.
Neutrophils
• Present in BM, blood and tissues
• Life span 2-3 days in tissues (hours in PB)
• 50% circulating neutrophils are marginated - adhere to endothelium, e.g. stress situation - marginated —> circulating (not counted in FBC)
Neutrophilia can develop in:
• minutes > demargination
• hours > early release from BM
• days > increased production (x3 in infection)
Neutrophilia (causes)
Infection
• Tissue inflammation (e.g.colitis, pancreatitis) , toxic granules
• Physical stress, adrenaline, corticosteroids
• underlying neoplasia
Malignant neutrophilia
• myeloproliferative disorders
• CML (chronic myeloid leukaemia)
Neutrophilia in infection
• Localised and systemic infections • acute bacterial, fungal, certain viral
infections - rare because usually cause lymphocytosis
Some infections characteristically do not
produce a neutrophilia e.g. brucella,
typhoid, many viral infections. - cause lymphocytosis
101
Describe monocytosis.
```
Rare but seen in certain chronic infections and primary haematological disorders
• TB, brucella, typhoid
• Viral; CMV, varicella zoster
• Sarcoidosis
• Chronic myelomonocytic leukaemia (MDS)
```
102
Describe lymphocytosis and link to glandular fever.
If mature is it :
•reactive to
•primary disorder
If immature it is:
•primary disorder (leukaemia/lymphoma)
Mature lymphocytes
•CLL
•autoimmune/inflammatory disease
Immature lymphoblasts
•Acute lymphoblastic leukaemia
Lymphocytosis (mature cells)
Is it primary or reactive ?
• Secondary (reactive); polyclonal response
to infection, chronic inflammation, or underlying malignancy.
• Primary; monoclonal lymphoid proliferation
e.g. CLL
```
Reactive lymphocytosis
Infection
• EBV - Mononucleosis syndrome, CMV, Toxoplasma
• infectious hepatitis, rubella, herpes
infections
Autoimmune disorders
neoplasia
sarcoidosis
```
```
Glandular fever
• EBV infection of B-lymphocytes via
CD21 receptor
• Infected B-cell proliferates and expresses EBV associated antigens
• Cytotoxic T-lymphocyte response
• acute infection resolved resulting in
lifelong sub-clinical infection.
```
•Elderly patients with lymphocytosis •mature lymphocytes (may be smear cells) •differential: reactive to underlying auto immune
disorder or chronic lymphocytic leukaemia
How can you distinguish?
•Morphology
•Immunophenotype
•Gene re-arragement
```
Evaluating lymphocytosis (B cells) Light chain restriction
polyclonal - Kappa & lambda
Monoclonal kappa only or lambda only - malignant
```
```
Evaluating lymphocytosis - gene rearrangement
Imuunoglobulin genes (Ig) and T cell receptor (TCR) genes undergo recombination in antigen stimulated B cells or T cells.
With primary monoclonal proliferation all daughter cells carry identical configuration of Ig, or TCR gene. This can be detected by Southern Blot analysis
```
103
Explain iron absorption.
How does the gut cell alter iron absorption?
```
Iron lost:
1) desquanted cells of skin and gut
2) bleeding - menstruation, pathological
Men need 1 mg/day
Women need 2 mg/day
```
Human diet provides 12-15mg iron/day Iron occurs in most natural foods
- Meat and fish(haem iron) - better absorbed iron
- Vegetables
- Whole grain cereal
- chocolate
Most iron eaten IS NOT ABSORBED
Can’t absorb ferric iron Fe3+ (Can only absorb ferrous iron Fe2+)
- Orange juice helps - Fe2+
- cups of tea make it worse - Fe3+
Factors affecting absorption
DIET: increase in haem iron, ferrous iron INTESTINE: acid (duodenum), ligand (meat) SYSTEMIC: iron deficiency,anaemia/hypoxia, pregnancy
Iron in diet —> intracellular iron through villi of duodenum —> ferritin —> iron in plasma, transferrin
Gut cell
Hepcidin binds to ferroportin (essential for absorption of iron at bottom of enterocytes) and destroys it so less absorbed
High iron - high hepcidin - low ferroportin - low absorption
```
Transferrin
Holds onto iron in the circulation
Different ways labs give details:
1) transferrin
2) total iron binding capacity, TIBC
3) transferrin saturation
```
Erythropoietin
Anaemia —> tissue hypoxia —> increase in erythropoietin —> red cell precursors
104
Describe anaemia of chronic disease.
Laboratory signs of being ill
1) C-reactive protein - increase in inflammation/ infection
2) erythrocytes sedimentation rate (ESR) high
3) Acute phase response - increases
- ferritin
- FVIII
- fibrinogen
- immunoglobulins
Associated conditions
1) Chronic infections e.g. TB/HIV
2) Chronic inflammation e.g. RhA/SLE 3)Malignancy
4) Miscellaneous e.g. cardiac failure
Pathogenesis
-Cytokines prevent the usual flow of iron
from duodenum to red cells
Block in iron utilisation - you have iron but flow blocked
Cytokines do several things
1. Stop erythropoietin increasing
2. Stop iron flowing out of cells
3. Increase production of ferritin
4. Increase death of red cells
Therefore
- make less red cells
- more red cells die
- less availability of iron (stuck in cells/ferritin)
Cytokines include
- TNF alpha
- interleukins
105
Describe iron deficiency including investigations.
What are the 3 causes of a low MCV.
Causes
1. Bleeding e.g. menstrual/GI
2. Increased use e.g. growth/pregnancy
3. Dietary deficiency e.g. vegetarian
4. Malabsorption e.g. coeliac
```
Full GI investigations if:
Good diet and no coeliac antibodies
Male
Women over 40
Post menopausal women
Women with scanty menstrual loss
```
Full GI investigations
• Upper GI endoscopy - oesophagus, stomach, duodenum e.g. stomach cancer, gastritis, colonic polyp
• Take duodenal biopsy
• Colonoscopy
IF FIND NOTHING - small bowel meal and follow through
Other investigations
Menstruating woman <40 ….if heavy periods OR multiple pregnancies and no GI symptoms do nothing
? Urinary blood loss
Antibodies for coeliac disease
```
Laboratory parameters
MCV (mean cell volume)
Serum iron
Ferritin
Transferrin
(= total iron binding capacity, TIBC)
Transferrin saturation
```
Causes of a low MCV
1. Iron deficiency
2. Thalassaemia trait
3. Anaemia of chronic disease (low or N)
In a patient:
If low MCV, check other parameters, check other parameters:
Serum iron can be low in iron deficiency, anaemia of chronic disease and thalassaemia trait so confirm thalassaemia trait:
- Haemoglobin electrophoresis
- confirms an additional type of
haemoglobin is present
Ferritin is low in iron deficiency and high in chronic disease (acute phase protein).
But someone can have iron deficiency and has a chronic underlying disease so ferritin can be normal despite iron deficiency E.g. RhA plus bleeding ulcer
Lab clues that ferritin not ideal:
Raised CRP
Raised ESR
Transferrin
Iron deficiency: transferrin increase
Chronic disease: normal or even low (may not make proteins as well)
Transferrin saturation
Iron deficiency: low saturation
Chronic disease: normal
Further investigations
• Endoscopy and colonoscopy
• Duodenal biopsy
• Anti-helicobacter antibodies • Anti-coeliac antiodies
? Abdo ultrasound to look at kidneys
? Dipstick urine
? Pelvic ultrasound to exclude fibroids
```
Man of ANY AGE (even your age) with
a low ferritin
This suggests iron deficiency and he
needs to have upper and lower GI
endoscopies to look for a source of
bleeding
```
Additional tests
- blood film - small, pale, strange
shapes including pencil cells
- bone marrow? Stain for iron
106
Give the ordinary parameters for:
Classic iron deficiency
Anaemia of chronic disease
Classic anaemia of chronic disease
```
Classic iron deficiency
Hb LOW
MCV LOW
Serum iron LOW
Ferritin LOW
Transferrin HIGH
Transferrin saturation LOW
```
```
Classic anaemia of chronic disease
Hb LOW
MCV LOW or N
Serum iron LOW
Ferritin HIGH or N
Transferrin normal/low
Transferrin saturation normal
```
107
What are B12 and folate required for?
Required for DNA synthesis, absence leads to severe anaemia which can be fatal
B12 required for:
1. DNA synthesis
2. Integrity of the nervous system
Folic acid required for:
DNA Synthesis
Homocystine metabolism
108
Explain the clinical features of B12 and folate deficiency.
```
ALL RAPIDLY DIVIDING CELLS ARE
AFFECTED
Bone marrow
Epithelial surfaces of mouth and gut
Gonads - spermatogenesis
embryos
```
Anemia: weak, tired, short of breath - can’t make RBC’s
Jaundice - break RBC’s down
Glossitis (inflamed tongue) and angular cheilosis (cracks at corner of mouth)
Weight loss, change of bowel habit
Sterility
Anaemia
Macrocytic or megaloblastic
Macrocytic - Average red cell size is above the normal range
Vitamin B12/folate deficiency
Liver disease or alcohol
Hypothyroid
Drugs e.g. azathioprine
Haematological disorders:
Myelodysplasia, aplastic anaemia, Reticulocytosis e.g. chronic haemolytic anaemia
```
Megaloblastic - Describes a morphological change in the red cell precursors within the bone marrow
Defined by asynchronous maturation of
the nucleus and cytoplasm in the
erythroid series
Maturing red cells seen in the bone
marrow
Nucleus makes lots of proteins so blueish, cytoplasm more pink, nucleus denser, DNA not working properly so no sequence
Peripheral blood in megaloblastic:
Anisocytosis
Large red cells
Hypersegmented neutrophils - granular cytoplasm, more than 5 segments
Giant metamyelocytes
```
109
Give tests that you would do if someone had a macrocytosis.
Folate and B12 test
Thyroid function test
Liver function test
Reticulocyte test
110
Describe intake of folate.
Describe the diagnosis of folate deficiency.
Fresh leafy vegetables
Destroyed by overcooking/canning/processing
```
Decreased intake:
Ignorance
Poverty
Apathy
Elderly
Alcoholics
```
```
Increased demand:
-Physiological
Pregnancy
Adolescence
Premature babies
-Pathological
Malignancy
Erythoderma - large percentage of body surface becomes red and inflamed e.g. eczema, psoriasis
Haemolytic anaemias - break down RBCs, need to make more so demand increased
```
```
Examples
- alcoholic admitted with a head injury
after a fight
- 30y old lady with infected whole body
eczema
- 90 y old lady who has a cup of tea and a
jam sandwich for each meal
```
Lab diagnosis
FBC and film
Folate levels in the blood
Assessing cause of decreased folate
EASY – history (diet/alcohol/illness) EXAMINATION – skin disease/alcoholic liver disease
111
Explain the consequences of folate deficiency.
1.Megaloblastic, macrocytic anemia
2.Neural tube defects in developing fetus
3.Increased risk of thrombosis in association with variant enzymes involved in
homocysteine metabolism
```
Neural tube defects
Spina bifida
Anencephaly
ALL PREGNANT WOMEN TAKE
FOLIC ACID 0.4MG PRIOR TO
CONCEPTION AND FOR FIRST 12
WEEKS
```
```
Folic acid and homocysteine
Very high homocysteine levels are
associated with
-atherosclerosis
-premature vascular disease
```
```
Mildly elevated levels of homocysteine
are associated with:
cardiovascular disease DEFINITELY
arterial thrombosis PROBABLY
venous thrombosis POSSIBLY
```
112
Describe presentation of B12 deficiency and examination.
What are the consequences?
1) 40 y old female with tingling in fingers - parasthesia
Hb 10g/dl and MCV 105
Family history of auto-immune disease MEASURE VITAMIN B12 and it is low
2) Patient with an inflamed tongue
(glossitis)
Premature grey hair
falls over when they close their eyes
Loss of proprioception = Romberg’s sign (stand, arms out, close eyes, fall over)
```
History of presentation:
Paraesthesiae
Muscle weakness
Difficult walking
Visual impairment
Psychiatric disturbance
```
Examination - Absent reflexes and upgoing plantar responses e.g. knee jerks, central and peripheral damage from B12 deficiency
```
Consequences
Neurological problems
-Bilateral peripheral neuropathy
-Subacute comined degeneration of the
cord - Posterior and pyramidal tracts of the spinal cord - become paralysed
-Optic atrophy
-dementia
```
113
Describe the causes of B12 deficiency.
```
POOR ABSORPTION
Reduced dietary intake
-Stores are large and last for 3-4 years
-Animal produce
-Vegans are at risk
Infections/infestations
-Abnormal bacterial flora (stagnant loops) -Tropical sprue
-Fish tapeworm
```
```
Absorption
Normally:
Occurs in small intestine – B12 is then
stored – when stores are saturated
excess B12 is excreted in the urine
```
2 methods of absorption
Method 1
- Slow and inefficient (1%)
- duodenum
Method 2 - most absorption this way.
B12 must combine with intrinsic factor Intrinsic factor is made in the stomach
(parietal cells)
B12-IF binds to ileal receptors
Three things essential for absorption:
Intact Stomach
Intrinsic factor
Functioning small intestine
Impaired B12 absorption
1) Reduction in intrinsic factor
- Post gastrectomy -stomach cancer, ulcer removal
- gastric atrophy
- antibodies to intrinsic factor or parietal cells - autoimmune
Pernicious anaemia - autoimmmune condition associated with severe lack of IF
60 years peak age, family history, males have a decreased life expectancy -stomach cancer
2) Diseases of small bowel (terminal
ileum)
- Crohns
- Coeliac disease
- surgical resection
```
Infections
H Pylori
Giardia
Fish tapeworm
Bacterial overgrowth
```
Drugs associated with low B12
Metformin
Proton pump inhibitors e.g. omeprazole
Oral contraceptive pill
114
How do you diagnose someone with low B12?
Antibodies to parietal cells and intrinsic factor
Anitbodies for coeliac disease
Breath test for bacterial overgrowth
Stool for H Pylori
Test for Giardia
OLDEN DAYS - Shilling test (part I and part II) - radioactivity
```
Shilling test
Prior to test, replenish stores
a) drink radiolabelled B12
b) measure excretion in the urine
6 injections of B12 given to overload
Absorbed through small intestine —> into bloodstream —> filtered by kidneys —> urine radioactive
If not radioactive - not absorbed, not given all 6 injections, so need B12
Not absorbing B 12
- pernicious anaemia
- small bowel disease
Hadn’t corrected B 12 deficiency before the test
Part II
Repeat test with addition of intrinsic
factor
Measure excretion of B12 in the urine
```
115
What is a classic case but normal B12?
Measure methylmalonyl acid
Measure homocysteine
Look for anti-intrinsic factor antibodies
Treat as B12 deficiency until you get all of
the results back
116
Describe treatment for B12 deficiency.
Injections of B12 - 1000ug (i.m) ! 3x/week for 2 weeks
Thereafter every 3 months
IF NEUROLOGICAL INVOLVMENT
B12 injections alternate days until no further improvement – up to 3 weeks
Thereafter every 2 months
