EXERCISE 2: PREPARATION OF 2% RBC • Increasing the ratio of serum to RBCs increases the

degree of antibody coating. A commonly used ratio

SUSPENSION is 2 drops of serum to 1 drop of a 2-5% RBC
suspension.
By: Sir Patrick Romero
• Alternately, reducing the RBC suspension from 5%
OUTLINE: to 2-3% could double the serum to RBC ratio.
• By increasing the ratio of serum to RBCs, weakly
• Introduction
reactive antibodies that were not demonstrable
• Principle
under standard test condition are detected.
• Materials & Equipment
Therefore, it is best to use the weakest cell
• Procedure
suspension that can be observed easily for
INTRODUCTION agglutination.
• Washed red blood cells are required in various • Agglutination is dependent on a minimal number of
laboratory procedures, for example, in serologic test antibody molecules/RBC. If too many cells are
for infectious mononucleosis, washed sheep RBCs present in the serum-cell mixture, weak antibodies
serve as in vitro antigen. maybe missed because too few antibody molecules
• In the ASO (Antistreptolysin) test, human are bound to each other. (AAB, Technical Manual,
erythrocytes are used in the procedure to indicate p.167, 1994)
whether or not hemolysis has occurred.
PRINCIPLE
• In Blood Banking tests such as blood typing and
• Plasma and serum must be essentially and carefully
compatibility testing for transfusion purposes, the
labeled since they cannot be distinguished or
red cells of donor and recipient are washed at
differentiated visually. Hemolyzed samples should
certain stages of the procedure.
not be used. Free serum hemoglobin may mask
THE PURPOSE OF WASHING RBCS ARE: antibody – induced hemolysis.
• Washed red blood cells are red blood cells
1. To remove the plasma or serum (if the RBCs are
remaining after washing with a volume of
obtained from clotted blood), hence prevent
comparable solution using a method to remove
formation of fibrin clots.
almost all of the plasma.
2. Washing removes serum factors that inhibit
• The efficiency of washing depends on the kind of
complement reactivity.
saline and the method used.
3. Furthermore, it removes any soluble antigen-
• The supernantant fluid should be clear after
antibody complexes that compete with the target
centrifugation and produce a well-defined cell
cell for complement.
button.
• The cell should be washed once or 3x and
resuspended to a 2-4% concentration in normal
• Washing RBCs removes unbound globulin by saline solution (NSS) or low ionic strength saline
dilution, so it is important to decant supernatant (LISS)
saline thoroughly at each wash phase.
• RBCs must be resuspended completely with each MATERIALS AND EQUIPMENT
new addition of NSS; the RBC button should be 1. Marking Pen
shaken briskly and saline added in a forceful stream. 2. Laboratory Tissue
• The tube should not be inverted against the finger 3. Venous collected EDTA Sample
or palm, partly because this endangers the worker’s
health and partly because it can introduce globulins
into the wash solution if the hands are contaminated
with blood or reagent.
• The reason why 2% RBC suspension gives the best
result is based on the fact that the serum: cell ration
will markedly affect the sensitivity of agglutination
tests.
 4. 0.85% - 0.90% Saline solution in plastic wash
bottle.

5. 10% NaOH in Large Container

PART II: PREPARATION OF RBC SUSPENSION

6. Pasteur Pipettes

7. 1.0 and 5.0ml serologic pipettes 2% RBC Suspension

8. Serologic centrifuge
9. Test tube rack
10. Safety Bulb
11. Puncture – proof biohazard container

PROCEDURE
PART I: WASHING OF RED BLOOD CELLS

Correction: Repeat 2 more times for a total of 3 washes

STUDENT ACTIVITY

• Part I: Washing of Red Blood Cells Activity

• Part II: Dilution Activity