Use of supernatant osmolality and supernatant refraction to assess the glycerol concentration in glycerolized and deglycerolized previously frozen RBC.
BACKGROUND
Human RBC are frozen at a mean temperature of -80 degrees C (with a range of -65 degrees C to -90 degrees C) with a mean concentration of 40% w/v glycerol (with a range from 36% w/v to 45% w/v) for at least 10 years. After thawing and deglycerolization the RBC should have a residual glycerol concentration of about 1%. We conducted three studies to measure the supernatant osmolality and supernatant refraction in RBC frozen with 40% w/v glycerol and stored at -80 degrees C for as long as 16 years. The measurements were made before and after deglycerolization.


METHODS
In the first study, one hundred and three (103) units of RBC were glycerolized to achieve a concentration of 40% w/v glycerol in an open system and frozen at -80 degrees C for as long as 16 years. In the second study, 106 units of RBC were glycerolized to achieve a concentration of 40% w/v glycerol and in an open system and frozen at -80 degrees C for a mean of 14 years. In the second study, the RBC were deglycerolized using the Haemonetics ACP215 instrument before being stored at 4 degrees C in the AS-1 or AS-3 additive solution. In the third study, fifty-five (55) units of RBC were glycerolized to achieve a 40% w/v glycerol concentration in the functionally closed system of the Haemonetics ACP215 instrument containing the high separation bowl and frozen at -80 degrees C for at least 2 months. These RBC also were deglycerolized using the Haemonetics ACP215 and were stored at 4 degrees C in the AS-3 additive solution. Before and after deglycerolization, measurements also were made of the freeze-thaw recovery and the freeze-thaw-wash recovery values, the percent hemolysis, supernatant hemoglobin level, supernatant osmolality and supernatant refraction.


RESULTS
The supernatant osmolality provided an accurate estimate of the glycerol concentration in the thawed RBC before deglycerolization but the supernatant refraction did not. However, after deglycerolization, both the supernatant osmolality and the supernatant refraction gave accurate estimates of the glycerol concentration in the RBC.


CONCLUSIONS
The osmolality measured in the osmometer of the thawed supernatant of the glycerolized RBC provided an accurate estimate of the glycerol concentration but the percent refraction measured in the Palm Abbe refractometer did not. Both the osmolality and percent refraction in the deglycerolized washed RBC provided accurate estimates of the residual glycerol.
Robert Valeri
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NBRL Inc., Plymouth, MA 02360, USA. navblood@nbrl.org
Ragno
Gina
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D001781:Blood Component Removal; D015925:Cryopreservation / Q000379:methods*; D004912:Erythrocytes / Q000737:chemistry*; D005612:Freeze Drying / Q000379:methods; D005615:Freezing; D005990:Glycerol / Q000097:blood*; D006454:Hemoglobins / Q000032:analysis; D006801:Humans; D009994:Osmolar Concentration; D012044:Regression Analysis
D006454:Hemoglobins; D005990:Glycerol