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DEGLYCEROLIZATION

Deglycerolization Procedure	56K 100K 300K	56K 100K 300K
Washing Procedure	56K 100K 300K	56K 100K 300K

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

 

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