Topic: Hemoglobin Estimation - Methods other than Sahli's Faculty: Pathology Course: MLT 1st years, ATOT, RDT

1. Hb estimation - Methods
other than Sahli’s
Dr. Salman Ansari(MBBS)
Tutor, Dept. of Pathology
Kanachur Institute of Medical Sciences

2. Intro
● Hemoglobin - main component of RBCs
● Conjugated protein
● 1 molecule of Hb contains 2 pairs of polypeptide chains
and 4 haem groups
● 34% of RBC by weight is Hb
● Function of Hb: transporting O2 from lungs to tissues
● Different forms of Hb: OxyHb, CarboxyHb, SulfHb, MetHb

3. ● Mass of RBCs can be measured by Hb
estimation
● Haemoglobinometry: “measurement of Hb
concentration in blood”
● Blood sample: via fingerprick(capillary blood) or
i.v sample(anticoagulated blood)

4. Normal values
Men: 13-17 g/dL
Women: 12-15 g/dL
Infants: 13.5-19.5 g/dL

5. Methods of estimation of Hb
I. Colorimetric method
II. Measurement of O2 carrying capacity of Hb
III.Measurement of iron content of Hb
IV.Specific gravity method

6. I. Colorimetric methods
Includes:
1. Cyanmet-haemoglobin method
2. Oxyhaemoglobin method
3. Electronic counter method
4. Direct reading electronic haemoglobinometer
5. Sahli’s method

7. Cyanmet-haemoglobin method
- Best method for Hb estimation - recommended by Int’l Council
for Standardisation in Haematology(ICSH)
Principle:
- Dilute blood in Drabkin’s fluid(potassium ferricyanide and
potassium cyanide)
- Oxy-, carboxy- and met-haemoglobin get converted into
cyanmet-haemoglobin, with pink colour
- Intensity of pink colour is measured in a colorimeter at 540 nm
and compared with a standard Hb solution

8. Reagents:
● Drabkin’s fluid can be prepared as under:
● Drabkin’s fluid should be clear and pale yellow, with a pH of
7.0-7.4
Potassium ferricyanide 0.2g
Potassium cyanide 0.05g
Dihydrogen potassium
phosphate
0.14g
Distilled water 1000 ml

10. Procedure
- Add 0.02 ml of blood to 5 ml of Drabkin’s solution in a test tube(
dilution of 1:250)
- Mix well and allow it to stand for 3-5 minutes
- Take reading of test and standard in a spectrophotometer or
photoelectric colorimeter at 540 nm

11. Calculations

12. Advantages
- No chance of visual error
- All forms of Hb except sulfhaemoglobin can be
measured
- Standard is very stable

13. Disadvantages
- Potassium cyanide is a potent poison and has to be
safely stored in the lab
- Incorrect results may be obtained if blood is turbid due to
plasma proteins or hyperlipidemia, as absorbance will be
higher
- Results are affected due to hyperbilirubinaemia

14. Oxyhemoglobin method
- Simple and quick method
- Results are not affected by hyperbilirubinemia
Principle:
Blood is diluted in a solution of ammonia. There is
development of reddish-pink colour which is measured in a
photoelectric colorimeter at 625 nm and compared with
that of a standard oxyHb solution

15. Procedure
- Add 20 µl (0.02 ml) of blood to 4 ml of 0.4 ml/L ammonia solution
in a test tube
- Use a tight fitting stopper and mix by inverting the tube several
times
- Take reading of test and standard in a colorimeter with a yellow or
green filter(625 nm)
Calculations: Same as cyanmet Hb method

16. Advantages
- Use of ammonia solution is safer compared to
Drabkin’s fluid
- Result is not affected by hyperbilirubinemia
- Most forms of haemoglobins are measured in this
method

17. Disadvantages
- Does not measure sulfhaemoglobin
- Standard is not stable
- Turbidity of sample may lead to increased absorbance

18. 3. Electronic counter method
Multiparametric determination by electronic equipment
Principle:
- Based on electrical impedance principle
- Blood is diluted with isoton and lysate which lyses the RBCs
converting Hb into cyanmet-hemoglobin and its concentration is
measured in the spectrophotometer at 540 nm
Disadvantages:
- High WBC count(>30,000/µL) produces false elevation of Hb

19. 4. Direct reading electronic haemoglobinometers
- These instruments have inbuilt filters
- Reading of Hb in g/dL is visualised on the screen directly
Principle:
Principle of cyanmet-hemoglobin or oxyhemoglobin method or colour
comparators in which colour of blood is compared without conversion to a
derivate and is compared against a range of colours which represent
hemoglobin concentration
Advantage: No calculations are required to be made
Disadvantage: Calibration of the instrument may be faulty

21. Other methods
Rarely used
1. Alkali hematin method
2. Haldane method(carboxyHb method)

22. II. Measurement of O2 carrying capacity of Hb
III. Measurement of iron content of Hb
- For research purposes

23. IV. Specific gravity method
- Very rapid
- Used for screening blood donors of anemia in blood donation camps
- Normal SG of blood: 1.048-1.066