Hemoglobin Estimation - Methods other than Sahli's - Pathology - MLT, ATOT, RDT

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 Wom en : 12-15 g/dL Infants : 13.5-19.5 g/dL

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

6. Colorimetric methods Includes: Cyanmet-haemoglobin method Oxyhaemoglobin method Electronic counter method Direct reading electronic haemoglobinometer 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

9. 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

10. Calculations

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

12. 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

13. 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

14. 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

15. 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

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

17. 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

18. 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

19. Other methods Rarely used Alkali hematin method Haldane method(carboxyHb method)

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

21. 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