STANDARD OPERATING PROCEDURE

GLYCEROLIZATION AND DEGLYCEROLIZATION OF RED BLOOD CELLS
IN A CLOSED SYSTEM USING THE HAEMONETICS ACP215

NAVAL BLOOD RESEARCH LABORATORY
PHONE: (508) 747-4472
FAX (508) 759-2317
info@nbrl.org

TABLE OF CONTENTS

INTRODUCTION
COLLECTION AND PREPARATION OF BLOOD COMPONENTS
GLYCEROLIZATION
THAWING
DEGLYCEROLIZATION
QUALITY CONTROL
SHIPPING INSTRUCTIONS
TROUBLESHOOTING
EQUIPMENT LIST
VENDORS
ENCLOSURE 1
COMPETENCY EVALUATION
SELF-ADMINISTERED QUIZ
ANSWER SHEET
RELATED REFERENCES

This work was supported by the U.S. Navy (Office of Naval Research Contract N00014-94-C-0149, with the funds provided by the Naval Medical Research and Development Command).

REVIEWED AND APPROVED: C. ROBERT VALERI, M.D., DIRECTOR

INTRODUCTION

This is a Standard Operating Procedure (SOP) to glycerolize and deglycerolize human red blood cells using the Haemonetics ACP215 automated, closed system. The red blood cells can be collected into any FDA-approved anticoagulant or anticoagulant-preservative system. Prior to freezing the whole blood or additive-stored red blood cells are concentrated to prepare a red blood cell concentrate with a hematocrit value of approximately 75 V%. If the blood was not collected into the 800 ml PVC plastic bag collection system, the red blood cells are transferred into a standard 1000 ml polyvinyl chloride plastic bag using a sterile docking procedure. The red blood cells are glycerolized using the ACP215 and the glycerolized red blood cells are concentrated and the supernatant glycerol removed prior to freezing. The glycerolized red cells are washed in the Haemonetics ACP215 and resuspended in AS3 solution for post-wash storage.

The ACP215 system provides a functionally closed system for both the glycerolization and deglycerolization procedure resulting in a reduced potential for bacterial contamination. This, in addition to the resuspension of the deglycerolized red blood cells in AS-3 solution, allows for storage of the red blood cells at 4 C for up to 14 days following washing.

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

NOTE: If red blood cells were collected into the 800 ml quadruple polyvinyl chloride plastic collection bag system, no transfer is required (see Figure 2 & Figure 2a).

EQUIPMENT

Haemonetics ACP215

Waterbath or Thermogenesis Model MT204 or MT210 Plasma

Thawer

NIST Certified Thermometer

 

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. Glycerolization harness (Haemonetics LN225)

3. Sterile docking wafers (Terumo 3NCC987)

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

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

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

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

8. Latex gloves

NOTE: The following 2 items are NOT required if the blood was originally collected in the 800 ml polyvinyl chloride plastic bag collection system (Fenwal #4R1243 or MedSep #746-74)

9. 600 ml polyvinychloride plastic bag (1-2) (Baxter 4R2023)

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

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.

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 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.

A. Turning on the ACP215

1. Plug the shaker cord into the ACP215 power outlet and place it on the left side of the machine.

2. Plug the ACP215 main power cord in and turn the machine on. The machine will perform a series of internal checks prior to allowing you to select a protocol and the following message will appear on the screen:

3. Use the 'MODIFY PROGRAM' key to select the GLYCEROLIZATION protocol and press YES using the UP-ARROW key. The screen will now appear as follows:

B. Loading the glycerolization harness

1. Weigh and record the weight of the unit. To obtain the gross weight of the unit, weigh only the bag containing the red blood cell concentrate (i.e. 1000 ml or 800 ml bag). Do not include any attached transfer packs. To obtain the net weight of the red cells, subtract 42 g (weight of empty 800 ml bag) or 44 g (weight of empty 1000 ml bag) from the gross weight.

2. Remove the glycerolization harness from the plastic pouch and visually inspect it for tubing kinks, missing parts, and package integrity.

3. Close the ratchet clamp on the spike connector portion of the tubing segment.

4. Load the portion of the glycerolization disposable set between the two blue pump stop markings into the blood pump of the ACP215. Be sure that the heat-sealed portion of the glycerolization line is toward the front of the machine near the blood pump air detector.

5. Load the pump outlet tubing into the blood pump air detector. The Gelman anti-bacterial filter will hang vertically on the pump tubing in the front of the ACP215.

6. Connect the 0.2 micron filter to the pressure sensor located on the front of the ACP215.

7. The ACP215 will respond with the following screen:

Press 'yes' to scroll. Enter your initials using the up/down arrow keys. Press 'save' to accept initials.

8. The ACP215 will then produce the following screen:

 9. You are now ready to begin the glycerolization procedure and the following screen is displayed.

c. Connecting the solutions and blood to the glycerolization disposable set

1. 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 spike end of the glycerolization harness into the outlet port of the glycerol bottle stopper. Assure that the air vent is open on the spike to allow air to flow into the bottle while it empties.

2. Suspend the glycerol bottle to the IV pole of the ACP215.

3. Sterilely dock the glycerolization disposable to the bag containing the concentrated red blood cells. Use the donor collection line if the red blood cell concentrate is in the 800 ml plastic bag system.

4. Place the bag containing the red blood cells on the shaker platform; allow the attached transfer pack to hang over the shaker platform.

5. Unclamp the ratchet clamp on the glycerol line.

6. The glycerolization disposable set is now loaded and the ACP215 will perform a set of verification questions prior to proceeding.The display will nowprompt the operator to enter his/her initials.

7. The ACP215 prompts the operator to start the procedure. Press the 'MODIFY' key to enter the weight of the red blood cell concentrate to be glycerolized.

8. Press the 'START' key to begin the procedure.

D. Glycerolization procedure

1. Once the 'START' key is pressed, the shaker begins to shake and the blood pump begins to rotate counter-clockwise.

2. The glycerol solution will begin to flow from the glycerol bottle through the blood pump air detector, the plastic tubing, and the anti-bacterial filter at a computed flow rate. If no fluid detection occurs within 10 ml, the pump stops and the message 'CHECK LINE OCCLUSION' is displayed.

3. Five ml after the blood pump air detector has sensed fluid, the anti-bacterial filter is primed and the volume of glycerol solution begins to be counted and displayed on the screen.

4. The glycerol flow rate will progressively increase at a computed rate until the computed volume of glycerol has been added to the red cell concentrate. If any air detection occurs during the glycerolization, the pump stops and the message 'AIR DETECTED' is displayed. Check to confirm that the disposable is installed properly and slide clamps on all solutions are open.

5. The pressure is monitored throughout the glycerolization procedure, and if it exceeds 200 mm Hg, the blood pump will stop and the following message is displayed 'CHECK FOR LINE OCCLUSION, PRESS START TO RESUME, STOP TO END GLYCEROLIZATION'.

6. The shaker will continue to shake for 30 seconds following addition of the glycerol and the message 'GLYCEROLIZATION COMPLETE' is displayed. The printer will print out information related to the glycerolization procedure (see enclosed sample, Figure 3). If the printout is not acceptable, the ACP215 will give you the option to reprint it.

7. Heat seal the tubing between the empty bottle of glycerol and the glycerolized red blood cells. Leave approximately 7 inches of tubing attached to bag containing the red blood cells.

8. Discard the empty glycerol bottle and the glycerolization harness.

9. Sterilely dock a 600 ml transfer pack to the 1000 ml bag containing the glycerolized red blood cells. Do NOT squeeze the weld open. If using the 800 ml PVC plastic bag system, no 600 ml transfer pack is required.

10. Roll the bottom 2-3 inches of the 800 or 1000 ml 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.

11. 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 ematocrit 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.

12. Place the unit back onto the plasma extractor. Unclamp the hemostat and allow mixed glycerolized red cells to fill the integral tubing. Then clamp the tubing and heat seal the transfer bag as close as possible to the base of the 600 ml bag. Discard the transfer pack containing the supernatant glycerol. Heat seal and detach segments of the tubing containing the glycerolized red cells. This will be used for future crossmatch segments. Freeze these segments alongside the cryogenic vials in the cardboard freezing box. Be sure to leave at least 6 inches of tubing attached to the 800 or 1000 ml bag to be used for sterile docking during the deglycerolization procedure.

13. Affix the following labels to the 800 or 1000 ml bag (see Figure 4):

A. Blood product overlay label to indicate that the product has been processed into "Red Blood Cells, Glycerolized and Frozen in a Closed System"

B. Freezing facility label label with the manufacturer's name and bag lot number readable.

C. ABO, Rh confirmation label.

D. Infectious disease testing label (affixed to the back-side of the freezing bag) (see Figure 5).

14. 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.

15. Fold over the top portion of the bag (@3-4 inches for the 1000 ml bag and 2 inches for the 800 ml bag) 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. 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 (see Figure 6).

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

17. Place the plastic bag containing the glycerolized red cells into the cardboard box and close the box. 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 (see Figure 7). Place the cardboard box in a -80 C freezer for freezing and storage.

18. 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 areremoved 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 hematocritof the glycerolized unit is approximately 60 + 5V%.

THAWING

A unit of glycerolized frozen red cells can be thawed using one of two methods: a) rapid immersion into a heated water bath maintained at 42 C for approximately 45 minutes; or b) by placement into one of the pouches of a plasma thawer maintained at 36 C for 35 minutes. Upon removal from the water bath or plasma thawer, the surface temperature of the red cells is measured using an infrared scanner or a NIST certified thermometer and should be between 30 and 34 C.

A. Water bath method

1. If using the waterbath, turn on the power switch located at the end of the water bath. Allow the water to warm to 42 C. This will take approximately 1 hour. Switch on the circulating pump in the water bath used to thaw the frozen red cells. Allow the pump to run for 1-2 minutes, then check the water temperature to ensure that it has stabilized at 42 C.

2. Using freezer gloves, remove the box containing the red cells from the freezer. Record the time on the Deglycerolization Logsheet when the frozen red cells are placed in the water bath as the beginning of the deglycerolizing time period. Processing must be completed and the deglycerolized red cells must be placed in a 1-6 C refrigerator within 2 hours of removal from the -80 C freezer.

3. Open the freezing container and remove the unit of

frozen red cells. Thaw the unit still in its plastic overwrap by immersing it in the water bath. Place lead weights on top of the units so that the units remain submerged during the thawing procedure.

NOTE: The thawed red cells inside the plastic overwrap should remain in the water bath until they reach atemperature between 30 and 34 C. This will normally take approx. 45 minutes.

4. Remove the unit from the water bath and check the temperature of the unit using an infrared scanner. If the temperature of the unit is not between 30 and 34 C, replace the overwrapped unit back into the water bath.As described above, recheck the temperature every 5 minutes until the desired temperature is achieved.

5. Remove the unit from the water bath and dry off the overwrap. Tear open the overwrap and discard it. Wrap the thawed unit loosely in a disposable white towel. Check the bag for any breaks by gently compressing the unit in the towel, wiping the entire bag surface with the towel and then inspecting the towel for blood stains. The presence of blood stains on the towel is evidence of bag breakage, and the unit must be considered contaminated. Units suspected of being contaminated should be disposed of in compliance with local Standard Operating Procedures for the disposal of liquid-stored blood products.

5. The thawed glycerolized red cells are now ready for deglycerolization.

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 (+ 1 C).

2. Using freezer gloves, remove the box containing the red cells from the freezer. Record the time on the Deglycerolization Logsheet when the frozen red cells are placed in the water bath as the beginning of the deglycerolizing time period. Processing must be completed and the deglycerolized red cells must be placed in a 1-6 C refrigerator within 2 hours of removal from the -80 C freezer.

3. Open the freezer container and remove the unit of frozen red cells. Remove the plastic overwrap and place the unit into one of the pouches of the plasma thawer.

NOTE: The thawed red cells without the plastic overwrap should remain in the pouch of the plasma thawer until they reach a temperature between 30 and 34 C. This will normally take approx. 35 minutes.

4. Remove the unit from the plasma thawer. Wrap the thawed unit loosely in a disposable white towel. Check the bag for any breaks by gently compressing the unit in the towel, wiping the entire bag surface with the towel and then inspecting the towel for blood stains. The presence of blood stains on the towel is evidence of bag breakage, and the unit must be considered contaminated. Units suspected of being contaminated should be disposed of in compliance with local Standard Operating Procedures for the disposal of liquid-stored blood products.

5. The thawed glycerolized red cells are now ready for deglycerolization.

DEGLYCEROLIZATION

I. INTRODUCTION

The Haemonetics ACP215 is a closed, automated system with an attached shaker and printer. The system is designed to both glycerolize and deglycerolize red blood cells. After the glycerol-frozen red cells have been thawed, the plastic freezing bag is secured to the platform on the Haemonetics ACP215 by means of magnetic mounting posts. The ACP215 first dilutes once with 12% sodium chloride, and then with 0.9% sodium chloride-0.2 gm% glucose solution, utilizing the shaking platform to ensure adequate mixing. After dilution of the red cells, the ACP215 delivers the diluted red cells from the primary collection bag into the bowl and the washing procedure begins.

At the completion of the wash cycle, the centrifuge is stopped, the ACP215 then washes the red blood cells once the AS-3 preservative solution and finally resuspends the washed red cells into AS-3 preservative solution. The deglycerolized-resuspended red cells are siphoned from the wash bowl into the blood product bag of the Haemonetics deglycerolization disposable set. The unit is labeled with the expiration date and time.

II. MATERIALS

EQUIPMENT

Haemonetics ACP215

Sterile connection device

Heat sealer

CONSUMABLES

1. Deglycerolization disposable set (Haemonetics LN235)

2. 12% Sodium chloride solution (150 ml plastic bag) (Fenwal 4B7874); each 100 ml contains: 12 g sodium chloride USP

NOTE: Only 50 ml of this solution are used for each unit of red blood cells.

3. 0.9% sodium chloride-0.2 gm% glucose solution (2-liter plastic bag (Fenwal 4B7878); each 100 ml contains: 200 mg dextrose (anhydrous) USP, 900 mg sodium chloride USP.

NOTE: Only 1.6 liters of this solution are used for each unit of red blood cells.

4. AS-3 Preservative solution (300 ml bag); Haemonetics Corp. or MedSep Corp.

5. Latex gloves

6. Sterile docking wafers (Terumo 3NCC987)

III. MACHINE SET-UP

A. Installing the disposable

1. Plug the shaker into the ACP215 power outlet and place it on the left side of the ACP215.

2. Plug in the ACP125 and turn the power on. The ACP215 will perform a series of checks and, when complete, the following message will appear on the screen:

3. Using the 'MODIFY PROGRAM' key, select the 'DEGLYCEROLIZATION' protocol and press 'YES' using the UP-ARROW key. All the valves will open and the screen will read as follows:

4. Verify the integrity of the disposable set packaging by visually inspecting for any tears or holes and that the plastic lid is properly sealed (see Figure 8).

5. Verify the integrity of the disposable set (no tubing kinks or other visible defects. Close all slide clamps.

6. Press on the knobs of the centrifuge chuck and turn counter-clockwise to open the lid.

7. Load the bowl in the centrifuge chuck so that the bowl header is oriented for the effluent port (the clear line) to exit the right and the blue port exits left. Press hard on the bowl shoulder to ensure that it is properly seated in the chuck. Close and secure the centrifuge cover and confirm that the centrifuge cover is in the locked position by turning the knob clockwise.

8. Load the two pump tubing segments into their respective pump rotor: the 0.22 um filter must be placed between the solution pump outlet (tubing guide closest to the blood pump outlet tubing guide) and the 'Y' connector to the bowl. In addition, properly load the tubing segments into the two air detector sensors.

9. Load the six (6) valves with their corresponding tubing segments. Each of the six (6) tubing segments have a colored stripe that matches the color of a valve stem: Red for the bag containing the thawed red blood cells; Clear for the bag containing the washed red blood cells; Green for the bowl effluent line; Blue for the hypertonic 12% sodium chloride solution; Yellow for the 0.9% sodium chloride-0.2 gm% glucose solution; and Orange for the AS-3 preservative solution.

10. Load the clear effluent line tubing into the line sensor (making sure to completely seat the tubing), and close the cover.

11. Hang the waste bag from the large bag pins provided on the right side of the front panel. Hang the red blood cell product bag upside down from the small bag pins provided on the left side of the front panel.

12. Connect the two hydrophobic 0.2 um filters, one to the blood pump pressure sensor (BPPS) and the other to the bowl pressure sensor (BPS). Press 'YES' to continue. After both pressure sensors are properly loaded, press 'YES' to continue. The valves will automatically close.

13. The machine will then ask if you want to calibrate the line sensor. Press 'YES' to calibrate. Press 'NO' to bypass the calibration. Haemonetics Corporation recommends that you calibrate the ACP215 line sensor at least once daily (see Operatorís Manual).

B. Validating the line sensor calibration

1. Following installation of the disposable, the ACP215 will prompt you to validate that the line sensor is calibrated.

2. The ACP215 will tell you to insert filter #1. Insert filter #1 into the line sensor. This filter allows for complete light transmittance.

3. The ACP215 will then tell you to insert filter #2. Insert filter #2 into the line sensor. This filter is for 50% light transmittance.

4. The ACP215 will then tell you to insert filter #3. Filter #3 completely obstructs the light transmittance. If the values are acceptable, the ACP215 will inform you that the values are within the acceptable range and permit you to continue with the deglycerolization procedure.

5. If the ACP215 informs you that the values are not acceptable, repeat the validation procedure described above. If the repeat validation is not acceptable, the ACP215 instrument cannot be used until Haemonetics service personnel have evaluated the line sensor.

C. Bowl Seating Test

1. Following line-sensor calibration, the ACP215 will perform a bowl seating test to ensure that the bowl is properly inserted in the machine.

2. The centrifuge will run for 30 seoncd and the machine will ask if you heard any unusual noises during the testing. If you did not, press NO and continue on with the procedure. If you hear a loud or high-pitched noise, press YES and follow the instructions on the machine.

D. Connecting the blood and solutions to the disposable set

1. Spike the hypertonic solution bag to the bag line.

2. Spike the saline-glucose bag to the yellow line.

3. Connect the luer lock connector to the preservative solution to the orange line and crack the breakable seal. Verify that the breakable seal is fully separated internally by bending the component back and forth and rolling it between your fingers.

4. Perform a sterile connector device (SCD) welding between the red striped line and the tubing of the thawed-frozen red blood cell unit. If the red cell bag has two tubing segments, select the tubing segment that does not have the breakable seal. Verify that the SCD welding is not occluded by squeezing the weld area between your fingers and rolling to fully open the weld.

NOTE: If the tubing segment without the breakable seal is not long enough for insertion into the SCD machine, you may use the tubing segment with the breakable seal. After SCD connection, verify that the breakable seal is fully separated internally by bending the component back and forth and rolling it between your fingers and that the weld is not occluded by squeezing the weld area between your fingers and rolling to fully open the weld. If you encounter problems with flow during the procedure, check this tubing again to be sure you have fully opened the breakable seal.

5. Place the thawed-glycerolized red blood cell unit securely on the mounting plate of the shaker.

6. One by one, the ACP215 will ask you to verify that all colored tubing segments are loaded properly in the appropriate valves. Open all slide clamps and again verify that the SCD weld is fully open and no tubing kinks are present.

7. At this point, the ACP215 acknowledges that the disposable set is correctly loaded and the screen displays the statement 'ENTER OPERATOR INITIALS' followed by 'PRESS MODIFY TO PROGRAM, START TO BEGIN'. Verify the programming parameters for the procedure.

8. Press the 'START' key to begin the procedure.

E. Deglycerolization procedure

 Once the 'START' key is pressed, the ACP215 will evaluate the intregrity of the sterile connection weld by holding and monitoring a negative pressure. The ACP215 will then automatically start the deglycerolization procedure and complete the following steps:

1. Remove air from the product bag. The machine should be operating as follows:

This tells you that the blood pump should be turning clockwise, and the solution pump is stopped, the centrifuge is stopped and the clear valve to the red blood cells and the green valve to the waste bag are both open.

2. The machine will begin adding the 50 ml of 12% sodium chloride (hypertonic) solution and operating as follows:

The blood pump is now turning counter-clockwise as is the solution pump. The shaker is on and the blue valve to the hypertonic 12% NaCl solution and the red valve to the red blood cells are both open, allowing for delivery of the 12% NaCl solution to the red blood cells with mixing.

3. The machine will now stop for an equilibration delay of 150 seconds.

4. Next the machine will add the first dilution of 340 ml 0.9% sodium chloride-0.2 gm% glucose solution to the thawed red blood cells and the following should be observed:

This indicates that both the blood and solution pumps are turning counter-clockwise, the shaker is on and the yellow valve to the 0.9% sodium chloride-0.2 gm% glucose and red valve to the red blood cells are open.

5. The machine will now stop for an equilibration delay of 60 seconds.

6. The ACP215 will now fill the centrifuge bowl by transferring the thawed-diluted red blood cells to the bowl. Effluent will exit the bowl to the waste bag and the line sensor will begin to monitor the free hemoglobin level.

The blood pump will now be turning clockwise, the solution pump is stopped, the centrifuge is spinning at 8000 rpm and both the green valve to the waste bag and the red valve to the red blood cells are open.

7. The ACP215 will then rinse the bowl with 0.9% sodium chloride-0.2 gm% glucose solution

The blood pump is stopped, the solution pump is turning counter-clockwise, the centrifuge is still spinning at 8000 rpm and the green valve to the waste bag and the yellow valve to the 0.9% sodium chloride-0.2 gm% glucose solution are open.

8. The red blood cells are then returned to the freezing bag from the bowl.

The blood pump is turning counter-clockwise, both the solution pump and centrifuge are stopped, and the green valve to the waste bag and red valve to the red blood cells are open.

9. The ACP215 will then perform the second dilution of the red blood cells with 400 ml of 0.9% sodium chloride-0.2 gm% glucose solution.

The blood and solution pumps should be turning counter-clockwise, the shaker will be one and the yellow valve to the 0.9% sodium chloride-0.2 gm% glucose solution and the red valve to the red blood cells are open.

10. The ACP215 will now stop for an equilibration delay of 60 seconds.

11. The thawed red cells will be added to the bowl, effluent will be transferred to the waste bag and the line sensor will again monitor the free hemoglobin level.

The blood pump will be turning clockwise, the solution pump will be stopped, the centrifuge will be spinning at 8000 rpm, and both the green valve to the waste bag and the red valve to the red blood cells will be open.

12. The centrifuge will stop and 30 ml of the red blood cells in the bowl will be transferred to the freezing bag. There will be an equilibration delay of 45 seconds, the centrifuge will restart and the 30 ml of red blood cells will be returned to the centrifuge bowl for a 60-second centrifugation period.

13. The ACP215 will then begin the first of 5 wash cycles by adding 60 ml of the 0.9% sodium chloride-0.2 gm% glucose solution to the bowl. The effluent exiting the bowl will be transferred to the waste bag and the line sensor will monitor the free hemoglobin level.

The blood pump will be stopped, the solution pump will be turning counter-clockwise, the centrifuge will be spinning at 8000 rpm and the green valve to the waste bag and the yellow valve to the 0.9% sodium chloride-0.2 gm% solution will be open.

14. The centrifuge will stop and transfer 30 ml of the red blood cells in the bowl to the freezing bag. There will be an equilibration delay of 45 seconds, the centrifuge will restart, return the 30 ml of red blood cells to the bowl and centrifuge for 60 seconds.

15. The ACP215 will then perform the second of 5 wash

cycles by adding 70 ml of the 0.9% sodium chloride-0.2 gm% glucose solution to the bowl. The effluent exiting the bowl will be transferred to the waste bag and the line sensor will monitor the free hemoglobin level.

The blood pump will be stopped, the solution pump will be turning counter-clockwise, the centrifuge will be spinning at 8000 rpm and the green valve to the waste bag and the yellow valve to the 0.9% sodium chloride-0.2 gm% glucose solution will be open.

16. The centrifuge will stop and transfer 30 ml of the red blood cells in the bowl to the freezing bag. There will be an equilibration delay of 45 seconds, the centrifuge will restart, return the 30 ml of red blood cells to the bowl and centrifuge for 60 seconds.

17. The ACP215 will then perform the third of 5 wash cycles by adding 100 ml of the 0.9% sodium chloride-0.2 gm% glucose solution to the bowl. The effluent exiting the bowl will be transferred to the waste bag and the line sensor will monitor the free hemoglobin level.

The blood pump will be stopped, the solution pump will be turning counter-clockwise, the centrifuge will be spinning at 8000 rpm and the green valve to the waste bag and the yellow valve to the 0.9% sodium chloride-0.2 gm% glucose will be open.

18. The centrifuge will stop and transfer 30 ml of the red blood cells in the bowl to the freezing bag. There will be an equilibration delay of 45 seconds, the centrifuge will restart, return the 30 ml of red blood cells to the bowl and centrifuge for 60 seconds.

19. The ACP215 will then perform the fourth of 5 wash cycles by adding 150 ml of the 0.9% sodium chloride-0.2 gm% glucose solution to the bowl. The effluent exiting the bowl will be transferred to the waste bag and the line sensor will monitor the free hemoglobin level.

The blood pump will be stopped, the solution pump will be turning counter-clockwise, the centrifuge will be spinning at 8000 rpm and the green valve to the waste bag and the yellow valve to the 0.9% sodium chloride-0.2 gm% glucose solution will be open.

20. The centrifuge will stop and transfer 30 ml of the red blood cells in the bowl to the freezing bag. There will be an equilibration delay of 45 seconds, the centrifuge will restart, return the 30 ml of red blood cells to the bowl and centrifuge for 60 seconds.

21. The ACP215 will then perform the fifth and final cycle by adding 300 ml of the 0.9% sodium chloride-0.2 gm% glucose solution to the bowl. The effluent exiting the bowl will be transferred to the waste bag and the line sensor will monitor the free hemoglobin level.

The blood pump will be stopped, the solution pump will be turning counter-clockwise, the centrifuge will be spinning at 8000 rpm and the green valve to the waste bag and the yellow valve to the 0.9% sodium chloride-0.2 gm% glucose solution will be open.

22. The centrifuge will stop and transfer 30 ml of the red blood cells in the bowl to the freezing bag. There will be an equilibration delay of 45 seconds, the centrifuge will restart, return the 30 ml of red blood cells to the bowl and centrifuge for 60 seconds.

23. The ACP215 will then transfer 240 ml of the AS-3 additive solution into the bowl. The effluent exiting the bowl will be transferred to the waste bag and the line sensor will monitor the free hemoglobin level.

The blood pump will be stopped, the solution pump will be turning counter-clockwise, the centrifuge will be spinning at 8000 rpm and the green valve to the waste bag and the orange valve to the AS-3 additive solution will be open.

24. The machine will trap some of the effluent fluid in the effluent line and freeze all operations. A message will be displayed on the screen prompting you to 'CHECK HGB LEVEL IN EFFLUENT FLUID--PRESS YES IF ACCEPTABLE. PRESS NO IF NOT'. Compare the color of the effluent fluid to a Haemonetics plastic color comparator. If the color is less than 5, the hemoglobin level is acceptable and press YES and continue on with the procedure. If the color is 5 or greater, it is not acceptable and the deglycerolized unit must be subjected to further quality control testing prior to being released for transfusion.

25. After you check the effluent fluid, the centrifuge will stop and the deglycerolized, AS-3 resuspended red blood cells will be transferred to the product bag.

The blood pump will be turning counter-clockwise, both the solution pump and the centrifuge will be stopped, and the green valve to the waste bag and the clear valve to the deglycerolized red blood cell product bag will be open.

26. The ACP215 will announce 'DEGLYCEROLIZATION COMPLETE'. The printer will print a PROCEDURE SUMMARY AND HEMOGLOBIN LEVEL PROFILE DIAGRAM (see Figure 9).

If the printout is not acceptable, the ACP215 will give you the option to reprint it.

SUMMARY

The machine will go through all the deglycerolization steps automatically with a delay at step 24, where the operator must make a decision based on the color of the effluent whether the unit is acceptable or rejected. Using the Haemonetics plastic color comparator, determine if the effluent is acceptable. Hold the comparator next to the tubing containing the effluent and determine the number on the color comparator to which the supernatant hemoglobin corresponds. If the color is less than 5 (i.e. 4 or less), the effluent is acceptable and you may accept the product hemoglobin level. If the color is equivalent to a number 5 or greater the product hemoglobin is not acceptable and the unit must be subjected to further testing.

To determine if the color is related to red cell spillage additional tests must be performed. See the Quality Control section of this Standard Operating Procedure for additional testing requirements.

A. Termination of the Procedure

1. When the deglycerolization procedure is complete, the printer will print a summary of the procedure.

2. Affix a unit number, ABO/Rh and deglycerolization facility ID label to the label on the red blood cell product. Note the date washed and expiration date on the label (see Figure 10).

3. Using the Sebra integral tubing sealer, seal the tubing three times between the bowl and the deglycerolized unit leaving as much integral tubing attached to the product bag as necessary.

NOTE: To use the Sebra heat sealer, place the tubing into the sealing head. Squeeze the sealing head handle completely. A pink light will illuminate. Release the handle when the light goes off. DO NOT RELEASE THE HANDLE WHILE THE LIGHT IS ON. Consult the users manual for additional instructions.

4. Detach the unit of deglycerolized red blood cells from the disposable set by cutting the middle one of the three heat seals. Make sealed crossmatch segments with the Sebra sealer as required.

5. Record the time that the deglycerolized red cells are placed into the 1-6 C refrigerator as the end of the deglycerolization time period. Compare this time to the beginning time recorded in Step 3 of the thawing procedure to verify that deglycerolization was accomplished within the required 2-hour time period.

STORAGE AND ISSUE

Place the deglycerolized red blood cells into a refrigerator maintaining at 1-6 C.

 

FIGURE 8 (CLICK PICTURE TO ENLARGE)
LN235 DEGLYCEROLIZATION DISPOSABLE SET

 

FIGURE 9
SAMPLE DEGLYCEROLIZATION PRINTOUT

HAEMONETICS 215
THAWED BLOOD DEGLYCEROLIZATION
PROCEDURE SUMMARY

1. In addition to the above, each of the units evaluated for monthly quality control must also be weighed.

2. A sample of the unit will be removed and the hematocrit value measured.

3. The volume of the unit will be determined by dividing the weight by density.

4. The red cell volume will be determined to confirm that minimum requirements are met.

A. BREAKAGE. Discard any unit that shows evidence of breaks or unintended openings at any point during processing.

1. Check each thawed unit for container breaks by gently compressing the unit against a white disposable towel, wiping the entire unit surface after compression, and examining the towel for blood stains.

2. Visually inspect the deglycerolization disposable set for evidence of breaks or leaks before, during, and after the deglycerolization process.

B. OBSERVATION OF EFFLUENT. Check the graph printout of the waste solution through the wash cycle for signs of excessive hemolysis or of red cell spillage.

1. Hemolysis. At the beginning of the wash cycle the supernatant manifests a pale pink tinge that fades until it disappears after about 1200 ml of wash solution is used. The color of the waste solution should be less than number 5 on the scale of the color comparator. If signs of excessive hemolysis persist, the unit must be studied to determine whether the unit is safe for transfusion (see below). Hemolysis results from a freeze-thaw lesion or from mishandling during red cell washing, and the following should be checked:

a. Check freezer temperature charts during the storage period.

b. Check to see if the units that exhibit hemolysis were frozen at the same time, by the same person or using the same machine. Isolate any suspect units and evaluate and discard as necessary.

c. Confirm technician understanding of pre-glycerolization handling.

d. Confirm technician understanding of temperature requirements of the glycerol and red cells.

e. In units with poor freeze-thaw and freeze-thaw-wash recovery values, studies should be done to determine whether or not the poor in vitro results were due to the quality of the red blood cells that were frozen. Sickle trait red blood cells (SA), hereditary spherocytosis (HS), paroxysmal nocturnal hemoglobinuria (PNH) red blood cells, and red blood cells with glucose-6-phosphate dehydrogenase deficiency do not tolerate the freeze-thaw and freeze-thaw-wash recovery procedures. Red blood cells with poor freeze-thaw and freeze-thaw-wash recovery values with no apparent reason should be tested for these red blood cell abnormalities.

2. Spillage. When washing is performed using continuous-flow centrifugation, intact red cells can be observed in the effluent waste line. Spillage of intact red cells looks similar to hemolysis except that when intact red cells are present, the effluent appears cloudy red whereas when hemolysis is present, the effluent is transparent with a pink tinge. To detect whether hemolysis or loss of intact red cells is present, the effluent must be inspected against a white background. Spillage of red cells into the waste not only represents a loss of red cells from the unit but may also mask the presence of supernatant hemoglobin in the waste. The principal cause of spillage is the presence of too many red cells in the unit at the time of glycerolization.

a. Isolated unit spillage. These units are acceptable for transfusion as long as they meet all other criteria. Units in which spillage persists should be studied further to determine whether they are suitable for transfusion.

b. Recurrent and uncontrollable spillage.

(1) Check scale used to weigh units prior to glycerolization;

(2) Confirm technician understanding of glycerolization process;

(3) Check labels and composition of wash solution.

CONSUMABLES:

1. Sterile docking wafers (Terumo 3NCC987)

2. Alcohol swab, 70% (B-D 6894) (3);

3. Syringe, 20 ml (B-D 5661) with 16 g needle (B-D 5198)

METHODS

1. Weigh the blood in the 600 ml transfer pack.

2. Sterilely dock a transfer pack onto the 600 ml bag containing the deglycerolized red blood cells.

3. Transfer approximately 2 ml of washed red blood cells from the 600 ml bag into the transfer pack.

4. Heat seal and remove the transfer pack.

5. Using a needle and syringe, remove the red cells from the transfer pack.

6. Measure the hematocrit value.

Total weight of blood: Weigh the blood in the 600 ml bag following deglycerolization. Subtract the weight of the empty 600 ml bag (32 gm) to determine the net weight of the red blood cell concentrate.

Hematocrit concentration. Measure the hematocrit value.

Determine the density of the blood as follows:

Post deglycerolization unit = (0.0693 X hct) + 1.005

NOTE: Use hematocrit as a decimal.

Divide the net weight of the red blood cell concentrate by the density to determine the volume of the red blood cell concentrate.

Multiply the total volume of the blood by the hematocrit value (as a decimal) to determine the volume of red blood cells.

All units tested for monthly quality control should have a hematocrit value of >38 V%. This will result in a red cell volume of at least 110 ml with a corresponding total hemoglobin of 36 gm.

CONSUMABLES:

1. Sampling site coupler (Fenwal 4C2405)

2. Alcohol swab, 70% (B-D 6894) (3);

3. Blood agar plates (2)

4. BBL Septi-Check system (Fisher RD43231)

5. Syringe, 30 ml (B-D 5662) with 16 gauge needle (B-D 5198)

6. Syringe, 20 ml (B-D 5661) with 16 g needle(B-D 5198)

7. Plastic test tube (Falcon 2059)

8. Plastic test tubes (Falcon 2063) (2)

9. 4 X 4 gauze or Kimwipes

10. Chemistrip 4 The OB (urine) (Boehringer-Manheim 417144)

During student training and facility validation studies, deglycerolized red blood cells must be evaluated for sterility, residual glycerol, supernatant hemoglobin, extracellular potassium and freeze-thaw-wash recovery as described below. These units are not sampled using sterile docking and, therefore, should not be used for transfusion, even if the results are acceptable.

STERILITY

After the unit has been deglycerolized, a sample of the red cells is obtained by inserting a sampling site coupler (Fenwal 4C2405) into one of the entry ports of the 600 ml bag containing the red cells. Aseptically remove a 20.5 ml sample with a 30 ml syringe and 16 gauge needle for testing as follows:

1. Place a drop of red cells on each of two plates in the 4 quadrants of a blood agar plate (aerobic) and tilt the plate to allow each drop to streak each quadrant (0.5 ml sample is required).

2. The remainder of the sample will be put into the BBL Septi-Check system (Fisher #RD43231). This system consists of 2 vials, one containing 70 ml of thioglycollate broth (anaerobic) and the other containing 70 ml of trypticase soy broth (aserobic). Aseptically place 10 ml of blood into each of the vials according to the instructions provided with the culture system.

3. Incubate the blood agar plates at 37 C for 3 days and the broth tubes at 37 C for 7 days; examine daily for growth.

DETERMINATION OF RESIDUAL GLYCEROL (OSMOLALITY), SUPERNATANT HEMOGLOBIN, AND EXTRACELLULAR POTASSIUM LEVELS

SAMPLE REQUIREMENTS: Aseptically insert a 16-gauge needle of a 20 ml syringe through the sampling site coupler (previously used for obtaining a sample for sterility testings) and withdraw a 15 ml sample of the deglycerolized red cells from the 600 ml transfer pack. Remove the 16-gauge needle from the syringe and discard. (Follow local guidelines for needle removal and disposal procedures). Transfer a 12 ml sample of deglycerolized red cells into a plastic test tube (Falcon 2059). Transfer the remaining 3 ml sample into a plastic test tube (Falcon 2063).

NOTE: Do not transfer the blood from the syringethrough the needle into the test tube. This may cause hemolysis.

Centrifuge the sample at 2200 X g for 10 minutes in a 22 C refrigerated centrifuge. Transfer the supernatant into another plastic test tube (Falcon 2063) using a transfer pipet. The supernatant is used to determine residual glycerol (osmolality or refractive index/refraction), supernatant hemoglobin, and extracellular potassium levels.

RESIDUAL GLYCEROL

Refraction

1. Palm-Abbe refractometer (Model PA200, Misco Products Division, Cleveland , OH ) may be used to estimate the glycerol concentration in the supernatant of the thawed glycerolized RBC and in the supernatant of the deglycerolized RBC. The refraction is a mathematically derived value from the refractive index.

2. Using a disposable transfer pipet, transfer a sample of supernatant solution into the measuring prism, as described in the manufacturer's instruction manual.

3. Hold the instrument up to a light source (e.g., fluorescent light, window). Focus the eyepiece and determine the refraction value of the sample according to the manufacturer's instructions. The refraction value should be greater than 400 for the supernatant glycerol in the thawed glycerolized RBC and refraction value should be less than 40 to insure that the glycerol level is less than 1 g%.

Osmolality:

1. Calibrate the osmometer (Fiske Model 2400) using the manufacturer's procedure manual.

2. The thawed supernatant of the glycerolized RBC is diluted one to ten with distilled water prior to testing for supernatant osmolality.

3. Using a Gilson adjustable volume pipettor and pipet tip, transfer 20 microliters of supernatant solution into an osmometer cuvette and determine the osmolality of the sample. Osmolality should not exceed 400 mOsm/kg H2O to insure a residual glycerol level of less than 1 g%.

EXTRACELLULAR (SUPERNATANT) POTASSIUM

1. Calibrate the IL 943 flame photometer using the flame standard 140 mEq/L Na+/5 mEq/L K+, according to the manufacturer's instructions.

2. Deglycerolized RBC resuspended in AS-3 solution can be stored at room tempe rature for up to 4 hours prior to concentrating th RBC by centrifugation to isolate the supernatant solution for testing for extracellular potassium.

3. Using a Gilson adjustable volume pipettor, add 300 microliters of supernatant into the sample cup of the flame photometer and measure the extracellular potassium level. The extracellular potassium level should not exceed 3 mEq/L on the day of washing.

SUPERNATANT HEMOGLOBIN

SPECTROPHOTOMETRIC METHOD

1. Set the Spectronic spectrophotometer at a wavelength of 540 nm.

2. Prepare a standard curve using a low, normal and high total hemoglobin standard (J.T. Baker 3074), Drabkin’s solution (Sigma D5941) and Brij 35 solution (Sigma B4184).

A. Reconstitute the Drabkin's reagent (one vial) with 1000 ml of distilled water. Add 0.5 ml of the 30% BRIJ-35 solution. The Drabkin's solution may be stored at room temperature (18-26 C) in an amber bottle for up to 6 months.

B.Reconstitute the lyophilized hemoglobin standard with 50 ml of Drabkin's solution to prepare an 18 g% solution.

C.Pipet the following solutions to prepare the standard curve:

*Hemoglobin standard concentration is approximate, use exact concentration reported with each vial.

NOTE:These diluted standards are stable for as long as 6 months when stored tightly capped, in the dark at 4 C.

D. Place tube 1 into the spectrophotometer and zero the absorbance value. Read and record the absorbance values for tubes 2 through 4.

E.Plot a calibration curve (absorbance values vs hemoglobin concentration).The curve should be linear and pass through the origin.

 

F.Using the standard curve, calculate the extinction coefficient, K, as follows:

Hemoglobin Concentration
(g%)=(K)(Absorbance)

G.Calculate the average K for using the three hemoglobin standard solutions (6.0, 12.0, 18.0 g%).

H.Total hemoglobin measurements are performed using 0.02 ml of whole blood diluted with 5.98 ml of Drabkin's reagent (1:251 dilution).

I. Measure the supernatant hemoglobinconcentration using a 0.3 ml sample in 4.7 ml of Drabkin's reagent. The overall increase observed is 18-fold for the supernatant hemoglobin samples. The following formula is used to construct the supernatant hemoglobin standard curve:

Supernatant
Hemoglobin = (K)(Absorbance)(1000 mg/gram)
Concentration 18

3. Using a Gilson adjustable pipettor, pipet a 0.3 ml sample of supernatant and dilute the sample with 4.7 ml of Drabkin's reagent into a 13X100 mm Kimax glass culture tube. Mix and equilibrate for at least 2 minutes for the reaction to occur. Measure the absorbance value for the sample using the Drabkin's reagent solution as the blank.

4. Refer to the supernatant hemoglobin standard curve and determine the hemoglobin concentration of the sample (mg%). The supernatant hemoglobin concentration of the day of washing should be less than 150 mg%.

CHEMISTRIP METHOD

(Boehringer Mannheim Corp. Chemistrip 4 The OB urine test strip, Cat. No. 417144)

NOTE:The Chemistrip Method is only recommended when no spectrophotometer is available. However, if a spectrophotometer is available, the Chemistrip Method should not be used in lieu of the spectrophotometric method for measurement of supernatant hemoglobin for quality control testing each month.

1.Prepare the supernatant sample as previously described.

2. Briefly (no longer than 1 second) dip the test strip into the supernatant sample. Ensure thatthe chemically impregnated patches on the test strip is totally immersed in the sample.

3. Draw the edge of the strip along the rim of the test tube to remove excess sample.

4.Turn the test strip on its side and tap once ona piece of absorbent paper to remove any remaining sample and to prevent the possible mixing of chemicals.

5.Wait 60 seconds, then visually compare the protein color patches on the test strip to the color scale printed on the vial label. The protein visual color scale bears five colors patches, ranging from light yellow to dark green:

The first patch, designated as negative, indicates a supernatant hemoglobin value of approximately 16 mg%, measured using the spectrophotometric method. The second patch, designated as trace, indicates a supernatant hemoglobin value of approximately 45 mg%,measured using the spectrophotometric method. The third patch, designated as +30, indicates a supernatant hemoglobin value of approximately 96 mg%, measured using the spectrophotometric method. The fourth patch, designated as ++100, indicates a supernatant hemoglobin value of approximately 221 mg%, measured using the spectrophotometric method. The fifth patch, designated as +++500 mg/dl, indicates supernatant hemoglobin value of approximately 428 mg%, measured using the spectrophotometric method.

6.Note the test result on the quality control worksheet. No further calculations are necessary.

Total volume of waste solution. Measure the total volume of waste solution using a graduated cylinder. Usually, the total volume of waste solution is 1,500 ml/unit.

Total volume of unit following deglycerolization. Weight the unit following deglycerolization. Subtract the weight of the 600 ml bag (@32 grams) to determine the net weight of the unit. Divide the net weight by the density to obtain the volume of blood following deglycerolization.

Hemoglobin concentration.

1. Waste: Obtain a 5 ml sample from the waste bag and, using the same method described for the measurement of supernatant hemoglobin (mg%), determine the hemoglobin concentration. The hemoglobin concentration in the waste (mg%) multiplied by the total volume of waste (ml), divided by 100,000, will equal the total grams of hemoglobin lost.

2. Post-wash unit: Obtain a 2 ml sample from the unit and determine the hematocrit value and hemoglobin concentration using an automated analyzer. Multiply the hemoglobin concentration by the total volume to determine the total hemoglobin in the washed unit.

Freeze-thaw-wash recovery calculation: Divide the total hemoglobin in the waste by the sum of the total hemoglobin in the washed unit and the waste to determine the percentage of hemoglobin lost in the waste. Subtract this value from 100 to determine the % freeze-thaw-wash recovery value.

Volume of waste solution = 1500 ml
Hemoglobin concentration in waste = 300 mg/dl
Total hemoglobin in waste = 4.5 gm
Net weight of blood following deglyc = 305 gm
Hematocrit post deglyc = 50%
Density post deglyc = (0.0693 X 0.50)+1.005 = 1.03965
Volume of blood post deglyc = 305/1.03965 = 293 ml
Hemoglobin concentration in deglyc unit = 16 gm/dl
Total hemoglobin in deglycerolized unit = 46.9 gm

% of hemoglobin lost in waste = [4.5/(4.5+46.9)] X 100 = 8.8%

Freeze-thaw-wash recovery = 100 - 8.8 = 91.2%

Twelve units of frozen red blood cells should be placed into a polystyrene foam shipping container. Approximately 1 inch of crushed dry ice is placed on the bottom of the container. The frozen units are then added to the container as shown in Figure 11. Place an elastic rubber band around one of the top layer units for the attachment of a temperature monitoring device.

One temperature monitoring device should be used to monitor the temperature of the blood products inside the container during shipment.

Immediately place the temperature monitoring device under the elastic rubber band which is around one of the top-layer units. This will insure that the temperature monitoring device remains at the top of the product load as the dry ice melts during shipment. Immediately add more dry ice on top of the units so that a total of 40 lbs. has been added to each shipping container.

The shipping container should have a gross weight of at least 55 lbs. prior to shipment to insure that the proper amount of dry ice has been added to the container.

Follow the established procedures for shipment of liquid blood products.

POTENTIAL CAUSE: Collection of more than 500 ml of blood.

RESULT: The unit may contain more red blood cells than the bowl can accommodate resulting in red blood cell spillage into the waste bag during the deglycerolization. The glycerolization procedure will not be affected. These red blood cell units may exhibit slightly reduced freeze-thaw-wash recovery values (due to the loss of red cells into the waste solution), however, the recovered red blood cells will be of high quality and acceptable for transfusion.

SOLUTION: Determine the net weight and hematocrit of the unit prior to glycerolization, following glycerolization and/or following thawing. If the red cell volume (total volume multiplied by the hematocrit value) exceeds 200 ml red blood cell spillage may occur.

a. POTENTIAL CAUSE: Red blood cells centrifuged incorrectly following glycerolization resulting in a hematocrit value less than the recommended value of 60+5 V%.

RESULT: The supernatant volume in the glycerolized red blood cells will be increased and the volume of wash solution may not be sufficient to adequately remove the glycerol solution.

b. POTENTIAL CAUSE: Red blood cells centrifuged incorrectly following glycerolization resulting in a hematocrit value greater than the recommended value of 60+5V%.

RESULT: Red blood cells will be irreparably damaged from the initial overconcentrating exhibiting increased hemolysis and extracellular potassium levels during and following the deglycerolization. Additionally, the 50 ml volume of 12% sodium chloride used in the pre-dilution may further damage the red blood cells as the volume of supernatant solution will be lower than expected.

c. POTENTIAL CAUSE: Red blood cells centrifuged incorrectly prior to glycerolization resulting in a hematocrit value less than the recommended value of 75+5V%.

RESULT: The calculated volume of 6.2M glycerol to be added to the red blood cells to achieve the 40% W/V glycerol concentration assumes the unit has been concentrated to a hematocrit value of 75+5V%. If the unit has a hematocrit value lower than required, the glycerol volume added may not be sufficient to achieve the recommended 40% W/V concentration.

d. POTENTIAL CAUSE: Red blood cells centrifuged incorrectly prior to glycerolization resulting in a hematocrit value greater than the recommended value of 75+5V%.

RESULT: Studies have shown that concentration of red blood cells to hematocrit values of >80 V% may damage the red cells. Therefore, the red blood cells may have been damaged prior to the glycerolization/deglycerolization procedure.

SOLUTION: Measure the hematocrit value prior to glycerolization, following glycerolization and/or following thawing to determine that the unit was centrifuged correctly with the brake of the centrifuge turned OFF.

POTENTIAL CAUSE: The correct volume of glycerol was not added to the unit. Confirm the volume of glycerol added with the enclosed nomogram to determine that the machine added the correct volume.

RESULT: Red blood cells may be underglycerolized and not able to tolerate long-term storage as well as properly glycerolized red blood cells.

POTENTIAL CAUSE: The unit may contain abnormal red blood cells (such as sickle trait (SA), hereditary spherocytosis (HS), paroxysmal nocturnal hemoglobinuria (PNH), or glucose-6-phosphate dehydrogenase deficiency). Red cells with these abnormalities do not tolerate the freeze-thaw-wash procedure well.

SOLUTION: If no other reasons for the poor results can be determined, the units can be tested for the presence of abnormal red blood cells.

COMPETENCY OF PERSONNEL ASSIGNED TO OPERATE THE ACP215

Personnel assigned to operate the Haemonetics ACP215 instrument to glycerolize, deglycerolize, and resuspend washed red blood cells in the AS-3 additive solution must understand that the red blood cell are protected by glycerol during freezing and storage at -80 C. The final concentration of glycerol in the red blood cells must be 40-42 W/V%. The glycerolized red blood cells are centrifuged to prepare a red blood cell concentrate with a hematocrit value of 60 + 5 V% prior to freezing. The centrifugation procedure removes supernatant glycerol solution prior to freezing in order to reduced the volume of wash solution required to deglycerolize the red blood cells following thawing. The glycerol must be removed from the thawed previously frozen red blood cells prior to transfusion. Residual glycerol of greater than 1% may produce hemolysis of the red blood cells following transfusion. The procedures to add glycerol to the red blood cells and to remove the glycerol from the red blood cells require that the temperature of the red blood cells, glycerol solution, thawed glycerolized red blood cells, and solutions to wash the glycerolized red blood cells must be controlled. The temperature of the red blood cells at the time the glycerol is added should be at room temperature (20-30 C), like the temperature of the glycerol solution and solutions used to deglycerolize the red blood cells. The temperature of the thawed glycerolized red blood cells should be maintained at 32-34 C at the time of deglycerolization.

Freezing of red blood cells requires the intracellular cryoprotective agent, glycerol. Extracellular glycerol is removed prior to freezing to reduce the volume of solution needed to deglycerolize the red blood cells. The glycerolized red blood cells are concentrated to achieve a hematocrit value of the glycerolized red blood cell concentrate of 60 + 5 V% by centrifugation at 1248 X g (2200 rpm) for 10 minutes. The brake of the centrifuge must be off to minimize the agitation of the glycerolized red blood cells during slowing of the centrifuge.

Quality control testing for the adequacy of glycerolization is to measure the hematocrit of the glycerolized red blood cells on the day of freezing or on the day of thawing prior to washing. In addition, the supernatant osmolality of the glycerolized red blood cells measured on the day of glycerolization prior to freezing on the day of thawing prior to deglycerolization should be measured. A supernatant osmolality of 4800-5000 mOsm/kg H20 indicates an appropriate concentration of glycerol to preserve red blood cells frozen at -80 C, which is the temperature of dry ice and alcohol.

The supernatant refraction value can also be measured to assess the concentration of glycerol. The refraction should be about 450. The refraction value is about 10% of the supernatant osmolality value. A hand-held refractometer is used to measure the refraction value. The measurement of refraction is a simple method to assess the adequacy of glycerolization. The removal of glycerol to a level of 1% is assessed by the measurement of supernatant osmolality of the deglycerolized red blood cells. Supernatant osmolality of the deglycerolized red blood cells of less than 400 mOsm/kg H20 or a refraction value of less than 40 indicates a residual glycerol concentration of 1% or less.

The mean in vitro recovery of red blood cells associated with addition and removal of glycerol should be 75% with at least 80% of the units having an in vitro recovery of greater than 65%. The disposable blow-molded polycarbonate bowl used in the ACP215 has a volume of 275 ml. This volume requires that only 200 ml of red blood cells can be washed in this bowl. The volume of red blood cells that should be glycerolized and deglycerolized should not exceed 200 ml of red blood cells. To achieve this volume of red blood cells, the hematocrit of the donor and the weight of the blood, not the volume of blood, must be controlled. In donors with hematocrit values of less than 46 V%, a volume of 450 ml of whole blood should be colleted into 63 ml of CPDA1 anticoagulant. When the hematocrit of the donor is greater than 46 V%, a weight of 430 g of whole blood should be collected into 63 ml of CPDA1 anticoagulant.

The blood processed in the ACP215 requires that either an 800 ml or 1000 ml PVC plastic bag be used. The 800 ml or 1000 ml bag is needed to permit the dilution of the thawed glycerolized red blood cells with 12% sodium chloride and 0.9% sodium chloride-0.2 gm% glucose solutions. The 800 ml or 1000 ml bags permit the dilution and proper mixing of the thawed glycerolized red blood cells with the 12% sodium chloride and 0.9% sodium chloride-0.2 gm% glucose solution.

The wash procedure in the ACP215 utilizes external dilution of the thawed glycerolized red blood cells with the wash solution on two occasions and the transfer of the diluted glycerolized red blood cells into the disposable blow-molded bowl that removes the supernatant solution into the waste bag. Following two external dilutions with wash solution, the glycerolized red blood cells in the disposable blow-molded bowl are washed on five separate occasions. The centrifuge is stopped during this procedure on five different occasions to permit mixing of the glycerolized red blood cells with the 0.9% sodium chloride-0.2 gm% glucose solution. Following the deglycerolization procedure using 1.5-1.6 liters of 0.9% sodium chloride-0.2 gm% glucose solution, the deglycerolized red blood cells are washed with AS-3 additive solution. A volume of 300 ml of AS-3 solution is used to wash and resuspend the deglycerolized red blood cells in the AS-3 additive solution and storage at 4 C for 2 weeks.

The technician must respond to the software program. Air in the plastic tubing will be identified by the air detector that the technician must respond to. The disposable set and the disposable blow-molded bowl must be placed into the ACP215 properly. The sterile connector device (SCD), in-line filters, and disposable blow-molded bowl with an external seal have been approved by the FDA to be a functionally closed system to glycerolize and deglycerolize red blood cells.

Validation of the integrity of the disposable set for glycerolization and deglycerolization of the red blood cells, validation of the line sensor used to detect hemolysis using filter, proper insertion of the disposable set to glycerolize the red blood cells, and the disposable set to deglycerolize the red blood cells, are required to certify the competency of the individual assigned to operate the ACP215.

The mixer and printer attached to the ACP215 are essential for the proper operation of the ACP215. The mixer, centrifuge, and pump to deliver the solutions are programmed to execute the deglycerolization procedure automatically. The printer provides the information to document that the centrifuge, mixer, and pump to deliver the blood and wash solutions are functioning properly.

Filters are used to validate the calibration of the line sensor to record the hemolysis during the deglycerolization procedure. The technician is responsible to observe the supernatant fluid in the plastic tubing at the completion of the deglycerolization procedure. A color comparator is used to assess the degree of hemolysis and/or presence of intact red blood cells. A value of 5 or greater on the color comparator requires that a sample of blood from the unit be obtained using a sterile connector device (SCD). The sample must be spun to evaluate the supernatant fluid to assess whether excessive hemolysis is present.

The 325 ml volume of the blow-molded bowl will accommodate 200 ml of red blood cells. To prevent spillage of intact red blood cells which will create a color comparator value in the plastic tubing of 5 or greater, a volume of approximately 50 ml of blood is now sequestered into the red blood cell transfusion bag at the completion of the deglycerolization procedure using 0.9% sodium chloride-0.2 gm% glucose solution with a supernatant osmolality of 340 mOsm/kg H20 prior to the addition of the AS-3 additive solution with an osmolality of 280 mOsm/kg H20. The addition of the AS-3 additive solution will produce swelling of the red cells and potential spillage of the red blood cells. The software program now sequesters a volume of deglycerolized red blood cells into the transfusion bag prior to addition of the AS-3 additive solution to prevent spillage of red blood cells.

Quality control measurements to assess glycerolization and deglycerolization procedures include in vitro freeze-thaw-wash recovery value, measurement of supernatant hemoglobin, supernatant osmolality or supernatant refraction value, and culture.

Freeze-thaw-wash recovery is assessed by measurement of the recovery of cellular hemoglobin. Total hemoglobin, supernatant hemoglobin, hematocrit value, volume of blood, volume of wash solution, and spun and non-spun waste solution are needed to calculate the recovery of red blood cells following the freeze-thaw-wash procedure. Two methods can be used to measure the in vitro recovery following the freeze-thaw-wash procedure. The thawed unit and deglycerolized unit or the deglycerolized unit and the waste solution can be used to calculate the in vitro freeze-thaw-wash recovery value. Testing of the waste solution processed as spun and non-spun will detect the loss of intact red blood cells into the waste solution (i.e. red blood cell spillage). Acceptable values for the deglycerolized red blood cells are as follows:

a. Supernatant hemoglobin level of 150 mg% or less on the day of deglycerolization is considered to be an acceptable value.

b. Freeze-thaw-wash recovery value of 75% with at least 80% of the units having a freeze-thaw-wash recovery value of 65%.

c. Supernatant osmolality of less than 400 mOsm/kg H20 or a refraction value of less than 40.

d. Negative culture for aerobic and anaerobic bacteria.

e. No breakage of the disposable set and the transfusion bag in which the deglycerolized red blood cells are stored.

f. Deglycerolized red blood cells must be labeled with ABO and Rh labels; labels to identify that glycerolization was done using a functionally closed system and deglycerolization was done using a functionally closed system; length of storage in the frozen state, length of storage following deglycerolization and resuspension in AS-3 additive solution at 4 C; infectious disease makers tested at the time of freezing.

Sera must be frozen with the red blood cells. The sera will be stored in two provials with the frozen red blood cells. In addition, two provials of sera will be stored at -80 C in a central laboratory. The provials will be used for testing for infectious disease agents that were not done at the time of freezing which may be required by the FDA. The current method of freezing permits storage of red blood cells frozen with 40% W/V glycerol at -80 C for 10 years. If new testing is required following red blood cell freezing, frozen sera must be available to perform this testing. A method to collect sera from donors whose red blood cells are frozen must be established so that frozen sera will be available both with the frozen red blood cells in the rigid cardboard box and in a central laboratory which will store the frozen sera for subsequent testing. All frozen blood products (red blood cells, platelets, plasma, mononuclear cells) should have sera frozen with the frozen blood product so that these sera can be used for testing of new FDA-required infectious disease markers. The frozen blood products must be labeled with infectious disease markers that were tested for in the unit. Details of this method used to test the blood must be identified on the label. Labels detailing the infectious disease marker testing performed must be applied to both the unit and to the cardboard box the unit is stored in.

ANSWER SHEET FOR SELF-ADMINISTERED QUIZ

RELATED REFERENCES

Valeri CR, Ragno G, Pivacek LE, Srey R, Hess JR, Lippert LE, Mettille F, Fahie R, OíNeill EM, Szymanski IO: A multicenter study of in vito and in vivo values in human RBCs frozen with 40-percent (wt/vol) glycerol and stored after deglycerolization for 15 days at 4C in AS-3: assessment of RBC processing in the ACP 215. Transfusion 2001;31:933-939.

Valeri CR, Ragno G, Pivacek L, OíNeill EM: In vivo survival of apheresis RBCs, frozen with 40-percent (wt/vol) glycerol, deglycerolized in the ACP 215 and stored at 4 C in AS-3 for up to 21 days. Transfusion 2001;41:928-932.

Valeri CR, Ragno G, Pivacek LE, Srey R, Hansson-Wicher M, Leavy ME: An experiment with glycerol-frozen red blood clels stored at ñ80 C for up to 37 years. Vox Sang 2000;79:168-174.

Valeri CR, Pivacek LE, Cassidy GP, Ragno G: The survival, function, and hemolysis of human RBCs stored at 4 C in additive solution (AS-1, AS-3, or AS-5) for 42 days and then biochemically modified, frozen, thawed, washed, and stored at 4 C in sodium chloride and glucose solution for 24 hours. Transfusion 2000;40:1341-1345.

Valeri CR, Pivacek LE, Cassidy GP, Ragno G: Posttransfusion survival (24-hour) and hemolysis of previously frozen, deglycerolized RBCs after storage at 4 C for up to 14 days in sodium chloride alone or sodium chloride supplemented with additive solutions. Transfusion 2000;40:1337-1340.

Valeri CR, Srey R, Tilahun D, Ragno G. In vitro quality of red blood cells frozen with 40% W/V glycerol at -80 C for 14 years, deglycerolized with the Haemonetics ACP215 and stored at 4 C in additive solution-1 or additive solution-3 for up to 3 weeks. Transfusion 44:990-995, 2004.

Valeri CR, Ragno G, Gennouai A, van Houten P, Rose L, Rose M, Egozy E, Popovsky M. Automation of glycerolization of RBC using the high separation bowl in the ACP215 instrument. Transfusion 45:1621-1627, 2005.