PRACTICAL-1, 2, 3

Blood Groups, Clotting Time, and Bleeding Time

Very important
Extra
information
Terms Physiology Practical
 Determine your
own Bleeding
and clotting
time, discuss
the normal
ranges

Understand and
Recognize the
practice the method
used in determining importance of
blood groups (ABO OBJECTIVES bleeding time
and (Rh) systems) and clotting
and explain their time in
importance in blood haemostasis.
transfusion.

Aims of the Practical :

To determine:
1. Blood groups.
2. Clotting time.
3. Bleeding time.
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 Blood Groups
ABO System
Group A: Antigen A on RBC membrane ,Anti B in
plasma.
Group B: Antigen B on RBC membrane, Anti A in
plasma.
GroupAB: Antigen A and B on RBC
membrane, no Antibodies in
plasma.
Group O: No Antigen on RBC, but there are
Antibodies in
plasma.
Rhesus Blood Group
Rh+ve(positi Antigen D on RBC (96-98%).
ve)
Rh- No antigen D on RBC (2-4%).
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 ve(negative)

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 General types of RBC

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 Lab Working materials

To identify the blood type in lab we have to bring

the
following materials:

1.Anti A (blue titer).

2.Anti B (yellow
titer).
3.Anti D (to identify the rhesus
antigen) 4.Microscobic slides and a
microscope 5.Alcohol swab.
6. Grease pencil.
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7. Tooth picks
8. Lancet or a pricker.

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 Blood group Procedure

1. Prick a finger using a lancet after sterilizing it with

alcohol swabs
2. place one drop of blood in each of the
compartments A, B and D
(these are clearly labeled on the microscope slides
provided).

3. Quickly add a drop of anti-A, anti-B and anti-D sera

to
compartments A, B and D respectively.

4. Mix the serum with the drop of blood by

moving the slides gently for a minute or two, or with
the help of different pieces of tooth picks

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 5.Examine the mixtures for signs of RBC
agglutination or clump formation. If there is a doubt,
examine the slides using the low power of a
microscope.

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 Blood samples on the
slides
Important visible
features
The same If there is a coagulation and reaction
type
between the anti titer (antibody =
agglutinins) and the blood sample
(antigens = agglutinogens).
Another If there is no coagulation and reaction.
type

Note: more than 30 blood group systems have been identified other than

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 Blood groups

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 Important Clinical Applications of blo

Hemolytic
Blood disease of Bloo
transfusi the d
on newborn produc
(HDN) ts

Hemolytic disease of the newborn (HDN)

*It is a blood disorder in a fetus or newborn infant. HDN may
develop when a mother and her unborn baby have different
blood types (called "incompatibility").
*The mother produces substances called antibodies that attack the
developing baby's red blood cells
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 HDN

Clinical conditions

The
The most least
common commo
n
Rh
ABO incompatibility,
incompatibility,
usually not severe. can cause very
severe anemia in
the baby

ther is given an injection of RhoGAM during the second trimester. If the baby is Rh- positive, the mother will ge

Prevention method Treating method

RhoGAM
injections
(anti-D
Infants with mild Rh Infants with severe
incompatibility Rh incompatibility
with exchange
Drugs used to treat allergic reactions (antihistamines) transfusion after birth
Drugs used to treat swelling and allergies (steroids) or intrauterine
Feeding and fluids (hydration)
Fluids given through a vein (intravenously)
14 Light therapy using bilirubin lights
MedicinesContact
to raise blood pressure if it drops too low
 Rh incompatibility

Rh incompatibility is a condition that develops when a pregnant woman

has Rh- negative blood and the baby in her womb has Rh-positive blood
inherited from the Rh-positive father.
During pregnancy, red blood cells from the unborn baby can cross into the
mother's bloodstream through the placenta.
Because the mother is Rh-negative, her immune system treats Rh-positive
fetal cells as if they were a foreign substance and makes antibodies against
the fetal blood cells. These anti-Rh antibodies may cross back through the
placenta into the developing baby and destroy the baby's circulating red
blood cells.
When red blood cells are broken down, they make bilirubin.This causes an
infant to
become jaundiced.
Because it takes time for the mother to develop antibodies, firstborn
infants are often not affected unless the mother had past miscarriages or
abortions that sensitized her immune system. However, all children she
has afterwards who are also Rh-positive may be affected.
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 Clotting Time
The Normal time forSubstances
clotting: used
Importance The as
source of heparin in the
anticoagulant:
Definition: of
identifying
the clotting
he time required the blood to form a clot. time:
1. Heparin. 1.Mast cells.

1. Almost 3-10 min. For surgery.

2.Warfarin. 2.Basophils.
2.For
Diagnosis
of
bleeding 3.Calcium
*The whole blood clotting time disorders. 3.Liver.
Oxalate.
is a rough measure of all
intrinsic clotting factors in the
absence of tissue factors. 4.Sodium
4.Lungs.
Citrate.
*Used in diagnosis of hemophilia, when the clotting time is higher than normal
5.EDTA
(Ethylene Diamine Tetra- butyric Acid

*Its chief application is in monitoring anti-

coagulant therapy.
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 Materials
petri- Alcohol
dish. swabs

Cotton
wool
Plastici
ne

water bath
37°C Capillary
tubes of
uniform size
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 Clotting time Procedure

1. Clean finger with alcohol swap, prick it with lancet

and note the time that the prick is made.

2. Wipe away the first drop of blood.Then while the

blood is still
flowing freely place one end of a capillary tube in
the blood. Holding the tube horizontally let it fill by
capillary action.

3. Close the end of the capillary tube with

plasticine. Place the tube in the water bath. Repeat
all the above steps with many capillary tubes.

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 Clotting time Procedure cont.

4. Two minutes after making the puncture, break a capillary

tube and separate the two halves slowly, and look for a
thread like clot between the two broken halves of the tube.

5.Repeat the procedure at 30 second intervals with the

remaining
tubes.

6.When the blood forms a continuous thread-like clot

between the broken ends of the tube, the end-point has
been reached, note the time.

7. The time from pricking the finger to the appearance of the clot
is the
clotting time

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 Results

Usually the clotting time

measured by this
method is in the range
5-15 minutes.

Prolong clotting time

seen in deficiencies in
the intrinsic coagulation
pathway.

Clinical condition: the clot

Hemophilia: a medical blo is
condition in which the ability of od sev
to erel
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 y reduced by the lack of
coagulation factor.
*Deficiency of factor 8 *Deficiency of factor 9 (IX )
(VIII) leads to disease or Christmas factor leads
known as Hemophilia A. to disease known as
Hemophilia B.

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 Clotting Time using Test Tube Metho

Place 2 ml blood into

non heparinized test
tube incubated in
water bath.
Every 30 second
invert gentle to
check for clot
formation.
Time from pricking
finger to clot
formation is clotting
time.
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 Bleeding time
*Definition:The time taken for bleeding to stop (time for a platelet plug to
form).

*Bleeding time is a test of platelet function.

* Methods of Bleeding Time, IVY’s Method, Duke’s Method, Template
Method

*The normal range of bleeding time is 2 – 7 minutes

Materials
A
Filter stylette/lenc
paper et to prick an
ear lobe

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 Alcohol stop-
swabs watch.

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 1. Bleeding time Procedure by Duke

1. Clean the lobe of the ear/finger tip with an alcohol swab.

2. When it is dry, make a single puncture with a lancet (about

3mm deep).
3. Note the time at which the puncture is made.
4. The skin of the ear/ finger tip should not be touched once the
puncture has been made until the experiment is over.

5. Apply a piece of filter paper to the blood-drop every 30

seconds until the bleeding stops.
6. The bleeding time estimated by this method of a normal range
subject is: 2-7 minutes.

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 2. Template Method

A sphygmomanometer cuff is applied to the subject’s arm and inflated to 40mmHg.

The volar surface (palm) is cleaned with 70% alcohol.

A sterile metal template with a linear slit (11mm long) is pressed firmly against the skin.

A scalpel blade, with a guard, is carefully introduced so that it protrudes 1mm through the
template slit. An incision, 1mm deep and 9mm long can then be made.

Blood is gently, but completely removed with filter paper at 15 second intervals until
the bleeding stops.

Normal bleeding times determined with this method are in the range 3-10minutes.

If the bleeding time exceeds 15 minutes:

Stop the procedure. Apply pressure to stop the bleeding. Report as greater than 15 min.

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 3.
IVY’s Method

A sphygmomanometer cuff is applied to the subject’s arm and inflated to

40mmHg.

The volar surface (palm) is cleaned with 70% alcohol.

3 Punctures are made with lancet. Wound Should be 3mm in depth and 2-3mm in
length.
Stopwatch started as soon as puncture is made. one stopwatch for each puncture.

Blood oozing from each puncture is gently blotted with filter paper at
30s interval. Continue till blood no longer stains filter paper.

Normal bleeding times determined with this method are in the range 2-9
minutes.

If the bleeding time exceeds 15 minutes:

Stop the procedure. Apply pressure to stop the bleeding. Report as
greater than 15 min.

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 Clinical Application

Bleeding • Platelet dysfunction.

time is • Blood vessel wall disorders.
prolonge • Haemophilia.
• Von Willebrand Disease.
d in the • Thrombocytopenia.
followin • Vitamin K deficiency.
g • Medications:Aspirin
conditions
:
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