Home
« Previous | TOC | Next »

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

 

« Previous | TOC | Next »
Home