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1. HUMAN ANTIHAEMOPHILIC
VACCINE
SUBMITTED TO:
DR. C. SREEDHAR
PROFESSOR AND HOD
DEPT. OF PHARMACEUTICAL
ANALYSIS
KARNATAKA COLLEGE OF
PHARMACY
BANGALORE
SUBMITTED BY:
S.GOKULRAJ
M PHARM 1ST SEMESTER
DEPT.OF PHARMACEUTICAL
ANALYSIS
KARNATAKA COLLEGE OF
PHARMACY
BANGALORE
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SUBJECT:APA

2. HUMAN ANTIHAEMOPHILIC
VACCINE
Introduction:
 It is also called as human coagulation factor VIII.
 This vaccine is used for treatment of haemophila
A.
 It is rich in clotting factor VIII
 Human Antihaemophilic vaccine which is
obtained from human plasma. It is rich in clotting
factor.
 Antihemophilic factor is a naturally occurring
protein in the blood that helps blood to clot.
 A lack of antihemophilic factor VIII is the cause
of hemophilia A.
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3.  Human antihemophilic factor works by temporarily
raising levels of factor VIII in the blood to aid in clotting.
 Human antihemophilic factor is used to treat or prevent
bleeding episodes in people with hemophilia A. It is also
used to control bleeding related to surgery.
Category
 Antihaemophilic to correct deficiencies of coagulation
factor Vlll
Description
 White or pale yellow powder or friable solid.
Storage
 Stored in atmosphere of nitrogen in light-resistant
containers at a temperature below 8°c. The containers
are sterile and sealed so as to exclude micro-organisms.
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4. SELECTION OF HUMAN
DONORS
 The plasma to be used for preparing Dried Human
Antihaemophilic vaccine
 It is obtained from blood of healthy human donors
who are, as far as can be ascertained after clinical
examination, laboratory tests on their blood and
consideration of their medical history, free from
detectable agents of infection transmissible by
blood transfusion.
 The examinations and tests to be carried out are
decided by the National Regulatory Authority.
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5.  In particular, the blood must be tested with negative
results for
(a) evidence of syphilis infection;
(b) hepatitis B surface antigen and
(c) HIV antibodies by suitably sensitive methods.
 The haemoglobin value of the donor's blood is not less
than 12.5 per cent w/v.
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6. PREPARATION OF HUMAN
ANTIHAEMOPHILIC VACCINE
 The blood is withdrawn aseptically through a closed
system of sterile tubing into a sterile container in
which a suitable anticoagulant solution has been
placed before sterilization.
 During the withdrawal there is no interruption in the
flow from the donor, and the container is gently
agitated.
 Immediately after the withdrawal is completed, the
blood is cooled at 4°c
 If the plasma is to be stored frozen it is separated
from the cellular components by centrifugation and
frozen to -300 or below, preferably within 12 hours
of collection.
 If the plasma is not to be frozen it is separated from
the cellular components by centrifugation as soon
as possible and not later than 18 hours after
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7.  Dried Human Antihaemophilic Fraction may be
prepared :Ii-om human plasma so obtained by
precipitation under controlled conditions of pH, ionic
strength and temperature with organic solvents, or by
freezing and thawing.
 The precipitate may be washed by extraction with
suitable solvents, dissolved in a solution of sodium
citrate adjusted to a pH of 6.8 to 7.2, which may also
contain sodium chloride.
 The solution is sterilized by filtration through a
membrane filter, distributed in sterile containers and
dried from the frozen state.
 The air is removed or replaced by oxygen-free
nitrogen and the containers are sealed so as to
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8. IDENTIFICATION TESTS
 PH
 Loss on drying
 Haemagglutinins, anti-A and anti-B.
 Hepatitis B surface antigen
 Abnormal toxicity
 Pyrogens
 Sterility
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9. BIOLOGICAL ASSAY
Principle
 The potency of antihaemophilic vaccine is determined
by comparing the amount necessary to reduce the
clotting time of a test mixture containing substances
that cause clotting of blood with the amount of the
Standard preparation necessary to produce the same
effect under the conditions of the following method of
assay.
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10. Standard preparation
 The Standard preparation is the 4th International
Standard for Blood coagulation factor
VIII:C,concentrate, human, established in 1989,
consisting of an intermediate purity concentrate of
human blood clotting factor VIII (supplied in ampoules
containing 6.3 Units of clotting factor VIII), or another
suitable preparation the potency of which has been
determined in relation to the International Standard
 The Unit is the specific antihaemophilic factor
contained in such an amount of the Standard
preparation as the Ministry of Health & Family Welfare,
Govt. of India may from time to time indicate as the
quantity exactly equivalent to the Unit accepted for
international use
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11. SPECIAL REAGENTS
 Normal serum reagent
 Phospholipid reagent
 Clotting factor V solution
 Substrate plasma
 Substrate plasma deficient in clotting factor V
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12. Preparation of the test and standard solution.
Add sufficient imidazole buffer pH 7.4 to produce solutions containing
betweens 0.5 and 2 units per ml (stable for 15 mins at 20c).
Three sucessive 2-fold dilutions in the range 1 in 16 to 1 in 256 using a
mixture of 1 volume of a 3.8%w/v solution of sodium citrate and 5 volumes
of saline solution as diluent.
(clotting times between 17 and 35 sec )
Introduce 0.1 ml each of clotting factor V solution, phospholipid reagent
and normal serum reagent into each of six glass incubation
tubes(75mm100mm).
To the first tube add 0.1 ml of the highest dilution of the standard
preparation place the tube in a water-bath at 37c, add 0.1 ml of 0.05M
calcium chloride and start stop-watch.
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13. During the next minute add 0.1ml of the second highest dilution of the standard to a
second tube, place it in the water, and add 0.1 ml of 0.05M calcium chloride at
exactly 1 minute by the stopwatch.
Repeat the procedure with lowest dilution of the standard and highest to lowest
dilutions of the preparation being examined so that the calcium chloride solution is
added at 2, 3, 4 and 5 minutes by the stop-watch ,respectively
Place in a water bath at 37c twelve glass tubes each containing 0.2 ml of 0.025M
calcium chloride and a further tube containing about 3 ml of substrate plasma.
At 14 minutes, 40 seconds by the stop watch, transfer 0.1 ml of the mixture from the
first incubation tube to one of the tubes containing 0.2ml of 0.025M calcium
chloride solution and mix
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14. At 15 minutes add 0.2 ml of the warmed substrate plasma and, using a second stop-
watch, record as the clotting time the time interval between the addition of the substrate
plasma and the first indication of fibrin formation which may be observed visually or by
mechanical means.
Repeat the procedure with the other incubation tubes at 1 minute interval and carry out a
second series of determinations at 21 to 26 minutes.
The period of incubation should, if necessary, be adjusted so that the clotting times
recorded in the corresponding tests in the two series of determinations do not differ by
more than 5%, showing that a stable plateau of prothrombin activator formation has been
reached.
Carry out a blank determination using in place of the preparation being examined.
An equal quantity of a mixture of 1 volume of a 3.8% w/v solution of sodium citrate and
5 volumes of saline solution.
The result of the Assay is not valid unless the clotting time in the blank determination is
more than 40 seconds .Calculate the result of the assay by standard statistical methods.
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15. Side effect
 Tingly feeling in your face, ears, or arms
 Headache
 blurred vision
 Fever
 Drowsiness
 Runny nose
 Skin rash
 Joint pain 2 weeks later
 Nausea
 vomiting
 Upper stomach pain, loss of appetite, dark
urine, clay-colored stools, jaundice (yellowing
of the skin or eyes).
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16. uses
 Used for treatment of haemophilia A.
 It is used for deficiency of clotting factor.
 Antihemophilic factor injection is used to treat
and control serious bleeding problem.
 It is used for surgery time.
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17. THANK YOU
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