Citrate-phosphate-dextrose (CPD) anticoagulant
Citrate-phosphate-dextrose-adenine (CPDA1) anticoagulant
CPD/AS-1 anticoagulant/preservative system
CP2D/AS-3 anticoagulant/preservative system
CPD/AS-5 anticoagulant/preservative system

CONSUMABLES

1. Glycerol:

A. Glycerolyte 57 (6.2 M glycerol, 500 ml bottle). Each 100 ml contains 57 g glycerin, 1.6 g sodium lactate, and 30 mg potassium chloride, buffered with 51.7 mg monobasic sodium phosphate (monohydrate) and 124.2 mg dibasic sodium phosphate (dried), pH 6.8 (Fenwal #4A7833)

B. 6.2 M Glycerolizing Solution (500 ml bottle). Each 100 ml contains 57.1 g glycerin, 1.6 g sodium lactate, and 0.03 g potassium chloride, buffered with 43 mg monobasic sodium phosphate and 220 mg dibasic sodium phosphate, pH 7.0 (Cytosol PN-5500)

2. Plasma transfer set with a coupler and needle adapter (Fenwal #4C2240)

3. Sterile docking wafers (Terumo 3NCC987)

4. Sterile filtered airway needle (B-D 5200)

5. Labels for the freezing bag and for the cardboard storage box

6. Heat-sealable plastic bags (3), 8" X 12" (Kapak/Scotchpak 404)

7. Corrugated cardboard storage box. (Dimensions: 7" X 5.25" X 2" outside)

8. Alcohol swab (70%) (B-D 6894)

NOTE: The following 2 items are not needed if the blood was originally collected into the 800 ml plastic bag system

9. 600 ml polyvinyl chloride plastic bag (Baxter 4R2023)

10. 1000 ml polyvinyl chloride plastic bag (Baxter 4R2032)

WARMING PROCEDURE

At the time of glycerolization, the red cells, glycerol solution and room temperature should be within a temperature range of 20 C (68 F) to 30 C (86 F). The temperature of a bottle of glycerol located in the storage area should be monitored by inserting a calibrated thermometer into the full bottle of glycerol or using an infra red scanner. If the glycerol is below 20 C, the glycerol can be warmed to a temperature of 20-26 C by incubation at 37 C for the appropriate time to achieve the desired temperature. The red cells can be warmed by one of the following manipulations:

A. Remove the liquid red cell concentrates from the 4 C refrigerator and place them in sealed, double plastic bags for protection against wetting. Immerse the double-bagged units in a 37 C water bath for 10-20 minutes.

B. Remove the liquid red cell concentrates from the 4 C refrigerator and place each unit in a Thermogenesis plasma thawer pouch for 5 minutes at 36 C.

C. Remove the liquid red cell concentrate from the 4 C refrigerator and store at room temperature for a period of about 2 hours.

The water bath and plasma thawer procedures are described below.

a. Water bath method:

1. If using the water bath, turn on the power switch located at the end of the water bath. Allow the water to warm to 37 C. This will take approximately 1 hour. Switch on the circulating pump in the water bath a few minutes prior to use to ensure a uniform temperature of 37 C throughout the bath. Temperature is measured with a thermometer that has been verified against a National Institute of Standards and Technology (NIST) certified thermometer.

2. Place the 800 or 1000 ml bag containing the red cell concentrate in a plastic bag and heat seal the bag. Place the sealed plastic bag inside a second plastic bag and heat-seal. Keep the unit submerged during incubation by adding lead weights on top.

NOTE: Each plastic overwrap bag must be flattened to remove all the air prior to sealing. If this is not done properly, the units will float on the surface of the water bath during incubation, and the desired temperature will not be achieved.

3. Incubate the unit in the 37 C water bath for 10-20 minutes. The temperature of the red cells should be 20 C-30 C. Measure the surface temperature of the unit with an infrared scanner or NIST certified thermometer.

4. Remove the bag from the water bath; wrap the unit loosely in a clean, dry disposable towel, dry the surface of the overwrap, and remove the plastic overwraps from the primary bag, assuring that the inner bag and unit are not contaminated with any water from the water bath.

5. The red cells are now ready for glycerolization.

b. Plasma thawer method

1. If using the plasma thawer, turn on the power to the plasma thawer and allow the system to warm to 36 C.

2. Place a unit in each pouch of the plasma thawer.

NOTE: Plastic overwrap bags are not required when the plasma thawer is used.

3. Set the timer for 5 minutes. At the end of 5 Minutes the temperature of the red cells should be 20-30 C. Remove the unit from the pouch and measure the surface temperature of the unit with an infrared scanner or NIST certified thermometer.

4. The red cells are now ready for glycerolization.

GLYCEROLIZATION PROCEDURE

1. Weigh the unit and record the weight on the enclosed glycerolization worksheet.

2. Remove a plasma transfer set from its box, slide the roller clamp to the coupler end of the plasma transfer set, and close the roller clamp.

3. Sterilely dock the plasma transfer set to the 800 ml or 1000 ml bag containing the red blood cells. Squeeze weld.

4. Place the bag containing the red cells on the shaker platform.

5. Remove the metal pull tab from the top of the glycerol bottle, swab the rubber stopper with an alcohol swab (70%), and then aseptically insert the coupler end of the plasma transfer set into the outlet port of the glycerol bottle stopper.

6. Insert a filtered airway needle into the vent port of the glycerol bottle stopper. As the bottle vents, invert the glycerol bottle and install it on the support stand hook provided on the shaker so that the rubber stopper on the bottle of glycerol is held 18 inches (45 cm) above the level of the bag on the shaker.

7. Using the nomogram (Table 2) and the previously recorded net weight, determine the volume of glycerol solution to be added to the red cells during each of the three glycerol addition steps. Using the factory graduations as a guide, mark the volume of glycerol to be added for each of the three steps.

8. Switch the modified Eberbach shaker on low speed (180 oscillations/minute).

9. Open the roller clamp of the plasma transfer set and add the first volume of glycerol from the solution
bottle directly into the bag containing the red cells.

10. Close the roller clamp, turn off the shaker and equilibrate the red cells for 5 minutes.

11. Switch the modified Eberbach shaker on low speed. Open the roller clamp and add the second volume of glycerol from the solution bottle directly into the bag containing the red cells.

12. Close the roller clamp, turn off the shaker and equilibrate the red cells for 2 minutes.

13. Turn on the shaker, open the roller clamp and allow the third, and final, volume of glycerol to enter directly into the 800 or 1000 ml bag.

14. Close the roller clamp and heat seal the tubing between the empty bottle of glycerol and 800 or 1000 ml bag. Leave approx. 7 inches of tubing on the bag containing the glycerolized red blood cells. If no transfer pack is attached to the bag containing the glycerolized red blood cells, sterilely dock a 600 ml transfer pack onto the bag containing the glycerolized red blood cells. Do not squeeze the weld.

15. Roll the bottom 2-3 inches of the bag containing the red blood cells and tape. Spin the glycerolized red cells at 1248 X g in a 22 C refrigerated centrifuge for 10 minutes (Table 1).

NOTE: The brake on the centrifuge should be set at zero. This brake setting will minimize red cell mixing which occurs as the rotor slows down from maximum to zero.

It is essential that these instructions be followed exactly as written. The centrifugation speed and time are specific to glycerolized red blood cells and should not be confused with centrifugation speeds used to concentrate liquid-stored red blood cells. An increase in the centrifugation speed will result in irreparably damaged red blood cells that will exhibit excessive hemolysis during the deglycerolization and post-wash storage period. A decrease in the centrifugation speed or use of the brake during centrifugation will result in an increased supernatant volume. If the glycerolized red blood cells contain more supernatant than expected, the volume of wash solution may not be sufficient to wash the red blood cells and adequately remove the glycerol.

16. Carefully remove the unit from the centrifuge and place it on a plasma extractor. Squeeze weld. Express all visible supernatant glycerol from the 800 or 1000 ml bag into the 600 ml transfer bag to achieve a hematocrit of 60 + 5 V%. When red cells appear in the cannula, clamp the integral tubing with a hemostat. Remove the bag from the plasma extractor and resuspend and mix the glycerolized red cells thoroughly by manual agitation. The glycerolized red cell concentrate must be resuspended completely before freezing to prevent hemolysis.

17. Heat seal and detach the 600 ml transfer pack leaving at least 6 inches of tubing attached to the 800 or 1000 ml bag.

18. Affix the following labels to the bag (Figure 3):

A blood product overlay label to indicate that the product has been processed into "Red Blood Cells, Frozen"

A freezing facility label label with the manufacturer's name and bag lot number readable.

An ABO, Rh confirmation label.

And an infectious disease testing label affixed to the back-side of the freezing bag (Figure 4).

19. Mark the label with the expiration date of the
blood product, which is currently 10 years from the day of collection. Weigh the unit just prior to freezing and record the gross weight of the glycerolized red cells.

20. Fold over the top portion of the 800 ml or 1000 ml bag (approx. 2-4 inches) and then place the unit into a plastic bag overwrap (8" X 12") and seal across the top using an impulse sealer so that there is as little trapped air as possible (Figure 3). The plastic bag will not break during freezing and the sealer will provide an air-tight and leakproof seal to ensure protection of the unit at the time of thawing. Make sure that the ports and tubing segments are folded beneath the unit so that they are protected from breakage when frozen.

21. Place two polyethylene cryogenic vials of plasma or sera into the cardboard box. The remaining two vials are frozen separately at -80 C

22. Place the plastic bag containing the glycerolized red cells into the cardboard box and close the box (Figure 5). Affix a product label, ABO/Rh label, expiration date, collection facility ID label, unit number label, freezing facility ID label, and label indicating infectious disease marker testing performed on the unit, on the outside of the box. Place the cardboard box in a -80 C freezer for freezing and storage (Figure 6).

23. Each unit should be frozen at the bottom of the -80 C freezer during the initial 24-hour period to ensure proper freezing. To avoid improper freezing, the units should not be stacked on each other. After the initial 24-hour period of freezing at the bottom of the -80 C freezer, the frozen units can be stacked and stored in other -80 C freezers.

NOTE: No more than 4 hours should be allowed to lapse between the time the red cells are removed from the 4 C refrigerator and the time they are placed in the -80 C freezer. The final concentration of glycerol is approximately 40% W/V and the hematocrit of the glycerolized unit is approximately 60 + 5 V%.