SOP ACP215 - Competency of Personnel Assigned to Operate the ACP215

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.

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