Method of Haemoglobin Estimation:Sahli's & Cyanmethemoglobin method, Specific gravity method

1. Hemoglobin
Estimation

2. Introduction
 A hemoglobin test measures the amount
of hemoglobin in your blood.
 Hemoglobin is a protein in your red blood cells that carries
oxygen to your body's organs and tissues and transports
carbon dioxide from your organs and tissues back to your
lungs.

3. Purpose of estimating
hemoglobin
 To detect the oxygen carrying capacity of blood.
 The result assists in detecting diseases, which
causes a deficiency or excess of hemoglobin.
 Studying changes in hemoglobin concentration
before or after operations and blood transfusions.
 To detect anemia and its severity and to monitor an
anemic patients response to treatment.
 To check hemoglobin level of blood prior to donating
blood.
 To calculate red cell indices.

5. Normal range of Haemoglobin
 The normal value depends on the age and sex of the individuals:
• Men: – 13-18 gm/dl
• Women :– 11-16 gm/dl
• Full term / cord blood :– 13-19 gm/dl
• Children 1 year :- 11-13 gm/dl
• Children 10 - 12 years :- 12-15gm/dl

6. Principle:
When blood is added to 0.1 N hydrochloric acid, hemoglobin
is converted to brown colored acid hematin. The resulting
color after dilution is compared with standard brown glass
reference blocks of a Sahli hemoglobinometer.
 Specimen
Capillary blood or thoroughly mixed anticoagulated
(EDTA or double oxalated) venous blood.
1. Sahli’s Method

7.  Requirements
1. Sahli hemoglobinometer: It consists of -
a) A standard brown glass mounted on a comparator
b) A graduated tube (Hellige tube)
c) Hb pipette (0.02 ml)
2. 0.1N hydrochloric acid
3. Distilled water
4. Pasteur pipettes

8. 1 By using a Pasteur pipette add 0.1 N hydrochloric acid in the tube
up to the lowest mark (20% mark)
2 Draw blood up to 20 mark in the Hb pipette. Adjust the blood
column, carefully without bubbles. Wipe excess of the blood on the
sides of the pipette by using a dry piece of cotton (or gauze).
3 Transfer blood to the acid in the graduated tube, rinse the pipette
well, mix the reaction mixture and allow the tube to stand for at
least 10 minutes.
4 Dilute the solution with distilled water by adding few drops at a time
carefully and by mixing the reaction mixture, until the color
matches with the glass plate in the comparator.
5 The matching should be done only against natural light. The level of
the fluid is noted at its lower meniscus and the reading
corresponding to this level on the scale is recorded (blood
hemoglobin, g/dl)
Procedure

9. • Easy to perform
• Quick
• Inexpensive
• Can be used as a bedside procedure
• Does not require technical expertise
Advantages

10. • For maximum colour, longer time is required
• Perfect matching with brown glass standard is not
possible
• Carboxyhemoglobin,methemoglobin and
sulfhemoglobin are not converted to acid hematin
• Developed of colour is slow and acid hematin is not
stable
• Source of light will influence the comparison of
colours Sahli’s Acid Hematin Method
Disadvantages

11.  A decrease in hemoglobin below normal range is an
indication of anemia.
 An increase in hemoglobin concentration occurs in
hemoconcentration due to loss of body fluid in
severe diarrhea and vomiting.
 High values are also observed in congenital heart
disease (due to reduced oxygen supply) in
emphysema and also in polycythemia.
 Hemoglobin concentration drops during pregnancy
due to hemodilution.
Clinical Significance

12. NAME OF METHOD:
CYANMETHEMOGLOBIN METHOD
 Most accurate method for estimation of Hb.
 Recommended by International Committee for
Standardisation in hematology because All
forms of Hb are converted to
cyanmethemoglobin (except sulfhemoglobin)
 Stable and reliable standard is available.
Cyanmethemoglobin Method
2. Colorimetric Method

13.  When blood Is mixed with Drabkin's reagent containing
potassium cyanide and potassium ferricyanide,
haemoglobin reacts with ferricyanide to form
methemoglobin which is converted to stable
cyanmethemoglobin (HiCN) by the cyanide.
 The intensity of the colour is proportional to
haemoglobin concentration in blood and it is compared
with a known cyanmethemoglobin standard at, 540 nm
(green filter)
Principle

14. 1. Drabkin's reagent (pH 7.0 -7.4)
It contains following components in 1000 ml of
distilled water -
a) Potassium ferricyanide: 400 mg
b) Potassium dihydrogen phosphate: 280 mg
c) Potassium cyanide: 100 mg
d) Nonionic detergent: 1 ml
This reagent is stable in a polythene container at 2-8°C.
2. Cyanmethemoglobin (HiCN) standard (Hb standard): It is commercially available.
This standard is directly pipetted in a cuvette and optical density measured at 540
nm (green filter): The reading obtained, corresponds to 15 g/dl, haemoglobin.
3. Hb pipette
4. Test tubes (15 x 125 mm)
5. Photometer or spectrophotometer
 Specimen
Capillary blood or thoroughly mixed anticoagulated (EDTA or double oxalated) venous
blood.
Requirements

15.  Pipette in the tubes labeled as follows
 Mix the contents in the tube labeled as 'Test' thoroughly
and wait for 5 minutes.
 Read absorbance of 'Test' by setting blank to 100% T at 540
nm (green filter). If reading of blank is equal to distilled
water, it is not necessary to keep a blank. In that case read
'Test' against distilled water,
 Read absorbance of standard (15 g/ dl) by pipetting it
directly in a cuvette
Procedure
NO Sample Test Blank
1 Drabkin’s Reagent(ml) 5.0 5.0
2 Blood(ml) 0.02 -

16. Calculation:
Preparation of a Standard Graph
Requirements:
1. Drabkin's reagent.
2. Cyanmethemoglobin (Hb standard )
standard : 15 g/ dl
Procedure
Pipette in the tubes, labeled as follows
 Mix well and read intensities of these standards by setting blank to
100% T at 540 nm (green filter). Prepare a graph by plotting 0.0.
readings on Y axis and concentrations of hemoglobin standards,
i.e. , 5.0 g, 10.0 g and 15.0 g on X ax is.
 A straight line passing through origin indicates agreement with
Beer's law. This graph can be used as a standard graph for
hemoglobin determination.
No Reagent Std.5 Std.10 Std.15 Blank
1 Drabkin’s
Reagent(ml)
3.34 1.67 0.0 5.0
2 Hb
Standard(ml)
1.66 3.33 5.0 0.0

17.  All forms of Hb except sulphemoglobin are converted to
hemiglobincyanide/cyanmethemoglobin (HiCN).
 Visual error is not there as no color matching is
required.
 Cyanmethemoglobin solution is stable and it’s color
does not fade with time so readings may not be taken
immediately.
 Absorbance may be measured soon after dilution.
 A reliable and stable reference standard is available
from World Health Organisation for direct comparison
Advantages

18.  Diluted blood has to stand for a period of time to
ensure complete conversion of Hb.
 Potassium cyanide is a poisonous substance and
that is why Drabkin’s solution must never be pipetted
by mouth.
 The rate of conversion of blood containing
carboxyhemoglobin is slowed considerably.
Prolonging the reaction time to 30min can overcome
this problem.
 Abnormal plasma proteins cause turbidity when
blood is diluted with Drabkin’s solution.
 A high leucocyte count also causes turbidity on
dilution of blood. Centrifuging the diluted blood can
help overcome the turbidity.
Disadvantages

19.  Hemoglobin being the largest single
constituent, affects the specific gravity of
blood more than other substances.
 Serum proteins are the next heaviest
constituents of blood.
 It is assumed that (which is not always true)
the level of serum proteins and other
smaller constituents remain the same, so
any change in the specific gravity of blood
is mainly due to change in concentration of
hemoglobin.
3. Specific Gravity
Method(Physical Method)

20. • Copper sulfate solutions of specific gravity 1.055 for male and
1.053 for female are taken into two 100 ml wide mouth
bottles.
• A drop of blood is collected by skin puncture and dropped
into the appropriate solutions.
• It is observed for 15 to 20 seconds. If the hemoglobin is
normal, the drop sinks to bottom. This indicates that the drop
is denser than the specific gravity of the solution.
• In the case of an anemic patient, the drop tends to hesitate
before going down or stays at the top. This indicates that the
drop is lighter than the solution.
• The specific gravities 1.055 and 1.053 correspond
approximately to the minimum normal hemoglobin levels of
men and women viz. 13 g/dl and 12 g/dl respectively
Specific gravity method
(qualitative)

21.  Hence this method uses the principle that when a drop
of whole blood is dropped into a solution of copper
sulphate, which has a given specific gravity, the blood
will maintain its own density for approximately 15
seconds.
 The density of the drop is directly proportional to the
amount of hemoglobin in that drop.
 If the blood is denser than the specific gravity of the
solution , the drop sinks to the bottom , if not it will
float.
 Hemoglobinemia is the presence of free hemoglobin in
the blood plasma. It is observed in conditions such as
severe Infections, severe burns, hemolytic blood
transfusion reaction, etc.
 Hemoglobinuria is the presence of free hemoglobin in
the urine and occur when the free hemoglobin in the
plasma reaches a concentration between 30 and 300
mg/dl

22.  The copper sulphate floatation method is based on
the principle of it he relative specific gravity of
hemoglobin.
 When a drop of blood is dropped into a copper
sulphate solution, the drop encases the hemoglobin
in a sac of copper proteinate, which prevents any
change in specific gravity for about 15 s.
 If the drop of blood has a satisfactory hemoglobin
concentration, and therefore a satisfactory specific
gravity, it will sink in the solution within 15 s. If not,
the drop will hesitate, will remain suspended or will
rise to the top.
 The copper sulphate solution used should have a
specific gravity of 1.053 (equal to 12.5 g/dL
hemoglobin).The solution should be stored at room
temperature and should be tightly capped to prevent
evaporation.
Principle

23. Maintain = equal to 1.053 = 12.5 gm%
Sink = > 1.053 = >12.5 gm%
Float = < 1.053 = <12.5 gm%
Interpretation