Estimation of Hemoglobin by Sahali’s Method

Aim: To determine the haemoglobin content in 20µl of the blood sample.

Principle: A hemoprotein composed of globin and heme that gives red blood cells their characteristic colour; functions primarily to transport oxygen from the lungs to the body tissues. The red blood cells are broken down with hydrochloric acid to get the haemoglobin into a solution. The free haemoglobin is exposed for a while to form hemin crystals. The solution is diluted to compare with a standard colour.

Requirements: Hemometer, Single mark pipette, Distilled water, Needle, Spirit, Cotton, HCl.

Procedure:

  1. Take 1/10 HCl in the Hb tube upto the lowest mark ‘2’.
  2. Prick the finger with needle and collect 20µl of blood sample with single mark pipette
  3. Place the Hb tube on the working table for five minutes for the formation of hemin crystals.
  4. Place the Hb tube in the compater/hemometer and add drop by drop of distilled water into it until the colour of the solution in the Hb tube coincides with the glass plates of the compater.
  5. If the colour coincides with the glass plates of the compater, observe the reading in the Hb tube. The percentage of Hb can be calculated from the reading.

Evaluation: Hb content in grams X 100 / 14.5

  • Normal Values:
    • Male: 14 to 18grams
    • Females: 13 to 14grams
    • Children: 10 to 13grams

Result: The hemoglobin content present in 20µl of blood sample is _____________________.

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