BLOOD EXPERIMENTS

BLOOD SAMPLING

INTRODUCTION: Most investigations on blood are done on venous blood, which is most easily
collected in moderated quantities. If only a very small volume is required for a test (0.1 - 0.2ml),
then a capillary sample obtained by stabbing the pulp of a finger with a lancet will suffice. Arterial
samples are more difficult to obtain so they are generally only used if there is a specific
indication. E.g. determination of arterial blood gasses.
1. CAPILLARY BLOOD: The commonly chosen sites for puncture of the skin are the ball of
a finger or a fleshy dependant part of an ear lobe in adults and the heel of an infant. The
skin of the selected site is cleaned with a spirit swab. When the cleansing fluid has
evaporated completely and the skin is dry - the skin is punctured to a depth of about 3mm,
using a sterile blood lancet. If the surface is wet, the blood will spread over it and will not
form a compact drop. A free flow of blood is essential and only the very gentlest squeezing
is permissible, ideally, large drops of blood should exude slowly but spontaneously after
pricking the site. If the finger is squeezed firmly in order to obtain blood, the results are
unreliable. If the poor flow is due to the part being cold and cyanosed, too high figures for
red cell count, Hb content and leucocyte count are usually obtained.

One method of ensuring free blood flow through the puncture site is to make a little ridge along the
long axis of the ball of the finger by gentle pressure on its sides, make the puncture on this ridge
and then release the pressure. If the blood is still slow a flowing, the thumbs may be used gently
to open the edges of the wound.
2. VENOUS BLOOD: Some of the experiments which you will do in the lab demand the use
of large volumes of blood which cannot readily be obtained by pricking. It is therefore
important to understand the method of withdrawing blood from the vein.
(i) Apply a tourniquet (which could be a blood pressure cuff or a rubber tube) to the
upper arm just tightly enough to block venous return from the forearm.
(ii) Choose a clean, sterile, sharp needle of size 22. Fix it to a 10ml. Syringe.
(iii) The subject should be seated comfortably on a chair or lying down in bed
with his forearm fully extended. Locate and palpate the veins that stand out after
the application of a tourniquet. A vein supported on either side by subcutaneous
fat is most suitable for venepuncture.
(iv) Clean the spot with soap and water or with a spirit swab. The vein is
steadied by
stretching the skin over it. With the plunger pushed into the barrel as far as it will
go, hold the syringe firmly and push the needle through the skin with the bevel of
the needle facing upwards. Rotate the needle slightly so that the vein is entered
from the side. As soon s the needle enters the veins, blood will enter the syringe.
(v) Collect the desired volume of blood. Remove the tourniquet and after
that remove the needle from the vein.
(vi) You will observe slight bleeding from the site of the puncture after the
needle is taken out. The best method of minimising this is to press a cotton swab
firmly on the site of the puncture and keep the arm elevated above the level of the
heart for about three minutes.

3. DANGERS OF VENEPUNCTURE: This is an inoccuous procedure when done properly

but there are various or potential dangers to bear in mind.

i. Infection:
a. From Needle to the subject. The use of sterile, prepacked equipment has
more or less eliminated this danger.
b. From Subject to subject. Obviously, you must never use the same needle
on more than one subject. Otherwise, any disease in which the infective
agent is present in the blood can be transferred e.g. hepatitis, malaria, or
syphilis.

c. From Blood-Contaminated Equipment to other people

 Classically the cleaner picks up the rubbish and contaminated needle
sticks into his hand. More recently it has been appreciated that the
hepatitis virus is released into the air from blood-contaminated products.
Therefore both needles and all contaminated products must be disposed
of with special care.

ii. Damage to other organs: The ante-cubital fossa is used partly because there are
few substantial nerves in the region.

iii. Air Embolism: (Only A Theoretical Danger in this context) If large amounts of air
are injected into the vein it will eventually collect in the pulmonary artery and
impede circulation. Amounts in excess of 50 - 100mls may be fatal.

iv. Subjects Faint: A small proportion of subjects (perhaps 1/20) fell faint when blood
is taken. This is not dangerous in itself but if standing and unsupported they may
hurt themselves on falling. Always have the subject seated. If the subject feels
faint, stop taking blood, stay with them and if more than transient, lie the subject
flat on the floor.

v. Incorrectly of inadequately labelled samples: In current hospital practice,

this is a serious danger. More transfusion reactions are caused by incorrect
labelling or identification than any other cause. The best practice is to label the
sample tube with the subject's full name before putting blood into it. Always check
and double-check where samples of blood for transfusion are involved.

ANTICOAGULANTS

For many investigations, unclotted blood is required. Without anticoagulant, blood clots within
minutes in a bottle (or in the syringe if left too long).
Ca++ ions are required for the clotting reaction to proceed. Any substance which combines with
Ca++ ions so that none remain free in the blood will prevent clotting. Sequestrene is the trade
name for a calcium chelator (whose chemical name is abbreviated to EDTA) which is widely used
for routine work. Oxalate can be used for certain special techniques and citrate is used for
anticoagulating blood for transfusions.

HEPARIN

Heparin is a mucopolysaccharide found in mast cells and prepared commercially from bovine
lungs. By definition 1 unit of heparin (about 0.1mg of a pure substance) is required to prevent the
coagulation of 1ml of blood. Generally, 5-10 units/ml are used to make sure.

It is a relatively strong organic acid and its anticoagulant action is thought to result from interference
with the enzymes, due to the powerful electronegative charges carried by the molecule.

Functionally, it appears to act as an antithrombin in the presence of its plasma cofactor

(antithrombin II).

ESTIMATION OF PACKED CELL VOLUME (PCV) OR HAEMATOCRIT

The estimation of the packed cell volume is often a valuable guide in diagnosing certain blood
disorders. It is expressed as the percentage volume of the blood cells. Two methods are
commonly available and both involve centrifuging a sample of blood so that the red cells become
packed at the bottom of the sample with the plasma on top which is normally of straw colour. In
the Wintrobe method, more blood is required; so it can only be performed on a venous sample of
blood. It requires a relatively long centrifugation time. Far more convenient is the capillary tube
method which requires a small blood sample and only minutes for centrifugation. This results in
the elimination of all trapped plasma.

The Technique is as follows:

1. Take a capillary tube which has been already heparinized to prevent the blood from
clotting.
2. Fill up the capillary tube from a freely flowing capillary puncture about three-quarters.
3. Seal off the end of the capillary tube in a bunsen flame or preferably use the
special plasticised sealer which requires no heat and always gives flat bottom.
4. Place the capillary tube in the microhaematocrit centrifuge and screw on the
safety cover.
5. Close the lid and spin at 10,000 rpm for 5 minutes.
6. Place the spun tube into the specially designed scale and read off the PCV as a
percentage.

QUESTIONS

1. What cells form the uppermost layer of the packed cell columns?

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2. In what disease is the haematocrit found to decrease significantly?

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3. List 2 conditions which lead to a significant increase in hematocrit.

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CLOTTING/COAGULATION TIME

- Withdraw from a vein using a disposable sterile 5ml syringe and needle
- Clean four small plain test tubes and rinse them out with a sterile saline solution.
- With all aseptic precautions withdraw blood from a vein and place 1 ml in each of the
four tubes
- Start a stop-watch the instant blood begins to flow into the syringe
- Two of the tubes are tilted to 45o at half-minute intervals. When coagulation has
occurred the tubes can
- Be inverted without spilling their content
- The remaining tubes remain undisturbed and serve as a check on the endpoint
observed in the other tubes
- The time interval between the moment of withdrawal of blood into the syringe and the
moment when the tube may be inverted without spilling the content is recorded and
taken as the clotting time

BLEEDING TIME

Duke’s Method
- Wipe the lobe of the subject ear gently with alcohol and allow it to dry.
- Use a disposable lancet and make a single puncture 4mm deep into the lobe of the
ear.
Note the time
- Thirty seconds later remove the drop of blood by applying a clean piece of filter
paper.
 - Repeat the procedure every 15 seconds using a fresh area of the paper on each
occasion until the bleeding ceases (Note the time bleeding ceases) count the spots of
blood.
- Collect results from the various groups to get an impression of the normal scatter.

1a. Average bleeding time:

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b. Average Clotting time:

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2. What does prolonged bleeding time signify?
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3. What aspect of hemostasis does clotting time assess?

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DETERMINATION OF BLOOD GROUPS

Blood groups refer to the presence on or in the red cell membrane of certain antigens, the so-
called blood group factors. There are a large number of different blood group antigens. They have
inherited characteristics and most of them have been shown to belong to genetically independent
blood group systems. The two systems which are of great importance in transfusion or blood are
the ABC and Rhesus systems. In the lab, you are going to determine which blood group of the
ABC system you belong to. If the antiserum is available your Rh typing can also be done.

BLOOD TYPING PROCEDURE:

(a) ABO - Place a drop of the antisera provided on a white porcelain tile and clearly mark an
A and B. Obtain a 1.20 dilution of your blood using a white cell pipette. Add a drop of the
diluted blood to each of the antisera. Mix them and at the end of 10 minutes note whether
agglutination of the red cells has taken place.

(b) Rh - Obtain a 1:20 dilution (with 0.9% saline) as in the ABC system. Place 1 drop of this
red cell suspension in a labelled tube and add 1 drop of anti-Rh (or more precisely anti-D)
serum. Incubate in the water bath for one hour at 37oC. After incubation for one hour,
examine tubes for agglutination as in ABO grouping and check microscopically.
(Agglutination is not nearly as complete in this test as in the ABO test. If cells are
agglutinated even in small groups of 3 or 4 cells, this is regarded as positive). The
 presence of agglutination indicates that your red cells contain the D antigen, i.e. your blood
group is Rh +Be.

The blood group can then be determined as follows:-

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Reaction of subjects cells to :

Anti sera A Anti sera B Blood Group

Agglutination Agglutination AB
Agglutination - A
- Agglutination B
- -
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If cells do not agglutinate with anti-Rho (anti-D) typing sera, the subject is Rh negative.

1. What is your blood group?

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2. List four(4) complication of Blood Transfusion

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