ESTIMATION OF HAEMOGLOBIN (Hb) CONCENTRATION
Whole blood haemoglobin concentration can be estimated by a number of methods. Most
commonly used methods are:
Cyanmethaemoglobin method
Alkaline haematin method
Acid haematin method
Each of these methods has its advantages and disadvantages. Most commonly used method is
cyanmethaemoglobin method. Major advantage of this method is the availability of a stable and
reliable standard preparation. This method, however, does not measure sulphhaemoglobin (SHb).
Acid haematin method has the advantage of being useful without a colorimeter (Sahli's
haemoglobinometer) but is the least accurate of all. Alkaline haematin method has the advantage
that it can measure carboxy- haemoglobin, methaemoglobin and sulph- haemoglobin but it does
not measure foetal haemoglobins (HbF and Hb Barts').
CYANMETHAEMOGLOBIN METHOD
The principle of this method is that blood sample is diluted in a solution containing potassium
cyanide and potassium ferricyanide (Drabkin's solution). It converts haemoglobin (Hb) and
methaemoglobin (Hi) to cyanmethaemoglobin (HiCN), which is a stable compound. The
absorbance of the solution is measured in a photoelectric colorimeter with a yellow green filter or
in a spectrophotometer at a wavelength of 540 nm and is compared with a standard solution of
HiCN.
Requirements
1. Diluent (Drabkin's solution) Potassium ferricyanide 200 mg Potassium cyanide 50 mg
Potassium dihydrogen phosphate 140 mg Nonidet P40 (Sigma) 1 ml Distilled water up to
1000 ml The pH should be between 7.0-7.4 and the solution should be clear and pale
yellow in colour. It should give zero absorbance against water at 540 nm. The reagent is
stored at room temperature in a brown borosillicate glass bottle. If Nonidet is not available
then reaction time is to be increased, as haemolysis may be slow.
Reagent can be obtained in prepared concentrate form. If stored properly, the reagent is fit for use
for several months. The reagent is discarded if it becomes turbid or the absorbance changes.
2. Cyanmethaemoglobin reference solution: The cyanmethaemoglobin reference preparation
is used for direct comparison with blood, which is also converted to HiCN. Solutions of
different concentrations are commercially available and if unopened are stable for years.
But once opened, it is only stable for few hours. It is therefore recommended that a
calibration curve should be prepared with the help of these solutions and future readings
should be taken from it. But it is necessary that with each batch of tests or at least few times
a day the calibration is checked by a fresh cyanmethaemoglobin reference solution or an
internal reference prepared against it. The manufacturer’s inset with the pack of standards
gives the Hb g/L equivalent of HiCN concentration of the standard.
Procedure Venous blood collected in EDTA or free flowing capillary blood can be used.
Measurement can be carried out on blood that has been stored at 4°C for several days, provided it
is free from infection and contamination. 20 µl of blood is added to 4 ml of diluent and well mixed
by inverting the tube several times. It is allowed to stand at room temperature for 3-5 min so that
all Hb is converted to HiCN. The absorbance is then measured in the spectrophotometer at 540
nm. Hb level can be directly read from previously prepared calibration curve or chart.
Alternatively, absorbance of known standard is also read in the spectrophotometer with each batch
of tests and Hb is calculated by the formula:
Hb (g/L) = Abs. of test×Conc of standard(g/l)
Abs. of standard
Preparation of calibration curve/chart commercially available standard solution of HiCN is diluted
in Drabkin's solution so as to give concentrations equivalent to Hb concentrations of 2.0, 4.0, 6.0,
8.0, 10.0, 12.0, 14.0, 16.0, 18.0 and 20.0 g/dl. Pre-diluted standards are also commercially
available.
Absorbance is read in a spectrophotometer at 540 nm. These readings are converted into Hb conc.
in g/dl with the help of conversion table provided by the manufacturer of the standard. Absorbance
is plotted against Hb concentration on a linear graph paper, absorbance being on vertical axis and
Hb conc. on horizontal axis. All points must join in a straight line. A ready reference chart can be
prepared from this curve
Precautions:
Performance of equipment and calibration curve should be quality controlled by testing
simultaneously a commercial or in-house reference preparation with each batch of tests
and maintaining quality control chart. For details see the chapter on quality control.
If Nonidet has not been added to the diluent, then 10-15 min should be given for reaction
to complete and reading should be taken immediately.
Abnormal plasma proteins and high white cell count may result in turbid reaction
mixture. This should be centrifuged and clear supernatant should be used for taking the
reading.
SAHLI'S ACID HAEMATIN METHOD
The method is based on the principle that haemoglobin is converted into acid haematin by addition
of 0.I N Hydrochloric acid. The resultant solution is then compared with a reference solution in a
colorimeter or coloured strip.
Details of procedure, if a photoelectric calorimeter is used, are the same as for
cyanomethaemoglobin method. Details of procedure, when Sahli's haemoglobinometer is used are
given below:
Requirements
Sahli's haemoglobinometer
Sahli's pipette
0.1N HCl
Dropping pipette Procedure
i. Fill the tube of Sahli's haemoglobinometer up to mark with 0.1N hydrochloric acid.
ii. Venous or capillary blood may be used. The Sahli's pipette is filled up to the 20 mark by
gentle suction. Wipe outer side of pipette clean. There should be no air bubbles in blood
column.
iii. Blow the blood into the graduated tube of the Sahli's haemoglobinometer and suck the
solution in and out of pipette 2-3 times.
iv. Allow to stand for 5 min, so that haemoglobin gets converted into acid haematin.
v. Compare the colour of the solution in the graduated tube with that of the reference strips
on either side of the haemoglobinometer.
vi. If the colour of the graduated tube is darker, add drop by drop either 0.1N HCl or distilled
water by the dropping pipette and mix with glass rod, until the colour matches with the
reference strips.
vii. Note the reading on the graduated tube. This is the haemoglobin level in g/dl. Some tubes
also give level in percentage. To convert percentage into g/dl multiply the percent figure
by 0.146.
Reference range: Adult male: 13.0-17.0 g/dl
Adult female: 12.0-15.0 g/dl
