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44. Application of theoretically determined human spermatozoa cryopreservation methods to the commercial sperm bank setting
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Abstracts / Cryobiology 53 (2006) 367–446
The time-critical factor for efficient treatment is the quick and effective response to injuries involving massive blood loss [OSullivan, OSullivan, Mil Med 166 (2001) 362–365]. Different approaches were done including the clinical evaluation of a system using frozen blood products (frozen red packed cells (FRPC) and frozen platelets (FPL)) [Erber, Tan, Grey, Lown, Med. J. Aust. 165 (1996) 11–13; Rosenblatt, Hirsch, Valeri, Mil. Med. 159 (1994) 392–397; Sputtek, Sputtek, in: Fuller, Lane, Benson (Eds.), Life in the Frozen State, CRC Press, Boca Raton, 2004]. In missions it is reported that elevated treatment levels causes the severity of injuries seen at admission to increase [Appenzeller, Mil. Med. 169 (2004) 187–191]. Besides blood, further stabilisation needs the maintenance of coagulation capabilities to secure patient transportation for final treatment at home. Two matters characterised the work at ISAF in the summer of 2003. The joint use of two blood banks (GE and NL) and two casualties in a short time—a bombing attack against a German bus and an accident to an Italian vehicle one week later. Following the bus attack, 24 injured soldiers were sent to the multinational GE field hospital. Four heavily injured patients were identified. The top-ranked patient had blood group 0 Rh negative. Massive transfusion was followed by known clinical complications as coagulopathy and ongoing microbleedings and further surgical treatment was necessary. The use of plasma, coagulation factors, as well as FRPC and FPL led to stabilization at a low level. Because of the ongoing haemostatic situation (haemoglobin 46 g/l, platelets 14 · 109/l, aPTT > 75 s, Quick 37%) the application of fresh whole blood was initiated. Supportive treatment with FRPC and FPL then led to a stable situation for transportation by Medical Evacuation (MedEvac). The second casualty was a severely injured Italian after a car accident. Based on the experience one week earlier a decision to give whole blood was made early and enough blood was available. The additional use of frozen platelets was without complications and increased their number by 25%. Here we report the use of NL frozen blood products with clinical effectiveness and the need for whole blood donation in case of shortage of non-substitutable blood groups. The combination of different blood banking systems in missions can back up the treatment of severely injured soldiers. Due to different time-tables of availability the delay of thawing when using frozen products can be solved only by sufficient supplies of immediately available cooled RPC. The urgent demand for platelet substitution could only be accomplished by frozen or fresh products. Frozen products should become the basis for sufficient trauma treatment in missions. Whole blood donation seems to be a basic necessity for missions if insufficient frozen products are available, even with delayed serology. It is possible to maintain blood support in missions with mass casualties by the use of this combination. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.044
44. Application of theoretically determined human spermatozoa cryopreservation methods to the commercial sperm bank setting. Erinn M. Flynn, Erin L. Calloway, Michael A. Byers, John K. Critser, Erik J. Woods, Genome Resources, 46202 Indianapolis, IN, USA Previous reports investigating the ability of various cryoprotective agents (CPAs) to permeate the human spermatozoa plas-
ma membrane have indicated that ethylene glycol (EG) is a superior permeant to glycerol (GLY). Further studies along similar lines have demonstrated that this effect is observed even at low temperatures, and theorized that for these reasons EG would be a superior CPA to GLY. These same studies ultimately validated this hypothesis under ideal laboratory conditions. However, to date most anonymous donor commercial sperm banks in the United States have been reluctant to move away from using GLY-based cryoprotectant media. Part of this reluctance is due to a lack of data comparing media systems under commercial sperm bank settings, and while the enhanced effect of EG is evident under specific controlled cooling rates coupled with controlled ice nucleation, these practices are usually not practical in the commercial sperm bank environment. For these reasons, this study was designed to test the use of EG-based cryoprotectant media systems using standard commercial sperm bank freezing protocols. To that end, human spermatozoa were first divided into control and experimental groups. The two control groups were processed either (1) using the standard freezing protocol used in our anonymous donor program, which consists of drop-wise addition over 10 min of an equal part of a commercially available 12% GLY solution in an egg yolk base (for a final concentration of 6% or 0.65 M GLY), slow cooling to 4 C in a refrigerator, followed by rapid cooling in a charged dry-shipper and ultimate plunging into LN2; or (2) using exactly the same protocol but substituting Hams-F-10 medium for the egg yolk base. The two experimental groups consisted of sperm processed the same way, except (1) a 12% EG solution prepared in Hams F-10 medium; or (2) a 12% EG solution prepared in egg yolk base was used (both corresponding to a final concentration of 6% or 0.97 M EG when added at a concentration of 1:1 to sperm suspensions). Both control and experimental samples were thawed using our standard protocol which consists of removing a sample from LN2 and placing on the bench top for 30 min, followed by analysis of motility as the end point. Initial results indicated no statistically significant difference (P > 0.05) between the experimental and control groups; however the GLY and EG samples frozen using the egg yolk base have exhibited higher overall motilities on average. These results suggest that any benefit of using EG over GLY may be obscured by damage imparted by the standard non-controlled cooling method. Results of further experiments are pending. (Conflict of interest: None. Source of funding: Genome Resources.) doi:10.1016/j.cryobiol.2006.10.045
45. Cryopreservation of CD34+ cells under controlled ice nucleus formation. Inga Bernemann a, Xing-Sheng Ju b, Martin Zenke b, Birgit Glasmacher a, a Institute for Multiphase Processes/Biomedical Engineering, University of Hannover, 30167 Hannover, Germany; b Institute for Biomedical Technologies/Cell Biology, Aachen University Hospital, 52074 Aachen, Germany Cooling down to storage temperature is the most critical process in cell cryopreservation. Extracellular ice formation causes enhancement of solute concentration in the cell surrounding medium. This increase in concentration is responsible for water flows following the osmotic gradient. Particularly two parameters affect the dehydration of the cells: the cooling rate and the nucleation temperature. In general the nucleation temperature is difficult to control because of the stochastic nature of ice nucleus
Abstracts / Cryobiology 53 (2006) 367–446
The time-critical factor for efficient treatment is the quick and effective response to injuries involving massive blood loss [OSullivan, OSullivan, Mil Med 166 (2001) 362–365]. Different approaches were done including the clinical evaluation of a system using frozen blood products (frozen red packed cells (FRPC) and frozen platelets (FPL)) [Erber, Tan, Grey, Lown, Med. J. Aust. 165 (1996) 11–13; Rosenblatt, Hirsch, Valeri, Mil. Med. 159 (1994) 392–397; Sputtek, Sputtek, in: Fuller, Lane, Benson (Eds.), Life in the Frozen State, CRC Press, Boca Raton, 2004]. In missions it is reported that elevated treatment levels causes the severity of injuries seen at admission to increase [Appenzeller, Mil. Med. 169 (2004) 187–191]. Besides blood, further stabilisation needs the maintenance of coagulation capabilities to secure patient transportation for final treatment at home. Two matters characterised the work at ISAF in the summer of 2003. The joint use of two blood banks (GE and NL) and two casualties in a short time—a bombing attack against a German bus and an accident to an Italian vehicle one week later. Following the bus attack, 24 injured soldiers were sent to the multinational GE field hospital. Four heavily injured patients were identified. The top-ranked patient had blood group 0 Rh negative. Massive transfusion was followed by known clinical complications as coagulopathy and ongoing microbleedings and further surgical treatment was necessary. The use of plasma, coagulation factors, as well as FRPC and FPL led to stabilization at a low level. Because of the ongoing haemostatic situation (haemoglobin 46 g/l, platelets 14 · 109/l, aPTT > 75 s, Quick 37%) the application of fresh whole blood was initiated. Supportive treatment with FRPC and FPL then led to a stable situation for transportation by Medical Evacuation (MedEvac). The second casualty was a severely injured Italian after a car accident. Based on the experience one week earlier a decision to give whole blood was made early and enough blood was available. The additional use of frozen platelets was without complications and increased their number by 25%. Here we report the use of NL frozen blood products with clinical effectiveness and the need for whole blood donation in case of shortage of non-substitutable blood groups. The combination of different blood banking systems in missions can back up the treatment of severely injured soldiers. Due to different time-tables of availability the delay of thawing when using frozen products can be solved only by sufficient supplies of immediately available cooled RPC. The urgent demand for platelet substitution could only be accomplished by frozen or fresh products. Frozen products should become the basis for sufficient trauma treatment in missions. Whole blood donation seems to be a basic necessity for missions if insufficient frozen products are available, even with delayed serology. It is possible to maintain blood support in missions with mass casualties by the use of this combination. (Conflict of interest: None declared. Source of funding: None declared.) doi:10.1016/j.cryobiol.2006.10.044
44. Application of theoretically determined human spermatozoa cryopreservation methods to the commercial sperm bank setting. Erinn M. Flynn, Erin L. Calloway, Michael A. Byers, John K. Critser, Erik J. Woods, Genome Resources, 46202 Indianapolis, IN, USA Previous reports investigating the ability of various cryoprotective agents (CPAs) to permeate the human spermatozoa plas-
ma membrane have indicated that ethylene glycol (EG) is a superior permeant to glycerol (GLY). Further studies along similar lines have demonstrated that this effect is observed even at low temperatures, and theorized that for these reasons EG would be a superior CPA to GLY. These same studies ultimately validated this hypothesis under ideal laboratory conditions. However, to date most anonymous donor commercial sperm banks in the United States have been reluctant to move away from using GLY-based cryoprotectant media. Part of this reluctance is due to a lack of data comparing media systems under commercial sperm bank settings, and while the enhanced effect of EG is evident under specific controlled cooling rates coupled with controlled ice nucleation, these practices are usually not practical in the commercial sperm bank environment. For these reasons, this study was designed to test the use of EG-based cryoprotectant media systems using standard commercial sperm bank freezing protocols. To that end, human spermatozoa were first divided into control and experimental groups. The two control groups were processed either (1) using the standard freezing protocol used in our anonymous donor program, which consists of drop-wise addition over 10 min of an equal part of a commercially available 12% GLY solution in an egg yolk base (for a final concentration of 6% or 0.65 M GLY), slow cooling to 4 C in a refrigerator, followed by rapid cooling in a charged dry-shipper and ultimate plunging into LN2; or (2) using exactly the same protocol but substituting Hams-F-10 medium for the egg yolk base. The two experimental groups consisted of sperm processed the same way, except (1) a 12% EG solution prepared in Hams F-10 medium; or (2) a 12% EG solution prepared in egg yolk base was used (both corresponding to a final concentration of 6% or 0.97 M EG when added at a concentration of 1:1 to sperm suspensions). Both control and experimental samples were thawed using our standard protocol which consists of removing a sample from LN2 and placing on the bench top for 30 min, followed by analysis of motility as the end point. Initial results indicated no statistically significant difference (P > 0.05) between the experimental and control groups; however the GLY and EG samples frozen using the egg yolk base have exhibited higher overall motilities on average. These results suggest that any benefit of using EG over GLY may be obscured by damage imparted by the standard non-controlled cooling method. Results of further experiments are pending. (Conflict of interest: None. Source of funding: Genome Resources.) doi:10.1016/j.cryobiol.2006.10.045
45. Cryopreservation of CD34+ cells under controlled ice nucleus formation. Inga Bernemann a, Xing-Sheng Ju b, Martin Zenke b, Birgit Glasmacher a, a Institute for Multiphase Processes/Biomedical Engineering, University of Hannover, 30167 Hannover, Germany; b Institute for Biomedical Technologies/Cell Biology, Aachen University Hospital, 52074 Aachen, Germany Cooling down to storage temperature is the most critical process in cell cryopreservation. Extracellular ice formation causes enhancement of solute concentration in the cell surrounding medium. This increase in concentration is responsible for water flows following the osmotic gradient. Particularly two parameters affect the dehydration of the cells: the cooling rate and the nucleation temperature. In general the nucleation temperature is difficult to control because of the stochastic nature of ice nucleus