SOP Blood Banking

1. Titration of Anti –D
Procedure‐
1. Label 10 test tubes as follows‐
Tube no 1 2 3 4 5 6 7 8 9 10
Dilution 1 2 4 8 16 32 64 128 256 512
2. Deliver 0.1ml of saline in each tube (except the first tube) with the help of a graduated
serological pipette.
Alternative Drop Method: use two identical droppers for dispensing saline and serum.
Count drops for dilution instead of measuring volume.
3. Add 0.1ml of test serum in tubes 1 and 2.
4. Mix the contents of tube 2 by blowing through the serological pipette and transfer 0.1
ml of diluted serum from tube 2 to tube 3.
Note: Return the residual fluid back to the tube in order to maintain constant volume in
all tubes.
5. Mix the contents of tube 3 by blowing and retransfer 0.1ml of diluted serum of tube 3 to
tube 4 and continue the process until the 10th tube is reached. Mix the contents of the
10th tube (512 dilution) and set aside 0.1ml of the diluted serum which may be needed
for further dilution.
6. Add 1 drop of 22% albumin in all tubes and mix.
7. Add 1 drop of 2‐5% saline suspension of Rh–positive red cells in all the test tubes (1 to
10) and mix.
8. Incubate the tubes for 30 munities at 37˚С
9. Examine for haemagglutination and record the result. The reciprocal of the highest
dilution that shows agglutination is the titer.
10. If the tube No 10(1:512) shows agglutination, use the diluted serum (step 5) of the 10th
tube for further dilution and repeat the procedure.
Tube no 01 02 03 04 05 06 07 08 09 10 Titer
Dilution 1 2 4 8 16 32 64 128 256 512
Example 1 4+ 4+ 4+ 3+ 3+ 2+ 1+ 0 0 0 64
Example 2 4+ 4+ 4+ 4+ 3+ 3+ 2+ 2+ 1+ 0 256
Example 3 0 0 2+ 2+ 1+ 1+ 0 0 0 0 32*
Example of antibody titer and score*
11. In order to obtain correct information regarding change in titer, freeze the test serum,
store at ‐20˚С and run the titration again in parallel with any fresh specimen that may be
received from the patient. A difference of two dilutions is taken as significant change of
the titer value.

2.
Du Testing :
An anti D reagent suitable for indirect antiglobulin test is used for Du testing i.e.
o IgG monoclonal anti D
o Polyclonal IgG anti D
o Blend IgG monoclonal & IgM monoclonal anti D
o Blend monoclonal IgM & Polyclonal IgG anti D
Materials
o 75X10 mm tubes
o Suitable anti D reagent ( as above)
o test red cells
o Antiglobulin ( AHG) reagent
o Control IgG coated red cells
Methods
i. One drop of 2‐4% suspension of test red cells, add 2 drops of anti D
ii. Mixed and incubate at 37˚С for 45 minutes
iii. Look for agglutination
iv. If positive test record the sample at D positive
v. If negative i.e. no agglutination, wash the cells 3 – 4 times with saline and decant the
last wash completely.
vi. Add two drops of AHG and mixed gently
vii. Centrifuge at 1000 rpm for 1 minute
viii. Re – suspend the cells bottom gently look for agglutination and record the result.
ix. All negative reactions should be confirmed by adding known IgG sensitized control cells
re‐centrifuged and look for agglutination. The presence of agglutination confirms the
test result and no agglutination indicates invalid tests.

3. Cross Matching
1. Albumin Method
• The RBC of both the donor (blood bag) and the patient is washed with normal saline
3 – 4 times.
• 2 ‐ 5 % red cells suspension of the donor (blood bag) and the patient is prepared.
• 19 drops of saline is added in the cells one drops for preparation of 5% cells.
• Two tests tube clean and dry is taken and labeled as Major and Minor
• In the Major tube Patient’s serum is taken 4 drops and then 2 drops of donors 5%
red cell is taken.
• In the Minor tube Donor’s serum is taken 4 drops and then 2 drops of patient’s 5%
red cells is taken.
• 2 drops of Albumin is added in both the Major and Minor tube.
• Incubate for 15 min at 37˚С. After incubation centrifuge the tube and look for
agglutination.
2. Saline Cross – Matching
• Wash the RBCs of patient and donor 3‐4 times with normal saline.
• Prepare 2‐5% cell suspensions of patients and donor with normal saline.
• Label two test tubes as MAJOR & MINOR.
• In the MAJOR tube, add 4 drops of patient’s serum and 2 drops of 5% saline
suspension of donor cells.
• In the MINOR tube add 4 drops of donor’s serum and 2 drops of 5% saline
suspension of patient cells.
• Mix and incubate both the tubes at room temperature for 45‐60min.
• After incubation centrifuge both the tubes at 1000rpm for 1min.
• Slightly dislodge the button and look for agglutination macroscopically as well as
microscopically.
• If agglutination is not seen in any of the tubes, donor’s and patient’s bloods are
considered to be compatible.
Preparation of 5% red cell suspension
• Take 2 drops of whole blood and wash with normal saline 3‐4 times
• Take 1 drop of washed red cells in a clean tube and then add 19 drops of Saline

4.
Preparation of Pooled O Positive Cells
• Select 2‐3 O Positive cells .Take pooled cells in a clean test tube and wash 3‐4 times
with normal saline.
• Make 2‐5% saline suspension I e 1 drop of cell and 19 drops of normal saline.
Preparation of Sensitized O Positive cells
• Dilute anti D ( IgG) 1: 50 with normal saline.
• Prepare 5% O positive cell suspension.
• Mix equal volume of diluted anti D and 5% O positive cell suspension.
• Incubate at 37˚С for 30 min.
• Look for agglutination. if there is no agglutination
• Wash the cells 3 times.
• Make 5% cell suspension ( Sensitized O positive Cells )
• Do the DCT. There should be 2 + agglutination.
•
3. COOMB’s Method:
• Wash the RBCs (both the patient’s & donors) 3‐4 times with normal saline.
• Prepare 2‐5% red cell suspension.
• Label two test tubes as MAJOR & MINOR.
• In the MAJOR tube add 4 drops patient’s serum and 2 drops of donor’s 5% cells.
• In the MINOR tube add 4 drops donor’s serum and 2 drops of patient’s 55 cells.
• Mix and incubate both the tubes at 37˚С for 30‐60 minutes.
• Centrifuge the tubes at 1000 rpm for 1 minute and look for agglutination.
• If there is no agglutination wash the cells of both the tubes 3‐4 times with normal
saline.
• Add 2‐3 drops of AHG serum to both the tubes.
• Incubate both the tubes at room temperature for 5 minutes.
• Centrifuge the tubes at 1000 rpm for 1 min and look for agglutination.
• If there is agglutination in any tube the cross matching is reported as incompatible.
• If there is no agglutination (crosshatching is compatible) add 2 drops of 5%
sensitized O+ (ve) cells to both the tubes. Mix and centrifuge. There should be 2+
agglutination. Otherwise the test is invalid and the whole procedure is to be
repeated.

5. Direct Coombs Test‐
• The red cell to be tested will be tested will be washed with normal saline at least 3‐4 times.
• 2‐5% red cell suspension will be prepared.
• A drop of this cell suspension will be put in a clean dry test tube.
• A drop of Coombs reagent (Anti –Human Globulin) will be added to it.
• The mixture will be incubated at room temperature for 5 minute.
• The mixture will be centrifuged at 1000 rpm for 1 minute.
• The cell button formed will be gently dislodged and observed for agglutination.
Indirect Coombs Test
Procedure
1. Positive Control Negative Test
control
Diluted anti D 1:50 4 drops ‐ ‐
Normal Saline ‐ 4 drops ‐
Serum Patients ‐ ‐ 4 drops
5% pooled O Positive 2 drops 2 drops 2drops
cells
2. Mix and incubate at 37˚С for 60 min.
3. Centrifuge hook for agglutination. If there is no agglutination wash the cells 3 times with
normal saline.
4. Add 2 drops of AHG serum to all the tubes.
5. Incubate at room tem for 5 min
6. Centrifuge for 1 min at 1000 rpm
7. Look for agglutination.

6. BIOSAFETY AND WASTE DISPOSAL:
The personnel working in the blood bank should follow bio‐safety measures and guide lines for
the protection against blood transmissible disease such as HIV, HBsAg, HCV etc. The risk of
laboratory acquired infection with blood borne diseases is primarily from contamination of
hand, mucosa of the eyes, mouth, and nose and infectious blood and other body fluids. It is
mandatory to note that even though the occupational risk is low the consequences of infection
of blood borne disease are dire.
Bio‐safety Guidelines:
Universal safety precautions for the Laboratory personnel will be strictly followed. They will be
advised to carry out this instruction:‐
1. To wear safety gloves whenever handling any samples.
2. Not to touch eyes, nose mouth and other exposed membranes and skin with gloved hands.
3. To wash hands thoroughly with soap/detergent and /or antiseptic solutions and water after
every procedure or any contamination.
4. To wear Laboratory coats when working in the Laboratory.
5. Entry to the Laboratory will be restricted.
6. The work surface will be disinfected after the procedures and also at the end of each working
day with 0.1% Sodium Hypochlorite solution.
7. The needles and other sharp materials will be placed in a puncture resistant container
containing 0.1% Sodium Hypochlorite solution.
8. PIPETTING BY MOUTH WILL BE STRONGLY RESTICTED.
9. In case of any spill the area will be covered with 0.1% Sodium Hypochlorite solution with
gloved hands .The area will be left as such for 10‐15 minutes and then cleaned .The surface will
be wiped again with the disinfectant.
10. The broken glass pieces will be swept away with dust pan and brush.
11. In case of any puncture/wound, the site(s) will be cleaned thoroughly with detergent /soap
and plenty of water.

7. The various major wastes generated from the Blood bank include:‐
1. Blood bags‐ After the expiry date, contaminated or puncture, incompletely or improperly
collected, clotted blood and with Serological tests for infectious diseases positive.
2. Sharp items‐Needles, blood lancets and blades.
3. Cotton, gauge pieces, gloves.
4. Needle covers, pipette tips, tubing.
5. Reagent samples of Positive, Controls supplied with the kits.
6. Other Chemicals used in the Laboratory.
Proposed Method of Disposal:
The Standard Operating Procedure for these biomedical wastes, color code and type of
container to be used for disposal is attached. All non‐infective waste materials such as
sweeping, waste papers, plastic covers of syringes, I/V sets etc will be collected in a covered
bucket labeled as “Non‐Infectious Waste” and sent to the nearest garbage bin from where the
source will be disposed to the land filling.
Intermixing of the different types of wastes will be prevented by adequate labeling of the containers.
The various containers will be cleaned after every use with appropriate disinfec

8. Selection of ABO group blood for exchange transfusion
Baby’s blood group Mother’s blood group Blood selected for E.T
A A , AB A or O
O , B O
B B , AB B or O
O , A O
O A ,O, B O
AB A A or B
B B or O
AB AB , A , B or O
If the mother’s antibody is reactive against a high frequency antigen and no compatible blood is
available:
a) The mother’s sibling can be tested for compatible blood.
b) A unit of blood can be cancelled from the mother, if the obstetrician agrees then it is
safe. Mother’s red cell is constituted in AB plasma.
c) If no compatible blood is available and the clinical situation is urgent ,exchange
transfusion with incompatible blood (for the high frequency antigens) is preferred to no
transfusion at all as it helps to remove antibodies and bilirubin .with holding of blood
may result in death of the infant.

9.
SOP For Hemoglobin
Estimation by Copper Sulphate Method
Dispense 30ml of copper sulphate solution in a clean dry
transparent container.
Select the middle figure or ring finger and clean the figure
tip with antiseptic solution and allow it to dry.
Using a disposable lancet, make a puncture, so that there
should be free flow of blood.
Wipe the first drop of blood
Collect the blood in the capillary tube and allow it to fall
freely from the height of about 1 cm in the Cuso4 solution.
If the drop of blood remains at the surface or it rises from
the bottom of the solution, then the drop is lighter than
the copper sulphate solution and the Hb is less than
12.5gm%. If the blood sinks within 12‐15 seconds, then the
Hb value is more than 12.5gm%.

10.
SICKLE CELL TEST
STEP 1
Making Sodium Meta bisulfate.
1. Na2S2O5___________________ 0.5gm
2. ______________25ml
STEP 2
1. Place a small drop of blood (20µl)
2. ‘’ ‘’ of Na2S2O5
3. Mix Carefully
4. Put a cover slip
5. Put DPX to create an airtight enclosure around the cover slip
6. Wait for 15min
7. See under microscope 40x
8. Wait for 24 hours put it in a Petri dish with moist filter paper
9. See under microscope

11.
ERYBANK ANTI‐ A1 LECTIN
TEST PROCEDURE
1. Slide Test
i. Prepare a 10% Suspension of the red blood cells to be tested in
isotonic saline.
ii. Place 1drop of Erybank Anti‐ A1 Lectin on a clean glass slide.
iii. Pipette 2 drop of the cell Suspension on the slide.
iv. Mix well with a mixing stick uniformly over the area
v. Rock the slide gently back and forth.
vi. Observe for agglutination macroscopically at one minute.
2. Tube Test
i. Prepare a 5% cell suspension to be tested in isotonic saline.
ii. Place 1 drops of Erybank Anti‐ A1 Lectin into a labeled test tube.
iii. Pipette 1 drop of the test red cell suspension into the test tube
iv. Centrifuge for 1 minute at 1000 rpm or 20 seconds at 3400 rpm.
v. Gently resuspend the cell button, observing for agglutination
macroscopically.
INTERPRETATION OF RESULTS
Slide and Tube Tests
Agglutination is a positive test result and indicates the presence of A1 antigen.
Don’t interpret peripheral drying or fibrin strands as agglutination. No
agglutination is a negative test result and indicates the absence of A1 antigen.

12.
ERYBANK ANTI‐ H LECTIN
1. TEST PROCEDURE :
i. Place 1 drop of Erybank Anti – H Lectin on a clean glass slide.
ii. Pipette 50µl of whole blood to be tested on the slide and mix well
with a mixing stick uniformly over the area.
iii. Rock the slide gently back and forth.
iv. Observe for agglutination macroscopically at two minutes.
2. TUBE TEST :
i. Place a 5% suspension of the red cells to be tested in isotonic saline.
ii. Place 1 drop of Erybank Anti‐ H Lectin into a test tube and mix well.
iii. Pipette 50µl of the test red cell suspension into the test and mix well.
iv. Centrifuge for 1 minute at 1000 rpm or 20 seconds at 3400 rpm.
v. Gently resuspend the cell button, observing for agglutination
macroscopically.
Interpretation of results
Slide and tube Tests
Agglutination is a positive test and indicates the presence of H antigen. No
agglutination is a negative test result and indicates the absence of H antigen and
the red cells being of Bombay phenotype (Oh).

13.
ERBALISA HIV 1+2
Test Procedure:‐
1. Bring all the reagents and test specimens at room temperature use.
2. Except the blank, add 100µl of sample dilute to each well. In each run,
there will be one blank, three negative controls and one positive control.
3. Add 10µl of control and test specimens to the respective wells. Mix
properly with pipette. Cover the plate with black cover and incubate for 15
minutes at 20‐30° .
4. Wash the plate as per microplate washing procedure.
5. Add 50µl of conjugate to each well (except blank well) cover the plate with
cover and incubate for 15 minutes at 20‐30 .
6. Repeat step no 3.
7. Add 50µl of color reagent to each well. Cover the plate with black cover and
incubate for 15 minutes in dark at 20‐30 .
8. Add 100µl of stopping buffer to each well.
9. Read absorbency at 450nm (using 620/630/650nm as reference
wavelength). Deduct blank absorbency from the control and test wells.
Micro plate Washing Procedure:‐
Dilute washing solution (1+19) in distilled or deionised water. Washing solution
may be crystallized at cool storage condition. If so, use it after thawing at 37
water bath. Total 6 cycles with at least 350µl wash buffer per well per wash to be
done and a soak time of 30secs. Invert the plate and tap it on absorbent pad to
remove the remaining washing solution.
Cut – off Value formula: ‐ NCX+0.20

14. ERBALISA HEPATITIS B
TEST PROCEDURE
1) Bring all the reagents and test specimens at room temperature before use.
2) Add 50µl of sample diluents to each well. In each run there will be one blank
(100 µl sample diluent plus 50µl conjugate), three negative controls and one
positive control. Add 50µl of control and test specimens to the respective
wells. Add 50µl of conjugate to each well. Cover the plate with black cover
and incubate for 60 minutes at 20‐37°C.
3) Wash the plate as per Microplate washing procedure.
4) Add 50µl of color reagent .Cover the plate with black cover and incubate for
15 minutes in dark at 20‐30°C.
5) Add 100µl of stopping buffer to each well.
6) Read absorbency at 450nm (using 620/630/650nm as reference wavelength)
Deduct blank absorbency from the control and test wells.
MICROPLATE WASING PROCEDURE
Dilute washing solution (1+19) in distilled or deionised water. Washing solution
may be crystallized at cool storage condition. If so, use it after thawing at 37ºC
water bath. We suggest that 6 cycles with at least 0. 35ml wash buffer per well
per wash and a soak time of 30seconds. Invert the plate and tap it on absorbent
pad to remove the remaining washing solution.
CUT‐ OFF VALUE FORMULA: 0.1 + NCX

15. ERBALISA SEN HBsAG
Test Procedure:
1. Bring all the reagents and test specimens at room temperature before use.
2. Add 25µl of sample diluents to each well. In each run, there will be one
blank (100 µl) conjugate), three negative controls and one positive control.
Add 75 µl of control and test specimen to the respective wells. Add 50 µl of
conjugate to each well. Cover the plate with black cover and incubate for
60 minutes at room temperature(20‐25 )
3. Wash the plate as per micro plate washing procedure.
4. Add 50µl of color reagent. Cover the plate with black cover and incubate for
15 minutes in dark at 20‐30 .
5. Add 100µl of stopping buffer to each well.
6. Read absorbency at 450nm (using 620/630/650nm as reference
wavelength). Deduct blank absorbency from the control and test wells.
Micro plate washing procedure:
Dilute washing solution (1+19) in distilled or demonized water. Washing solution
may be crystallized at cool storage condition. If so, use it after thawing at 37
water bath. Total 6 cycle with at least 350µl wash buffer per well per wash to be
don with a soak time of 30 seconds. Invert the plate and top it on absorbent pad
to remove the remaining washing solution.
Cut – off value formula: NCX+0.15

16. ERBALISA HEPATITIS C
Test Procedure:
1. Bring all the reagents and test specimens at room temperature before use.
2. Except the Blank, add 100µl of sample diluent to each well. In each run,
there will be one blank, three negative controls and one positive control.
Add 10µl of control and test specimen to the respective wells. Mix properly
with pipette. Cover the plate with blank cover and incubate for 45 minutes
at 20‐30 .
3. Wash the plate as per micro plate washing procedure.
4. Add 50µl conjugate to each well (except blank well). Cover the plate with
black cover and incubate for 15 minutes at 20‐30 .
5. Repeat step no 3.
6. Add 50µl of color reagent to each well. Cover the plate with black cover and
incubate for 15 minutes in dark at 20‐30 .
7. Add 100µl stopping buffer to each well.
8. Read absorbency at 450nm (using 620/630/650nm as reference
wavelength). Deduct blank absorbency from the control and test wells.
Micro plate washing procedure:‐
Dilute washing solution (1+19) in distilled or demonized water. Washing solution
may be crystallized at cool storage conditions. If so, use it after thawing at
37 water bath. Total 6 cycles with at least 350µl wash buffer per well per wash
to be done with a soak time of 30 seconds. Invert the plate and tap it on
absorbent pad to remove the remaining washing solution.
Cut – off value Formula:‐ NCX+ 0.30

17. GRADING OF
AGGLUTINATION
/CLUMPING
REACTIONS:
++++ = Single clump of agglutination with no free
cells.
+++ = 3-4 individual clumps with few free cells.
++ = Many fairly large clumps with many free cell.
+ = Fine granular appearance visually, but definite
small clumps (10-15 cells) per low power field.
W = 2 to 3 cells sticking together per low power field,
with uneven distribution
- = ALL the cells are free.
NOTE: Haemolysis (Partial or Complete) will be
interpreted as POSITIVE and marked ‘H’.

18. In areas where sickle cell anemia is prevalent the donor’s blood
should be screened for sickle cell trait before the blood is used
for exchange transfusion.
Volume and haematocrit of blood for exchange transfusion
Ideally an exchange transfusion equal to twice the newborn
infant’s blood volume is recommended. The blood volume of a
full term newborn is approx 85 ml/kg. Exchange transfusion
with partially concentrated red cells having haematocrit of 50-
55% is preferable to whole blood.
Special considerations in compatibility testing for E.T.:
(1)If ABO group of mother and infant are compatible it is
convenient to obtain the serum from mother than from the infant
for compatible or the ABO
(2)If mother’s blood is not available or the ABO group of the
mother is not known, or the ABO group of the mother and infant
are incompatible, the baby’s serum or the elute from the cord
cell is used for cross-matching.
(3) Blood should be cross-matched against mother’s serum by
IAT and enzyme method.

19. FIRST AID
ACCIDENTS CAUSED DUE TO ACIDS
a. BODY BURNS
Wash with plenty of water first and then with sodium bicarbonate solution.
After the wash, apply a paste sodium bicarbonate –petroleum jelly mixture on the
burnt part for 10-15 minutes.Again wash with plenty of water and apply Carron oil
(An equal quantity of lime water and linseed oil.)
b. SPILLAGE IN THE EYES
Wash with plenty of water first and then with 2 %sodium bicarbonate
solution followed by water again. Wipe out the water from your eyes and then put
2-3drops of Olive oil.
c. SPILLAGE ON CLOTHES
Use dilute Ammonia solution to neutralize the acid. Then wash with plenty
of water.
d. ORAL INTAKE
Gargle with water first and then drink at least 1 liter of milk or water
followed by lime juice.
ACCIDENTS CAUSED DUE TO ALKALI
a. BODY BURNS
Wash with plenty of water first and then with boric acid solution .Make a
paste of Boric acid, apply on the burnt part and after 10-15 minutes wash it off.
b. SPILLAGE IN THE EYES
Keep open the eyelids and wash with plenty of water first and then with
dilute boric acid solution (0.5%).Again clean your eyes with water .Wipe out the
water and put 2-3 drops of Olive oil.
c. SPILLAGE ON THE CLOTHES
Neutralize the alkali with dilute Acetic acid solution (3%).Then wash with
plenty of water.

20. d. ORAL INTAKE
Gargle with water first and then drink atleast 1 liter of milk or water.Eat
oranges.
BURNS CAUSED BY BROMINE
Wash the burnt part with either Alcohol or petrol and then apply Olive oil.
BURNS CAUSED DUE TO FIRE
Apply either Petroleum jelly or Burnol on the burnt part.
WOUNDS
First remove any foreign particle (like glass pieces, etc) from the wound.
Clean well with water and apply Tincture Iodine, put a bandage and seek medical
attention.
GENERAL ADVICE
• For severe burns caused due to acid , keep the burnt part in Sodium
bicarbonate-Petroleum jelly mixture and for those burns caused due to alkali,
keep in Boric Acid solution for at least half an hour.
• While packing compounds of Cobalt, Chromium, Cadmium, Bismuth,
Barium, Lead, Tin etc.., it is necessary to wear the respective safety
equipments /guards (like gloves, goggles, face mask etc.). In case these
chemicals are accidently consumed try to spit it out even by vomiting.
• While packing Formaldehyde or hydrogen Peroxide, if it falls on the body or
clothes, wash with plenty of water.
• If Copper Sulphate is consumed accidently, drink a cup of warm water
containing a spoon of Sodium Chloride or Zinc Sulphate.
• It is the duty of everyone to ensure that chemicals do not come in contact
with the body through their mouth, eyes, ears and nose. Also ensure that the
necessary safety equipments are used.
• After providing First Aid, seek medical attention, if necessary.

21. FIRE
• Do not use water to extinguish an electric fire as it could give you a shock.
Use dry powder fire extinguishers.
• If solvents lighter than water (like Benzene, Acetone, I.P.A., methanol,
Petroleum ether, Diethyl ether, etc.) catch fire use a wet blanket to cover the
fire or use a suitable fire extinguisher. Splashing water on such fires could
spread the flame. Be familiar with the usage of various types of fire
extinguishers.

22. DONATH LANDSTEINER ANTIBODY
Step 1 Collect 4ml blood
Step 2 put 2ml in put 2ml in test tube
Tube marked marked 4 ºC which
37 ºC is kept in beaker with ICE
↓ ↓
Step 3 bring to lab and keep in fridge
put in 37ºC incubator Not in freezer
↓ ↓
Step 4 Wait for 11/2 hour wait for 1 hour
↓ ↓
Step 5
put it in 37⁰C
incubator for 20 minutes
↓ ↓
Step 6 Centrifuge Centrifuge
↓ ↓
Check hemolysis Check hemolysis
in supernatant in supernatant.

23. AGTROL
STARTER PACK FOR PREPARING COOMBS CONTROL CELLS
SUMMARY
Anti‐human globulin reagent is used in blood group serology for performing compatibility testing,
antibody screening, antibody detection and detection of D’ red cell type. Usage of Coombs control cells
is advocated for functional validation of anti‐human globulin reagent.
REAGENT
AGTROL starter pack for preparing Coombs control cells contains:
1.Ready to use, standardized pre‐diluted Anti‐D (IgG) monoclonal antibody reagent
2. Red blood cell preserving solution for serological applications. Each batch of reagents undergoes
rigorous quality control at various stages of manufacture for its performance characteristics.
REAGENT STORAGE AND STABILITY
Store the reagent at 2‐8⁰C DO NOT FREEZE.
The shelf life of the reagent is as per the expiry date mentioned on the reagent vial label.
PRINCIPLE
Human ‘O’ Rho (D) positive cells in presence of AGTROL pre‐diluted monoclonal do not agglutinate but
are sensitized with IgG antibodies. After processing, these sensitized red blood cells are resuspended in
red blood cell preserving solution for long term storage and use .When anti‐ human globulin reagent is
added to these sensitized cells the incomplete Anti‐D (IgG) antibodies are agglutinated by the anti‐
human IgG component. The agglutination reaction validates the serological activity of the anti‐ human
globulin reagent and confirms that the anti‐human globulin reagent was added in the test procedure.
NOTE
1. In vitro diagnostic reagent for laboratory and professional use only Not for medicinal use.
2. AGTROL Anti‐D (IgG) reagent is not from human source, hence contamination due to HBsAg and HIV is
practically excluded.
3. AGTROL Anti‐D (IgG) reagent contains 0.1% sodium azide as preservative. Avoid contact with skin and
mucosa .On disposal flush with large quantities of water.
4. Extreme turbidity in both AGTROL Anti‐D (IgG) and red blood cell preserving solution reagent may
indicate microbial contamination .Such reagents must be discarded.

24. SAMPLE COLLECTION AND STORAGE
No special preparation is required prior to sample collection by approved techniques .Samples should be
stored at 2‐8⁰C if not tested immediately. Do not use hemolysed samples. Anticoagulated blood using
various anticoagulants should be tested within the below mentioned time period: EDTA or Heparin ‐
2days, Sodium citrate or sodium oxalate ‐ 14 days, ACD or CPD ‐28 days.
ADDITIONAL MATERIAL REQUIRED
Test tubes (12x75mm), Pasteur pipettes, isotonic saline/ isotonic buffered saline, Anti‐ human globulin
reagent(Available from TULIP: ERYCLONE® Anti‐ human globulin reagent, (Cat.No:10180002,10180005),
Rho (D) positive red blood cells, Incubator at 37⁰C, laboratory centrifuge, optical aid.
PROCEDURE
Bring all the reagents to room temperature (25‐30⁰C) before testing.
Preparation and validation of Coombs control cells
Preparation of 5% Coombs control cell suspension
1 Collect fresh O Rho (D) positive red blood cells preferably with citrate as an anticoagulant.
2 Wash 1ml of freshly collected O Rho (D) positive red blood cells with isotonic saline at least three
times.
3 After the third wash, thoroughly decant the supernatant .To the cell button add 5ml of AGTROL
Anti‐D (IgG) reagent and gently resuspend the red blood cells.
4 Incubate the mixture at 37ºC for 15minutes.
5 After incubation wash the sensitized red blood cells thoroughly at least 4 to 5 times with isotonic
saline.
6 Decant the supernatant thoroughly after the last wash. Resuspend the cell button gently with
about 1‐2 ml of AGTROL red blood cell preserving solution. The complete resuspended red cells
should be added back to the balance AGTROL red blood cell preserving solution in the dropper
vial .A stabilized suspension of 5% Coombs control cells is thus obtained .Label appropriately with
the date of preparation.
7 Store the Coombs cells at 2‐8ºC. Use within 4 weeks of preparation.
Validation of prepared 5% Coombs control cell suspension.
1. Add one drop of Coombs control cells into a test tube.
2. Add two drops of Anti‐human globulin reagent and mix well.
3. Centrifuge for 1 minute at 1000 rpm or 20 seconds at 3400rpm.
4. Very gently resuspend the cell button and observe for agglutination macroscopically.

25.
Confirmation of negative Antiglobulin test reactions
1. Add one drop of Coombs control cells to the samples negative during Direct or Indirect
Antiglobulin test.
2. Centrifuge for 1 minute at 1000 rpm or 20 seconds at 3400 rpm.
3. Very gently resuspend the cell button and observe for agglutination macroscopically.
.
I NTERPETATION OF RESUTS:
Agglutination reaction indicates that the anti‐human globulin reagent is functional and the test is valid.
No agglutination indicates that the anti‐human globulin reagent does not have sufficient activity and the
test is invalid.
REMARKS:
1. As under‐centrifugation or over‐centrifugation could lead to erroneous results, it is
recommended that each laboratory calibrate its own equipment and the time required for
achieving the desired results.
2. Erroneous results may also occur due to improper red blood cell concentration, improper
temperature while performing the test.
3. Store the Coombs control cells at 2‐8⁰C with cap tightly closed.
4. Do not contaminate the prepared Coombs control cell suspension as it may subsequently
effect the stability.
5. Glassware used to retrieve the AGTROL reagents and Coombs control cell suspension should
be scrupulously clean and sterile.

26. CARBOGEN
RAPID PLASMA REAGIN (RPR) CARD TEST /CARBON ANTIGEN FOR SYPHILIS
TESTING
SUMMARY
Syphilis is a sexually transmitted (venereal) disease caused by the spirochete Treponema
pallidum. After infection the host from Treponema pallidum, in addition, the host also from
Non‐Treponemal anti‐lipoidal antibodies in response to the lipoidal material released from the
damaged host cell. These antibodies are traditionally referred to as ‘Reagins’.
The Rapid Plasma Reagin (RPR)/ CARBON Antigen test is a macroscopic non‐Treponemal
flocculation test for the detection and quantitation of antilipoidal antibodies. Non‐Treponemal
test like CARBOGEN® are of great value when used for screening and follow up of therapy.
PRINCIPLE
During the test procedure, the specimen, serum or plasma is mixed with CARBOGEN® reagent
forming visible black floccules. If anti‐lipoidal antibodies are not present in the specimen, there
will be no flocculation.
SAMPLE COLLECTION AND STORAGE
1. No special preparation of the patient is required prior to sample collection by approved
techniques. Hemolysed or lipemic samples are not suitable for testing.
2. Fresh serum or plasma should be used for testing.
3. Samples not tested immediately may be stored at 2‐8°C for up to 48 hours.
4. Hazy samples should be centrifuged. Use the clear supernatant for testing.
TEST PROCEDURE
Bring reagent and samples to room temperature before testing.
Thoroughly mix the CARBOGEN® reagent suspension by gentle agitation before testing.
Qualitative Method
1. Pipette one drop (50µl) of the test specimen, positive control and negative
control onto separate reaction circles using a sample dispensing pipette.
2. Add one drop of well mixed CARBOGEN® reagent next to the test specimen,
positive control and negative control by using the reagent dropper provided
with the kit. Do not let the dropper tip touch the liquid on the slide.
3. Using a mixing stick, mix the test specimen and the CARBOGEN® reagent
thoroughly spreading uniformly over the entire reaction circle.
4. Immediately start a stopwatch. Rotate the slide gently and continuously either
manually or on a mechanical rotor at 180 rpm.
5. Observe for flocculation macroscopically at 8 minutes.

27. Quantitative Method
1. Using isotonic saline prepare serial dilutions of the test specimen positive in the
qualitative method 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and so on.
2. Perform the qualitative procedure using each dilution as a test specimen.
3. The titre is reported as the reciprocal of the highest dilution which shows a
positive result.
INTERPRETATION OF TEST RESULTS
Qualitative Method
1. Large and medium black floccules against white background : Reactive
2. Small black floccules against white background : Weakly Reactive
3. No floccules, even grey background : Non Reactive
Quantitative Method
The titre of anti‐lipoidal antibodies is the highest dilution of the test specimen giving a
positive result.

28. ERYBANK
BOVINE SERUM ALBUMIN
22% SOLUTION FOR SEROLOGICAL APPLICATIONS
SUMMARY
Bovine Serum Albumin is mainly used to enhance the reactivity of blood grouping and typing
antibodies in direct agglutination tests. Bovine Albumin also enhances the reactivity and
sensitivity of indirect Antiglobulin test which is used for compatibility testing, antibody
screening, identification and titration.
REAGENT
ERYBANK® BOVINE SERUM Albumin is manufactured from selected raw Bovine serum, its
protein concentration and pH adjusted to 22% and 7.1(±0.2)respectively Its conductivity is
controlled specifically for serological application.
REAGENT STORAGE AND STABILITY
1. STORE THE REAGENT AT 2‐8°C. DO NOT FREEZE
2. The shelf of the reagent is as per the expiry date mentioned on the reagent vial label.
PRINCIPLE
Agglutination of antibody coated red cells depends upon the class and type of the antibody
involved and the characteristics of the reaction medium such as ionic strength and pH.
Incomplete antibodies of IgG class, especially those with Rh specificity, agglutinate red cells if
the zeta potential between the red cells is adjusted by addition of salts and colloids such as
Bovine Serum Albumin. Addition of BSA enhances such immunological reactions and increases
test sensitivity.
NOTE
(1) In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.
(2) The reagent contains sodium azide 0.1/% as preservative. Avoid contact with skin and
mucosa. On disposal flush with large quantities of water.
(3) Extreme turbidity may indicate microbial contamination or denaturation of protein due to
thermal damage. Such reagents should be discarded.

29.
SAMPLE COLLECTION AND PREPARATION
No special preparation of the patient required to prior to sample collection by approved
techniques. Samples should be stored at 2‐8°C if not tested immediately. Do not use hemolysed
samples. Donor units can be tested up to the end of their dating.
For the Indirect Antiglobulin test, serum from fresh clotted whole blood should be used.
ADDITIONAL MATERIAL REQUIRED COMPATIBILITY TESTING
Test tubes (12x75 mm), 0.2 ml serological pipettes, Pasteur pipettes, Human red blood cells
with specific antigen reacting with the antibody to be titrated, Centrifuge, Incubator, Isotonic
saline, Anti Human globulin reagent, regent, such as ERYCLONE® Anti human globulin reagent,
Coombs control cells: (Refer ERYCLONE® Anti Human Globulin pack insert), AB neutral human
serum.
BROAD SPECTRUM COMPATIBILITY TEST
Major cross match procedure
Initial Phase:
1. Label two test tubes as A (for albumin) and B (for saline); depending upon the number of
donors to be cross matched, as many pairs of such labeled tubes would be required.
2. Prepare a 5% suspension of the red cells to be tested in isotonic saline.
3. Pipette two drops of recipient serum in both the labeled test tubes.
4. Pipette one drop of donor red cells in both the labeled test tubes and mix well.
5. Only to the albumin tube (A) add two drops of ERYBANK® Bovine Serum Albumin reagent and
mix well.
6. Centrifuge both tubes for one minute at 1000 rpm (125g) or for 20 second at 3400 rpm (1000
g).
7. First observe for hemolysis. Resuspend the cell button and observe for agglutination
macroscopically.
8. Proceed to incubation phase.
Incubation phase
1. Incubate the saline tube at room temperature and the albumin tube at 37°C for fifteen
minutes.
2. First observe for hemolysis. Resuspend the cell button and observe for agglutination
macroscopically.
3. Proceed to Antiglobulin phase.

30.
Antiglobulin phase
1. Only the albumin tubes (A) are tested in the anti globulin phase.
2. Wash the mixture of red blood cells and serum thoroughly with isotonic saline for a minimum
of three times. Decant completely after the last wash.
3. Place two drops of ERYCLONE® Anti HUMAN globulin into the test tube containing the
sedimented cells and mix well.
4. Centrifuge for one minute at 1000 rpm (125 g) or 20 seconds at 3400 rpm (1000 g)
5. Very gently, resuspend the cells and observe for agglutination macroscopically.
Antibody Titration Test
1. a) Prepare a 5% suspension of red blood cells with specific antigen reacting with
antibody to be titrated, in ERYBANK® Bovine Serum Albumin reagent.
b) Also prepare a 5% suspension of patients red cells in ERYBANK® Bovine Serum
Albumin reagent
2. Label ten test tubes (1 to 10) and make progressive dilutions of the patient serum as
indicated below:
a) Pipette 0. 1ml of AB neutral serum into each test tube except the first tube.
b) Pipette 0. 1ml of the patient serum into first two tubes only.
c) After mixing the contents of the second tube thoroughly, transfer 0.1ml of the
mixture to the third tube. Continue the serial dilution by transfer up to tube No.
Ten. Discard 0.1ml of the mixture from the last tube.
3. To tubes No. One through Nine, add one of Albumin suspended selected red blood cells, (as
prepared in point No. 1 (a) above) and mix well.
4. To tube No. Ten add one drop of patient red cells suspended in albumin (as prepared in
point No. 1 (b) above) and mix well.
5. Incubate all the tubes 37°C for a minimum of fifteen minutes.
6. Centrifuge all the tubes for one minute at 1000 rpm or 20 seconds at 3400 rpm (1000 g).
7. Very gently, resuspend the cell buttons and observe for agglutination macroscopically.
8. Antiglobulin test should be performed on all tubes which do not show a very strong
agglutination.

31. INTERPRETATION OF RESULTS
Compatibility test
In all phases of the compatibility test, if on agglutination or hemolysis is observed then the
patient and the donor may be considered compatible.
If haemolysis or agglutination at any point till the completion of the Antiglobulin phase is
observed the patient and donor are considered incompatible.
Antibody titration test
The end point of the titration is the reciprocal of the last tube showing agglutination.
REMARKS
1. If plasma is used in the indirect Antiglobulin test, the complement dependent antibodies may
not be detected due to the absence of calcium.
2.To all negative test results, after the Antiglobulin test phase, one drop of Coombs control cells
should be added .If the Coombs control cells do not agglutinate, then the compatibility test
must be repeated.
3. Red blood cells showing a positive dire Antiglobulin test should not be used for the indirect
Antiglobulin test.
4Bovine serum albumin will not bring about agglutination of red cells by all IgG BLOOD
GROUPING TYPING ANTIBODIES.
5 As under centrifugation or over centrifugation can lead to erroneous results, it is
recommended that each laboratory calibrate its own equipments and the time required for
achieving the desired result
6. After usage the regent should be immediately recapped and replaced to2‐ 8°C storage.

32. ANTI‐D(RhO)
(IgM)
MONOCLONAL BLOOD TYPING ANTIBODIES FOR SLIDE AND TUBE TEST
SUMMARY
Monoclonal antibodies are derived from hybridoma cell lines , created by fussing mouse
antibody producing B lymphocytes with mouse myeloma cells or are derived from a human B
cell line through EBV transformation. Each hybridoma cell line produces homogenous
antibodies OF only one immunoglobulin class, which are identical in their chemical structure
and immunological activity.
Human red blood cells are classified as Rho (D ) Positive depending upon the presence or
absence of (Rho) antigen on then. Approximately 85% of the Caucasia population are Rho
(D) positive. The D” Phenotype is a variant of D (Rho) About 60% of the D” s, now classified as
weak or partial D’s, may react with Anti‐D (IgM) in slide tests and about 90% may be detected
by the tube technique.
REAGENT
Anti‐D (Rho) (IgM) is ready to use reagent, prepared from supernatants of cell cultures with
antibody producing B lymphocytes obtained through EBV transformation and is blend of
monoclonal antibodies of immunoglobulin class IgM. These antibodies are a mixture of several
monoclonal antibodies of the same specificity but having the capability of recognizing different
epitopes of the human red blood cell antigen D (Rho).
Anti‐ D (Rho) (IgM)does not detect all weak and prepared D’s For the confirmation of negative
reactions with Anti‐D (Rho) (IgM) further testing with an incomplete Anti D‐ (Rho) of IgG class is
strongly recommended to confirm the presence or absence of weak/ partial D’s.
Each batch of reagent undergoes rigorous quality control at various stages of manufacture for
its specificity, avidity and performance.
REAGENT STORAGE AND STABILITY
1.Store the reagent at 2‐8°C.do not freeze.
2. The shelf life of the reagent is as per the expiry date mentioned on the reagent vial label.

33. PRINCIPLE
Human red blood cells possessing the D(Rho) antigen will agglutinate in the presence of
antibody directed towards the antigen Agglutination of red blood cells with Anti‐ D(Rho) (IgM)
reagent is a positive test result and indicates the presence of D (Rho) antigen .No should be
further tested for D” (Presence of weak/ partial D’s) by performing the D” test procedure using
incomplete Anti‐D (Rho) of IgG class, as described later.
NOTE
1. In vitro diagnostic reagent for laboratory and professional use only .Not for medicinal use.
2. Anti‐ D (Rho)(IgM) reagent is not from human source, hence contamination due to HBsAg
and HIV IS practically excluded.
3. The reagent contains sodium azide 0.1% as preservative. Avoid contact with skin and mucosa
On disposal flush with large quantities of water.
4. Extreme turbidity may indicate microbial contamination or denaturation of protein due to
thermal damage. Such reagent should be discarded.
SAMPLE COLLECTION AND PREPARATION
No special preparation of the patient is required prior to sample collection by approved
techniques. Samples should be stored at 2‐8°Cif not tested immediately. Do not use
heamolysed sample.
Anticoagulated blood using various anticoagulants should be tested within the below
mentioned time period:
EDTA or Heparin ‐2 days
Sodium citrate or sodium oxalate ‐14 days
ACD or CPD ‐28 days
ADDITIONAL MATERIAL REQUIRED FOR SLIDE AND TUBE TESTS
Glass slides (60x85mm),test tubes (12x75mm), Pasteur pipettes, isotonic saline, Centrifuge,
Timer, Mixing sticks, ERYCLONE®
Anti Human Globulin (Coombs) reagent, ERYCLONE® Anti‐D(IgG)or RHOFINAL® Anti‐D (IgM+
IgG).

34. TEST PROCEDURE
Bring reagent and samples to room temperature before testing.
Slide Test
1 place one drop of Anti‐D (Rho)(IgM) reagent on a clean glass slide.
2. Pipette one equal drop of whole on the slide.
3. Mix well with a mixing stick uniformly over an area of approximately 2.5 cm².
4. Rock the slide gently, back and forth.
5. Observe for agglutination macroscopically at two minutes.
Immediate Spin Tube Test
1. Prepare a 5% suspension of red cells to be tested in isotonic saline.
2. Place one drop of reagent Anti‐D (Rho)IgM)into correspondingly labeled test tube.
3. Pipette into the test tube one drop of the 5% cell suspension and mix well.
4. Centrifuge for 1 minute at 1000 RPM (125g)OR 20 SECONDS AT 3400 RPM (1000g)
5. Gently resuspend the cell button, observing for agglutination macroscopically.
Du TEST PROCEDURE
1. Prepare a 5% suspension of the red cells to be tested in isotonic saline.
2. Place one drop of any incomplete Anti‐D (Rho)IgM class )reagent such as ERYCLONE® Anti‐D
(Rho) IgM ) into a labeled test tube.
3. Add to test tube one drop of the 5% cell suspension and mix well. Incubate at 37°C for 15
minutes.
4. Wash the contents of the tube thoroughly, at least three time, with isotonic saline and
decant completely after the last wash.
5. Add two drop of ERYCLONE® Anti Human Globulin reagent and mix well.
6. Centrifuge for 2minute at 1000 RPM(125g) or 20 seconds at 3400 RPM (1000 g).
7. Very gently, resuspend the cell button, observing for agglutination macroscopically.

35. INTERPRETATION OF RESULTS
Slide and Tube Tests
a) Agglutination is a positive test result and indicates the presence of D (Rho) antigen. Do not
interpret peripheral drying or fibrin strands as agglutination is a negative test result and
indicates the absence of D (Rho) antigen.
b) Cord cells heavily sensitized with Anti‐D (Rho) may give a false negative immediate spin test
result.
Du Test procedure
(a) Agglutination indicates the presence of Du antigen (Presence of weak/ partial D’s).No
agglutination indicates the absence of the Du antigen (Absence of weak/ partial D’s).(b)Mixed
field agglutination in the Du test on red cells from a recently delivered woman may indicate a
mixture of maternal Rho (D) negative and fetal Rho (D) Positive blood.(c)Red cells
demonstrating a positive direct Antiglobulin test cannot be accurately tested for Du antigen
(Presence of weak/ partial D’s).
REMARKS
(1) As under centrifugation and/ or overcetrifugation could lead to erroneous results, it is
recommended that each laboratory calibrate its own equipment and the time required for
achieving the desired results
(2) It is strongly recommended that as a routine quality control measure known as Rho
(D)positive and Rho (D)negative red cells be occasionally run, preferably on a daily basis so as to
control reagent performance and validation of test results.
(3) After usage, the reagent should be immediately recapped and replaced to 2‐8°C storage.
(4)The label minimum titre claim is based on Rho (D)positive red cells (phenotype :R¹ r) for
ANTI‐d monoclonal (IgM) reagent. This is based on titration procedure as recommended by the
manufacturer .Any deviation in test procedure could result in variable results.

36. ANTI‐ A, ANTI‐B. ANTI –A, B
MONOCLONAL BLOOD GROUPING ANTIBODIES FOR SLIDE AND TUBE
TESTS
SUMMARY
Monoclonal antibodies are derived from hybridoma cell lines, created by fusing mouse antibody
producing B lymphocytes with mouse myeloma cells. Each hybridoma cell line produces
homogenous antibodies of only one immunoglobulin class, which are identical in their chemical
structure and immunological activity.
Human red blood cell antigen can be divided into four group A, B,AB and O depending on the
presence or absences of the corresponding antigens on the red blood cells.
Approximately 41% of the Caucasian population have the A Antigen, 9% have the B Antigen, 4%
have both A and B antigens, while the remaining have neither the A nor the B antigen.
REAGENT
Anti‐A, Anti‐B, and Anti‐A,B are ready to use reagents prepared from supernatants of mouse
hybridoma cell cultures. These antibodies of immunoglobulin class IgM are mixture of several
monoclonal antibodies of the same specificity but having the capability of recognizing different
epitomes of the human red blood cell antigens A and B. Each batch of reagent undergoes
rigorous quality control at various stages of manufacture for its specificity, avidity and
performance.
REAGENT STORAGE AND STABILITY
1. Store the reagent at 2‐8°C. DO NOT FREEZE.
2. The shelf life of the reagent is as per the expiry date mentioned on the reagent vial label.
PRINCIPLE
Human red blood cells possessing A and/or B antigen will agglutinate in the presence of
antibody directed towards the antigen. Agglutination of red blood cells with Anti‐ Anti‐B, Anti‐
A,B reagents is a positive test result and indicates the presence of the corresponding
antigen.

37. NOTE
1. In vitro diagnostic reagent for laboratory reagent for laboratory and professional use only.
Not for medicinal use.
2. The reagent contains sodium azide 0.1% as preservative .Avoid contact with skin and mucosa.
On disposal flush with large quantities of water .
3. Extreme turbidity may indicate microbial contamination or denaturation of protein due to
thermal damage. Such reagents should be discarded.
4. Reagents are not from human sources, hence contamination due to HBsAg, and HIV IS
practically excluded.
SAMPLE COLLECTION AND PREPARATION
No special preparation of the patient is required prior to sample collection by approved
techniques. Samples should be stored at 2‐8°C if not tested immediately. Do not use hemolysed
samples.
Anticoagulated blood using various anticoagulants should be tested within the below
mentioned time period:
EDTA or HEPARIN : 2 days
Sodium citrate or sodium oxalate : 14 days
ACD or CPD : 28 days
ADDITIONAL MATERIAL REQUIRED FOR SLIDE AND TUBE TESTS
Glass slides (60 x 85 mm), Test tube (12 x75 mm), Pasteur pipettes , Isotonic saline, Centrifuge,
Timer, Mixing sticks.
TEST PROCEDURE
Bring reagents and samples to room temperature testing.
Slide Test
1. Place one drop of Anti‐A or‐B or Anti‐A,B reagent on a clean glass slide.
2. To each reagent drop, add one small drop if whole blood
3 Mix well with a mixing stick uniformly over an area of approximately 2. 5cm².
4. Rock the slide gently, back and forth.
5. Observe for agglutination macroscopically at two minutes.

38. Tube test
1. Prepare a 2‐3% suspension of the red cells to be tested in isotonic saline.
2. Place one drop of Anti‐A, Anti‐B, Anti‐A,B into corresponding labeled test tubes.
3. Pipette into each of the test tubes , one drop of the test red cell suspension and mix well.
4. Centrifuge for 1 minute at 1000 RPM (125 g) or 20 seconds at 3400 RPM (1000g) or incubate
at room temperature for 20‐30 minutes.
5. Gently resuspend the cell button, observing for agglutination macroscopically.
INTERPRETATION OF RESULTS
Slide and tube tests
Agglutination is a positive test result and indicates the presence of A and /or B antigen. Do not
interpret peripheral drying or fibrin strands as agglutination. No agglutination is a negative test
result and indicates the absence of A and /or B antigen.
REMARKS
1. (a) Anti‐A, Anti‐B and Anti‐A,B reagent do not show a reaction with crypt antigens (T, Tn
,activated cells).
(b) Anti‐B is truly negative reacting with acquired B characteristics.
2. In the tube test procedure ,it is recommended that tubes with negative reactions should be
re‐centrifuged and results read again after 5 minutes so that weak antigens are not overlooked.
3. As under centrifugation or over centrifugation could lead to erroneous results. it is
recommended that each laboratory calibrate its own equipment and determine the time
required for achieving the desired results.
4. Results of forward grouping obtained by using Anti‐A, Anti‐B reagent and Anti‐A,B should
always be reconfirmed by performing reverse grouping with known red cells.
5. It is strongly recommended that red cells with known ABO characteristics should be
occasionally run, preferably on daily basis so as to control reagent performance and validate
test results.
6. After usage the reagent should be immediately recapped and replace to 2‐8°C storage.
7. The label minimum titre claim is based on A1 group cells for Anti‐A reagent, B group cells for
Anti‐B reagent and A1B cells for Anti A1B reagent. This is based on titration procedure as
recommended by the manufacturer .Any deviation in test procedure could result in variable
results.