LESSON 3: Preparation of Red Cell Suspensions with Different Concentrations

LEARNING OBJECTIVES

At the end of this lesson, the students will be able to:

1. Prepare red cell suspension with different concentrations (2%-5%)

2. Observe the proper pipetting techniques.

INTRODUCTION

Numerous procedures that is done inside the laboratory requires demonstration of antigen and
antibody reactions in vitro; thus, the addition of indicator cells to the system is needed. The best example s
the testing for naturally occuring ABO antibodies in the plasma donors and receipients that requires the
utilization of such suspension. There are commercially available cells that are packed in different
combinations, but the most common is A1 and B combination.

Two to five percent red cell suspensions are the universally employed indicator cells displays such
reactions. These suspensions are prepared by using previously washed anticoagulated blood.

A 2-5% red cell suspension is used for the following tube examination procedures:
• ABO and Rh typing
• Direct antiglobulin test and auto control
• Donor unit compatibility (crossmatch)
• Red cell phenotyping

PREPARATION OF RED CELL SUSPENSION WITH DIFFERENT CONCENTRATION

A. Materials

1. Graduated conical tubes with screw cap cover (10-mL capacity)

2. Gloves
3. Marking pen
4. Gum label
5. Centrifuge
6. Pasteur pipette
7. Test tube rack
8. Wasserman test tubes
9. Nescofilm
10. Aspirator bulb
11. Automatic pipette/serologial pipette (0.1 mL/2.0 mL/5.0 mL )

1|P a g e I M M U N O H E M A T O L O G Y L A B O R A T O R Y - 2 0 2 2 - 2 0 2 2
 LESSON 3: Preparation of Red Cell Suspensions with Different Concentrations
B. Reagents/Sample

1. Normal saline solution (0.85%-0.95%) in wash bottle

2. Anticoagulated blood(EDTA)

C. Procedure

WASHING
1. Pipette 3mL of whole blood into 10-mL conical tube.
2. Fill the tube with NSS until it reached the 10 mL.
3. Cover the tube with screw cap and gently mix by inversion.
4. Centrifuge for 5 minutes at 3,400 rpm
5. Decant by aspirating the supernatant using a pasteur pipette. Be sure that the packed red cells
are not disturbed.
6. Resuspend the cells with another volume of NSS until it reaches the same initial measurement.
7. Repeat steps 3-5
Note: washing the cells is preferably done 3 times
8. Set wash red cells aside

RED CELL SUSPENSION

1. Label 4 Wasserman tubes as 2% RCS, 3%RCS, 4%RCS and 5% RCS.
2. Using calibrated pasteur pipette, add _______ of washed packed red blood cells into
Wasserman tubes
3. Using a 2.0 mL or 5.0 mL serological pipette/automatic pipette, deliver the exact amount of NSS
to the following volume for each prepared tube:
2% TUBE 1=5ml = 4.9 mL
3% TUBE 2=4ml = 3.9 mL
4% TUBE 3=3ml = 2.9 mL
5% TUBE 4=2ml = 1.9 mL
4. Cover the prepared tube with Nescofilm and mix gently (if the suspension is correct the

(Formula: TV x % suspension = mL of WPRBC)

Note: For best results red cell suspensions should be used for testing on the day of preparation.

Variations in red cell concentration can markedly affect the sensitivity of tests results. If red
cell suspensions are too concentrated, it may mask weak agglutination. When red cell suspensions
are too low in concentration, they become difficult to visualize, and in extreme cases, a weak
positive can fail to be detected.

2|P a g e I M M U N O H E M A T O L O G Y L A B O R A T O R Y - 2 0 2 2 - 2 0 2 2
 LESSON 3: Preparation of Red Cell Suspensions with Different Concentrations

Exercise 3

1. Why is NSS used to wash the red blood cells?

___________________________________________________________________
___________________________________________________________________
_______________________________________________________

2. Why is Pasteur pipette used to deliver the WPRBC rather than the serological pipette?
___________________________________________________________________
___________________________________________________________

3. What is the importance of using red cell suspension in the laboratory?

___________________________________________________________________
___________________________________________________________

Using the formula given above complete the table in preparing red cell suspension with
desired concentration.

Total volume= amount of WPRBC + amount of NSS to be added

Desired % of Amount of washed Amount of NSS to be Total Volume

suspension packed red blood added
cells
0.24mL 8mL
2.5% 10mL
0.12mL 2.88mL

3|P a g e I M M U N O H E M A T O L O G Y L A B O R A T O R Y - 2 0 2 2 - 2 0 2 2
 LESSON 3: Preparation of Red Cell Suspensions with Different Concentrations
References:

-Cardona, C.C, Martin , G.L, and Garcia- Meim, R. (2016) Laboratory Manual in Blood Banking
2nd Edition

-Whitlock, S. A., Immunohematology for Medical Laboratory Technicians 2010

4|P a g e I M M U N O H E M A T O L O G Y L A B O R A T O R Y - 2 0 2 2 - 2 0 2 2