Blood grouping,Direct anti globulin test,Reverse grouping requires 3-5% RBC suspension

1. WASHED CELL SUSPENSIONS

2. Red cell suspensions provide the appropriate
serum to cell ratio to allow for grading and
interpretation of tests results
3% Red Blood Cell Concentration in Saline

3. The purpose of washing the red blood cells is to remove plasma,
which contains substance that may interfere with antigen-
reaction. The following may be in the plasma and may interfere
testing:
• Soluble antigens such as A and B may be present and neutralize
your reagent.
• Interfering proteins such as Wharton's jelly that is seen in
newborn cord blood, cold-acting autoimmune antibodies, and
increased levels of immunoglobulins that may cause either
agglutination or rouleaux..
• Hemolyzed red blood cells due to a difficult draw will interfere in
your grading interpretation of hemolysis
• Fibrinogen can result in fibrin strands forming that makes
Washing Red Blood Cells Before Making the 3% Suspension

4. In the process of blood typing, blood samples are mixed with
known anti-A and anti-B antibody serum, and are then
monitored for agglutination.
If agglutination occurs, then the particularABOantigen(s) on
the red blood cell surface have reacted with the antibodies
present in the serum.
Wharton’s jelly [ contain hyaluronic acid & chondroitin
can contaminate blood samples by coating the red blood cells
making the cells polyagglutinable, leading to a false positive
reaction.
When possible, cord blood should be collected using the
syringe method rather than the gravity and glass tube method
WHARTON’S JELLY

5. SPECIMEN
The preferred specimen is an EDTA (lavender top) tube of venous
blood.
Un-anticoagulated glass (red top) tubes may be used if
completely clotted. Prior to preparation of a cell suspension, the
specimens should be centrifuged except cord blood specimens and
specimens incorrectly provided in “serum separator” tubes. Patient
plasma/serum is separated from the red cells and placed into an
appropriately labeled tube with the patient's name and medical record
number or date of birth, and the date the sample was drawn.
RBCs are removed directly from cord blood specimens without centrifugation. For
serum separator tubes, remove RBCs prior to centrifugation and wash them as

6. Washed 3-5% patient or donor red cell suspension for tube tests starting with
packed cells
1. Properly label a 12 x 75 test tube.
2. With a pipette, remove approximately 3-5 drops of blood from the patient or
donor sample and add to the test tube. For donor tubing segments, cut open
the segment and squeeze 1-2 drops of blood into the test tube.
3. NOTE: Since antibody coated RBCs are heavier than uncoated cells
and settle to the bottom of the sample the contents should be mixed well before blood
is removed. If a clotted specimen is used, blood should be removed from the bottom of
the clot for the same reason.
4. Fill the test tube approximately 3/4 full with saline by positioning the tip of
the wash bottle directly over the tube and squeezing it. This will mix the blood
and the saline, increasing the efficiency of washing.
5. Centrifuge the tube for the appropriate time calibrated for cell washing.
6. When the centrifuge stops, remove the test tube and decant the supernatant
saline.
7. Shake the cells loose from the bottom of the tube.
8. Add the volume of saline necessary to prepare 3 5% suspension,
approximately 40 drops.

7. Method to defibrinate a clot
Materials
1. Sieve and pestle
2. Clotted blood specimen
Procedure
1. Spin the specimen until a good separation occurs between the clot and serum (3,500 rpm for 5 minutes).
2. Pipette the serum into a clean test tube labeled with the patient's name, account number, and date of the
sample.
3. Place the sieve over a Petri dish.
4. Pour the remaining clot onto the sieve and mash well with the pestle. Rinse the sieve with saline.
5. Transfer red cells suspended in saline from the Petri dish into clean 13x100 mm test tubes labeled with
patient's name and date of sample. Rinse the Petri dish and transfer the saline to the labeled tubes.
6. Spin these tubes at 3,500 rpm for l minute. Aspirate the supernatant saline and pool the packed cells, rinsing
remaining cells from the tubes with saline.
7. Continue pooling and centrifugation until all the cells are contained in one test tube.
8. Wash the cells until the supernatant is free of hemoglobin.

8. https://www.youtube.com/watch?v=cx1Gd5Gov0g
http://www.health.gov.nl.ca/health/bloodservices/pdf/prep_red_cell_susp.pdf
https://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=18&cad=rja&uact=
8&ved=0ahUKEwj7nJ_dwt7YAhVEoZQKHdXtDhYQFgh-
MBE&url=http%3A%2F%2Fwww.indianinitiative.org%2Fwp-
content%2Fuploads%2F2011%2F06%2F202-Preparation-of-Red-Blood-Cells-for-
Analysis.doc&usg=AOvVaw2Dk9RTrbpHd9nrqz_EFXGm
http://faculty.madisoncollege.edu/mljensen/BloodBank/lectur
es/Basic_Laboratory_techniques&Reagents.htm