(Blood Bank Name and address) / Procedure number: 278 / Page 1 of 2
Subject: Thawing Red Cells Frozen in 40% Glycerol

THAWING RED CELLS FROZEN IN 40% GLYCEROL

PURPOSE

To deglycerolize red cells which are frozen in a 40% glycerol-citrate solution.

PRINCIPLE

This procedure is used when preparing in-house reagent red cell antibody detection and identification panel for the week. After thawing the desired tubes of RBCs, solutions of 16%, 8%, 4%, and 2% glycerol are added to the RBCs in succession, allowed to equilibrate, and then removed. Finally the RBCs are washed multiple times with saline. These deglycerolized red cells can then be used to prepare red cell suspensions, or stored in liquid preservative solution for use on subsequent days.

POLICY

If hemolysis persists after saline washes, do not use the red cells.

MATERIALS AND EQUIPMENT

1.Tubes containing 3 – 4 mL of RBCs frozen in 40% glycerol solution.

2.16% glycerol citrate thawing solution, stored at 2-6C

3.8% glycerol citrate thawing solution, stored at 2-6C

4.4% glycerol citrate thawing solution, stored at 2-6C

5.2% glycerol citrate thawing solution, stored at 2-6C

6.0.85% saline

7.Red Cell Preservative Solution

PROCEDURE

1.Remove appropriate frozen red cell aliquots from the freezer and thaw at 37C (approximately 5 minutes).

2.Centrifuge the tubes at least 1-2 minutes on high speed and aspirate the supernatant.

3.Add approximately 2 mL of 16% glycerol citrate thawing solution to each tube, and mix thoroughly.

4.Let the tubes equilibrate at least 5-10 minutes at room temperature.

5.Centrifuge the tubes at least 1-2 minutes on high speed, and aspirate the supernatant.

6.Repeat steps 3, 4, and 5 using the 8% glycerol-citrate thawing solution, followed by the 4% and 2% solutions.

7.Wash the red cells with 0.85% normal saline until the supernatant saline is clear (usually 2-3 times).

8.Remove an aliquot of each tube of the washed RBCs, and prepare cell suspensions for testing for the current day.

9.Add an equal volume of Red Cell Preservative Solution to the remaining red cells. Mix and cap the tubes. Store at 2-6oC.

RECORD KEEPING

Record which vials were thawed on the appropriate page in the Reagent Preparation Log.

REFERENCES

Adapted from the Blood Center of Wisconsin Immunohematology Reference Laboratory procedure manual.

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