Hemoglobin estimation by Sahli's
method
(Sahli’s haemoglobinometer)
DR. MOHAMMED CHYAD
AL-NOAEMI
Hemoglobin Estimation
• Haemoglobin concentration provides
information about the status of anaemia in
the population.
Normal adult Hb:
• In male, it is 14-18 g/100ml of blood (dl).
• In female it is 12-16 g/100ml of blood.
Hemoglobin is estimated By either
•
•
•
•
Sahli’s Method
Colorimetric Method.
Autoanalyzer.
Hemoglobin and Hematocrit (HCT) Test Meter Kit
• Sahli’s method were compared to
Haemiglobincyanide (HiCN) method of same
subjects. Results: Sahli’s method is less accurate and
has lower values than Haemiglobincyanide method
Sahli’s Method
• Hermann Sahli (May 23, 1856 – April 28, 1933)
was a Swiss internist.
Sahli,s apparatus (1930)
Materials used
• 1. Sahli’s haemoglobinometer.
• 2. Two Pasteur pipettes (one for HCl and one for
distilled water).
• 3. Glass rod to stir ( stirrer )
• 4. 0.1 N - Hydrochloric acid
• 5. Distilled Water
• 6. Comparison tube.
• 7. Pipette ( Hemoglobin pipette with rubber tubing
and mouthpiece )
Procedure
• 1. Add 0.1N.hydrochloric acid (1: 10 diluted) to the
haemometer tube (comparison tube) up to lowest
graduation ( 0.02 gramme ).
• 2. Sterilize the fingertip with Isopropyl alcohol,
surgical spirit and allow it to dry
• 3. Using sterile lancet prick the finger tip
• 4. Wipe away first few drops of blood
• 5. Suck blood into the hemoglobin pipette ( capillary
pipette ) up to 20 cu.mm( avoid air bubbles coming
into a tube )
• 6. Wipe the outside tip of pipette, clean with tissue
Cont. Procedure
• 7. Immediately transfer the blood to the comparison
tube
• 8. Suck blood back into the pipette several times and
blow out again into the tube(to mix blood with HCl )
• 9. Place the haemometer tube in the stand and allow
for 5-10 minutes (during this period HCl lysis red
cell, and released hemoglobin on reacting with HCl
forms a dark brown colored Acid Hematin ).
• 10. Now using a Pasteur pipette to add a few drops
of distilled water (or 0.1N HCl) and stir the contents
with a glass rod .
Cont. Procedure
11. Continue to add water (or acid) drop by drop and stir the
contents each time until the solution is just darker than the
standard .
12. Carefully add one or two drops of water till the color
exactly matches with that of the Standard and note the reading
13.while taking the reading hold the haemoglobinometer
against good daylight at arms length
14. The comparison tube represent 100 percent haemoglobin
with reference to a standard which is 14. 8 g Hb / 100 ml of
blood
Summary-Sahli’s Method
• It measures acid hematin. A sample of 0.02 mg of
whole blood taken with a pipette, is mixed with a
small quantity of 0.1mol/litre HCl. After few
minute, distal water or 0.1 mol/lit HCl is added drop
by drop and mixed, until the color of the solution
matches the color of the two identical standards
placed to the left and right of the dilution tube.
Hemoglobin concentration is then read from the
graduated scale on the dilution tube.
Drabkin's is more accurate than
sahli’sMethod
• Colorimetric Method: Using potassium
ferricyanide will produce Cyanmethemoglobin.
• Comparative study of hemoglobin estimated by
Drabkin's and Sahli's methods.
• P Balasubramaniam, A Malathi.
• Year : 1992 | Volume : 38 | Issue : 1 | Page : 8-9
• http://www.jpgmonline.com/article.asp?issn=00223859;year=1992;volume=38;issue=1;spage=8;epag
e=9;aulast=Balasubramaniam
Hemoglobin
• The name hemoglobin is the combination of
heme and globin, reflecting the fact that each
subunit of hemoglobin is a globular protein
with an embedded heme (or haem) group.
Each heme group contains one iron atom, that
can bind one oxygen molecule. The most
common type of hemoglobin in mammals
contains four such subunits.
Hemoglobin (Hb):
• Each RBC contains 200-300 millions molecules of Hb, which
represents about one third of the RBC mass. Each Hb
molecule consists of globin [globular protein; consists of 2
alpha (141aa and 2 beta (146aa) subunits] and four heme
molecules. Each heme molecule is attached to one subunit.
Each heme contains an iron atom to which an oxygen
molecule could be combined. Hence, one Hb molecule can
carry maximally four oxygen molecules.
• [If Valine replace Glutamine → Sickle cell anemia].
• When blood is pumped to the lungs, Hb becomes saturated
with oxygen, forming oxyhemoglobin (bright red in color).
When this blood is pumped to the body tissues, some its
oxygen dissociates from Hb, and this reduced Hb is dark red
in color.
Hemoglobin
green = heme groups
red & blue = protein subunits
Types of Hemoglobins
• HemoglobinA (Hb A), (α2alphaβ2beta), about 97%.
• Hemoglobin A2 (HbA2), (α2δ2 delta), . About 2%.
• Hemoglobin F (HbF), (α2γ2Gamma), or fetal Hb. It is
less than 1% in adults but it is the dominants Hb in
fetal life.
Glycosylated hemoglobin (HbA1c): normally it is
about 5-6%. In which Hb A is combined with
glucose. It is increase in diabetes mellitus.
• [Alpha-α , Betaβ-, Delta-δ, Gamma- γ-globin.]
Glycosylated Hemoglobin
• Long-term control of blood sugar concentration can be
measured by the concentration of Hb A1c. Measuring it
directly would require many samples because blood
sugar levels vary widely through the day. Hb A1c is the
product of the irreversible reaction of hemoglobin A
with glucose. A higher glucose concentration results in
more Hb A1c. Because the reaction is slow, the Hb A1c
proportion represents glucose level in blood averaged
over the half-life of red blood cells, is typically 50-55
days. An Hb A1c proportion of 6.0% or less show good
long-term glucose control, while values above 7.0% are
elevated. This test is especially useful for diabetics.[23]
Hemoglobin and,
Hematocrit (HCT) Test Meter Kit
HIGH PRECISION PROFESSIONAL AND HOME
USE PORTABLE METER
Glucometer