Determine the amount of hemoglobin in the blood

Determine the amount of hemoglobin in the blood

Hemoglobin (Hb or Hgb) is the iron-containing oxygen-transport metallo-protein in the red blood cells of vertebrates and the tissues of some invertebrates.

Different types of hemoglobin

1. Hemoglobin A (95%)
2. Hemoglobin A2 (1.5-3.5%)
3. Hemoglobin F
4. Hemoglobin H
5. Hemoglobin S
6. Hemoglobin C
7. Hemoglobin AS
8. Hemoglobin SC

Methodology for estimation of Hb

• Sahlis method
• Cyanmethaemoglobin method (spectrophotometric method)
• Haldanes method
• Talliquist method
• Alkaline haematin method
• Oxyhaemoglobin method
• Determination of specific gravity
• Estimation of iron content of blood (van slyke and stadie method)
• Gowershaemoglobinometer
• Von fleischlshaemometer
• Colorimetric method
  

Hemoglobin concentration measurement is among the most commonly performed blood tests, usually as part of a complete blood count. Results are reported in g/L, g/dL or mol/L (1 g/dL equals about 0.6206 mmol/L). 

Normal values of hemoglobin are

Men: 13.5 to 16.5 g/dl
Women: 12.1 to 15.1 g/dl
Children: 11 to 16 g/dl
Pregnant women: 11 to 12 g/dl .

Sahlis method

1. This method is commonly used to estimate Hb at laboratory level.
2. Dil-HCl (N/10) is used to convert Hb to acid hematite causing brown colorization of test sample which is matched with standard. Reading is recorded as gm/dl of blood.

Procedure

1. Fill graduated tube with N/10 HCl upto 20 mark
2. Take a sprited cotton and wash ring finger ball then prick the finger with strile needle
3. Discard first drop of blood that oozes out after pricking
4. Take Hb pipette with one end touching oozed blood and fill it upto 20 mark
5. Transfer blood to graduated tube containing dilHCl and suck small amount and expel it. Repeat process for 3-4 times
6. Stir the solution and leave for 10 min undisturbed
7. Now add distilled water drop by drop with gentle stirring
8. Continue dilution till it matched exactly with standard plate. Note the result.

Observation

1. Three parameters can be assessed by this method.
2. Hb concentration in gm/dl in blood.
3. Percentage of Hb (14.5 gm/dl Hb is considered 100%).
4. O2 carrying capacity (1.34 ml of O2 is carried by 1 gmHb).

Error limit: - 8-10 %

Precaution

1. Pricking should be appropriate so that no extra bleeding may occur.
2. After blood is drawn using pipette, tip of   pipette should be wiped properly to prevent wrong results and clotting.
3. Wait for exact 10 min after mixing blood with Dilute HCL else it will result in wrong interpretation.
4. Matching with standard should be done against well lightened area otherwise manual error may occur.
5. Read lower meniscus to get a proper interpretation.