Determination/Estimation of hemoglobin

A. Estimation of hemoglobin through sahli’s hemoglobinometer (Acid-haematin method)

It is visual comparator method for estimation of hemoglobin. As visual comparison method leads to inaccuracy, this method is not used nowadays but was widely used before. the use of spectrophotometric methods like Cyanmethemoglobin method is preferred to it.

Principle:

When the blood is added to dilute hydrochloric acid (HCl), hemoglobin present in the RBCs is converted into brown-colored acid haematin. The acid haematin solution is further diluted until it’s color matches exactly with the permanent standard brown glass compared by direct vision.

Materials Required:

• Sahli’s hemoglobinometer set consisting comparator, hemoglobin tube, hemoglobin pipette and stirrer.
• N/10 hydrochloric acid (HCl)
• Distill water

Procedure:

• Hemoglobinometer tubes and pipettes should be ensured to be dry.
• Hemoglobinometer tube is filled with N/10 HCl upto its lowest mark 2g% or 10% mark with help of dropper.
• 20µL blood is taken in Sahli’s pipette. Extra blood is wiped out and blood is added to hemoglobin tube containing HCl
• Blood and acid are mixed and leaved for about 10 minutes for complete conversion of hemoglobin to hematin
• Distill water is then added drop by drop and stir till the color matches with standard glass of comparator.
• Reading at lower meniscus is then recorded, which directly gives hemoglobin concentration in 100 ml of blood.

B. Colorimeter method of hemoglobin estimation:

• 20 ml of blood mixed with oxalate or E.D.T.A is drawn with help of hemoglobinometer pipette
• These blood are mixed with 5 ml of Drabkin’s solution (cyanide-ferrocyanide) in test tube.
• After 10 minutes, it is compared with standard with help of colorimeter and recording is recorded.
• It gives value in g per 100 ml of blood.

C.    Spencer Hb-method (Photometer):

• Blood chamber is filled with drop of blood
• Blood is then hemolysed with help of hemolysis stick for 30 seconds.
• Chamber are then inserted in slit on left side of instrument.
• Instruments are hold to look through in left hand. Light switch button is pressed with help of left thumb- green split field will appear in instrument.

Normal level of hemoglobin in different animals:

Decreased in hemoglobin is observed in case of anemia and increased level occurs in polycythemia and hemoconcentration.