Jack Roberts 2Nd Year Revision Notes 2008-09 MCD - Haematology

Jack Roberts – 2nd Year Revision Notes 2008-09 – MCD - Haematology

MCD – Haematology

Iron Deficiency

1.Describe the role of iron in erythropoiesis.

Iron – essential component of many haem-containing molecules including enzymes, haemoglobin and myoglobin
Iron → Haem + Globin → Haemoglobin (carries oxygen)
Low iron = Low haemoglobin = anaemia
Haem consists of a protoporhyrin ring with a ferrous iron (Fe2+) at its centre
Anaemia → Tissue Hypoxia → Increase in erythropoietin → red cell precursors (survive – grow – divide)

2.List the dietary sources of iron, the factors influencing the absorption of iron, and the causes of iron deficiency.

Red celles live for 120 days – to re-make huge numbers on daily basis, 20mg/day is required – most iron is recycled
Loss – desquamation of gut/skin cell; bleeding – menstrual/pathological
Men – 1mg/day; Women – 2mg/day
Diet provides 12-15mg/day – most not absorbed
Sources – meat, offal and fish (haem iron), vegetables, whole grain cereals, chocolate
Haem is better absorbed than free iron (up to 10% absorption) – not adversely affected by other food components
Unable to absorb Fe3+; Fe2+ 1-2%
Improve – Acid pH, ascorbic acid, digestive enzymes
Inhibit – phytates and phosphates (tea)
Systemic factors affecting absorption – iron deficiency, anaemia/hypoxia, pregnancy
Total iron – 3-5g

Metabolic pool

Haemoglobin2500 mg

Myoglobin500 mg

Storage pool

Ferritin and haemosiderin0-1000 mg

Transit pool

Plasma protein-bound iron3 mg e.g.transferrin bound

Transferrin – holds to iron in circulation – glycoprotein made in the liver (2 iron binding sites)
Total iron binding capacity (TIBC)
Interacts with a transferrin receptor on the surface of erythroblasts
The complex is then internalised, the iron removed, and then the transferrin re-circulated
Iron itself acts as a positive regulator of erythropoiesis and for the expression of the gene that codes for ferritin
Iron is a negative regulator for the expression of the gene that codes for the transferring receptor

3.Describe the clinical and haematological features of iron deficiency anaemia, and the diagnosis and management of iron deficiency.

Hypochromatic microcytic anaemias – red cells have less haemoglobin than usual
Low MCH
Low MCHC (hypochromia)
Low MCV (small, microcytic)
IDA, ACD, Thalassaemias
Iron deficient anaemia (IDA) – most common cause of anaemia worldwide
Blood loss - the main sources of blood loss are uterine in women of childbearing age group, followed by gastrointestinal blood loss, which may be overt or occult.
Dietary deficiency occurs in vegans and vegetarians with unbalanced diets poor in iron but can also occur in non-vegetarians.
Increased needs occur during childhood, especially during the pubertal growth spurt, and during child bearing.
Malabsorption is a less common cause of iron deficiency
Clinical
General – tiredness/lethargy, pale, short of breath (dyspnoea), palpitations
Specific – koilonychias (spoon shaped nails)
Treatment – oral iron compounds (commonly ferrous sulphate). Side effects may include constipation and indigestion.

In cases of difficulty, iron can be given parenterally (outside alimentary canal) – IM, IV

4.Describe the clinical and haematological features of anaemia of chronic disease and explain how this is distinguished from iron deficiency.

No obvious cause except that the patient is ill
Associated conditions – chronic infections (TB/HIV), chronic inflammation (RhA/SLE (lupus)), malignancy, miscellaneous e.g. cardiac failure
Laboratory signs of being ill:
C-reactive protein (raised)
Erythrocyte Sedimentation Rate (raised)
Acute phase response- increases inferritin; FVIII; fibrinogen; immunoglobulins
Pathology – cytokines:
Stop erythropoietin increasing
Stop iron flowing out of cells
Increased production of ferritin
Increased death of red cells
Accumulation of excess iron in the bone marrow storage pool but with a block in iron incorporation into erythroblasts, which may lead to reduced haemoglobin synthesis and hypochromia
Therefore - make less red cells; more red cells die; less availability of iron
Cytokines include TNF alpha, interleukins
The main pathological difference with iron deficient anaemia is the presence of raised marrow iron stores, a normal or raised serum ferritin and a normal to low serum transferrin/Total Iron Binding Capacity (TIBC)

5.Assessment of iron status – haematological features

Parameter / Comment / Iron deficiency / ACD / Thalassaemia Trait
Hb / Non-specific screening / Low / Low / Normal or low
MCV / Differential diagnosis of type of anaemia / Low (normal in early stages) / Low or normal / Low
Serum Iron / Low / Low / Normal
TIBC (transferrin) / Raised / Normal or Low / Normal
Transferrin saturation / Low / Normal
Ferritin / Specific to IDA when low / Low / Normal or raised / Normal
Soluble TfR / Dependant on erythroid activity / High / Normal / Normal or slightly raised
BM iron stores / Gold standard for iron status / Absent / Increased / Normal or increased

Confirmation of thalassaemia trait – haemoglobin electrophoresis - additional type of haemoglobin is present
Ferritin is not perfect – iron deficiency plus underlying chronic disease e.g. bleeding stomach ulcer (ferritin will be normal despite deficiency). Additional tests:
Blood film – may see changes associated with iron deficiency e.g. ellipocytes
Bone Marrow aspirate – slides can be stained to look for iron stores
Look for soluble transferring receptors
Transferrin
IDA – increases
ACD – normal or even low
Therefore saturation will be low in IDA compared to normal in ACD

Vitamin B12 and Folic Acid

1.Macrocytic anaemia

Increase in MCV or the red cells (cells larger than normal) – measured by automated full blood count machine
Causes –
Vitamin B12 or folate deficiency
Liver disease
Hypothyroidism
Excessive alcohol consumption
Drugs e.g. azathioprine, zidovudine
Haematological disorders
myelodysplasia
aplastic anaemia
reticulocytosis e.g. chronic haemolytic anaemias

2.Megaloblastic anaemia

Abnormal but distinct morphological appearance of early and developing blood cells – discernable by light microscopy
Normal:

Proerythroblast – dark blue cytoplasm – high RNA content; nucleus – slightly condensed chromatin
Erythroblast – progressively less RNA, more haemoglobin
Late erythroblast – pink rather that blue cytoplasm – confined to bone marrow
Nuclear chromatin becomes more condensed until extruded completely → reticulocyte – may be found in peripheral blood – precursors of RBC
Megaloblastic anaemia:
Megaloblast (vs. normoblast)
Anisocytosis
Large red cells (MCV high)
Hypersegmented neutrophils - >5 abnormal
Giant metamyelocytes
WBC and platelet count may also be low
Delayed maturation of the nuclei – many blood cells die in the bone marrow – R cell production increases to compensate – ineffective erythropoiesis

3.Vitamin B12 Deficiency

Haematinic deficiencies – consider intake; demand; absorption, loss or utilisation
Inadequate intake is rare
Vitamin B12 is found in animal products, so vegans are at risk
Abnormal bacterial flora in the small bowel (e.g. associated with stagnant loops) can consume vitamin B12
Increased demands are usually readily covered by the vitamin B12 stores, which are relatively large in relation to daily needs and usually sufficient to last for many years.
Absorption of B12 is complicated and failure of absorption is the commonest cause of B12deficiency
B12 is absorbed in the small bowel following combination with intrinsic factor.
Intrinsic factor is made in the stomach (parietal cells).
B12 absorption may be impaired in the following situations:
reduction in active intrinsic factor
post gastrectomy
autoimmune gastric atrophy (“pernicious anaemia”)
small bowel disease
surgical resection
Crohn’s disease
coeliac disease
Excessive losses: this is not a common cause of B12 deficiency

Consequences of vitamin B12 deficiency:
Megaloblastic anaemia
Neurological problems:
peripheral neuropathy
sub-acute combined degeneration of the spinal cord
optic neuropathy
dementia
Laboratory diagnosis of B12 deficiency:
Blood count and film
Serum B12 level
Shilling test may be necessary to determine the cause of the deficiency.
Radiolabelled B12 is given orally and its excretion in the urine is measured, after first having saturated the serum B12-binding proteins by giving an intramuscular injection of non-radio-active B12.
Any radiolabelled B12 detected in the urine must have been successfully absorbed in the small intestine.
If the excretion is low then the test is repeated with the addition of intrinsic factor.
If this restores the excretion of B12 to normal it is possible to conclude that the defect lies with a lack of intrinsic factor secretion.
The detection of anti-parietal cell and anti-intrinsic factor antibodies in the blood, particularly the latter, would be additional evidence that a patient had pernicious anaemia.

4.Folate Deficiency

Inadequate intake is common – through either ignorance, poverty or apathy
Folate is found in animal and plant products but is readily destroyed by cooking, canning and processing
Daily requirements = 100µg, store = 10mg (3-4 months)
Common in elderly and alcoholics
Increased demand is also a common cause of deficiency:
Physiological – pregnancy, lactation, adolescence, premature babies
Pathological – an excessive turnover on cells as may occur with haemolytic anaemias, malignancy or erythroderma
Absorption occurs in the duodenum and jejunum
Failure of absorption is rare unless these is widespread disease of the small bowel such as in coeliac disease
Excessive loss is not a normal cause of folate deficiency
Consequences of deficiency:
Megaloblastic anaemia
Neural tube defects in developing fetus – spina bifida, anencephaly
A possible chance of increased risk of coronary artery disease if associated with variant enzymes in the folate metabolic pathway
Required for homocysteine metabolism – high levels of homocysteine is associated with atherosclerosis and premature vascular disease
Lab Diagnosis:
Full Blood Count and film
Serum folate
Red cell folate – gives a better indication of body stores of folate whereas serum folate reflects recent intake

5.Explain that synthesis of DNA requires both vitamin B12 and folate

Both are required for the metabolic pathway that ultimately ends in DNA synthesis
DNA requires folic acid in the tetrahydrofolate (FH4) form to act as a co-factor in synthesis, therefore folic acid deficiency hinders this process.
Vitamin B12 is required to produce FH4 and so indirectly affects DNA synthesis.
All cells which are dividing are thus affected, but the actively proliferating bone marrow cells (i.e. where haemopoiesis occurs) are particularly affected.
The slowdown of DNA synthesis and the prolonged cell cycling (of cells already in the circulation) causes the discharge of blood cells before they have undergone the full number of divisions (i.e. premature blood cells are released into the circulation). Consequently, these prematurely released cells tend to be enlarged (red blood cells) – this leads to anaemia that is both macrocytic and megaloblastic.

The Haemoglobin Molecule and Thalassaemia

1.Describe the structure and function of the haemoglobin molecule and list the normal haemoglobins in the fetal, neonatal and adult periods.

Each haemoglobin molecule contains:
4 globin protein chains (two pairs 2+2)
4 haem groups (protoporphyrin rings)
4 molecules of iron
Up to 4 molecules of oxygen
The main function of Hb is the carriage of oxygen from the lungs to the tissues.
Hb molecules can exist in two spatial configurations:
Deoxy haemoglobin exists in a tight (T) configuration and has a relatively low affinity for oxygen.
Oxygen molecules are taken up sequentially by the 4 haem groups and at some point the partially liganded Hb molecule switches.
Relaxed (R) configuration has a markedly higher affinity for oxygen.
This can be represented diagrammatically by the oxygen dissociation curve.
H+ ions, CO2 and 2,3-DPG (an organic phosphate compound) all stabilise the T form of the oxygen molecule by forming H bonds and thus decrease the oxygen affinity of the molecule.
This is represented on the oxygen dissociation curve as a shift to the right i.e a higher concentration of O2 is needed for maximum O2 saturation if the concentration of CO2, H+ ions or 2,3-DPG are high.
Thus in metabolically active tissues where the concentration of H ions and CO2 are high, oxyhaemoglobin will assume the T configuration and give up oxygen readily.
Conversely in the lungs where CO2 is exhaled, oxygen affinity is higher. This effect of CO2 on the affinity of Hb for oxygen is called the Bohr effect.

Alpha Cluster – Chromosome 16 / Beta Cluster Chromosome 11
Adult / α / β,δ
Foetal / γ
Embryonic / ζ / ε

Six different types of globin exist, three of which are transient embryonic haemoglobins. The genes that code for the globins are located in two clusters.

There are two alpha genes for the alpha globin protein. Two areinherited from each parent so adults have four alpha globin genes in total.
Foetal haemoglobin – predominantly Hb F (α2γ2)
Adult haemoglobin
>95% Hb A (α2β2)
1-3.5%Hb A2(α2δ2)
Trace Hb F (α2γ2)
If someone has a reduction in the number of beta chains (e.g. thalassaemia trait) then HbA2 would be proportionately increased and detected as a higher percentage on haemoglobin electrophoresis.
(δ2γ 2) and (β2δ2) do not exist

2.Describe the genes controlling haemoglobin synthesis and explain how genetic defects lead to  and  thalassaemias.

Thalassaemias are disorders in which there is reduced production of one of the two types of globin chains in haemoglobin leading to imbalanced globin chain synthesis
5% of the world population estimated to be carriers
Underproduction of a globin chain may be the result of
Gene missing completely (deletion)
Gene abnormal
start signal; no transcription
mRNA unstable; no translation
protein abnormal/dysfunctional

3.Describe briefly the clinical and haematological features of α thalassaemia

Alpha chains are found in HbA and HbF so alpha thalassaemia may present clinically in utero
Alpha thalassaemia is usually (>80% cases) due to a deletion of one or more alpha genes
Each alpha cluster (one on each chromosome) has two alpha genes - four syndromes are possible, each with an increasing degree of anaemia and associated morbidity
α+ trait (ααα)Mild anaemia
α0 trait (αα)Mild anaemia
Hb H disease (α)Significant anaemia
Hb Bart’s hydrops fetalis Death in utero
Genetic screening – all women screened for [--αα] → partner [--αα]
Could give baby of [-- --]
α+ trait common in Africa and α0 trait particularly common in SE Asia.

4.Describe briefly the clinical and haematological features of  thalassaemia major and the principles of management.

Most types of β thalassaemia are due to point mutations and over 100 different mutations have been described
Severe defect in BOTH beta chains
no problems in utero because HbF is alpha and gamma chains
at 2-3 months, become profoundly anaemic with Hb 3 or 4g/dl
failure to thrive and general malaise
In the absence of beta chains, alpha chains accumulate and precipitate in the bone marrow causing cell death; this is called ineffective erythropoiesis
Cells which do manage to mature and enter the circulation contain β-chain inclusions and are removed by the spleen which subsequently enlarges
The anaemia stimulates erythropoietin production and this causes expansion of the bone marrow in the skull and long bones
A patient with thalassaemia major has profound anaemia and requires regular blood transfusions to survive
A patient with thalassaemia intermedia, has anaemia but does not require regular blood transfusions
No transfusions - die aged 7
Blood transfusion - die aged 25 from iron overload or viral transmissione.g. hepatitis B and C and HIV
Each unit of blood contains 200mg iron and this accumulates in the liver, heart and endocrine glands - the effects of this start to appear by the end of the first decade
Secondary sexual development may be delayed or absent
hypoparathyroidism and adrenal insufficiency may become apparent
progressive liver and cardiac damage occur and liver damage from the iron overload may be exacerbated further by infectious hepatitis
Removal of iron is difficult. Currently the most successful drug is an iron-chelating agent called desferrioxamine
Not orally active
Subcutaneous infusion
Expensive
90% get into 30’s if well chelated – death usually (60%) a result of cardiac failure 2° to iron overload
Bone marrow transplantation has the potential to cure thalassaemia major and should be considered in transfusion-dependent thalassaemics under the age of 16 years who have an HLA-identical sibling greater than 18 months of age

5.Describe the haematological features of  thalassaemia trait, how it is diagnosed and why this is important.

Carrier state for abnormal β globin gene, usually clinically silent
Hb may be normal
MCV low (microcytosis)
MCH low
Normal MCHC
Red cell count increased
HbA2 increased [electrophoresis]
There are two situations in which identifying patients as having β thalassaemia trait (DNA analysis) is of value:
Microcytosis may be misinterpreted as iron deficiency if the raised red cell count and normal MCHC are not noted If these patients are then put on long term iron they can become iron overloaded
It is important to identify pregnant patients with thalassaemia trait so that their partners can be tested and the couple can be counselled about their chance of having a baby with clinically significant thalassaemia and can be offered further testing.

6.Describe how  thalassaemia trait can be differentiated from iron deficiency anaemia and the anaemia of chronic disease.

See previous table

Abnormal White Cell Counts

1.In a leucocytosis (increased white cell count) explain the importance of the differential count and peripheral blood morphology in planning further investigation.

White cells consist of two main groups which are present throughout body tissues and play a central role in the response to infection mediated via phagocytosis and soluble proteins of the immunoglobulin and complement system:
Phagocytes
Monocytes
Granulocytes → neutrophils, basophils and eosinophils
Immunocytes
T and B lymphocytes and NK cells
Differentiation and maturation:

Myeloblast → Promyelocyte → Myelocyte → Metamyelocyte → Neutrophils

present in the peripheral blood

Bone marrow is the principal source of WBC’s where they proliferate and differentiate from common stem cells
WBC’s mature in the peripheral blood
Cytokines influence differentiation and proliferation, processes which are directed by DNA (damage to which leads to cancer →leukaemia - lymphoma/myeloma)
Lymphoid cell proliferation is governed by IL2
Myeloid differentiation is governed by G-CSF and M-CSF
Erythroid differentiation is governed by erythropoietin

When an elevated WBC count is identified it is necessary to first look at the automated differential
Is the leukocytosis due to elevated numbers of a particular cell type such as in lymphocytosis, neutrophilia or eosinophilia or alternatively due to an increase in all cell types
A blood film will determine whether only mature cells are present in the peripheral blood, or whether immature forms such as myeloblasts or lymphoblasts are present
The morphology of white cells will also identify other reactive changes such as toxic granulation of neutrophils
By looking at elevate WBC’s in this fashion allows one to identify the type of underling problem