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Practical On Blood 1

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7 views

Practical On Blood 1

Uploaded by

Charles Ozochukwu
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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BLOOD I

ESTIMATION OF HAEMOGLOBIN AND PACKED CELL VOLUME


INTRODUCTION
q Estimates of haemoglobin concentration of whole blood and the
packed cell volume are most commonly used to determine the
presence or absence of anaemia.

q The Haemoglobin and PCV provide adequate information for


routine practice.
AIMS OF THE EXPERIMENT

1. To Estimate the Haemoglobin content of the blood by:


a. The Colorimetric method
b. The Sahli’s method
2. Determination of the Packed Cell Volume
3. Calculation of the Mean Corpuscular Haemoglobin Concentration
(MCHC)
PRINCIPLE

• When blood is mixed with a solution containing potassium


ferricyanide and potasium cyanide, the potassium ferricyanide
oxidizes iron to form methaemoglobin.
APPARATUS
• Colorimeter
• Test tubes
• Automated micropipette
• Drabkin’s solution
• Haemoglobin paper
COLORIMETER
OPTICAL FILTER
• TEST TUBES
• AUTOMATED MICROPIPETTE
DRABKIN’S SOLUTION
• HAEMOGLOBIN PAPER
PROCEDURE:
Diluent – Drabkin’s solution
Constituents:
1. Potassium ferricyanide – 200mg. This oxidises Haemoglobin to
methaemoglobin.
2. Potassium cyanide – 50 mg. Methaemoglobin reacts with potassium
cyanide to form cyanmethaemoglobin.
3. Potassium dihydrSogen phosphate – 140mg.
4. Distilled water – make up to 1000ml
5. PH 7.0 – 7.4 (1 Litre)
PROCEDURE CONTD.
1. Pipette 5mls of the diluent (Drabkin’s solution) into the test tube.
2. Add 20µl (0.02mls) of blood.
3. Seal the tube using the thumb and mix by inversion.
4. Stand the diluted test solution at room temperature for 10 minutes.
5. Use the blank drabkin’s solution to zero the the colorimeter.
6. Read the optical density of the solution in a photoelectric
colorimeter.
7. From the calibration graph provided, read off the haemoglobin
concentration directly in g/dl.
SAHLI’S METHOD

APPARATUS
• Complete Sahli’s apparatus – calibrated tube, 0.1N HCl, automated
micro pipette, dropper.
• SAHLI’S APPARATUS

Standard Glass Comparator


• SAHLI’S APPARATUS CONTD

2. 0.1N HCL
3. PASTEUR PIPETTE
• AUTOMATED MICROPIPETTE
METHOD
1. Fill the graduated tube to the mark 20 with freshly prepared 0.1N HCl.
2. Pipette 0.02mls ( 20µl) of blood sample provided.
3. Mix by swirling.
4. Allow to stand for 5 minutes.
5. The dark brown acid haematin is formed.
6. Using your pasteur pipette, add distilled water drop by drop. Stir with the
glass rod after each addition.
7. Continue until you get the colour in the standard comparation.
Note: Average haemoglobin for male is 14 g/dl while that of female is
12.5 g/dl.
PACKED CELL VOLUME

APPARATUS

• Heparinised capillary tube


• Microhaematocrit centrifuge
• Plasticine
• Microhaematocrit reader
HEPARINISED CAPILLARY TUBE
SLIDE 12 MICRO HAEMATOCRIT CENTRIFUGE
MICRO HAEMATOCRIT READER
• Plasticine
PRINCIPLE

• This involves centrifuging a sample of blood so that


the blood cells will be thrown to the bottom of the
tube while the plasma remains on top.
PROCEDURE
• A capillary tube of 7.5cm long and 1mm internal diameter containing
an anticoagulant (heparin) is provided.
• Blood (well mixed venous blood or directly from a capillary puncture)
is allowed to enter the tube by capillary action (2/3 of the tube).
• Seal one end of the capillary tube with plasticine or sealant.
• Place the tube in a groove of micro-haematocrit centrifuge and spun
at 12000 rev / min for 5 minutes.
• Read the packed cell volume as a percentage using Hawksley
Haemocrit reader.
• Normal Range : Men : 40 to 52 %

• Women : 37 to 47 %
CALCULATION OF MEAN CORPUSCULAR
HAEMOGLOBIN CONCENTRATION(MCHC)

• MCHC = Hb in g/dl/pcv % x 100


CLINICAL SIGNIFICANCES
Haemoglobin is Decreased:
ØAnaemia
ØSickle cell disease
ØPregnancy.
ØDrugs like chloramphenical and penicillin
ØRenal disease
ØCirrhosis
ØHaemorrhage
ØMalignancies eg.leukemia
ØHemolysis
ØMalnutution
• Increased in:
• Bone marrow disease that causes abnormal increase in red blood
(Polycythemia vera)
• People from high altitude .
• In children.
• Low oxygen level in blood.
• Too little water in the blood (dehydration)

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