1. The document discusses various methods for estimating hemoglobin concentration, including the cyanmethemoglobin method, oxyhemoglobin method, and Sahli's acid hematin method. 2. The cyanmethemoglobin method is recommended, involving converting hemoglobin to cyanmethemoglobin with potassium cyanide and measuring absorbance at 540nm. 3. Errors can occur from insufficient mixing, inaccurate pipetting, inadequate mixing with reagent, or exposing the solution to light for too long. The document provides details on performing the cyanmethemoglobin method and calculating hemoglobin concentration.

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3. Haemoglobin variants:
 Hemoglobins with altered oxygen-carrying
capacity
 In these hemoglobins a different molecule
replaces the O2 molecule.
Carboxyhemoglobin:
 O2 has been replaced by CO (carbon
monoxide).
 CO preferentially binds to hemoglobin over
O2 by an affinity 210 times greater than O2.
Background
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4. Sulfhemoglobin:
 Sulfur has replaced the oxygen.
 This is also a stable form of hemoglobin It may
result in denatured hemoglobin.
Methemoglobin:
 The oxidized state of hemoglobin with iron in the
ferric state.
 Hemoglobin is unable to bind to O2 in this state.
 This is an inherited or acquired state.
Background
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5.  The haemoglobin concentration (Hb) of a
solution may be estimated by:
1. By its power of combining with oxygen or
carbon monoxide:
 The oxygen-combining capacity of blood is 1.34 ml O2
per g haemoglobin.
 Ideally, for assessing clinical anaemia, a functional
estimation of Hb should be carried out by
measurement of oxygen capacity.
 This is hardly practical in the routine haematology
laboratory.
 It gives results that are at least 2% lower than those
given by the other methods, probably because a small
proportion of inert pigment is always present
Principles of Haemoglobin Estimation
Methods
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6. 2. By its iron content:
 The iron content of haemoglobin can be
estimated accurately, but again the
method is impractical for routine
purposes.
 Iron content is converted into
haemoglobin by assuming the following
relationship:
0.347 g iron = 100 g haemoglobin
3. Measurement of its colour:
Principles of Haemoglobin
Estimation Methods
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7.  Two methods are in common use:
1. Cyanmethaemoglobin (HiCN) method,
2. Oxyhaemoglobin (HbO2) method.
 Other methods that have been used include:
1. Sahli‘s acid haematin method
2. Alkaline-haematin method
SPECTROMETER
(SPECTROPHOTOMETER) OR
PHOTOELECTRIC COLORIMETER
method

8. Principle:
 the haemoglobin is converted to acid haematin
by diluting with weak acid.
Technique:
 The graduated tube is filled to the mark 20 with
10N HCl
 0.02 ml blood added
 After 5 minutes the brown solution is diluted
drop by drop with distilled water until the color
matches that of the standard
 Read the result g/dl or percent from the
graduated tube
Sahli‘s acid haematin method
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10. Disadvantages :
 Less accurate because:
1. Individual’s error
2. Only Hb-O2 (but not HbCo, SHb and Hi) is
converted to acid haematin.
3. The colour develops slowly, is unstable, and
begins to fade almost immediately after it
reaches its peak
 For these reasons usually give result
approximately 5% less than actual results
Sahli‘s acid haematin method......
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11.  The alkaline-haematin method gives a true
estimate of total Hb even HbCO, Hi, or SHb is
present
 a modified method has been developed in
which blood is diluted in an alkaline solution
with non-ionic detergent and read in a
spectrometer at an absorbance of 575 nm
against a standard solution of chlorohaemin.
 Some studies has shown a bias of 2.6% when
compared with the reference method.
alkaline-haematin method
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12.  Venous blood into EDTA
 Free flowing capillary blood may be
measured and added to the diluting fluid
without anticoagulation.
Test Sample
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13.  The haemiglobincyanide method is the
internationally recommended method for
determining the haemoglobinconcentration of
blood.
Principle:
 The basis of the method is dilution of blood in a
solution containing potassium cyanide and
potassium ferricyanide.
 Haemoglobin, Hi, and HbCO, but not SHb, are
converted to HiCN.
 The absorbance of the solution is then measured
in a spectrometer at a wavelength of 540 nm or a
photoelectric colorimeter with a yellow–green
filter.
CYANMETHAEMOGLOBIN)
METHOD
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14.  The original (Drabkin's) reagent had a pH of 8.6
 A modified solution, Drabkin-type reagent is
recommended by the ICSH:
 has a pH of 7.0–7.4
It is less likely to cause turbidity from precipitation of
plasma proteins
Requires a shorter conversion time (3–5 min) than
the original Drabkin's solution
It has the disadvantage that the detergent causes
some frothing
Diluent: Drabkin’s
solution
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15. Constituent Quantity
Potassium ferricyanide (0.607
mmol/l)
200 mg
Potassium cyanide (0.768
mmol/l)
50 mg
Potassium dihydrogen phosphate
(1.029 mmol/l)
140 mg
Nonionic detergent 1 ml
Distilled or deionized water To 1 litre
Preparation of Drabkin’s solution
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16.  The diluent should be clear and pale yellow in
colour
 The pH should be 7.0–7.4 and must be checked with
a pH meter at least once a month.
 When measured against water as a blank in a
spectrometer at a wavelength of 540 nm, absorbance
must be zero.
 If stored at room temperature in a brown glass
bottle, the solution keeps for several months.
 If the ambient temperature is higher than 30°C, the
solution should be stored in the refrigerator but
brought to room temperature before use.
 It must not be allowed to freeze
Preparation of Drabkin’s solution

17. Drabkin’s reagent should be discarded if:
 Becomes turbid.
 The pH is found to be outside the 7.0–7.4
range.
 It has an absorbance other than zero at 540
nm against a water blank.
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18. Drabkin's storage?

20.  1. Haemiglobincyanide Reference Standard:
 Haemiglobincyanide reference standard is
commercially available.
 The HiCN reference preparation is intended
for direct comparison with blood that is
converted to HiCN.
2. Secondary standard of preserved blood or
lysate:
 Should be treated as the sample, diluted with
Drabkin’s reagent, incubated and read
photometrically
Haemoglobin Reference Standards
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21.  Make a 1 in 201 dilution of blood by adding 20 μl
of blood to 4 ml of diluent.
 Stopper the tube containing the solution and
invert it several times.
 Let the test sample stand at room temperature
for at least 5 min (to ensure the complete
conversion of haemoglobin to
haemiglobinocyanide),
 then pour it into a cuvette and read the
absorbance in a spectrometer at 540 nm or in a
photoelectric colorimeter with a suitable filter
against a reagent blank.
Procedure

22.  Absorbance of the test sample must be
measured within 6 hours of its initial dilution
 The absorbance of a commercially available
HiCN standard (brought to room temperature
if previously stored in a refrigerator) should
also be compared to a reagent blank in the
same spectrometer or photoelectric
colorimeter as the patient sample.
 The standard should be kept in the dark, and
contamination should be avoided.
Procedure
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23.  Hb (g/dl)= O.D of test X Conc. of STD X D.F
O.D of STD
Calculation of Hb
concentration
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24.  Insufficient mixing of blood specimen.
 Inaccurate pipetting and the use of badly
calibrated pipettes.
 Inadequate mixing of blood with Drabkin's
solution.
 Exposure of the preparation
(Cyanomethaemoglobin solution) to the
direct light for long period.
Source of errors
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25.  The HbO2 method is the simplest and quickest
method for general use with a photometer.
 Its disadvantage is that it is not possible to
prepare a stable HbO2 standard, so the
calibration of these instruments should be
checked regularly using HiCN reference solutions
or a secondary standard of preserved blood or
lysate ( p. 27 ).
 The reliability of the method is not affected by a
moderate increase in plasma bilirubin, but it is
not satisfactory in the presence of HbCo, Hi, or
SHb
OXYHAEMOGLOBIN METHOD
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26. A measured quantity of blood is mixed with
dilute ammonia solution and the intensity of
the red color is measured photometrically.

Principle:
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27.  Wash 20 µl of blood into a tube containing 4
ml of 0.4 ml/l ammonia (specific gravity 0.88)
to give a ×201 dilution.
 Use a tightly fitting stopper and mix by
inverting the tube several times.
 The solution of HbO2 is then ready for
matching against a standard in a
spectrometer at 540 nm or a photometer
with a yellow–green filter against a water
blank.
 Fresh ammonia solution must be made up
each week. Once diluted, the blood sample is
stable at 20°C for about 2 days.
Procedure

28.  A standard should be prepared from a specimen of
normal anticoagulated whole blood.
 Its haemoglobin is first determined by the HiCN
method ( p. 27 ).
 The blood is then diluted 1:201 by pipetting 20 µl of
the well-mixed blood into 4 ml of ammonia
 Sequential dilutions are made in ammonia, and
absorbance is read in a spectrometer at 540 nm or
photometer using a yellow–green filter.
 The readings are plotted on arithmetic graph paper.
 Linearity of response is checked, and absorbance is
related to haemoglobin from the measurement
obtained in the original sample by the HiCN method.
Standard

29.  Direct calculation from a STD:
 Hb (g/dl)= O.D of test X Conc. of STD X D.F
O.D of STD 1000
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30.  Adult male: 13-17 g/dl
 Adult female: 12-15 g/dl
 Children:15-22 g/dl
Range of Haemoglobin in
Health
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31. Questions