Practical No : 02
Hb, ESR, PCV ASSESSMENT AND OSMOTIC FRAGILITY TEST
Haemoglobin (Hb) Measurement
Objectives :The student should be able to,
1. Define normal Hb levels
2. List the conditions that
a. Decrease Hb levels
b. Increase Hb levels
3. Identify the instruments that are used to measure Hb levels
4. Describe the steps involved in performing the test
5. Read and interpret the results
6. Describe the advantages and disadvantages of the test
Can be done using the Sahli's method or the Talliquest method, which both employ the method of
estimating haemoglobin by measurement of its colour.
The Talliquest method is not used routinely as errors may occur.
Sahli's Method
Principle :
The blood is diluted in an acid solution, converting Hb to acid haematin. The test
solution is matched against a coloured glass reference.
Instruments : Surgical Spirit, cotton wool & Sterile Lancet
Micropippette (Sahli pipette) graduated upto 20 mm3 (20l)
Calibrated tube with glass rod and sucker
Sahli Haemoglobinometer
0.1 N (0.1 mol/l) HCl acid
Calibrated
Distilled water and Absorbant paper
tube with 0.1
mol/l HCl
Method : Fill the calibrated tube to the '20' mark with 0.1mol/l
HCl
- Clean the fingertip with surgical spirit and prick
with a sterile lancet
- Using the micropipette, suck blood upto the 20 l
mark. Do not allow air bubbles to enter.
- Wipe the outside of the pipette with absorbant paper
- Blow the blood from the pipette into the calibrated
tube containing the acid solution.
- Rinse the pipette by drawing in and blowing out the
acid solution 3 times.
- The mixture of blood and acid will now give a
brownish colour
- Allow to stand for five minutes
Blood
converted to
acid haematin
- Now place the calibrated tube in the comparator
and stand facing a window
- Compare the colour of the calibrated tube with
the reference tubes
If lighter, the Hb value is 4g/l or less
If darker, continue to dilute the blood acid
mixture with distilled water drop by drop, all
the while stirring with the glass rod.
- Stop when the colours match
- Now, note the mark that is reached.
Results :
Depending on the type of tube, the result can
be read as g/dl or g%
Interpretation :
The normal Haemoglobin values for males and females are as follows,
Male
14 - 15 g/dl
Female
13 - 14 g/dl
A value less than the above will indicate Anaemia and a value much higher than the above
will indicate polycythaemia.
Further tests have to be performed, however, to identify the nature / cause of the anaemia.
Advantages and Disadvantages :
Advantages
A very simple method
Easy to perform, with minimum technical
expertise
Can transport apparatus easily
Disadvantages
Colour comparison may vary from
observer to observer.
Obstruction of micropipette by air
bubbles
Addition of extra distilled water to tube
Due to tight sucking, ECF may enter the
micropipette
Incomplete transformation of haem to
acid haematin
Packed Cell Volume (PCV) - (Haematocrit / Erythrocyte volume fraction)
Objectives :The student should be able to,
1. Describe what is Packed Cell Volume (PCV)
2. Define normal levels
3. List the conditions that
a. Increase the PCV and b. Decrease the PCV
4. Identify the instruments used to measure PCV
5. Describe the steps involved in performing the procedures
6. Read and Interpret the results
Packed cell volume estimation can be done by two methods
1. Wintrobe method (Macro)
2. Microhaematocrit method (Micro)
Wintrobe Method
Instruments : Ordinary electric centrifuge
Special graduated tubes (Wintrobe tube)
(Bore - 0.6 mm / Length - 9.5 cm /
Calibrations - 0 - 100)
Long fine capillary Pasteur pipette with rubber teat
Sterile syringe and needle, cotton wool and surgical
spirit
Anticoagulant (EDTA)
Method :
-
Result :
Draw venous blood, add it to a tube of
anticoagulant (EDTA) and mix well
Using the capillary pipette, fill the wintrobe
tube with blood upto the '100' mark.
Make sure that there are no air bubbles.
Centrifuge for 30 minutes at 3600 rpm
Read the level at which the red cells meet
the level of leukocytes
The graduation should be read from down upwards
(towards the 100 mark)
Microhaematocrit method
Instruments : Microhaematocrit centrifuge (special, high speed)
Capillary tubes containing a dried deposit of Heparin (anticoagulant)
(length - 75 mm / bore - 1.5 mm)
Plastic modeling clay
Sterile blood lancet, surgical spirit and cotton wool.
Method :
Clean fingertip with surgical spirit and prick with a sterile lancet.
- Wipe away the first drop with filter paper
- Apply the tip of the heparinized capillary tube to
the drop of blood.
- The blood will flow into the tube by capillary
action
- Fill about 3/4ths of the tube.
- Plug the other end of the tube with plastic
modeling clay
- Place the capillary tubes in the centrifuge slots.
(the sealed end of the tube should point
outwards)
- Centrifuge at 15000 rpm for 3 minutes.
- The blood will have separated into
layers.(Plasma / white cell coat / red cell layer)
Results :
The reading is made at the junction between red cells
and white cells using the haematocrit meter (reader).
100
50
0
Interpretation :
The normal values for PCV are,
Males
Females
-
40 - 54
37 - 47
PCV is increased in
-
Polycythaemia
Spherocytosis
Severe dehydration
PCV is decreased in
-
Anaemia
Erythrocyte Sedimentation Rate (ESR)
Objectives :The student should be able to,
1. Describe what is Erythrocyte Sedimentation Rate (ESR)
2. Describe the physiological basis of ESR
3. Define normal ranges
4. List the conditions that
a. Increase the ESR
b. Decrease the ESR
5. Identify the instruments that are used to measure ESR
6. Read and interpret the results.
Erythrocyte Sedimentation Rate (ESR) measures the rate at which red blood cells in anticoagulated
blood settles to the bottom of a calibrated tube.
Instruments : Westegren ESR tube
(internal diameter - 2.5 mm / graduated from 0 -200 mm)
Westegren stand
Anticoagulant : 38g/l (3.8%) trisodium citrate solution
5 ml graduated syringe
Timer
Small tube or bottle
Method :
- Place 0.4 ml of 3.8% tri sodium citrate in a
small tube or bottle
- Collect venous blood applying the tourniquet
as loosely as possible
- Collect 2 ml of blood.
- Remove the needle from the syringe and add
1.6 ml blood to the bottle containing
anticoagulant
- Mix well by shaking the bottle gently.
Tri Sodium
Citrate
Anticoagulated
blood
- Measurement of ESR should begin within 2
hours of collecting blood !
- Draw the citrated blood into the westegren tube
using a rubber bulb upto the '0' mark
3-way pipette
filler
Westegren tube
- Place the tube in the Westegren stand making
sure that the tube is completely upright.
- Check that there are no air bubbles in the tube
- Check that the stand is level and is away from
the windows
- Start the timer after setting it to one hour.
ESR
1 hr
Results :
Are read one hour later
Note the height of the column of plasma in
mm graduations starting from the '0' mark at
the top
The results are expressed as
ESR - ………….mm/ 1st hour
Interpretation :
The normal values for ESR are,
Males
1 - 10 mm/ 1st hour
Females
3 - 14 mm/ 1st hour
ESR depends on
The ratio of specific gravity of RBC : Plasma
F
F
g
Rouleaux formation
ESR is also known as a test of the Acute Phase Response.
Here, a deviation from the normal values will be seen when there are increased amounts of
acute phase proteins (Caeruloplasmin, Haptoglobin) in the blood.
ESR increased in,
Chronic inflammations & infections
Eg. TB
Acute inflammations & infections
Normal Pregnancy (Physiological)
Malignancies
Severe anaemias
Q:
ESR decreased in
Polycythaemia
Sickle cell disease
Cryoglobinaemia
Why do females have a slightly higher ESR than males ?
Osmotic Fragility Test
Objectives :The student should be able to,
1. Describe what is 'Osmotic Fragility Test'
2. Define the normal osmotic fragility range
3. Describe how to perform the test
4. Describe the conditions that
a. Increase osmotic fragility
b. Decrease osmotic fragility
5. Read and interpret the results
This measures the fragility of red blood cells when put into solutions of hypotonic saline of varied
tonicity.
Principle :
When red cells are placed in buffered saline solutions of varying tonicity, there is a
variation in the net amount of fluid entering or leaving the cells.
In an Isotonic solution
(0.9% saline)
No net fluid shift
In a Hypotonic solution
Net fluid shift into the
red cell
In a Hypertonic solution
Net fluid shift out of
the red cell
No change in the RBC
The red cell shrinks
The red cell swells
volume
Thus, at very low tonicity, the red cell will absorb water to such an extent that it will
lyse (haemolysis).
The osmotic fragility of freshly taken red cells reflects their ability to take up a
certain amount of water before lysing. This is determined by their volume to surface
area ratio.
Instruments / materials :
Test tube stand
10 conical test tubes
NaCl solutions of varied concentration ranging from 0.9% saline to 0.1%
Sample of anticoagulated venous blood (using heparin as EDTA contains
osmotically active particles)
Pipette filler
Method :
Obtain a sample of venous blood and mix it with anticoagulant.
- Take 10 conical test tubes and place in the stand
- Put 5ml each of the solutions of varied tonicity into these tubes and label them
- Using a pipette filler put 1 drop of blood each into the tubes.
- Mix the blood and saline in each tube
- Allow the tubes to stand for one hour or centrifuge them
- Now, observe for changes.
Results :
The following changes will be seen in the test tubes
Slight reddish
solution with red
button
Clear solution
with red button
0.90
0.75
0.65
0.60
0.55
0.50
0.40
Reddish solution
no red button
0.35
0.30
0.20
% solutions
- Observe the concentration of the saline where red colour first appears in the solution
- Observe the concentration of the saline where the red button completely disappears from
the bottom of the tube.
- This will be the normal osmotic fragility range.
- Plot these results on a graph.
Interpretation:
The graph shows the normal range
of osmotic fragility. (
)
This ranges from 0.45 - 0.55 with
initial lysis at 0.55 and completion of
lysis at 0.45
Osmotic fragility is
Increased in, (curve shifted to right)
Hereditary Spherocytosis
Autoimmune Haemolytic anaemia
100
80
60
40
20
0
0.2
0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.75 0.9
Decreased in, (curve shifted to left)
Iron deficiency anaemia
 - Thalassaemia minor
Sickle cell anaemia
Q. : Explain why the osmotic fragilty differs from the normal range in the above
conditions.