STANDARD OPERATING PROCEDURE

FREEZING OF RED BLOOD CELLS WITH 40% W/V GLYCEROL IN AN 800 ML OR 1000 ML POLYVINYLCHLORIDE PLASTIC BAG WITH DEGLYCEROLIZATION USING THE HAEMONETICS MODEL 115 INSTRUMENT

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
ENCLOSURE 1
SHIPPING INSTRUCTIONS
EQUIPMENT LIST
VENDORS
COMPETENCY
SELF TEST
ANSWER SHEET
RELATED REFERENCES
TROUBLESHOOTING

 

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

INTRODUCTION

This Standard Operating Procedure describes the procedures to glycerolize and deglycerolize units of human red cells using a manual method in either the 800 ml primary polyvinyl chloride plastic bag system or in a 1000 ml polyvinyl chloride (PVC) plastic bag.

The red blood cells can be collected into any FDA-approved anticoagulant or anticoagulant-preservative system such as CPD, CPDA-1, CP2D/AS3, CPD/AS1, or CPD/AS5. 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 originally collected into the 800 ml plastic bag system, the red blood cell concentrate must be transferred to a standard 1000 ml polyvinylchloride plastic transfer pack using a sterile docking procedure. The red blood cells are glycerolized 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 115. The Haemonetics 115 cell washer is considered an "open" system and the FDA allows for storage of these cells at 4 C for only 24 hours 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

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

CONSUMABLES

1. Glycerol:

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

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

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

3. Sterile docking wafers (Terumo 3NCC987)

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

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

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

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

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

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

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

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

WARMING PROCEDURE

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

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

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

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

The water bath and plasma thawer procedures are described below.

a. Water bath method:

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

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

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

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

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

5. The red cells are now ready for glycerolization.

b. Plasma thawer method

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

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

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

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

4. The red cells are now ready for glycerolization.

GLYCEROLIZATION PROCEDURE

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

An ABO, Rh confirmation label.

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

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

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

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

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

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

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

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.

DEGLYCEROLIZATION

A. INTRODUCTION

The Haemonetics Blood Processor 115 is a gravity flow, non-programmed, continuous-flow washing system which has a mixing platform of fixed oscillation rate and excursion distance integrally attached. After the red cells have been thawed, the plastic freezing bag is secured to the platform on the Haemonetics 115 by means of adjustable magnetic mounting posts. The cells are first diluted once with 12% sodium chloride, and then twice with 0.9% sodium chloride-0.2% glucose solution, utilizing the shaking platform to ensure adequate mixing. After dilution of the red cells, the bag containing the thawed-diluted red blood cells is removed from the platform and suspended in an inverted position on a support hook above the wash bowl and the red cells are permitted to flow into the spinning wash bowl until the first effluent is noted in the waste line. As soon as the waste appears, the flow of 0.9% sodium chloride-0.2% glucose solution is initiated. This solution flows simultaneously with the remaining red cells entering the bowl and then continues until a total volume of 1.5 liters has entered the bowl. At the completion of the wash cycle, the centrifuge is stopped and the deglycerolized red cells are siphoned from the wash bowl into a 600 ml transfer pack. The unit is labeled with the expiration date and time. Just before transfusion the washed red cells are concentrated by centrifugation, and the supernatant solution is removed into an attached 300 ml transfer pack and discarded.

NOTE: At the present time, red cells not used immediately after washing may be stored at 4 C for up to 24 hours.

B. CONSUMABLES

1. Double blood spike harness, washing bowl and waste bag (Haemonetics 7497)

2. 600 ml polyvinyl chloride plastic bag (Baxter 4R2023)

3. 300 ml polyvinyl chloride plastic bag (Baxter 4R2032)

4. 12% Sodium Chloride Solution (150 ml plastic bag) (Fenwal 4B7874); Each 100 ml contains: 12 g sodium chloride USP.

5. 0.9% Sodium Chloride-0.2% Glucose Solution (2-liter plastic bag) (Fenwal 4B7878). Each 100 ml contains: 200 mg dextrose (hydrous) USP, 900 mg sodium chloride USP.

6. Sterile docking wafers (Terumo 3NCC987)

Since the Haemonetics Blood Processor 115 operates on the principle of gravity flow, the heights at which the blood and solutions are hung will determine the flow rate (Figure 7). The four support hooks should be positioned as follows:

Upon installation of the Haemonetics 115, the operator should check to see that the recommended heights actually yield the expected flow rates (see Quality Control Section). The solution hook should be adjusted so that the desired flow rate is achieved. Excessive flow rate can result in red cell spillage. In such cases the wash solution should be lowered to reduce the flow rate until spillage ceases.

1. Remove the disposable wash set from its box and CLOSE ALL SIX SLIDE CLAMPS on the harness tubing (Figure
8).

2. Check to see if all slide clamps were provided on the harness tubing and that all four bag spikes and the component bag receptor port are properly covered. Install bowl, harness, and waste bag on the machine according to the manufacturer's instructions.

3. Remove the 600 ml transfer pack from its box. Aseptically insert the spike of the 600 ml bag into the component bag receptor port on the cell wash harness. Place the 600 ml bag on the hooks provided on the front of the cell wash stand. Alternatively, the 600 ml quadruple plastic bag recovery system can be used to recover two (2) units of thawed, glycerolized red blood cells that are washed in the same polycarbonate bowl.

4. Aseptically insert the spike on the blue color coded harness line into the bag containing the 12% sodium chloride solution. Invert the bag and hang it on the middle solution support hook.

5. Aseptically insert the spike on the yellow color- coded harness line into the bag of 0.9% sodium chloride-0.2% glucose solution. Invert the bag and hang it on the uppermost solution support hook.

6. Insert the spike on the red color-coded harness line into the port of the 800 or 1000 ml bag containing the thawed red cells and place the bag on the shaker.

7. Arrange the shaker magnets and the unit on the shaker platform so that the ports of the bag containing the thawed red blood cells point toward the operator. The bag should be stretched flat so that the maximum surface area covers the shaker. This will insure proper mixing during the dilution steps. The blood bag label should face down so that the operator can observe mixing of the wash solution with the thawed red cells.

DILUTION OF THE THAWED RED CELLS BEFORE WASHING

1. The volume of 12% sodium chloride solution to be added will be determined by the volume of red blood cells frozen as follows:

CAUTION: Damage to the red cells may occur if more than the recommended volume of the 12% NaCl solution is added to the thawed unit.

2. Using the factory suggested graduations as a guide, mark the bag of 12% sodium chloride solution at the level expected when the required volume of the solution has been added to the unit.

3. Turn the shaker on, open the slide clamp on the tubing leaving the blood bag and open the slide clamp on the tubing leaving the 12% sodium chloride and allow the recommended volume of 12% sodium chloride to enter the red blood cells (approx. 30 seconds). Close both slide clamps and turn off the shaker. Allow the red cells to equilibrate with this solution for at least 2 minutes.

NOTE: The shaker must be on until all of the 12% sodium chloride solution is added. Visually check the unit for signs of localized solution pooling as indicated by deep reddish-to-black-colored areas, caused by inadequate mixing. This can be remedied by repositioning the bag on the shaker platform before continuing the dilution process.

4. Using the factory suggested graduations as a guide, mark the 0.9% sodium chloride-0.2% glucose solution bag at the points where the solution level should be when one dilution of approximately 100 ml and a second dilution when an additional 150 ml have been added to the unit. Mark a third point on the bag (for the wash cycle) at a point 1250 ml below the 150 ml dilution mark (Figure 9).

NOTE: The total amount of this solution used is 1500 ml.

5. Turn the shaker on, unclamp the slide clamp on the tubing leaving the blood bag and the slide clamp on the tubing leaving the bag of 0.9% sodium chloride-0.2% glucose solution and allow approximately 100 ml of this solution to enter the unit. Watch the unit for signs of localized solution pooling as described above. Close both slide clamps. Turn the shaker off. Allow the red cells to equilibrate with this solution for at least 2 minutes.

NOTE: Flow rate should be no faster than 100 ml/minute. This can be estimated by timing the rate of solution level fall across the factory graduation marks. If the flow rate is too rapid, it can be reduced by lowering the height of the solution support hook.

6. Turn the shaker on, reopen the slide clamp on the tubing leaving the blood bag and the slide clamp on the tubing leaving the bag of 0.9% sodium chloride-0.2% glucose solution, and allow approximately 150 ml of this solution to enter the unit. Clamp the tubing leaving the 0.9% sodium chloride-0.2% glucose solution bag and close the clamp on the tubing leaving the blood bag. Turn the shaker off. Allow the red cells to equilibrate with this solution for at least 2 minutes.

WASH CYCLE

1. Remove the unit from the shaker platform. Insert the bottom grommet of the blood bag onto the blood bag support hook to permit the unit to hang in an inverted position.

2. Relocate the 0.9% sodium chloride-0.2% glucose solution bag from the uppermost to the lowermost solution support hook.

3. Check all tubing for occluding kinks and straighten as necessary. Check the tubing attached to the cell wash bowl; it must not touch the centrifuge.

CAUTION: Be sure that the waste tubing never becomes occluded during this procedure. Occlusion of the waste tubing may generate back pressure in the cell wash bowl that could cause the rotating seal to vent to atmosphere.

4. Check to make sure that the feed tube support arm properly engages the feed tube of the cell wash bowl and that the centrifuge cover is properly placed onto the cell washer. Turn centrifuge on. Set timer for 5 minutes.

NOTE: If a power failure occurs when the centrifuge is on, IMMEDIATELY close all the slide clamps. This will prevent gross spillage of red blood cell into the waste bag. When power is re-established, wait for the centrifuge to spin for 1 to 2 minutes to ensure resedimentation of the red cells in the bowl. Then, reopen the slide clamps to finish the procedure.

5. Open the slide clamp on the tubing leaving the blood bag and the slide clamp on the tubing entering the cell wash bowl to permit the diluted red cells to enter the spinning bowl. Visually check the flow of red cells into the bowl. The flow rate into the bowl should be approximately 75 ml per minute. If red blood cell spillage occurs, lower the blood bag support hook. Normally, the bowl should fill in 5 to 7 minutes. If an extended fill time is observed, check the tubing for kinks or aggregate materials which may clog the tubing.

NOTE: The flow of red cells from the blood bag can checked by inverting the blood bag momentarily, allowing air from the blood bag to enter the tubing. If the air bubbles do not move through the tubing to the bowl, the flow has stopped. Check for an occlusion in the tubing. Straighten tubing kinks or squeeze the bag containing the red blood cells to dislodge microaggregate material.

6. As soon as the first effluent appears in the waste line, unclamp the tubing leaving the 0.9% sodium chloride-0.2% glucose solution bag to permit this solution to flow into the bowl along with the remaining diluted red cells. INSPECT THE INLET TUBING ATTACHED TO THE BOWL. THIS TUBING MUST ALWAYS CONTAIN RED CELLS AS LONG AS RED CELLS ARE DRAINING FROM THE BLOOD BAG. If the tubing appears void of red cells at this point, look for tubing kinks or aggregates and then reestablish the flow of red cells into the bowl.

NOTE: A pale pink tinting with free Hemoglobin (hemolysis) in the effluent waste is normal at this point. Using the free hemoglobin reference scale as a guide, estimate the degree of hemolysis in the waste. The degree of hemolysis should be equal to or less than the number 6 at this point in the wash cycle. If color of the waste appears to be equal to or darker than the number 7, check to see if the proper volume of 12% NaCl solution was added to the unit, otherwise consult the Quality Control section.

7. When all the diluted red cells have been transferred from the bag to the bowl, clamp the tubing leaving the blood bag.

8. Check the flow rate of the 0.9% sodium chloride 0.2% glucose solution to be sure it does not exceed 120 ml/minute. The flow rate can be checked by timing the rate of fall of the solution across the factory graduation marks. One hundred ml will take 50 seconds to flow out of the bag at a rate of 120 ml per minute.

NOTE: An excessive flow rate will result in spillage of the red cells into the waste during the wash cycle. Spillage is detected by the examination of the waste which exits the cell wash bowl. If red cell spillage occurs, the red color in the waste line will appear cloudy red as opposed to transparent red as will be the case with hemolysis. If spillage is observed, lower the height of the blood bag and the bag of 0.9% NaCl 0.2% Glucose.

9. Normally, the pale tinge of hemolysis in the effluent waste line should disappear after delivery of 1,000 to 1,200 ml of 0.9% sodium chloride-0.2% glucose solution in the wash cycle.

NOTE: If the red color of effluent has not disappeared when 1,000-1,200 ml of wash solution has been used, check the waste effluent for spillage of red cells and reduce the flow rate as necessary.

10. Clamp the tubing leaving the wash solution and the tubing leaving the bowl when the 1500 ml volume of 0.9% sodium chloride-0.2% glucose solution is empty.

NOTE: The color of the waste should be equal to or less than the number 5 using the free hemoglobin reference scale at the completion of this procedure. Continued discharge of hemolysis after this point indicates that the unit of blood is washing abnormally and should be studied prior to transfusion There is very little variation from unit to unit with this wash protocol.

11. Turn the centrifuge off. Just before the bowl stops, clamp the effluent line when red blood cells begin to appear. Open the clamp to the 600 ml bag immediately once the bowl has stopped rotating. Unclamp the effluent line. If necessary to initiate flow into the bowl, squeeze air from the waste bag into the bowl to force the washed red cells out of the bowl and establish a siphon flow of red cells into the 600 ml bag. When siphon flow begins, stop squeezing the waste bag. DO NOT SQUEEZE ANY LIQUID OUT OF THE WASTE BAG AND INTO THE BOWL. Air bubble gaps will appear in the tubing between the bowl and the 600 ml bag, and the siphon flow will stop when the bowl has been emptied. Alternatively, the dry quadruple plastic bag system can be used to collect two units of deglycerolized red blood cells processed in the same disposable polycarbonate bowl.

NOTE: As the bowl drains, sterile air trapped in the waste bag leaves the waste bag and enters the bowl. CAUTION: WASTE SOLUTION SHOULD NOT RETURN TO THE BOWL.

12. Remove the 600 ml transfer pack containing the red cells from the cell washer stand. Turn the transfer pack to the upright position and squeeze the trapped air from the transfer pack into the bowl. Continue squeezing to fill the integral tubing with red cells. Clamp the tubing between the bowl and the 600 ml transfer pack.

13. Affix unit number, ABO/Rh and deglycerolization facility ID labels to the label on the 600 ml transfer pack, and note the date washed and expiration date and time on the label, currently 24 hours from the time the frozen red cells were placed in the water bath for thawing (Figure 10).

14. 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 600 ml transfer pack as necessary.

15. Detach the unit of deglycerolized red cells from the harness, by cutting the middle one of the three heat
seals. Make sealed crossmatch segments with the Sebra sealer as required.

16. Record the time that the deglycerolized red cells are placed into a 1-6 C refrigerator as the end of the deglycerolizing 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.

CAUTION: If at any time during this procedure the cell wash disposable system becomes vented to room air, the unit and disposable set must be discarded.

PROCEDURE CONTINUATION FOR SECOND UNIT

1. Remove the overwrap from the second bag of 0.9% sodium chloride-0.2% glucose solution. Aseptically, withdraw the yellow color-coded spike from the used 2-liter bag of 0.9% sodium chloride-0.2% glucose solution and discard.

2. Aseptically insert the yellow color-coded spike into the full 2-liter bag of 0.9% sodium chloride-0.2% glucose solution and reposition the bag on the uppermost solution hook.

3. Remove the empty bag from the first unit processed from the blood bag support hook. Insert the remaining red color-coded harness spike into the port of the second unit of thawed glycerolized red cells to be deglycerolized, and place the second unit on the shaker. Place the empty bag from the first unit processed on the shaker, underneath the second unit to be deglycerolized. Alternatively, sterilely dock the red color-coded harness line to the plastic tubing on the bag of thawed glycerolized red cells. Arrange the shaker magnets and the unit on the shaker platform so that the ports of the primary bag point toward the operator. The bag should be stretched flat so that the maximum surface area covers the shaker. This will insure proper mixing during dilution steps.

4. Continue processing the second unit by repeating the steps described above.

Note: During the washing of the second unit, during the pre-dilution phase, be sure to unclamp the correct slide clamp which enters the blood bag of the second unit.

STORAGE AND ISSUE

1. Since units washed in the same disposable bowl must be transfused to the same recipient, secure the two units of deglycerolized red cells with an elastic band or tie tag during storage at 4 C.

2. Place the deglycerolized red cells into a refrigerator maintained at 1-6 C for up to 24 hours (24 hours from the time the frozen red blood cells were place in the water bath for thawing). Because the Haemonetics Model 115 is considered an open system, the deglycerolized red blood cells may be stored at 4 C for only up to 24 hours prior to transfusion.

3. Immediately prior to transfusion, the deglycerolized red blood cells must be centrifuged to concentrate the red cells and remove the supernatant solution. Sterilely dock a 300 ml transfer pack onto the 600 ml bag containing the deglycerolized red blood cells. Alternatively, when the dry quadruple 600 ml plastic bag system is used, each of two units of deglycerolized red blood cells is collected into 2 of the 4 600 ml plastic bags. Following centrifugation of the deglycerolized red blood cells resuspended in 0.9% NaCl-0.2 gm% glucose, the supernatant solution is expressed into the empty 600 ml transfer pack integrally attached to each of the 600 ml plastic bags containing the deglycerolized red blood cells. Spin the deglycerolized red cells at 3000 X g in a 22 C refrigerated centrifuge for 4 minutes. Transfer the supernatant 0.9% sodium chloride-0.2 gm% glucose solution from the unit into the attached 300 ml transfer pack. Heat seal the tubing and detach the transfer pack containing the supernatant solution.

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.

 

FIGURE 7 (CLICK PICTURE TO ENLARGE)
PRE AND POST DILUTION HEIGHTS

 

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

 

FIGURE 9 (CLICK PICTURE TO ENLARGE)
VOLUMES OF SOLUTIONS USED IN DILUTION AND DEGLYCEROLIZATION OF RED BLOOD CELLS

 

FIGURE 10 (CLICK PICTURE TO ENLARGE)
600 ml Transfer Pack

 

 

QUALITY CONTROL

INTRODUCTION

Following describes a recommended quality control for units of red blood cells frozen and deglycerolized using this method. The quality control section is divided into 3 separate sections.

a. All Units: All units must be inspected for breakage, wet ports, and observation of the effluent (waste solution) throughout the wash cycle.

b. Monthly Quality Control: For each 90 units of red cells deglycerolized, 7 units must be evaluated for quality control. Enclosure 1 defines the method utilized to determine the number of units to be evaluated for monthly quality control. Quality control must be performed as follows:

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.

c. Student Training and Facility Validation Studies: When new students are being trained to perform this procedure or during facility validation, quality control should include the following: visual inspection for breakage, wet ports, and effluent; weight, hematocrit and red cell volume; and measurements of sterility, residual glycerol, supernatant hemoglobin, and supernatant potassium of the deglycerolized units will be determined. Freeze-thaw-wash recovery values will be calculated. Flow rates of wash solutions will be verified.

Units used for student training and facility validation studies should not be used in vivo, even when results are satisfactory. When personnel are being trained to freeze, thaw and wash red cells, the units should be quality-controlled as described and results recorded on the enclosed Red Blood Cell Deglycerolization and Quality Control Record.

A. ALL UNITS

VISUAL OBSERVATION AND INSPECTION (ALL UNITS)

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 wash harness, wash chamber, waste bag and interconnecting tubing for evidence of breaks or leaks before, during, and after the deglycerolization process.

B. WET PORTS. Before opening the port seal to connect the thawed unit to wash harness, visually inspect the port areas for any evidence of residual water droplets. Carefully and thoroughly wipe these areas dry with a clean cloth as necessary.

C. OBSERVATION OF EFFLUENT. Check the appearance of the waste solution through the wash cycle for signs of excessive hemolysis or of red cell spillage. Record on the enclosed Red Blood Cell Deglycerolization and Quality Control Record.

1. Hemolysis. At the beginning of the wash cycle the supernatant manifests a pale pink tinge which fades until it disappears after about 1200 ml of wash solution is used. The color of the waste solution should be less than the 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 or by the same person. Isolate any suspect units and evaluate and discard as necessary.

c. Confirm technician understanding of pre-glycerolization handling, the three glycerolization steps and of the need for proper manual mixing of the glycerol with the red cells during the final addition, i.e., use of the table provided to determine the volume of glycerol to red cell weight.

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

e. Check accuracy of balance used for glycerolization.

f. Confirm technician understanding of the predilution requirements prior to deglycerolization.

g. 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. Red cell spillage can also occur if the proper wash solutions are not used and if the spindle speed of the Haemonetics 115 is too slow. The following action is recommended if red cell spillage occurs:

a. Isolated unit spillage. Gradually lower the brackets supporting the sodium chloride-glucose solution and red cells until spillage ceases; 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 spindle speed of 5800 rpm of the cell washing bowl using a hand-held tachometer;

(4) Check labels and composition of wash solution.

B. MONTHLY QUALITY CONTROL

For each 90 units of red blood cells deglycerolized each month, 7 units must be evaluated for weight, hematocrit and red cell volume as described below.

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 the transfer pack and remove.

5. Using a needle and syringe, remove the red blood 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 >28 V%. This will result in a red cell volume of at least 110 ml with a corresponding total hemoglobin of 36 gm.

C. STUDENT TRAINING AND FACILITY VALIDATION STUDIES

During training and facility validation studies, units should be studied extensively for quality control. Units used for student training and facility validation studies should not be used in vivo, even when the quality control results are satisfactory. When personnel are being trained to freeze, thaw and wash red cells, the units should be quality-controlled as described and results recorded on the enclosed Red Blood Cell Deglycerolization and Quality Control Record.

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 (aerobic). 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 an 16-gauge needle of a 20 ml syringe through the sampling site coupler
(previously used for obtaining a sample for sterility testing) 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 syringe through 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

Refractive Index/Refraction

Hand Held Refractometer:

1. For field use, a Cambridge Instruments hand-held refractometer (TS meter, Model 10400A) may be used to estimate the residual level of glycerol. The refractometer contains a liquid prism which is self-temperature correcting. The meter has three scales; urine specific gravity, serum or plasma protein, and refraction. The refraction scale should be used; 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 less than 30 to insure that the glycerol level is less than 1 g%.

ABBE Refractometer:

1. Turn on the ABBE refractometer (American Optics Corp. Model 10480) and the constant temperature water bath (Haake Model A80) and allow to equilibrate to 20 C.

2. Calibrate the equipment with a known liquid material (e.g., absolute methanol).

3. Using a disposable transfer pipet, transfer 2 drops of supernatant solution into the refractive prism surface.

4. Close the refractive prism and determine the refractive index of the sample according to the manufacturer's specifications. The refractive index should not exceed 1.3355 to insure a residual glycerol level of less than 1 g%.

Osmolality

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

2. 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. 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 1.5 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 total hemoglobin standard kit (Sigma 525-A) according to the manufacturer's instructions. Included in the kit are: Drabkin's reagent, 30% BRIJ-35 solution, and a lyophilized hemoglobin standard (18 g%).

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:

TUBE#	HEMOGLOBIN SOLUTION (ml)	DRABKIN'S SOLUTION (ml)	HEMOGLOBIN CONCENTRATION (g%)
1	0.0	6.0	0.0
2	2.0	4.0	6.0
3	4.0	2.0	12.0
4	6.0	0.0	18.0
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 hemoglobin
concentration 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 = Concentration

(K)(Absorbance)(1000 mg/gram) 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 200 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. 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 that the chemically impregnated patches on the test strip are 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 on a 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 color 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.

ESTIMATION OF FREEZE-THAW-WASH RECOVERY (%)

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.

Sample calculation

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 = 425 gm
Hematocrit post deglyc = 35%
Density post deglyc = (0.0693 X 0.35)+1.005 = 1.029
Volume of blood post deglyc = 425/1.029 = 413 ml
Hemoglobin concentration in deglyc unit = 10 gm/dl
Total hemoglobin in deglycerolized unit = 41.3 gm

% of hemoglobin lost in waste = [4.5/(41.3+4.5)] X 100 = 9.8%

Freeze-thaw-wash recovery = 100 - 9.8 = 90.2%.

VERIFICATION OF FLOW RATE OF 12% NACL AND 0.9% NACL-0.2% GLUCOSE WASH SOLUTIONS AND DILUTED RED CELLS

The flow rates of the 12% NaCl and 0.9% NaCl-0.2% glucose wash solutions and of the diluted red blood cells should be checked every 6 months. The recommended flow rates are as follows:

Hang the wash solutions and a bag of saline (in lieu of diluted red cells) on the appropriate hooks. Using a stopwatch, time the delivery of the solutions into an empty plastic bag. Adjust the hooks to achieve the proper flow rates as outlined above, if necessary. Make the appropriate corrections in your Standard Operating Procedure Manual.

RED BLOOD CELL GLYCEROLIZATION WORKSHEET
(Printable Version)

RED BLOOD CELL DEGLYCEROLIZATION RECORD
(Printable Version)

RED BLOOD CELL GLYCEROLIZATION/DEGLYCEROLIZATION QUALITY CONTROL RECORD
(Printable Version)

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. 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 (Figure 9).

Follow the established procedures for shipment of liquid blood products.

FIGURE 11 (CLICK PICTURE TO ENLARGE)
SHIPPING CONTAINER

COMPETENCY OF PERSONNEL ASSIGNED TO OPERATE THE ACP215

Personnel assigned to follow this procedure to glycerolize red blood cells and operate the Haemonetics 115 cell washer instrument to deglycerolize red blood cells must understand that the red blood cells 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 reduce 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 bowl used in the Haemonetics 115 has a volume of 425 ml volume. This bowl allows for volumes of blood up to 500 ml to be washed in this bowl.

The blood processed in the 115 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 115 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 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. 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 resuspended in the sodium chloride-glucose wash solution.

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.

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 deglycerolization was done using the Haemonetics 115 cell washer, a functionally open system; length of storage in the frozen state, length of storage following deglycerolization; 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.

a. 45 V%
b. 75 V%
c. 90 V%

a. hematocrit is measured
b. hematocrit is estimated from the method used to concentrate the red blood cells prior to removal of the plasma to prepare the red blood cell concentrate (1615 X g for 4 minutes)

a. CPD
b. CP2D
c. CPDA-1
d. all of the above

a. 24 hours
b. 3 days
c. 6 days
d. 8 days

a. 4 C
b. 20-30 C
c. 34-37 C

a. 4 C
b. 20-30 C
c. 34-37 C

a. hematocrit of the unit
b. weight of the red blood cell concentrate
c. volume of red blood cells
d. all of the above

a. 1248 X g for 10 minutes with brake off
b. 1248 X g for 10 minutes with brake on
c. 1248 X g for 8 minutes with brake off
d. 1248 X g for 8 minutes with brake on

a. 45+5 V%
b. 60+5 V%
c. 70+5 V%
d. 80+5 V%

a. ABO and Rh
b. infectious disease markers
c. expiration date following storage at -80 C
d. all of the above

a. supernatant osmolality level of 4800-5000 mOsm/kg H20
b. supernatant refraction level of 480-500
c. supernatant glycerol assay of 40-42 W/V
d. all of the above

a. to avoid wetting the PVC bag containing the red
blood cells in the water bath during the thaw procedure.
b. to protect the PVC bag during storage in the frozen state
c. a and b

a. water bath with a mechanical pump maintained at 42 C for 45 minutes
b. ten-pouch Thermogenesis thaw bath maintained at 36 C for 40 minutes
c. four-pouch Thermogenesis thaw bath maintained at 36 C for 40 minutes
d. all of the above

a. 20-22 C
b. 28-30 C
c. 30-34 C
d. 36-38 C

a. to reduce the glycerol level to less than 5%
b. to reduce the glycerol level to less than 2%
c. to reduce the glycerol level to less than 1%

a. avoid hemolysis of the red blood cells following transfusion
b. avoid contamination of the red blood cells
c. avoid incompatibility of the red blood cells
d. all of the above

a. 50 ml of 12% sodium chloride and 2 liters of 0.9% sodium chloride-0.2 gm% glucose solution
b. 100 ml of 12% sodium chloride and 2 liters of 0.9% sodium chloride-0.2 gm% glucose solution
c. 150 ml of 12% sodium chloride and 2 liters of 0.9% sodium chloride-0.2 gm% glucose solution

a. external dilution of the thawed glycerolized red blood cells with 12% sodium chloride and 0.9% sodium chloride-0.2 gm% glucose solution
b. external dilution with 0.9% sodium chloride-0.2 gm% glucose solution alone

a. color comparator - less than 5
b. color comparator - less than 3
c. color comparator - greater than 5
d. color comparator - greater than 3

a. the deglycerolized unit should be discarded
b. the deglycerolized unit should be sampled using the sterile connector device and the blood sample spun to assess the supernatant hemoglobin level
c. the color comparator of greater than 5 indicates the presence of intact red blood cells
d. the color comparator of greater than 5 indicates excessive hemolysis.
e. none of the above
f. all of the above

a. evaluate the color of the fluid in the plastic tubing connecting the disposable bowl to the waste
solution
b. evaluate the integrity of the disposable set used to deglycerolize the red blood cells.
c. document that the color comparator of less than 5 was observed for the supernatant solution in the plastic tubing connecting the disposable blow-molded bowl to the waste solution
d. all of the above

a. sera obtained from the donor at the time of collection of the blood
b. plasma obtained from the anticoagulated blood on the day of collection of the unit of blood
c. plasma obtained from the anticoagulated blood on the day of freezing of the red blood cells

a. antibodies to HIV 1/2
b. antibodies to HTLV 1/2
c. hepatitis B surface antigen
d. antibody to hepatitis B core antigen
e. antibody to hepatitis C
f. P24 antigen
g. STS
h. nucleic antibody testing (NAT) for hepatitis C and HIV virus
i. none of the above
j. all of the above

ANSWER SHEET FOR SELF-ADMINISTERED QUIZ

RELATED REFERENCES

Valeri CR, Pivacek LE, Gray AD, Cassidy GP, Leavy ME, Dennis RC, Melaragno AJ, Niehoff J, Yeston N, Emerson CP, Altschule MD. The safety and therapeutic effectiveness of human red cells stored at -80 C for as long as 21 years. Transfusion 1989;29:429-437.

Valeri CR, Ragno G, Pivacek LE, Srey R, Hansson-Wicher M, Leavy ME: An experiment with glycerol-frozen red blood clels stored at -80 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, Pivacek LE, Cassidy GP, Ragno G: In vitro and in vivo measurements of human RBCs frozen with glycerol and subjected to various storage temperatures before deglycerolization and storage at 4 C for 3 days. Transfusion 2001;41:401-405, 2001.

Valeri CR, Pivacek LE, Cassidy GP, Ragno G: In vitro and in vivo measurements of gamma-radiated frozen, glycerolized RBCs. Transfusion 2001;41:545-549.

TROUBLESHOOTING/ADDITIONAL COMMENTS

1. OBSERVATION: Increased hemolysis during the deglycerolization procedure and/or post-wash storage

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.

2. OBSERVATION: Increased hemolysis observed in the thawed unit.

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.

3. OBSERVATION: Extensive hemolysis observed during the deglycerolization procedure with significantly reduced red blood cell recovery.

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.