HAEMOGLOBIN AND HAEMOGLOBIN ESTIMATION METHODS

BY PROF. OLUFEMI AKANNI

Haemoglobin is a component of red blood cell. It is a respiratory

pigment which enables the red blood cell to carry oxygen (O 2) to
the tissue in order to provide energy and to return carbon (iv)
oxide (CO2) and other waste products to the lungs.

It is a large complex protein molecule with a molecular weight of

65,000 Daltons (64.8 – 75.5). It consists of 2 parts - the globin
which is the protein portion and about 4% of the molecule and the
haemoglobin which is the combination of a molecule of Fe and
porphyrin and about 96% of the molecule. The haem portion is
synthesized in the mitochondria and the cytosol of immature
cells. Globin portion is synthesized by the ribosomes from the
pro-erythroblast stage to the reticulocytes.

The porphyrin is composed of the ring of four or five pyrole units.

It is the haem portion that is responsible for the carriage of O 2.
When the Fe (iron) is oxidized from the ferrous to ferric state the
O2 carrying capacity of Haemoglobin is lost and when
Haemoglobin combines reversibly with O 2, Oxy-haemoglobin is
formed.

Haemoglobin plays an important role in the transportation of CO 2

to the lungs. The Haemoglobin doesn’t bond with the CO 2 but
sticks to the red blood cell forming bicarbonate (HCO 3-) ion. About
90% CO2 is removed this way and the remaining CO 2 as HCO3- in
the plasma.

At the end of the life span of the red blood cells, the haemoglobin
gets broken down and is removed from the circulation by
Reticuloendothelial system (RES). The haemoglobin which is
broken down to Amino Acids (AA) enters the general A.A pool of
the body. The Fe (iron) from haem is removed and stored as
ferritin for re-use in the future cells. The rests of the haeme is

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converted to the yellow pigment bilirubin and green pigment
biliverdin.

Fe Fe
∝ Β

β ∝

Fe Fe

TYPES OF NORMAL HAEMOGLOBIN

There are three (3) different types of normal haemoglobin which

differ slightly in the structure of the globin chain attached to the
Haeme.

Haemoglobin A: forms about 98% of adult haemoglobin. It is

made up of 2 ∝ chain and 2 β chain with structural formular ∝2 β 2.
The ∝ chain contains 141 AA while β chain contain 146 AA.

Haemoglobin A2. A small portion present in all individuals

usually less than 2%. It has structural formular ∝2∂2 . The d
chain contains 146 AA . The ∝ chain as in haemoglobin A i.e 141
AA. In diseased condition such as thalassemia, haemoglobin A 2 is
high as well as in infants.

Haemoglobin F (HB F): Foetal haemoglobin forms more than ½

haemoglobin of the new born. It is the major respiratory pigment
from early intra – uterine life up to term. The structure is ∝2φ 2. φ
chain consists of 146 AA. The hb F disappears almost completely
during infancy. i.e. haemoglobin synthesis is gradual as the foetus
is still in the uterus.

HAEMOGLOBIN ESTIMATION

Haemoglobin is estimated in order to measure the O 2 carrying

capacity of blood and as well as the erythropoietic activities in the

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RES. The result of this enables us to determine the level of
haemoglobin.

When the haemoglobin level is below normal for the same age
range, sex and people living within the same environment, the
patient is said to be anaemic. When it is above normal the
patient is said to be polycythaemic.

Anaemia may be qualitative or quantitative.

Quantitative anaemia: is a deficiency of red cell in the circulating

blood which leads to the reduction in the blood volume while
qualitative anaemia is the deficiency of the red cell which is as a
result of insufficient elements that are necessary for the synthesis
of normal blood formation e. g. Fe, Vit B12, foliate and such
anaemias are referred to as nutritional.

Normal values and variation

1 molecule of haemoglobin will carry 4 molecules of O 2.

1 mole of haemoglobin contains 67,000g.

1 mole of O2 has a volume of 22.4 dm 3. Thus, 67,000g of

haemoglobin containing 4 molecules of O 2 will carry 4 x 22.4 dm3
of O2.

: 1g of haemoglobin will carry 4 x 22,400 mls = 1.34ml of O2

67,000
This is the Huffner’s factor

The above is for the active form of the haemoglobin only. When
the inactive forms are considered such as the met-hb and sulph-
hb, a more correct figure for use as the Huffner’s factor is 1.36ml
of O2.

The average normal value for female is 13.7g/dl

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The average normal value for male is 15.6 g/dl

The average normal value for cord blood is 20g/dl

There may be variation in the same individual at different time of

the day but such physiological variation appears to have no
clinical significance.

Methods of estimation of Haemoglobin

Generally, the haemoglobin relies on comparison of colour and

this can be achieved by:

1. Estimation of its ability to combine with Oxygen on

estimation of the amount of O2 contained in haemoglobin.

2. Measurement of Fe content of the haemoglobin.

All the methods depend upon matching the colour produced by

the test sample with the colour produced by the standards
sample of known haemoglobin.

The colour pigment produced for measurement should be

prepared easily and quickly and gave optically clear solution.

Methods of haemoglobin estimation are classified as

follows:

Physical: Based on specific gravity e,g Copper sulphate method

Chemical: Based on iron content of haemoglobin

Gasometric: Based on Oxygen combining capacity of

haemoglobin.

Calorimetric: Based on color

Visual: Tallquist method, Sahlis apparatus

Photoelectric: Cyanmethaemoglobin etc

Automated method.
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The methods are:

1. Cyanmethaemoglobin

2. Oxy-haemoglobin method

3. Carboxyl haemoglobin.

4. Alkaline haematin

5. Acid-haematin or Sahlis acid haematin

6. Pyrimidine chromogen

7. Copper Sulphate (CuS04) method.

1. Cyanmethaemoglobin Method

Principle: Haemoglobin–Fe is converted to the ferric state by

ferric-cyanide thus forming met-hb which is then combined with
KCN to form cyanmethaemoglobin which is them measured by the
colorimeter.

Reagents: KCN = 5g

K ferri-cyamide (K3 Fe(CN)6 = 20.0g

KH2PO4 or Na2CO3 = 14.0g

D/H20 = 1 litre

For use - dilute 1.00. The stock solution must be kept in dark
bottle

(Drabkin’s reagent)

Method:

To 4 mls of Drabkin’s reagent/solution in a clean test tube + 0.02

ml well mixed

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Anticoagulated blood. Mix very well by inversion. Allow to stand
at room temperature (R.T) for 10mins for total conversion. Read
at of 540nm in photo-electric colorimeter.

The cyanmethaemoglobin standard is stable for a long period. It

is worked out on the basis that haemoglobin has a molecular
weight of about 65,000 and a milli-molar co-eff extinction of 44.
This standard is regarded as the dilution of whole blood and the
original haemoglobin that it represents can be worked out and its
only 14.6g/100ml.

Hb concentration is calculated as follows:

O. D of test Concentration of std
x x the dilution factor
O . D of std 1000

e.g.
.28 14.6 g/l
x x 200
.35 1000

57.2mg

Merits of the Method

 The standard can be easily prepared and it is more stable

and convenient for routine use.

 All forms of haemoglobin are converted to met – hb except

sulph hb

 It eliminates colour blindness

 It doesn’t request artificial standard.

Demerits

1.The diluent is poisonous due to the Potassium cyanide (KCN).

2.Abnormal plasma protein or high Wbc may result in turbidity but
this can be avoided by centrifugation.
3.Time is taken for conversion.

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2. Acid – Haematin Method

Haemoglobin is converted to acid-haematin by the addition of

0.1N Hcl

Reagents + apparatus:

0.1N, Hcl, graduated Sahli’s tube and glass standard

Method: fill the graduated sahlis tube to the 20ml with 0.1N Hcl,
+ 0.02 ml of blood, mix well and leave for 5 – 10 mins.

At intervals of 5, 10, 15 minutes, + 0.01 N Hcl drop by drop,

mixing between each time until the colour matches that of the
standard. The % haemoglobin.

concentration is then read off by reading the amount of solution

in the graduated tube.

Calculation:

Sahli’s standard contains 17.2g/100ml as 100%

Assuming the quantity of diluent is 80%

Therefore, the concentration of hb is 80/100 x 17.2

= 13.94g/dl.

Merit:

It is simple and the apparatus is cheap.

Demerits:

-Full colour development takes a very long time

-it doesn’t estimate all forms of haemoglobin such as met-hb,

carboxyl hb and sulph-hb

-the glass standard needs regular re-calibration.

Oxy - Heamoglobin
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Principle: Haemoglobin is converted to Oxy-hb by dilution with
ammoniated H2O and Oxy-hb is estimated using a photo-electric
colorimeter.

Reagent and Apparatus:

0.04% NH3 in d/H20, standard Hb and photo-electric colorimeter.

Method:

1. 0.02ml of b/d to 4ml HH3 and H20

2. Mix severally by inversion

3. Read the standard solution first, then the test using a yellow-
green filter I/for the 625

4. If the O.D is higher than .7, dilute with volume of d/H 20 and
read again. The same way for test.

Calculation:
O. D .test
x concentationof std
O . D . Std

Merits:

 It is the simplest, quickest method use.

 Suitable for use as visual colorimeter
 Its reliability is not affected by colorimeter in plasma bilirubin

Demerits:

 The solution of oxy. hb is not stable

 It is not possible to prepare a stable oxy-hb.
 Artificial standards need accurate calibration.
 Not reliable in the presence of carbon dioxide, met-sulph-hb

Alkaline Heamatin

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Principles: Haemoglobin is converted to Alkaline haematin by the
solution of diluted NaOH. The conversion is accelerated and may
be completed by boiling the mixture.

Reagent + App.

1. Gibson & Harrison standard: this is equivalent to hb

concentration of 16g/100ml of blood. The standard is made up of

(a) Chromium potassium sulphate 16.61g

(b) Anhydrous cobaltous sulphate 13.10g

(c) Potassium dichromate 0.69g

(A- C) are dissolved in 500ml d /H 2O, then 1 N H2SO4 1.8ml is

used. Heat the mixture to boiling 1 min and when cooled the
solution is made up to l litre with d/H 20. The solution is quite
stable.

2. 0.1N NaOH

3. Photometer or colorimeter

4. 0.01ml pipette

5. Test tubes

Procedure: 0.05ml of blood is used to 4.95ml of 0.1N NaOH

1. Mix well and heat in the boiling H20 bath for 4 minutes

2. Boil 5mls of the standard solution

3. Cool the tube in cold water and match the test against the
standard in the colorimeter using the green filter.

If the test gives too high a reading, + 5ml of water to both test
and standard and read again. If there is turbidity, + 1 or 2 drops
of teepol turbidity will clear.

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Merits:

 All the pigments are converted.

 A true solution is obtained
 Accurate and reliable
 Photoelectric method using a green filter eliminates personal
error.
 The artificial standard is easy to prepare and it is stable.

Demerits:

 Foetal blood is resistant to alkaline denaturation unless

exposed at boiling for 15 mins.
 A fresh sample of standard is prepared for the text with each
batch of hb – estimation.
 Evaporation can’t be avoided
 The brown colour may mask the turbidity formed.

Acid – Alkaline Method

This is a modified haematin method. The blood is first treated as

the haematin method adds NaOH.

Procedure: 0.05ml of blood is used to 4ml of 0.1N HCL and

allowed to stand for 30 minutes 0.95mls of 0.1N NaOH, td, mix
and read along with the standard in the photometer.

Demerits:

 Time wasted for the conversion.

CuSO4 Method:

This method is often used as the screening test for blood donor. It
is also in used in ANC clinic.

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Principle: Haemoglobin protein in the blood combines with copper
forming proteinates and this then coats the blood. Depending on
the specific gravity of the blood, it remains stationary, rises or
falls. The strength of the CuSO4 solution used vary from S.G
1.010 – 1.060.

Procedure:

1. A drop of capillary or anticoagulated blood is allowed to fall

from a height of about l cm into a solution of CuSO 4 of
known S. G.

2. The drop penetrates about 2cm into the liquid and it is either
remain stationary, rises or fall.

3. Observe the movement for about 15 seconds

4. If the drop is stationary, it is of the same as the CuSO4

solution. If it falls, it is heavier than the S. G. of CuSO 4
solution. It is rises, it is lighter. The S.G. of 1.010
corresponds to 70% haemoglobin.

Merits:

- Ideal for field work since no gas or electricity is required.

- Cheap and easy to perform.

Demerits:

1. It is not reliable for abnormal blood e.g. polycythaemia

and solution needs frequent changing.

Errors of haemoglobinomoetry

1. Pipettes: although may be standardized, they show some

variations, chipped pipettes or blocked should not be used.

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2. Reading: personal errors should be noticed using visual
colorimetric methods, this can be minimised by using
photoelectric colorimeter.

Physiological variation:

It has been established that Hb level varies only daily but from
one time of the day to the other.

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