Preparation of Complement Sensitized Red Cells


Incubating whole blood with a low ionic sucrose solution enhances the uptake of complement components onto red cells. Trypsin will cleave C3b to expose the C3d and C4d sites.

2.0Scope and Related Policies

2.1Complement coated cells will be prepared on an as needed basis for the evaluation of AHG reagents.

2.2These cells may be frozen in LN2 for future use for up to 10 years.

3.0Specimen – N/A



Block for test tubes

Mechanical stirrer and stirring magnet


Erlenmeyer flask

Squeeze bottle

Metal basin

Supplies:Ice cubes

Test tubes – 10 x 75 mm

Serological pipettes

Weigh boats

pH strips

Reagents:0.9% Normal saline


Na2 EDTA 2H20

NaH2 PO4 H20

Distilled Water

Na2H PO4

MgCl2 6H20

0.1% Trypsin

Anticoagulated whole blood (CPD or ACD)*

* See Procedural Note 8.1

Solution A

Sucrose - 23.1g

Na2 EDTA 2H20 - 0.395g

NaH2 PO4 H20 - 0.173g

Distilled H20 - Made up to a final volume of 250 mL

Solution B

Sucrose - 23.1 g

Na2 EDTA 2H20 - 0.395 g

Na2H PO4 - 0.178 g

Distilled H20 - Made up to a final volume of 250 mL

Solution C

MgCl2 6H20 - 0.18 g in 10 mL Distilled H20

0.1% Trypsin (DIFCO Lab)

5.0Quality Control – N/A


Procedure 1 - For Preparation of C3b coated RBC

6.1Prepare the above solutions, chill "squeeze" bottle containing 9% normal saline to 0-1°C (in ice bucket).

6.2To 250 mL of Solution A, add a sufficient amount of Solution B, (usually 15-25 mL) to produce a mixture at pH 5.1 (use pH meter or strips to measure pH).

6.3Place 195 mL of the mixture in Erlenmeyer flask and add mechanical stirrer.

6.4Place water and a few ice cubes in metal basin.

6.5Put flask into basin.

6.6Once mixture is chilled, add 1 mL of ACD or CPD anticoagulated blood.

6.7Immediately add 0.1 mL of solution C.

6.8Place on Magnetic Stirrer and stir gently for 1 hour maintaining temperature at 0-1°C with ice in basin (measure temperature periodically).

6.9Transfer the mixture to test tubs and wash and suction 3 times with 0.9% cold normal saline (squeeze bottle kept in ice bucket).

6.10Prepare appropriate cell suspension 3-5%.

Procedure # 2: For Preparation of C3d (and C4d) coated RBC

6.11Pack C3b coated RBCs prepared in Procedure # 1, suction off saline.

6.12For each drop of packed C3b coated cells, add 1 drop of 0.1% Trypsin in PBS (pH 7.3) ratio 1:1. See Procedural Note 8.2.

6.13Incubate at 37° C for 30 minutes.

6.14Wash RBCs 3 times in 0.9% normal saline.

6.15Make appropriate 3-5% cell suspension.

7.0Reporting – N/A

8.0Procedural Notes

8.1EDTA inhibits the uptake of complement therefore EDTA anticoagulated blood must not be used for this procedure.

8.20.1% Trypsin is prepared from 1% stock solution diluted 1 in 10 in PBS pH 7.3.


9.1Issitt PD, Anstee DJ. Applied Blood Group Serology, 4th ed. Durham, NC: Montgomery Scientific Publications, 1998: 97.

/ Ontario Regional Blood Coordinating Network
Standard Work Instruction Manual / RP.003
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