LESSON FOUR: BLOOD CELL COUNT AND HAEMOGLOBIN

Lecture overview

Welcome to lecture four where we shall look at manual blood cell count for the three cellular
components of blood. When either of the blood cells is affected (increased or decreased), there
may be an underlying pathological condition which may be diagnosed by cell counts in addition
to other diagnostic techniques. Further, we will also learn about haemoglobin formation, types of
normal and abnormal haemoglobins, as well as haemoglobin estimation techniques.

Lecture objectives

After completion of this lesson, you will be able to:

1. Perform manual cell counts

2. Understand the requirements needed for the cell count
3. Know how to interpret the cell count results
4. Appreciate haemoglobin, its synthesis, and functions
5. Differentiate between normal and abnormal haemoglobins
Lecture subtopics

1. Red cell, white cell and platelet counts

2. Sources of counting errors
3. Haemoglobin synthesis (Haem and globin chains)
4. Normal and abnormal haemoglobins
5. Haemoglobin degradation
6. Haemoglobin estimation
3.1 MANUAL CELL COUNT
To obtain a reliable cell count using the manual methods. It is important that: -
a) Blood dilution is carried out accurately using a appropriately prepared diluting fluids
b) Special slides for cells counts are understood and used correctly – The slides are called
counting chambers.
c) Calculation is carried out carefully.
These three factors require much exposure in practice to develop the skill.

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3.2 Diluting fluids.
- Because of presence of large numbers of cells in the whole blood, it’s necessary to dilute the
samples to ease the count.
- A good diluting fluid will ensure a reliable dilution and count. Such a fluid will:-
a) Ensure that cells to be counted are not destroyed
b) Obscure or destroy cells that are not to be counted
c) Ensure that the cells to be counted remain homogenously suspended, so that they do not
sediment too fast.
❖ There are two methods of diluting blood
1. Bulk method - Where large volumes are prepared
2. Micropipette method - Where small volumes are prepared using specially prepared
micropipette that give specific dilutions.
3.3 PIPETTES.
3.3.1 RBC Pipette
- This is a bulbous pipette used for diluting blood for RBC (T)
- It’s made to achieve a standard dilution of 1:100 and 1:200
- Its stem has two markings 0.5 and 1.0 while the back of the bulb has mark 101
- The bulb of this pipette has a red bead which acts as an identification as well as helping blood
and diluting fluid to mix during dilution.
- To achieve 1:200 dilution, well mixed blood is drawn up to the 0.5 mark and then the outside of
the pipette is cleaned /wiped with a piece of a clean gauze and then diluting fluid drawn up to fill
the bulb and up to 101 mark.
- The contents of the bulb are mixed by closing the two ends of the pipette with fingers and then
rocking the pipette, or rotating it in horizontal manner. Place the pipette on the bench and allow to
stand for 2 minutes.
- To charge the neubauer chamber, discard the 1st 3 drops or 1/3 of the bulb content and then charge
the chamber by allowing blood to flow under the cover slip until it has filled the entire sunken
surface.
- To achieve a dilution of 1:100, well mixed blood is drawn up to the 1.0 mark and dilution fluid
drawn up to the 101 mark.

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Diagram of a red cell pipette:

3.3.2 WBC Pipette

- This is also a bulbous pipette used for diluting blood for WBC count and platelet count.
- It is made to achieve a dilution of 1:10 and 1:20 but 1:20 is the standard dilution
- Its stem has similar markings as RBC pipette while the back of the bulb is marked 11.
- The bulb of this pipette has a white bead – for identification and mixing the bulb content.
- To achieve 1:20 dilution, well mixed blood is drawn up to the 0.5 mark and dilution fluid is
then drawn up to the 11 mark.
- To achieve 1:10 dilution, well mixed blood is drawn up to 1.0 mark and then diluting fluid
up to 11 mark.
NB: For bulk dilution, the same dilution factors are used for the specific cell type. Pipette 0.38 ml
of diluting fluid into a test tube using a graduated 1ml pipette. Label the test tube with a grease
pencil or adhesive tape and place in a test tube rack. Attach the plastic tubing to a 20µl pipette
(0.02ml); make a knot in the tubing. When using capillary blood, allow the blood to flow freely
without excessive squeezing. When using venous blood, mix it thoroughly, squeeze the plastic
tubing attached to the 20ml pipette and draw the blood up to the mark by gently releasing the
tubing and be careful not to introduce air bubbles and then add into 0.38mls of dilution fluid, this
is 1:20 dilution.
Diagram of the white cell pipette:

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3.4 COUNTING CHAMBERS.
- These are specially prepared heavy glass slides with special surfaces that are sunken
(depressions, wells)
- The sunken surfaces are also provided with very fine and accurate rulings. When a special
cover glass is placed over the sunken surface, space is created underneath the cover glass and
that space is called or known as a chamber
- Diluted blood is carefully layered into the chamber for the purpose of counting cells.
- Different counting chambers are known by the depth of sunken surface and by the rulings on
the sunken surface.
1) Neubauer counting chamber.
- This chamber is divided into nine (9) squares of 1mm2 each.
- For the purposes of cell count, only square A, B, C, D, & E as indicated are of significance.
- Squares A,B,C,D,and E are sub divided 16 times each
- Squares A, B, C, &D are subdivided with single line while E by triple lines. Each of the
subdivisions of square E is further subdivided 16 times but now in single lines.
- This chamber has a depth of 0.1mm or 1/10 mm
- The total no. of squares(E)S 16 X 16 = 256
Diagram:

2) Improved neubauer counting chamber

- This chamber is similar to neubauer except that the central square E is subdivided into 25
squares with triple lines and further subdivided 16 times each i.e. 25 x 16 = 400 tiny
squares.
- The depth of the chamber is 0.1mm / 1/10 mm

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 - This chamber is the one mostly in use and it’s used for all cell counts.
Diagram:

3.5 RED BLOOD CELL COUNT

3.5.1 Requirements.
1. Diluting fluids.
a) Hayems fluid
- They should not destroy RBCs.
It contains:-
• Mercuric chloride – 0.5g
• Sodium sulphate – 5.0g
• Sodium chloride – 1.0g
• Distilled H2O to dissolve up to 200mls.
✓ Mercurial chloride – provides a high S.G and also for fixing
✓ Na2So4 – nourishment to the RBC
✓ NaCl – provides isotonic medium
b) Toisson’s fluid
o NaCl – 1.0g – provides isotonic medium
o Na2SO4 – 8.0g – High S.G
o Glycerin 30mls - Nourishment
o D.H2O - 160ml - diluent.
c) Formal citrate
- Sodium citrate - 3.0g – anticoagulant and high S.G
- Formaldehyde – 1.0ml – preserves RBC integrity

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 - D.H2O - 200ml - diluent.
2. RBC Pipette with sucking tube & mouth piece or appropriate tube for bulk dilution
3. Counting chamber and cover slip
4. Dry pieces of gauze
5. Microscope will x 10 and 40 objectives
6. Specimen
3.5.2 Procedure
- Dilute the blood appropriately
NB. If the blood appears normal, make 1:200 but if the blood appears anaemic make 1:100 dilution
❖ Prepare the counting chamber for charging by:-
Wash the chamber thoroughly with soap and water, rinse with plenty of H2O.
Dry it thoroughly with dry gauze
Do the same for the cover slip
Place the cover slip on the raised surface/part above the sunken surface and make a firm
forward movement of the cover glass with pressure until rainbow colours appear (Newton
rings) which is an indication that the cover slip is firmly attached onto the chamber.
After discarding 3 drops of diluted blood, charge the chamber by allowing blood to flow
under the cover slip until it has filled the entire sunken surface.
Care should be taken not to overcharge the chamber which is indicated by an overflow of
diluted blood into the provided trough. Over charged chamber is rectified by touching the
edge of the cover slip with a filter paper.
It is important to avoid air spaces during charging which may be an indication of dirt,
working quickly or carelessly.
Allow diluted blood to settle by standing it on the bench for 1 – 2 minutes.
Place the slide on the microscope and scan using x 10 objective to identify the central
square E
Switch to power x 40 and count cells in 5 of the 25 squares preferable the four corner
squares and the central for accuracy. RBC appear large than WBC when they are in power
x 40 than when WBC are in x 10.
• Cells that overlie on the outer left line and the upper line are counted while the one’s that
overlie on the right and bottom are not counted or vice versa.

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3.5.3 Calculation
- Calculate the no. of RBC’s counted per cubic mm3 in diluted blood.
- Take N to be the no. of cells counted in 5 squares of the central square.
- Dilution factor will be 200
- Therefore No: of cells counted in the 1mm3 of diluted blood is:-
N ÷ D factor ÷ volume = N ÷ 1/200 ÷ (0.2 x 0.2 x .0.1) 5
▪ The area of each of the 25 squares is 0.2 x 0.2 = 0.04.
N ÷ 1/200 ÷ (0.2 x 0.2 x 0.1) 5
N X 200 ÷ (0.004) 5
1
N x 200 ÷ (0.02)
N x 200 ÷ 0.02
1
N x 200 x 1
0.02
= 200 N x 100 = 10,000N
2
3.5.4 Normal / reference range
❖ Adult males (12 years and above) - 4.5 - 6.5 x 10 12/l or 4.5 – 6.5 × 106/mm3
❖ Adult females (12 years and above) - 3.8 - 5.6 x 1012/l or - 3.8 - 5.6 x 10 6/mm3
❖ Full term fetus - 4.0 - 6.0 x 1012/l
❖ Infants (3 months) - 3.2 - 4.8 x 1012/l
NB: RBC rises gradually after 3 months to reach adult levels by 12 years of age.
3.5.5 Decrease of RBC (T)
- Multiple myeloma (cancer of B. marrow)
- Blood disorders (leukemia and anaemia)
- Physical factors like altitude.
3.6 WHITE BLOOD CELL COUNT
- This is the estimation of the number of white cells in a given volume of whole blood or other
body fluids e.g. CSF, pleural fluid, joint fluid, peritoneal fluid etc.
3.6.1 Requirement.

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1. White blood cell pipette (Thoma pipette) with sucking tube and mouth piece or relevant glass
ware for bulk dilution.
2. Diluting fluids.
a). Toissons fluid – as of RBC but tinged with methyl violet or Gentian Violet to stain the
WBC enhancing their accountability.
b). Turk’s fluid
▪ 1 – 2% glacial acetic acid – fixing WBC
▪ D.H2O – 100ml lyses the RBCS and also used as a diluent
▪ G.V – few drops of 0.5% concentration - stains WBC
c) Acetic acid 2% v/v
d) Hcl 1% v/v
e) Breachers fluid
3. Counting chamber and courtship
4. Microscope with x 10 objective
5. Dry pieces of clean gauze
3.6.2 Procedure
➢ Dilute blood approximately
➢ Prepare the chamber appropriately and charge it
➢ Allow for 1 – 2 min for cells to settle
➢ Count WBCs on 4 corner squares together with cells overlapping margins of the lower and
left hand side of the chamber or vice versa.
3.6.3 Calculations.
- Area of one large corner square is 1mm2
- Area of 4 large squares is 4mm2
- Depth of the chamber is 0.1mm; therefore, the volume of 4 squares is 0.1 x 4 = 0.4mm3 or µl
- Since the white cell count is expressed as per 1µl, the number of cells is denoted as N.
N – No: of cells counted x dilution factor which is 20.
N x 20 = 0.4mm3
N x 20 = 0.4mm3

X 1mm3
= 20 x 1 = 50
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 0.4
N x 50 = 50N
N = 89, therefore, cells counted = 50 x 89 = 4450.
▪ After the test, clean the counting chamber immediately and the cover glass in clean H2O,
then disinfect with spirit, dry with gauze and store carefully in a box in a safe place.
▪ If micro – pipettes are blocked, soak in Potassium dichromate (K2 Cr2O7) and solution or
neat HCl before washing.
3.6.4 Ways of reporting WBC
- 4450/mm3, 4.45x 109/l, 4450/ml and 4450 WBC /mm3

3.6.5 Reference range

Adults – 4000 – 11,000/µl or 4 – 11 x 109/l
Full term foetus – 10 – 26 x 109 / l
Children – 600 – 15,000/µl
- Leucocytes numbers decrease gradually to reach adult levels by 12 years of age.
Quality control
- Perform counts in duplicate (charge both areas.)
- External – send it to someone else to do the counting and compare
- Coulter counter
3.6.6 Causes of increased WBC
1. Infections e.g. bacterial infection e.g. Staphylococci, Streptococci, Pneumonococci,
Gonococci, Meningococci, E.coli, Mycobacterium tuberculosis, B. pertusis
- Viral infections – viral hepatitis.
- Mononucleosis infection
2. Inflammations. – i.e. burns, fractures and other injuries, arthritis, tumours.
3. Metabolic disorders – uremia, diabetic coma, eclampsia
4. Suffering from acute haemorrhage
5. Leukemia (chronic or acute)
3.6.7 Causes of decreased WBC (T)
1. Infections like typhoid fever, borellia, brucellosis, viral – measles, chicken pox, rubella,
influenza, HIV or protozoa (parasitic) leishmaniasis overwhelming military TB/Septiceamia

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 2. Drugs and chemical – for therapeutic purposes e.g. sulphonamides, chloramphemical, phenyl
butazone, metronidazole or flaggyl & anti TB drugs
3. Vit. Deficiency – folic acid (folates), vit. B12
4. Splenomegally – due to pitting process in the spleen
5. Diseases of the B.marrow - acute leukemia, aplastic anaemia.
3.7 PLATELET COUNT
3.7.1 Requirements
1. Diluting fluids.
a. 2% ammonium oxalate solution: Weigh 2g of the oxalate and dissolve in 100ml of
D.H2O. Always, prepare small amounts not more than 100ml and always store it at 40c. If
precipitate occurs in the solution, discard
b. Baar’s fluid – This fluid incorporates formalin as a platelet fixative so that they are not
disintegrated saponin used to haemolyse RBC
• Saponin – 0.25g – destroys RBC
• Sodium citrate. 3.5g – anticoagulant.
• Formalin – 1.0mls – fixative
• Brilliant cresy1 blue – 0.1g – stains the platelets
• D.H.2O – 100ml – diluent
NB: Filter before use. If brilliant cresyl blue is omitted, filtration is not necessary. Platelets can be
seen as small refractile unstained bodies.
2. WBC pipette 7. Counting chamber
3. Dry cotton gauze swabs 8. Blood sample
4. Sucking tube and mouth piece 9. Microscope x 40 object
5. Moist compartment (Petri dish with wet gauze & lid)
6. Timer
3.7.2 Procedure
❖ As for the other counters except:-
❖ If capillary blood is used, diluting fluid if drawn 1st up to 0.5 mark. This is done to prevent
the platelet from coming into direct contact with the glass surfaces and hence prevent
clotting /clumping together
❖ Blood is then drawn until initial level of diluting fluid raises up to 1.0 mark

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 ❖ Diluting fluid is then drawn up to 11 mark
❖ After charging the chamber, it is placed in moist compartment for 20 minutes before
performing the count. The count is performed and the same square (central) as RBC.
3.7.3 Calculation
Let N be the no. of cells counted.
Therefore, N ÷ Dilution factor ÷ vol.
= N ÷ 1/20 ÷ (0.2 x 0.2 x 0.1) 5
= N x 20 ÷ (0.004) 5
1
N x 20 ÷ (0.02)
N x 20 x 1/0.02 = N x 20 x 100 = 1000 N
2
3.7.4 Reference range
150, 000 - 450,000 ml
150 - 450 x 109/l
3.7.5 Sources of counting errors
There are two categories of errors
1. Faulty technique errors
2. Inherent error.
3.7.6 Faulty technique
At working due to ignorance/carelessness may be due to:
a) Not mixing the sample adequately
b) Use of inaccurate / poorly calibrated pipettes
c) Badly charged chamber – due to dust particles
d) Wrongly perform the count – faulty filing of chamber, blood is inadequately mixed with
anticoagulant.
e) Excessive squeezing after pricking due to - haemodilution
f) Air bubbles in pipettes
g) Prolonged use of tourniquet – haemoconcentration occurs
h) Clots in Venus blood - inadequate mixing.
i) Miscalculate the final count

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❖ All these can be avoided or minimized by strictly following instruction on the method of
dilution, the performance of the count, the calculation and also by ensuring that faulty
apparatus are not used.
3.7.7 Inherent errors.
➢ These are errors dictated by the laws of random distribution. They can only be minimized
(not avoided) by counting large numbers of cells or counting cells in large areas and obtain
averages (since more cells settle in some areas than others).
3.7.8 Physiological variations
-These are the variations in normal values that are observed in normal persons.
3.7.8.1 RBCs
▪ Age - Infants are born with high counts which drops suddenly in 3 months to begin rising
slowly up to adult levels by the age of 12 years.
▪ Sex – female (adults) generally have lower count than adult males due to
❖ Hormonal influence on haemopoiesis
❖ Menstrual blood loss
▪ Muscular activity – more of this activity causes arise in the count.
▪ Altitude – The higher the altitude, the greater the count as O2 requirements increase
▪ Drugs. – (Smokers) - they tend to have high counts but essentially (a non-physiological
variation).
3.7.8.2 WBC.
Age – in differential count
At birth – neutrophils – predominate, 10th day to 5 – 6 – 7 years, lymphocytes predominates.
After 7 years – neutrophils predominate. And at 12 years, one has values of an adult
Sex – No difference until after 50 years when women have a high count than men during
menopause
Diurnal variation – eosinophils counts show much variation at different times of the day.
Environmental variations. – In tropics, there is a decrease in the leucocytes count and difference
in lymphocyte and neutrophil counts
Hour to hour variations – Total counts are lower in the morning while subject is resting and
increase to a peak in the afternoon.
NB: platelets have no any physiological variations except in pathological circumstances

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 - In pregnancy and a short while after birth, the cell count is high in that the mother is
creating some immunity for herself and the new born.
3.8 HAEMOGLOBIN
- Haemoglobin is the red pigment found in the RBCs accounting for 90% of the dry weight
of a RBC.
- It is the active component of RBC therefore is responsible for carrying out O 2 from the
lungs to the tissues and CO2 from tissue to the lungs.

3.8.1 STRUCTURE OF HAEMOGLOBIN

- Haemoglobin is a very large molecule with molecular weight of 64500 daltons. 96% of
haemoglobin molecule comprises the protein molecule called globin while the rest is a
coloured iron complex called haem 4%.
- Haemoglobin has a tetrameric structure i.e. it has four units (monomers) which function as
one with almost similar structure.
- It has an ellipsoidal shape
- It is made up of two units (parts) each having an alpha and non – alpha chains
(polypeptide)
- The non-alpha polypeptide includes.
• Beta (β) – Polypeptide chairs
• Gamma (γ) “
• Delta (δ) “
• Epsilon (ε) “ and Zeta chains
- The alpha (α) and non – alpha are polypeptide and each has a haem attached to it. These
chains are referred to as globin chains.
- The alpha has 141 amino-acids and chromosomes 16 controls 2 alpha chain manufacture
- The non-alpha has 146 amino acids and chromosome 11 controls – 2 non-alpha
manufacture
- A mature RBC has 640 million molecules of hemoglobin
3.8.2 NORMAL HAEMOGLOBLINS
- Normal haemoglobins are distinguished by the kind of non - α - chains that they have
which is also determined by age from conception to adulthood.

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1. Haemoglobin Gower2
- Has two alpha and two epsilon chains (2α 2ε)
- It is the 1st Hb encountered after conception.
2. Haemoglobin F (fetal Hb)
- This Hb has two alpha and 2 gamma chains (2α 2γ)
- It is encounter in latter fetal development and comprises about 96% of the Hb of the full
term fetus – ready for delivery.
- It also forms about 1% of adult it Hb.
3. Haemoglobin A (adult Hb)
- This Hb has 2 alpha and 2 beta chains (2α 2β)
- This is the most abundant Hb in adult life making up 96 – 98% of adult Hb.
4. Haemoglobin A2
- Has 2 alpha and 2 delta chains (2α 2δ)
- This Hb is present in all ages but comprises a small percentage of 1 – 4%.
3.8.3 HAEMOGLOBIN SYNTHESIS
- Hb is synthesized in the RBC as it matures. All the intermediate (polychromatic) normoblast
stage becomes obvious that the maturing RBC has formed substantial amount of Hb.
- Hb consists of 2 major portions, iron containing haem and the protein portion containing globin.
3.8.3.1 HAEM SYNTHESIS
- It’s synthesized in the mitochondria in a series of biochemical reactions. The building
materials used to synthesize haem within a developing red cells are amino acids (glycine)
and succinic acid (succinyl co-enzyme A). One molecule of glycine is condensed with one
molecule of succinyl – co-enzyme A to form delta amino levunilic acid (δ – ALA) in the
presence of δ-ALA synthetase enzyme.
- Two δ– ALA molecules condense in presence of δ-ALA – dehydrase enzyme to form por-
phobilinogen, which is a mono pyrole ring.
- Four mono pyrole ring of porphobilinogen condense in presence of the above enzyme to form
uroporphobilinogen which is decarboxylated in to co-proporphyrinogen and then oxidized
in to protoporphyrinogen. Enzyme haem synthetase (ferrochalataze) catalyses the insertion
of ferrous iron (fe2+) into the protoporphyrin to from haem. The process begins in
basophilic erythroblast and competed within 9 – 10 days in reticulocyte.

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 - The 4 rings (tetra pyrole ring) are joined by C – atoms (= CH - ) referred to as methane bridges
and the ring is called protoporphyrin ring and has additional structures (with 1,2, or 3
carbon chains)
- All enzymes caring out this reaction are present in the mitochondria in the normoblast and
this is the site of haem synthesis. More than 300mg of haem are made each 24hrs.
- A mature RBC no longer has mitochondria and hence cannot make haem nor incorporate iron
into Hb. The reticulocyte can still do so.
- As it matures (RBC), it diminishes in size.

Activity
1. Explain the new improved Neubaur chamber in details
2. Outline Hayems fluid

3.8.3.2 GLOBIN SYNTHESIS

- This is done in polyribosomes and it dependent on the genetic make-up of an individual.
Amino acids are linked up as the genetic code dictates inheritance according to Mendelian
laws of inheritance
- There are two autosomal genes that control globin synthesis. The alpha (α) chains – structured
genes with coding of amino acids sequence and then non alpha chain structural genes where
the gamma, beta, delta, and zeta (ζ) loci are close together each will coding for the respective
amino acid sequence.
- There is also regulator genes that controls the rate of synthesis in early embryonic developed.
Epsilon – chains are mostly formed but are gradually replaced by gamma chains. Gamma
chains are replaced after girth gradually by β-chains. Regulator genes are responsible for this
transformation. The basic information on the sequence of amino acids in the globin chain is
held in the Normoblast nucleus on the DNA. This information is then passed on to the
cytoplasm by reproduction of nucleic acid (RNA) that mirrors the DNA chain through a
process called transcription). This is the massager RNA and it moves to the ribosomes and
their appropriate amino acids are brought along by a transfer RNA. This t– RNA is specific
for each amino acid. The amino acid is attached to the ribosomes, which moves along the m-
RNA, when further amino acid is attached in this way, a chain of amino acid in predetermined

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 sequence is formed and when the chain length is complete, it drops off the ribosome and
associates with another chain forming a dimmer (either α, β, δ, γ, and ζ dimmers).
- Normal dimmers – Hb A, HbA2 , Hb F, Hb Gower i and ii, Hb Portland)
- Abnormal dimmers – HbS, HbC, HbH, HbE, HbA1, HbM, HbD, Hb – Barts.
(Iron absorption is increased in – iron deficiency, pregnancy, idiopathic haemochromatosis, and
any conditions in which there are active erythropoiesis.
Deceased in – chronic infection and after large amounts of iron are ingested. Diffuse disease of
duodenum as non-tropical sprue)
3.8.4 HAEMGLOBINS
(HB synthesis begins during the 2nd month of gestation.
The predominant Hbs produced are Hb Portland (ζ2γ2), Hb Gower I (ζ2ε2), and Hb Gower II (α2ε2)
but small amounts of HbF and HbA can be detected even of these early stages. About 10 – 11
weeks of gestation, erythropoiesis moves to the liver and the spleen. Coincidentally, the embryonic
Hbs decline to be replaced by fetal haemoglobin (Hb F) a mixture of two Hbs differing in the
composition of the γ chain component. Aγ chains have alanine and Gγ chains have glycine at
position 136. At six to seventh months of gestation, red cell production shifts to the B.marrow but
Hb F predominates until late in the 3rd trimester, synthesis of Hb switches to Hb A, and also
synthesis of Hb A2 begins at the sometime as Hb A. Hb A2 – minor adult Hb
NB: Fetal red cells tend to be larger (macrocytes) and have shorter circulating lifespan. They
express the i, rather than the I (adult) surface antigen. Lack of the β isoenzyme of carbonic
anhydrase present in adult cells, and produce the Gγ and Aγ forms of Hb F at a Gγ:Aγ ration of 7:3,
whereas the adult Gγ:Aγ ration is 2:3.)
3.8.5 SYNCHRONISATION OF HAEM AND GLOBIN SYNTHESIS.
Haem and globin are formed in equivalent amounts. In some circumstances, haem synthesis may
be restricted e.g. in iron deficiency anaemia. This results in reduction of globin synthesis. This
indicates that haem actually controls globin synthesis. Polyribosomes that form globin dis -
aggregate and stop synthesis in absence of haem and re-aggregate to synthesize on introduction of
haem.
3.8.6 FUNCTIONS OF HB
- The primary function of Hb is the transport of O2 from the lungs to the tissues and Co2 from
the tissues to the lungs. At saturation Hb molecule carries 1.3ml of O2 gas.

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3.8.7 Factors enhancing Hb function
1. Each RBC has a very large no. of Hb molecules about 640 million and therefore O2 carrying
ability of the cell is maximized.
2. The tetrameric structure of Hb enhances its affinity as opposed to the monomeric unit (e.g.
myoglobin). Acquisition of O2 by one monomer excites another monomer to acquire O2
and so on, until all the monomers have carried O2. This is called the haem-haem
interaction.
3. Chemical changes within and outside the all affect Hb function favourably:-
o PH – changes have a direct effect bearing on the function due to large amounts of
Co2 in the tissues, the PH tends to be lower than in arterial blood (PH is more acidic
) this facilitates O2 transfer i.e. Hb O2 affinity is reduced this is called Bohr’s effect.
o 2,3DPG – In the RBC causes reduced O2 affinity of Hb by combining with
deoxygenated Hb. The high affinity of O2 Hb F appears to be related to it’s inability
to bind 2, 3-DPG
4. Other indirect factors enhancing transport of O2 are: -
a. The larger no. of RBC in circulation
b. The biconcave disc shape of RBC’s which increases the surface area of O2 up take
and the mobility of blood cells through tiny capillaries.
3.9 HAEMOGLOBINOPATHIES.
Definition - These are inherited abnormalities traceable to hemoglobin synthesis. They are
grouped into two groups
- Qualitative abnormalities – associated with structurally abnormal haemoglobin variants
- Quantitative abnormalities – associated with markedly reduced production of normal Hb
polypeptide chains.
3.9.1 Qualitative abnormalities.
- The quality of Hb here is abnormal due to synthesis of an Hb molecule that is structurally
abnormal. The major cause of abnormality is the alteration of the correct sequence of amino
acids i.e. substitution abnormalities.
3.9.1.1 Pathology
- The resultant haemoglobin may be insoluble / because of being crystalline ( as in HbS,
HbC, Hb D and HbE) or being unstable (as in Hb Genoa )

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 - The resultant Hb may have an altered O2 transport capability (as in HbM)
3.9.2 Quantitative abnormalities.
- The production of polypeptide chains of the globin component is markedly reduced
resulting in lack of specific chains to enable formation of normal Hb.
- An example is α - thalassemia and β - thalassemia
3.9.2.1 Hemoglobin S
- This Hb is caused by substitution of amino acid glutamic acid (negatively charged) by
valine (hydrophobic) on position 6 of the β chains. This Hb is functional until other factors
come into play (GAA becomes GTA)
Pathogenesis
- There is decreased Hb solubility and increased Hb viscosity when de-oxygenated. This
results in gelling and precipitation of Hb in the cell. The cells then crumble to ovoid and
finally sickle (banana) shape. This crumbling is reversible but repeated deoxygenations
eventually causes cell distortion (refer sickle cell anemia trait.)
3.9.2.2 Hb C (qualitative)
- Caused by substitution of β – chain glutamic acid on position 6 by lysine. It is functional.
Pathogenesis – there is decreased solubility when exposed to drying ending up with formation of
HbC crystals, cells ends up with Targeting phenomenon.
3.9.2.3 Haemoglobin D & HbE
- These are also substitution haemoglobins but of much rare occurrence.
3.9.2.4 Haemoglobin M.
This is a substitution Hb despite having normal methaemoglobin reduction enzyme function. it’s
unable to reduce methaemoglobin to haemoglobin. It has a stable ferric (Fe3+) ion that is not
reversible.
3.10 Alpha Thalassemia
- This is characterized by varying degree of reduction of α - chain synthesis. There’s varying
degree of normal Hb and increase Hb Barts (4γ) HbH (4β)
3.11 Beta Thalassemia
- This is characterized by varying degree of reduction of β – chain synthesis. Foeti are not affected
after birth, there is varying increase of HbA and Hb F.
NB: Haemoglobinopathies can be differentiated by Hb electrophoresis test.

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3.12 Haemoglobin pigments.
- These are Hb that are normal in structure but whose function has been altered by certain
chemical bonding which is not reversible. The pigments are usually associated with cyanosis
(a condition characterized by blueness of the skin due to lack of sufficient O2 supply).
a. Methaemoglobin
- Some chemical substances will cause the oxidation of ferrous iron to ferric ion in an
irreversible manner, despite the presence of normal methaemoglobin reduction pathway.
The chemicals include some nitrates and chlorates. The resultant Hb is functionally dead.
b. Sulphaemoglobin / carboxy haemoglobin
- Some chemical substances compete for Hb alongside O2 and unfortunately form
irreversible bonds with Hb. The resultant Hb are functionally dead
- Sulphur drugs are good examples of causative agents of sulphaemoglobin (SHb) while
carbon monoxide causes Carboxyhaemoglobin (HbCO)
c. Heinz bodies – refer to RBC inclusions.
3.13 HAEMOGLOBIN DEGRADATION
After expiry of erythrocytes life span (physiological and pathological), the cell lyses results in the
release of its contents Hb. If haemolysis has occurred in a RES cell, the process of breakdown of
the Hb is direct while when the haemolysis is intravascular the Hb has to be transported out of
circulation to an RES cell for its catabolism. 80% - 90% of red cell lyses occurs in macrophages
of the RES, the rest occurs intravascularly. Hb is broken down to its two components. Globin and
haem. The globin amino acid linkages are broken up and these reutilized or kept in protein stores.
The haem Fe++ ion is separated from the protoporphyrin and either reutilized in Hb synthesis in
another cell or stored. The protoporphyrin is catabolized to release CO2 and free bilirubin.
This is a yellow pigment and is transported to the liver for conjugation bound to albumin which is
a protein. This compound gives an indirect Reaction in Van- den- Bergh’s test (used to measure
bilirubin).
In the liver, glucoronic acid is attached to bilirubin by an enzyme called glucoronyl transferase.
This process is called conjugation. The bilirubin is now conjugated and gives a direct reaction
with Van den berg’s test. It is excreted into the bile and hence into the small gut. In the gut, the
bilirubin is converted to stercobilinogen. Some stercobilinogen is reabsorbed into the plasma and
re-excreted by the kidney in the urine and it is called urobilin.

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 HAEMOGLOBIN

Haem globin

Amino acids
(Re-utilized /stored)
Bilirubin
Iron
(Reutilized / stored)

Conjugated bilirubin

Bile (faeces) Reabsorbed (kidney / urine)

Stercobilinogen (Small gut)

Urobilinogen
Stercobilin (Excreted in face)

Urobilin (excreted in urine)

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3.14 Pathology of Hb degradation
a. Excessive breakdown leads to liver overload as too much indirect bilirubin (I.B) requires to
be converted to D.B ( direct bilirubin) some un -conjugated bilirubin goes back to tissues and
its dangerous (jaundice)
b. Obstruction of ducts leading to gallbladder from liver means conjugated bilirubin cannot be
excreted via stool and urine. Resultant conjugated bilirubin is deposited in tissues
c. Pathological liver unable to perform its normal conjugation function means unconjugated
bilirubin is deposited in tissues.
3.15 Hb normal values.
Adult’s males 130 – 180 gmll / 13 – 18 gldl
Females 115 – 165 gm/l 11.5 -16.5gldl
Infants (one day old and full term foeti – 135 – 195gm/l
Children:
3 months – 95 – 135gm/l
1 -2 yrs – 105 – 135gm/l
3 – 10 yrs – 115 – 145gm/l
After 10 yrs – begins to correct to adult levels.
3.16 Physiological variations
Follows much the same trend as for RBCs - age, sex, altitude (where every climb of
1500m accounts for an increase of 10mg/0.01g/l or 0.001g%.

3.17 Haemoglobin estimation

This generally depends on chemical conversions of Hb and comparison of colours using known
standards
STD – is a solution prepared as test with a known conc. of the substance under test
Blank – solution prepared in all aspects as test and STD but lacks the test substance.
- The aim of doing Hb estimation is to determine the O2 carrying capacity of blood.
3.17.1 Methods of Hb Estimation
1. Spectrophometric (colorimeter)
- Drabkin’s method.

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 - Oxyhaemoglobin method
- Alkaline haematin method
- Singer’s method (specific for HbF)
• Filter used is 540nm (green filter)
2. Electronic method e.g. coulter machine using colorimetric principle.
2. Drabkin’s / cyanmethaemoglobin
Requirement – Colorimeter, 0.02ml pipette, two bijou bottles / test tubes, 5ml pipette,
drabkin’s solution, piece of gauze, STD solution, green filter (540nm), blood sample
Composition of drabkin’s Solution (should be clear and pale yellow)
- Potassium cyanide (KCN) – 50mg / 0.05g
- Potassium ferricyanide (K3Fe (CN) 2 – 0.2g/200mg
- Sodium bicarbonate – 1g and D.H2O – 1000mls
Haemiglobincyanide – previously called cynmethaemoglobin.
Principle
Hb (including HbCO and Hi but not SHb) is converted to HiCN in this Reaction.
K Fe CN + Hb = Hi
KCN + Hi = HiCN
• D.H20 lyses the cells to release the Hb which is acted upon by potassium ferricyanide
to form methaemoglobin which is then converted to cyanmethaemoglobin by the action
of Potassium cyanide. Cyanmethaemoglobin is a stable pigment and it’s read
colorimetrically; the intensity of the colour is directly proportional to the Hb content of
that sample.
NB: Drabkin’s solution has a P.H of 9.6 and reacts rather slowly (requires 10mins for the
completion of the reaction). The P.H can be converted to 7.0 – 7.4 by addition of 140mg of
KH2 PO4 (potassium dihydrogen phosphate) and the Reaction time is reduced to less than 3
minutes. - It should be pale yellow and clear.
Procedure
- Switch on the colorimeter and give it 5 – 10 minutes to warm up to stabilize
- Pipette 5mls of drabkin’s solution in a big T. tube
- Add 0.02mls (20µl) of blood and mix well
- Allow it to stand for 10min for colour development

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 - Treat the STD in the same way as test i.e. 5mls of drabkin’s and 20µl of STD.
- Take OD of the STD and of the Test and use drabkin’s solution for blanking. Calculate the
Hb concentration using OD of T x (STD concentration).
OD of STD
Or take OD of test and compare with standard graph or chart to obtain the concentration of the test
sample.
NB:
❖ ODs can be read as many as 6hrs later because cyanmethaemoglobin is stable.
❖ STD concentration is always given.
❖ We have different types of STDs which are commercially available e.g. 15, 16, 18, 20gm
%. It is good to use the STD having the highest Hb value e.g. 20g%
❖ If the STD is not available, you can use the patient’s haemoglobin of known value e.g.
17.5g%
❖ When Hb is reduced, the person could be having anaemia.
❖ When Hb is increased the person could be having polycythaemia.
Advantages
- Hb need not be determined immediately as the dilution keeps for long .
- All forms of Hb except, SHb are converted to HiCN.
- It’s colorimetric and therefore accurate/ reliable (2% error)
- Commercial STDs are available, so the procedure is standardized
Disadvantages
- Potassium cyanide is very poisonous.
- Longer period (up to 10min) may be required.
- Apparatus are not portable.
- Machines use electricity, so they can’t be used in areas without electricity supply.
3. Alkaline haematin method.
-Also referred to as Gibson and Harrison.
Principle - Hb is converted to alkaline haematin by the action of 0.1 NaOH.
Requirement
- Photoelectric colorimeter with yellow green filter (IL ford no.625)

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-Cuvettes, T-tubes , 0.05ml pipette, 5ml pipette ,0.1NaOH,Gibson and Harrison STD, equivalent
to 160 g/l ,dry gauze swab , blood sample .
Procedures
➢ Label 2 T. tubes as test and STD.
➢ Into STD tube, put 4.95mls of NaOH plus 0.05 mls of the standard.
➢ Into the tube labeled test, put 4.95 mls of 0.1 N NaOH
➢ Then add 0.05ml of blood and mix well (but ensure excess blood is wiped off the
tip of pipette before adding)
➢ Heat the 2 tubes in a boiling water bath for 4min.
➢ Cool the tubes rapidly in cold H2O
➢ Read the OD of the test and of the STD and calculate the conc. of Hb.
Advantages
- Results are colorimetric and therefore more accurate
- No turbidity is observed because NaOH dissolves proteins – turbidity interferes with OD
readings.
- All Hb pigment variants are converted to alkaline haematin by NaOH, so the results obtained
are for almost all Haemoglobins in the individual
- The procedure is standardized.
Disadvantages.
- The method cannot be used where there is no electricity (except where the colorimeter can
use buttery)
- Boiling makes the procedure cumbersome
- Too much blood is used (50µl ) as compared to Sahli
- Some Hbs are resistant to alkaline denaturation e.g. HbF and Hb Barts and therefore are not
quantified together with the rest (this would be overcome by further heating for 4 minutes).
Cord blood is not used, why?
4. Oxyhaemoglobin method
Principle – haemoglobin is converted to oxyhaemoglogin (HbO2) by ammoniated water
(Ammonium Solution)
Requirements
- Photoelectric colorimeter and yellow/green filter (625).

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- Test tubes with tight fitting rubber bungs
- Cuvettes, 0.02ml pipette 0.4ml/l aq ammonia solution (0.04%).
- HbO2 STD (usually a neutral grey filter) equivalent to 146gm/l

Procedure
- Place 4mls of ammonia solution into a test tube
- Add 0.02mls of well mixed blood, cork tightly with rubber bung and mix thoroughly by
inverting the tube severally
- Take colorimetric readings (OD) of the test against of STD (treat STD as test)
- Calculate the conc. of test sample
OD of T x conce. Of STD (146g/l)
OD of S
Advantages
- It’s the easiest test to perform
- It’s not affected by moderate amount of bilirubin levels.
- Colorimetric hence accurate and reliable
Disadvantages
- It does not measure Hi, HbCO and SHb – because they are not acted upon by ammonia
solution.
- HbO2 fades with time so it has to be read immediately
5. Singers method
Principle – HbF resist alkaline denaturation, as well as Hb Bart’s.
i. The purpose of this test is to estimate an increase in HbF as opposed to other Hb’s like in
cases of thalassemia or sickle cell disease
ii. Persistent HbF in adults also referred to as hereditary persistent of foetal haemoglobin
Requirements.
- 0.2N NaOH, Half saturated acidified (NH4)2 SO4 solution, Haemolysate, colorimeter with
green filter, filter paper (what man)
Preparation of Haemolysate
- Wash the cells of the test sample 3 – times with normal saline
- On the packed cells, add an equal amount of D. H2O and mix to lyse the cells.

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 - Add 1/3 volume carbon tetrachloride/chloroform/toluene and mix. After mixing, centrifuge
slightly at 2000 rpm for 5 minutes.
- Chloroform / tetrachloride – removes cell stroma or membrane
- Haemolysate is bright red liquid
Procedure for HbF
✓ Take 3.2mls of NaOH and add 0.2mls of haemolysate – This ensures that all other Hbs are
denatured apart from HbF / Hb Barts
✓ Mix for 1 minute then add 6.8mls of the acidified (NH4)2SO4 to stop the Rxn
✓ Filter and take the O.D of the filtrate and use D.H2 O as blank
✓ Prepare a STD by taking 10mls of ½ sat. (NH4)2SO4 solution and then add 0.2mls of
haemolysate, take OD of (No filtering) STD. All the haemoglobins are intact
OD of T x 100% HbF - Don’t filter the STD
OD of STD
Normal range
✓ Adults – 1 – 2%
✓ Children > 1 year – 50 – 90%
✓ Cord blood - 97 – 98%
Self-Assessment Questions
1. Discuss haem synthesis in details
2. Explain the sources of errors in cell counts
3. List five abnormal haemoglobins

Summary
We have come to the end of lesson four whereby we have learnt about manual blood cell count,
physiological variations of blood cells, haemoglobins and haemoglobin formation among other
subtitles of the main titles.
Recommended Textbooks
1. F.A. Davis,(2001),Clinical Haematology and Fundamentals of Haemostasis; 4th
Edition D. Harmening,
2. Clinical Haematology Atlas, J.H. Carr, and B.F. Rodak W.B. Saunders (1998)

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 3. Lewis ,S. Mitchell (2001) Practical haematology Churchill Livingstone
4. Hoff brand, A.V (2001)Haematology (Essential) 4th ed. Blackwell Science
5. Hoff brand, A.V (2005) Haematology At A Glance Black well Publishing
Further reading
1. Ronald Churchill (2000)Hematology. Hoffman
2. Lewis. S. Mitchell(2006) Dacie and Lewis practical haematology 10th ed.
Churchill Livingstone
3. Hottman Ronald (2000) Hematology. harcourt International
4. Greer. P .(2003) Wintrobe's Clinical Hematology 11ed. Lippincot Williams &
Wilkins

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