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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.

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