Estimation of Haemoglobin Concentration

ASSIGNMENT HEMATOLOGY
Group # 05
GROUP MEMBERS:
M.ALI TAHIR, MAIRA QASIM, ZIRWA ZUBAIR, M.
YOUSAF ISHAQ, HAFIZ ABDUL WAHEED.
TOPIC:
 “Estimation of
Haemoglobin
Concentration”
INTRODUCTION
Hemoglobin is the protein molecule in red blood
cells that carries oxygen from the lungs to the
body’s tissues and returns carbon dioxide from
the tissues back to the lungs.
Hemoglobin is made up of four protein

molecules (globulin chains) that are connected
together.
STRUCTURE OF HEMOGLOBIN
Each hemoglobin molecule is made

up of four heme groups surrounding a
globin group, forming a tetra-hedral
structure.
Each subunit have one polypeptide

chain and one heme group.
STRUCTURE OF HEMOGLOBIN
Heme, which accounts for only 4 percent of the
weight of the molecule, is composed of a
ringlike organic compound known as a
porphyrin to which an iron atom is attached.
 The structure of the protein part can be studied
at four levels; primary structure,
secondary structure, tertiary structure, and
quaternary structure.
A haemoglobin molecule can transport four
oxygen molecules at a time.
FUNCTION OF HEMOGLOBIN
Each of the four subunits contains a heme (contains

iron) molecule, where the oxygen itself is bound
through a reversible reaction.
The heme transports oxygen from the lungs to the
tissues as well as takes carbon dioxide from the
tissues to the lungs.
About 70 percent of our body’s iron is found in the
red blood cells of our blood called hemoglobin and
in muscle cells called myoglobin.
FUNCTION OF HEMOGLOBIN
 Hemoglobin imparts
red color to the
blood and buffers
the blood pH and
maintains it to the
tolerable limits.
Myoglobin, in
muscle cells,
accepts, stores,
transports and
releases oxygen.
NORMAL HEMOGLOBIN
CONCENTRATION IN HUMANS
The normal range for hemoglobin is:
For men, 13.5 to 17.5 grams per deciliter.
For women, 12.0 to 15.5 grams per deciliter.
As a result, the hemoglobin level in a near-term
fetus or term infant is relatively high.
The normal hemoglobin concentration for a term
newborn is 19.3±2.2 g/dL (193±220 g/L), with a
hematocrit of 61%±7.4% (0.61±0.074), values that
continue to rise until they reach a maximum at
about 2 hours after birth.
PHYSIOLOGICAL VARIATIONS IN
HAEMOGLOBIN CONCENTRATION
Elderly renal Insufficiency,
Inflammation, testosterone deficiency ,
diminished erythropoiesis,
Myelodysplasia – reduce Hb
concentration.
Exercise – increases Hb concentration.
Altitude – increases Hb concentration.
Smoking – increases Hb concentration.
Posture – lying to sitting increases Hb
concentration.
COLLECTION OF BLOOD
Blood can be collected from three

different sources :
1. Capillary blood
2. Venous Blood
3. Arterial Blood
INDICATIONS FOR HEMOGLOBIN
ESTIMATION
Determine presence and severity of anemia.
Screening for polycythemia.
Response to specific therapy in anemia.
Estimation of red cell indices.
Selection of blood donors.
COLORIMETRIC METHODS
Color comparison between standard and test sample
is done by:
Visual Methods
1. Sahlis Acid Hematin
2. Tallqvist Hemoglobin Chart
3. WHO Hemoglobin Colour Scale
4. Specific Gravity Method
Photoelectric Methods
1. Cyanmethemoglobin Method
2. Oxyhemoglobin Method
3. Alkaline Hematin Method
1. SAHLI’S ACID HEMATIN METHOD
Type:
Visual comparative method of determining Hb
concentration.
Principle:
1. Mix the collected blood sample with an acid
solution so that Hb is converted into brown
coloured acid hematin.
2. Dilute with water till brown colour matches that
of brown colour started.
3. Hb value is read from scale
SAHLI’S ACID HEMATIN METHOD
Equipments:
1. Sahli’s haemoglobinometer
2. Sahli pipette(marked at 20 microliter)
3. Stirrer
4. Dropping pipette
 Reagents Used:
1. N/10 hydrochloric acid(HCL)
2. Distilled water
Procedure:
1. Place N/10 HCL into
Hb tube upto 2 grams.
2. Blood sample in sahli’s
Hb pipette upto 20
microliters.
3. Add blood sample to
acid’s solution.
4. Mix with stirrer.
Procedure(continue):
5. Allow to stand for 10
minutes.
6. Add distilled water drop
by drop until the colour
matches to the brown
glass standard.
7. Take the reading of the
lower meniscus from
graduated tube in
grams.
 Advantages:
1. Easy to perform
2. Quick
3. Inexpensive
4. Can be used as a bedside procedure
5. Donot require technical expertise
 Disadvantages:
1. For maximum colour longer time is required.
2. Perfect matching with brown glass standard is not
possible.
3. Source of light will influence the comparisons of colours.
4. Not so recommended because of inaccuracy.
2. TALLQUIST HEMOGLOBIN CHART
METHOD
 Explanation
1. Tallquist paper is used to determine the concentration
of hemoglobin in blood
2. The Tallquist method makes possible a quick estimation
of a patient’s hemoglobin levels.
 Principle:
1. A drop of blood is absorbed into a test paper, and the
colour of the blood is compared to the colours on a
chart.
2. The Tallquist method of measuring haemoglobin is used
to compare a small piece of Tallquist paper that has
been saturated with a sample of blood with a Tallquist
colour chart .
TALLQUIST HEMOGLOBIN CHART
METHOD
Equipment:
1. Tallquist paper (Filter paper)
2. Blotting paper
3. Hemoglobin scale
4. Sterile lancet or needle
5. Alcohol swabs
Reagents:
No reagents use in this method because it is direct
method of hemoglobin estimation.
TALLQUIST HEMOGLOBIN CHART
METHOD
 Procedure:
1. The procedure of this method is
very simple.
2. Take the blood on the blotting
paper.
3. Let it dry.
4. Then compare it with the given
chart.
5. Start from the light color and move
to dark color.
6. When the color match then write
the hemoglobin of sample.
TALLQUIST HEMOGLOBIN CHART METHOD
 Advantage:
1. Inexpensive
2. Rapid
3. Simple
4. No reagents required
5. Portable
6. No electricity required
 Disadvantage:
1. Only filter paper supplied.
2. Limited quantity can be used chance of faulty results.
3. Results influence by lightning ,size and thickness of blood spot.
4. Temperature and humidity also effect.
3. WHO HEMOGLOBIN COLOUR SCALE
METHOD
The World Health Organization (WHO) color
scale method is an inexpensive method for
estimating hemoglobin concentration from a
drop of blood by means of a color scale.
The color scale comprises a small card with six
shades of red that represent hemoglobin levels
at 4, 6, 8, 10, 12, and 14 g/dL, respectively.
WHO HEMOGLOBIN COLOUR SCALE
METHOD
The procedure, equipment, advantage and
disadvantage of WHO hemoglobin colour scale are all
same as of Tallquist hemoglobin chart method.
The WHO color scale was primarily designed for
anemia screening in obstetrical management,
pediatric clinics, malaria and hookworm control
programs, blood transfusion donor selection, and
epidemiological surveys.
WHO HEMOGLOBIN COLOUR SCALE
VIDEO
• https://youtu.be/ZTHCAX3MRlk
4. SPECIFIC GRAVITY METHOD
 Reagent used:
CuSO4
 Procedure:
1. In this method, a drop of the blood sample is
allowed to fall in the solution of copper sulfate
having specific gravity 1.053 from the altitude of 1
cm.
2. The hemoglobin concentration of 12.5 g/dl is
equivalent to the specific gravity of 1.053.
3. The drop of blood gets covered with copper
proteinate and remains separate and distinct for
15-20 seconds.
SPECIFIC GRAVITY METHOD
4. If the drop of blood sample

sinks within 15-20 seconds,
the specific gravity of copper
sulfate solution is lower than
the specific gravity of blood
sample and the approximate
value of hemoglobin is more
than 12.5 grams/dl and
hemoglobin level is
acceptable for the donation
of blood.
SPECIFIC GRAVITY METHOD
5. If the drop of blood sample floats, hemoglobin

value is less than 12.5 grams/dl and
unacceptable for blood donation.
6. However, the concentration of plasma proteins

and total leukocyte count also influence the
specific gravity of whole blood which may lead
false-positive result.
5. CYANMETHEMOGLOBIN METHOD
The method of choice for hemoglobin

determination is the cyanmethemoglobin
method. This is a type of colorimetric
method.
Requirements:
1. Prabkins solution
2. Photometer
3. Colorimeter
4. Cuvette
5. Sample
CYANMETHEMOGLOBIN METHOD
Principle:
When blood is mixed with apropriate reagent the
potassiun ferricyanide present in the reagent
tranforms Hb to methemoglobin and
methemoglobin combines with potassiun cyanide
and form hemoglobinocyanide. eg ,
cyanmethemoglobin.
Procedure:
1. Take a test tube and add 5ml drabkinns
solution and add 20 micro litre sample.
2. Mix well and incubate for 5 minutes.
3. Take calorimeter on it . Set to 0.
4. Take reading of test sample.
5. Measure optical density or absorbance
of standard.
Calculation:
Hb (gm/dl) = optical density of test/optical

density of standard × conc. Of standard.
Concentration of standard is fixed ie , 15g/dl.
But can vary accordind to rate kit.
6. OXYHEMOGLOBIN METHOD
 For the estimation of hemoglobin by

oxyhemoglobin method, blood sample is mixed
with a weak ammonia solution and then
absorbance of this solution is deliberated in a
photometer using a yellow-green filter or
measured in a spectrophotometer at 540
nanometer.
 Absorbance of the test sample is corresponded
with that of the standard solution.
OXYHEMOGLOBIN METHOD
• This method is much similar to

cyanmethemoglobin (hemoglobin-
cyanide) method.
OXYHEMOGLOBIN METHOD
Advantages:
Very simple and rapid.
Disadvantages:
This method is not much reliable as
compared to cyanmethemoglobin method
because there is no stable standard solution
is available, derivatives of hemoglobin
except oxyhemoglobin are not measured,
and color of oxyhemoglobin solution swiftly
dims.
7. ALKALINE HEMATIN METHOD
Hb is converted to the

alkaline hematin by
mixing the NaOH and
the colour measured
in a calorimeter at
540 nm.
Procedure is same as
oxyhemoglobin &
cyanmethemoglobin.
ALKALINE HEMATIN METHOD
Advantages:
It gives a true estimate of Hb and is not
affected by the presence of lipids and
plasma proteins.
 Disadvantages:
It is not used routinely as it is less accurate
than the cyanmethemoglobin method.
REFERENCES
Www.ncbi.nlm.nih.gvt
www.slideshare.net
Text Books 📚
Monica part-2
Practical Physiology (Edition, 2012)
VIDEO LINKS
 https://youtu.be/mWAEIvu1mV8 (Sahli’s Acid
Hematin Method)
 https://youtu.be/yfcs1n2P8ak (Tallqvist
Hemoglobin Chart Method)
 https://youtu.be/ZTHCAX3MRlk (WHO Hemoglobin
Colour Scale Method)
 https://youtu.be/XXpo_Wgeba0 (Hemoglobin
Specific Gravity Method)
 https://youtu.be/43kGbTtzcQA
(Cyanmethaemoglobin Method)
 https://youtu.be/fBVJTOjhpNs (Oxyhemoglobin
Method + Alkaline Hematin Method)
THANK
YOU🌹

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ASSIGNMENT HEMATOLOGY

Hemoglobin is made up of four protein

Each hemoglobin molecule is made

Each subunit have one polypeptide

Each of the four subunits contains a heme (contains

Blood can be collected from three

4. If the drop of blood sample

5. If the drop of blood sample floats, hemoglobin

6. However, the concentration of plasma proteins

The method of choice for hemoglobin

Hb (gm/dl) = optical density of test/optical

 For the estimation of hemoglobin by

• This method is much similar to

Hb is converted to the

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