Haem & Haemoglobin

Key Concepts
1. The oxygen carrying capacity of the blood is based on haemoglobin
2. Erythrocytes carry haemoglobin
3. Haem is an essential component of haemoglobin
4. Some of the genetic disorders that affect haemoglobin are relatively
common and can be serious

Haemoglobin Characteristics
Haemoglobin is a roughly spherical
molecule with the molecular weight of
64.4 kilodaltons.

It is a tetramer that consists of two

pairs of similar polypeptide chains
called globins.

To each of the four globin chains is a

haem group, which is a complex of iron
and protoporphyrin, thus allowing 4 O 2
molecules to attach to each
haemoglobin molecule.

Normal adult haemoglobin consists of

2 - and 2 -chains.

Haem is a protoporphyrin ring with a central iron atom.

Different Haemoglobins
There are 2 main families of globin genes: and non-. -like genes are
situated on chromosome 16 and include: (alpha) and (zeta). Non- -like
genes are situated on chromosome 11, and include: (beta), (delta),
(gamma) and (epsilon). The different types of genes are required to produce
the different globins necessary during various stages of development.
 On chromosome 11, there is only one gene controlling the production
of -globin, which contributes to adult haemoglobin (HbA). Also contributing are
two genes controlling the production of -globin in HbA.

Different haemoglobins are produced during embryonic, foetal, and

adult life. Each consists of a pair of -like chains and a pair of non--like
chains. The major adult haemoglobin is HbA, which consists of 2 - and 2 -
globin chains. There is no substitute for -globin in the formation of any of
the normal haemoglobins following birth, therefore the absence of all 4 copies of
the gene ( thalassaemia major Hydrops Foetalis) is incompatible with
extrauterine life. Alternative genes can be produced for a lack of -globin
production.

Embryonic haemoglobins are primarily the product of yolk sac

erythroblasts and are detectable only during the very earliest stages of
embryogenesis. In early development (between weeks 4 and 14 of gestation),
the human embryo synthesises 3 distinct haemoglobins:
- Hb Gower I 22
- Hb Portland 22
- Hb Gower II 22
Haemoglobin Function
Haemoglobin has the capacity to survive and function for 120 days.

The function of haemoglobin is to carry oxygen and deliver oxygen to

tissues. Oxygenation and deoxygenation of haemoglobin occur at the haem iron
group. When one haem takes oxygen, the affinity of the remaining haem
groups of the tetramer increases markedly. The oxygen-dissociation curve has
a sigmoid shape:
- At high oxygen tension in the lungs, oxygen is rapidly taken up
- At low oxygen tension, oxygen is rapidly released
 P50 is the partial pressure of oxygen at which haemoglobin is 50%
saturated. P50 is normally 27mmHg at 37C and pH 7.4. A shift of the curve to
the right, with a higher P50, indicates that a larger partial pressure of oxygen is
required to saturate haemoglobin (reduced affinity). Oxygen affinity is modulated
by pH, CO2, 2,3-diphosphoglucose, and temperature.

Haemoglobin Synthesis
Haemoglobin synthesis begins in the proerythroblast, which is one of
the very earliest stages of RBC development. At this point, RBCs are blue in
colour with little haemoglobin. The synthesis of globin chains and haem groups
is carefully coordinated and involves feedback processes. The formation of
haemoglobin by the insertion of haem into the pockets of 22-globin takes
place in the cytoplasm as the globin chains are released into the ribosomes.

RBCs
Normal haemoglobin values and RBC parameters:
Parameter Adult Adult Male
Female
RBC Count 3.8-5.8 x 4.5-6.5 x
1012/L 1012/L
Haemoglobin (Hb) 115-165 g/L 130-180 g/L
Haematocrit (HCT) 0.37-0.47 0.40-0.54
Mean Cell Volume (MCV) 76-96 fL
Mean Cell Hb (MCH) 27.0-32.0 pg
Mean Cell Hb 320-360 g/L
Concentration (MCHC)
RBC Distribution Width 11.5-14.5%
(RDW)
Reticulocytes 20-110 x 109/L (about 1%)
Reticulocytes are immature RBCs that increase in presence during
stressful situations.
Normal RBCs are palour in 1/3 of the
diameter of the entire RBC.

There are various haemoglobin disorders:

- Thalassaemias: reduced rate of production of one or more globin chains
- Structural change in a globin chain leading to instability or abnormal
oxygen transport
- Harmless mutations which interfere with the normal switching of foetal to
adult haemoglobin production

RBC Parameter Analysis

From haemoglobin, red cell parameters and blood film, various conditions
can be identified:
- Anaemia: low haemoglobin
- Hypochromia: pale red blood cells (low MCH)
- Microcytosis: small RBCs (low MCV)
- Reticulocytosis
- Nucleated RBCs
- Target cells
- Teardrop poikilocytes
It is important to differentiate between iron deficiency and thalassaemia,
as they may present with similar signs and symptoms. Iron deficiency can be
excluded via serum iron, TIBC and ferritin levels.

Another diagnostic tool is high pressure liquid chromatography (HPLC),

which is a specialised, rapid technique for analysing different haemoglobins
using automated instruments.
 Haemoglobin electrophoresis is a
technique that separates different
haemoglobin bands on a gel, using
the electrical properties of the
molecules.

Thalassaemia
Thalassaemias are the most common single gene disorders, and are
usually heterogenous. They were first recognised in 1925 by Cooley, who
observed that there was a series of infants who became profoundly anaemic and
developed splenomegaly over the first year of life. Many patients came from the
Mediterranean region. Their classification depends on which globin changes are
not being produced sufficiently: , and others.

Most thalassaemias are inherited in a Mendelian recessive fashion.

Heterozygotes are usually asymptomatic, but can be recognised by simple
haematological analysis. More severely affect patients are either homozygous or
have co-existing structural haemoglobin variant. Thalassaemias are
clinically divided into:
- Minor: asymptomatic carrier state
- Intermediate: anaemia and splenomegaly, however not transfusion-
dependent
- Major: severe, transfusion-dependent disorder

The pathophysiologies of thalassaemias begin with a genetic lesion,

leading to a lack of production of either - or -globin chains. If one type globin
chain is not being produced sufficiently, the other is usually produced at the
normal rate. This results in an excess of free globin but a deficiency of
haemoglobin. The presence of excess free globin can lead to the damage of
cells.
-Thalassaemia syndromes occur
with the loss of one or more -
globin genes.

The loss of one or two -globin

genes is clinically
inconsequential:
- Single gene deletions are
virtually undetectable
- Loss of two genes results in
microcytosis only

HbH disease occurs when only a single -

gene is active, leading to mild to
moderately severe haemolytic anaemia
with microcytic erythrocytes and
splenomegaly.

Total loss of four functional -genes is

incompatible with life and causes
stillbirth of hydropic foetuses.

HbH inclusion bodies are RBC precursors

that possess excessive amounts of free -
globin, but also a lack of HbA.

-Thalassaemia syndromes occur when one of the two -genes are

compromised. The signs and symptoms are similar to that of -thalassaemia. In
those affected, the erythrocytes have a reduced MCV and MCH, but there is
normal RDW in contrast to iron deficiency anaemia, where it is high
(anisocytosis). The RBC count is also often elevated, as well as reticulocyte
counts and HbA2 levels (4-6%), which are made of - and -globins. The HbF
level may also be elevated. Blood film may show target cells and basophilic
stippling.

Heterozygous -thalassaemia (thalassaemia trait/minor) is usually

innocuous and often contributes to microcytosis and hypochromia. Anaemia
is not often found.

Homozygous -thalassaemias (thalassaemia major/Cooleys anaemia) is

characterised by intense chronic haemolytic anaemia, which becomes
evident within months of birth and leads to dependency on transfusion to
sustain life. If untreated, the condition may develop marked intramedullary and
extramedullary expansion of bone marrow, thus causing deformity of the
skull. Massive enlargement of the liver and spleen can occur. Also, iron
overload produced by excessive intestinal absorption and transfusion, leads to
cardiac failure and endocrine problems.

Haemoglobin Variants
Haemoglobin variants involve conditions where abnormal globins are
produced. This is in contrast to thalassaemias where insufficient normal globin
is produced. A common haemoglobin variant is sickle haemoglobin (HbS),
which results from a GAG to GUG mutation in the codon for the 6 th amino acid of
-globin. There is a high prevalence in selected ethnic groups, due to its
protection against malaria.
HbS polymerises when
deoxygenated, distorting the
cell and injuring its
membrane.

The distorted cells then travel

through the microcirculation,
where the capillary diameter
is narrow, and is then slowed
are lodged into the
microvasculature.

The lodging of the cells

retards blood flow, reducing
oxygen tension and causing
further haemoglobin
polymerisation and tissue
ischaemia or infarction.

Sickle cell disorders include:

- Sickle cell trait (HbAS): is asymptomatic and is present in 8% of African-
Americans
o Carriers have none of the haemolytic or vaso-occlusive events
typical of patients with HbSS or other severe sickling disorders
- Sickle cell disease (HbSS): causes recurrent, painful crises secondary to
sickling
o Small vessel occlusion and tissue infarction occurs, as well as
severe haemolytic anaemia
- Other sickling disorders: HbS/-thalassaemia or HbS/other abnormal Hb

Iron Deficiency
Iron deficiency is a common cause of microcytic hypochromic anaemia.
The key to diagnosing iron deficiency is the ferritin level of the individual.
Ferritin is the storage form of iron in the body; therefore if there is a low level of
ferritin, the patient is deficient of iron.