Practical No 3

ESTIMATION OF HEMOGLOBINE (Hb) CONCENTRATION

( Sahli’s Method )
A) Introduction:
Structure of Hemoglobin
 Hemoglobin (hem + globin) is a conjugated protein with a globular molecule
composed of four subunits (two α and two β chains);
 Each unit contains a red iron-porphyrin (heme group).

Structure of Hemoglobin

Functions of hemoglobin
 Carriage of oxygen to form Oxyhemoglobin
 Carriage of carbon dioxide to form Carbaminohemoglobin
 Buffer

Normal values in adults

Males: 14 – 18 g/dl
Females: 12 – 16 g/dl

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B) Principle
Hb in the RBCs is converted into acid hematin by the action of HCl
Hb + 0.1N HCl  Acid Hematin (Brown in color)
The brown color so developed is matched against standard brown glass in the
comparator by direct vision.
Reading is taken directly as g Hb/dl blood.

C) Apparatus and Reagents

i. Hemoglobinometre: The box contains:
1. Hb tube (Sahli Adam’s tube)
2. Hb pipette (20 μl) or Micropipette
3. Stirring rod
4. Dropper
5. Brush
6. Bottle for HCl (0.I N)
7. Comparator

ii. Distilled Water

iii. Lancets, Alcohol Swab, Cotton & Gloves

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 D) Procedure
1- Fill the Hb-tube with N/10 HCl solution up to the mark 20% or 3g and keep it
inside the comparator.
2- Fill the Hb pipette with blood up to the mark 0.02ml (20 μl) by gentle sucking.
(Wipe the tip of the pipette with cotton to remove extra blood).
3- Expel the blood into the Sahli's tube containing the HCl solution
(Suck a small amount of acid into the pipette and expel it again into the tube,
making sure that no solution remains on or in the pipette).
4- Mix the contents by shaking the tube before any clotting occurs.
5- Put the tube back in the comparator and wait for about 10 minutes
(During this time, the red cells rupture, liberating the Hb into the acid solution,
the acid reacts with Hb and converts it into acid hematin).
6- Take out the tube from the comparator, add a few drops of D/W and stir the
contents with the glass rod. → continue to add water drop by drop, stirring the
contents each time, till the color of the solution matches the standard.
7- Read the upper meniscus (colored solution) and note the reading.

E) Student's result
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Comments (if any) _______________________________________________________

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Dr. Amir Elhadi

Sig _______________ Date ____________

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☻ Review questions:
1. Why is 0.1 N HCL used in the Sahli method of hemoglobin measurements?
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2. What is the relationship of the hemoglobin content to age?

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3. Name the conditions in which HB content of the blood is above or below its
physiologic level.
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4. What is the clinical significance of determination of HB concentration?

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