EXPERIMENT # 2

Venous blood collection

Larger volume of blood in adults can b obtained from the cubidal vein. The person taking the
blood sample must wear gloves on both hands. When the procedure is terminated the gloves
should be thrown in a container and then should be thrown off .

The subject should be in align or sitting position. A rubber sheet is placed under the subject’s
elbow and a tourniquet is put on the upper arm. The fist is clenched. The tourniquet has
disadvantages i.e. hypoxia in the army may damage the RBCs resulting in hemolysis. Pressure
may alter the concentration of substance i.e.lipids,hormone,protein,in the blood. Yet the
tourniquet make veni-puncture easier. The skin over selected vein is disinfected with the piece of
cotton ball socked in the elbow. The subject elbow is held either with the thumb placed on the
skin over the vein below the veni – punctured site. The thumb fixate the vein a sharp end of the
needle pointed towards the skin. A steroid needle is inserted in the vein at an angle of
approximately 35. The needle is pushed towards 1cm in the lumen of the vein. The tourniquet is
removed as soon as the blood flow is established. The blood is collected in special tubes. The
blood collecting tubes are prepared with required anti-coagulant.

FINALLY, a piece of cotton ball is placed on the site of veni – punctured . The needle is
withdrawn, the cotton is pressed on the skin until the bleeding stops.

ANTI – COAGULANTS:
 EDTA : (Ethylene diamine tetra acidic acid)
 SODIUM CITRATE
 HEPARIN

These are used as anti – coagulant in the laboratory practice. The EDTA forms chelate complex
with calcium. The removal of free ionic Ca+2 inhibits coagulation. Practically , EDTA – K2 is
dried in test tube. EDTA is used in the following test i.e. Cytomorphological examination , cell
count, hemotoxic, haemoglobin measurement.

HEPARIN:
It is an injectable that is also used in vitro – anti – coagulant. It has anti thrombin activity . The
heparin is that it does not alter the size of RBCs ,therefore it can be used in the haemotoxylin
test. Since the heparin does not induce remarkable haemolysis . It is used when testing the
osmotic legibility of RBCs.

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 Tube cap colour Additive Function of additive Laboratory test

Light bulb 3.2% NA nitrate Prevents blood from coagulation

clotting by binding
Ca2+.

Sodium tube with or Clot activator promotes Chemistry

without clot activator blood clotting with immunology
Red\Gold gel. glass or silica particles. serology

Green Na\lithium ,heparin Prevents clotting by Routine

with or without gel inhibiting thrombin and chemistry
thromboplastic

Lavender\pink K+ EDTA Prevents clotting by Haematology

binding calcium and blood bank

Grey Na+ Fluoride , Na+ Fluoride inhibits Glucose or

or K+ oxalate. glycolysis and oxalate blood, alcohol
prevents clotting by and lactic acid
precipating calcium

BLOOD TYPING AND COMPATABILITY TESTS:

Blood group serology techniques are based upon antigen- antibody reactions. Blood group
antigens are present on the membranes of thr RBCs whereas the antibodies are found in the
serum. In vitro(i.e within glass) the antigen-antibody reaction results in agglutination of the
RBCs. This phenomena is called hemagglutination. ABO blood typing; There are four major
ABO groups (A,B,AB,and O).

Blood typing is based on the Landsteiner’s principle; the naturally occurring

antibodies(agglutinins) in the serum of the subject (anti-A,anti-B)never correspond to the
subject’santigens(A,B)

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Before recipient receives transfusion, a compatibility test between donor and recipient blood
must be done. The one sided test determines the antigens on the RBCs. The two – sided test
determines both the RBCs antigens and anti-A and anti –B agglutinin contents of the serum and
therefore it is much more reliable than one sided test. In the one sided test, RBCs with weak
antigenic character may remain unnoticed . For example, a weak type A antigen may not
agglutinate with antibodies in the O or B sera,and the blood is falsely typed as group O . A two
sided test, however, will discover the anti-B agglutinin in the serum.

ONE- SIDED ABO BLOOD TYPING:

Completely clotted venous blood is used. The blood is diluted with isotonic saline(0.9%)the
suspension should contain 10% blood. The test sera containing the agglutinins for the blood
typing are supplied in glass capillaries or in 1ml vials. Remember ,agglutinin against antigen A is
coming from a person having blood group B ,agglutinin against antigen B is coming from a
person having blood group A etc. The tests are performed at room temperature. Different areas
of a white tile are marked “A” , “B” and “S”( S for the serum of the subject= autoserum for
control).

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 ABO BLOOD SYSTEM

One drop of serum O (containing antibody against A and B) is placed in the area O one drop of
test sera A (containing antibody against B ) and B (containing antibody against A) are placed in
the areas “A”and”B”of the tile, respectively. One drop of the 10% suspension of the patient’s
blood (diluted with physiological saline) is addedto each drop of test sera and to the auto serum.
Each serum – blood suspension is mixed by using a glass rod or the corner of the slide. After
each test site, the glass rod must be wiped off carefully. When using a slide change the corner
before mixing the subsequent blood serum suspension. This is important because mixing of
different antibodies carried by the rod or the slide from one place to the other leads to false
positive reaction. After about 0.5 min, the tile is tipped back and forth at 30 degrees. The blood
group is determined only after 5 mints. The reason for the 5 mints waiting period is to decide if
agglutination btw the patient’s RBCs and serum(autoagglutination) develop in the “S”area . In
the case of autoagglutination , the blood typing is likely to give false positive result and
therefore the blood group cannot be determined with the method describe here. The agglutination
is chked by naked eye. Agglutinated RBCs form cluster, i.e, whenever there is a match btw
antigen and antibody , blood mixed with the antibody no longer forms a homogenous drop, but
there will be an immunological reaction, an agglutination . With this method the blood group is
determined as follows;

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Blood type Anti A & Anti B Anti A Anti B Non(on serum)

A Agglutination Agglutination No reaction No reaction

B Agglutination No reaction Agglutination No reaction

AB Agglutination Agglutination No reaction No reaction

O No reaction No reaction No reaction No reaction

Rh BLOOD TYPING :
Using the patient serum(clotted blood) 50% RBCs suspension is produced the test is performed
on a while tile at room temperature by using an anti D test serum(mono clonal antibody).
Different areas of white type are marked as anti D, S. One drop of anti D test serum and one drop
of 50% suspension prepared from clotted blood are prepared from clotted blood are placed next
to each other in anti D test area . The drops are mixed by using a glass rod at the corner of glass
slide . In the S area one drop of 50% suspension of the patient blood is mixed with the
autoserum. The tile is tipped back and forth for 3 min then the drops are examined. Clumps
RBCs indicate the agglutination . The Rh blood remains homogenous. If agglutination is formed
in the S area then the Rh type cannot be determined using the method describe here.

 Cross Agglutination:
The test must be performed before transfusion because the ABO and Rh typing cannot
determine all the possible miss matches btw of the donor and blood recipient. The major
tests checks wether the donor RBCs are agglutinated by the serum of the recipient . The
minor test uses the recipients RBCs and the donor serum. The anti bodies of the donor
blood is greatly diluted in the plasma of the recipient and therefore, the mjor test is

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 performed before transfusion. The test is performed on the bottle filled with warm
water(39 – 42c) 2 test slides are selected on the surface of the bottle , 1 drop of the
recipient serum is placed in each teat area.

The serum is obtained by centrifugation of the blood sample at the 2nd test slide. The
bottle is tipped back and forth and agglutination is examined after 5 mints of incubation.
If agglutaion is noted on any of the test area .This donor’s blood preparation shouldn’t be
transfused. Hemolysis (vanish colour) also indicates incompatability. The same
procedure as a major test except that a suspension is prepared from RBCs of recipient ‘s
blood and plasma of donor RBCs are used. If agglutination occur blood shouldn’t be
transfused.

Biological test before blood transfusion:

This is final test to notice incompatability. A volume of 25ml of blood is infused to an adult
patient at a high speed. Then the infusion is continued at a lower speed and a patient is observed
for 3 – 5 mints(general condition,respiration,circulation).The infusion at high speed followed by
observed is repeated until 75ml of blood is infused. If pathological symptoms are not observed
the test is –ve ,the transfusion can be continued at a normal speed.

Blood group serology with serafol bedside card:

Serafol ABO and serafol ABO+ D are used to confirm patient blood type immediately before a
blood transfusion . The bedside test is used to confirm the recipient’s ABO or D blood group
antigens previously determined and thus ensure that there is a match btw a recipient ‘s blood
group and the conserved product. This identifies any mix- ups that may occur . With the serafol
bedside card the ABO identity can be decided can be checked in a very quick and simple way.
Monoclonal test reagent is applied to the surface of the plastic card in four cicular areas which
are spatially separated from each other and died in other to avoid additional labeling
mistakes.These monoclonal reagents provide strong and rapid agglutination.

Test Procedure:
1. Place one drop of isotonic saline solution on each reaction field and auto control field.
2. Add one drop of blood to each field of the card.
3. Stir each field with the stick for approx. 30 seconds. The reagents must dissolved
completely .
4. Gently rock the card back and forth approx.30 to 60 seconds ,then check each field for
agglutination.
5. Dry the reaction mixture and covered with self adhesive film.

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  Bleeding time:
Most basic test to evaluate thrombocyte function.

 DUKE Method:
A sterile disposable needle are special used for priocking. The fingertip is wiped of with a piece
if filter paper every 30s until the blood no longer stains the paper. Normal value is 1,3 mints.

IVY Method:
A blood pressure is cuff it inflated to 40mmHg on the subject’s upper arm. The surface of the
lower arm is cleaned with the alcohol swap and a super – facial incision(10mmHg , 1mm
depth)is made with the sterile lanced avoiding visible blood vessel.

The blood drops are soaked up every 30s with the filter paper until the blood no longer stains the
paper. Normal value is 3,.5 mints. Prolonged bleeding time indicate a deficiency or decreased no
of thrombocyte impaled blood vessel. The depth of the puncture or incision can be source of
error.

Clotting Time:

 Le – Void Method:
Approximately 5 – 6 ml of venous blood is collected. The blood is immediately distributedin
3 test tubes tat were pre warmed in a 37c water bath . Blood collecting is measured when the
blood doesn’t flow out of test tube when tilted horizontally. The collecting time is calculated
by average the result obtained within the three test tubes . The normal value is 5-5 min.
Blood clotting time longer than 10 mint is pathological. This test measures the endogenous
way of blood coagulation. The sensitivity is low.

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