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PubMed1988

A comparative fine structure study of rat cerebral cortex following ultra-rapid freezing and conventional chemical fixation procedures.

Adult rat (Sprague-Dawley) cerebral cortex was processed by ultra-rapid freezing at liquid helium temperature followed by freeze-substitution, osmium fixation and by other chemical, post-osmication procedures at 0-4 degrees and ambient temperatures as a comparative study for purposes of identifying differences and/or similarities in fine structure following these techniques. Five methods of processing were used: 1) rapid, slam-freezing at liquid helium temperature followed by osmium tetroxide/acetone freeze substitution; 2) perfusion with buffered, 2% glutaraldehyde at ambient temperature followed by post-osmication (2%); 3) en-bloc, buffered 2% glutaraldehyde fixation at 0-5 degrees centigrade and post-osmication (2%); 4) buffered, 2% osmium tetroxide perfusion at ambient temperature; and 5) en-bloc, buffered 2% osmium tetroxide fixation at 0-5 degrees centigrade. In ultra-rapid-frozen cortex good preservation was seen to a depth of 10-15 microns from the surface of the initial, copper-block contact. The tissue processed by ultra-rapid-freeze, freeze substitution demonstrates a general 'smoothness' of plasmalemmal and organelle membranes not observed in tissue prepared by chemical fixation alone. Cellular and organelle morphological differences were minor beyond the general 'smoothness' of membranes and a more intense background, electron density found in tissue prepared by rapid-freeze. Of particular interest was the practically identical images found in the four, chemical techniques not preceded by ultra-rapid freezing. High magnification images also revealed rather minor differences following ultra-rapid-freezing compared to tissue fixed by chemical fixation alone. Although these morphological differences are minimal, there can be no question of the fact that ultra-rapid-freeze followed by freeze-substitution is morphologically superior to chemical fixation alone. Ultra-rapid-freeze, eventually utilizing other substitution agents than osmium tetroxide, will offer several advantages and should be particularly useful for investigators involved in cytochemical and immunochemical methods.

Animals↗
PubMed1993

Effect of freezing method, thawing temperature and post-thaw dilution/washing on motility (CASA) and morphology characteristics of high-quality human sperm.

Sixteen semen samples, 12 donor and four patient samples of high initial quality, were processed to compare the effect of two freezing methods, two thawing temperatures and the effect of dilution and washing on sperm motility and morphology characteristics. Sperm samples were divided in two equal parts and frozen either by fast vapour freezing or by slow computer-controlled freezing. For each freezing method, half of the straws were thawed at room temperature (22 degrees C), the other half were thawed at 37 degrees C. From each freeze-thawing treatment, one straw was evaluated immediately post-thawing; another straw was washed to remove the cryoprotectant solution. In this way, each semen sample was subjected to eight freeze-thawing treatments. No effect of the freezing method and thawing temperature was observed on motility characteristics evaluated by computer-assisted semen analysis, nor on light-microscopical morphology parameters. Post-thaw dilution and washing, however, exerted a deleterious effect on sperm motility, by reducing percentage motility by 50% compared to unwashed thawed specimens. Linearity and percentage of morphologically normal spermatozoa were obviously impaired, while percentage of abnormal tails and beat cross frequency increased significantly. In general, freeze-thawing was most successful when rapid vapour freezing was followed by 37 degrees C thawing, and when slower computer-controlled freezing was combined with 22 degrees C thawing, causing significant interactions between the freezing method and the thawing temperature. For semen samples of high initial quality, vapour and computer-controlled freezing were equally effective in terms of recovery of morphologically normal, motile spermatozoa.

Cryopreservation↗
PubMed1992

Xylem embolism in response to freeze-thaw cycles and water stress in ring-porous, diffuse-porous, and conifer species.

Vulnerability to xylem embolism by freeze-thaw cycles and water stress was quantified in ring-porous (Quercus gambelii Nutt.), diffuse-porous (Populus tremuloides Michx., Betula occidentalis Hook.), and conifer species (Abies lasiocarpa Nutt., Juniperus scopulorum Sarg.). Embolism was measured by its reduction of xylem hydraulic conductivity; it was induced by xylem tension (water-stress response) and by a tension plus a freeze-thaw cycle (freeze response). Conifers showed little (Juniperus) or no (Abies) freeze response even to repeated cycles. In contrast, Quercus embolized more than 90% by freezing at tensions below 0.2 MPa, whereas similar embolism without freezing required tensions above 4.5 MPa. Diffuse-porous trees (Betula, Populus) showed an intermediate freeze response. The magnitude of the freeze response was correlated with conduit volume but occurred at higher tensions than predicted from theory. Large early-wood vessels (2.8 x 10(-9) m(3)) in oak were most vulnerable to embolism by freezing, small vessels in Populus and Betula were intermediate (approximately 7 x 10(-11) m(3)), and tracheids in conifers (about 3 x 10(-13) m(3)) were most resistant. The same trend was found within a stem: embolism by freeze-thawing occurred preferentially in wider conduits. The water-stress response was not correlated with conduit volume; previous work indicates it is a function of interconduit pit membrane structure. Native embolism levels during winter corroborated laboratory results on freezing: Quercus embolized 95% with the first fall freeze, Populus and Betula showed gradual increases to more than 90% embolism by winter's end, and Abies remained below 30%.

Journal Article↗
PubMed1977

Freezing Injury in Onion Bulb Cells: II. Post-thawing Injury or Recovery.

Onion (Allium cepa L.) bulbs were subjected for 12 days to either a moderate freeze (-4 C) or a severe freeze (-11 C). They were then thawed slowly over ice. During 7 to 12 days following the thaw, the injury progressed with time in the severely frozen bulbs, but appeared completely repaired in the moderately frozen bulbs. This was shown by the following post-thawing changes.Infiltration of the intercellular spaces increased from 80 to 90% to 100% after the severe freeze, and decreased from 30 to 50% to zero after the moderate freeze. All of the cells were alive immediately after thawing whether the freeze was moderate or severe. Corresponding to the infiltration results 7 to 12 days later, many to most were dead following the severe freeze, all were alive following the moderate freeze.The conductivity of the effusate from pieces of bulb tissue increased after the severe freezing, and decreased after the moderate freezing. The concentration of K(+), total solutes, and sugars in the effusate paralleled the conductivity changes. Neither the pH of the effusate nor the permeability of the cells (as long as cells were living) to water was changed following either the severe or the moderate freezes. Some treatments of the thawed tissue following the severe freeze halted the progress of injury.The above results indicate that the semipermeable properties of the cell are uninjured but that the ion and sugar transport mechanism is damaged by freezing. Most likely the primary injury is to the active transport mechanism involved in their transport. It must be concluded that the final injury following freezing and thawing cannot be evaluated from the degree of infiltration or the conductivity of the effusate immediately after thawing, since injury may progress or recede following the thawing.

Journal Article↗
PubMed2001

Freezing of gait in PD: prospective assessment in the DATATOP cohort.

OBJECTIVE: To study the development of freezing of gait in PD. BACKGROUND: Freezing of gait is a common, disabling, and poorly understood symptom in PD. METHODS: The authors analyzed data from 800 patients with early PD from the Deprenyl and Tocopherol Antioxidative Therapy of Parkinsonism (DATATOP) clinical trial who were assigned either placebo, deprenyl, tocopherol, or the combination of deprenyl and tocopherol. The primary outcome measure was the time from randomization until the freezing of gait score on the Unified Parkinson's Disease Rating Scale (UPDRS) became positive. RESULTS: Fifty-seven patients (7.1%) had freezing of gait at study entry and 193 (26%) of the remaining patients experienced the symptom by the end of the follow-up period. Those with freezing of gait at baseline had significantly more advanced disease than those without the symptom, as measured by total UPDRS and Hoehn and Yahr stage. High baseline risk factors for developing freezing of gait during the follow-up period were the onset of PD with a gait disorder; higher scores of rigidity, postural instability, bradykinesia and speech; and longer disease duration. In contrast, tremor was strongly associated with a decreased risk for freezing of gait. At the end of follow-up, the signs most strongly associated with the freezing phenomenon were gait, balance, and speech disorders, not rigidity or bradykinesia. Deprenyl treatment was strongly associated with a decreased risk for developing freezing of gait; tocopherol had no effect. CONCLUSIONS: Freezing of gait is directly related to duration of PD. Risk factors at onset of disease are the absence of tremor and PD beginning as a gait disorder. The development of freezing of gait in the course of the illness is strongly associated with the development of balance and speech problems, less so with the worsening of bradykinesia, and is not associated with the progression of rigidity. These results support the concept that the freezing phenomenon is distinct from bradykinesia. Deprenyl, in the absence of L-dopa, was found to be an effective prophylactic treatment and should be considered for patients with PD who have an onset of gait difficulty.

Aged↗
PubMed1999

Memorization of contextual and CS conditioned fear response (freezing) in a one-trial acquisition paradigm.

In fear-conditioned Wistar rats freezing was induced by the delivery of a series of footshocks paired to tones (CS) in a specific conditioning chamber (context). CS and contextual fear were acquired in the same single conditioning session without preexposition to the conditioning chamber (day 1). Different groups of animals were conditioned employing three increasing US (footshock) intensities (0.25, 0.5, 0.75 mA). During the retention sessions context and CS conditioned freezing (fear response) were measured using a paradigm that fulfilled the following conditions: i) CS freezing retention was measured in a context different from the conditioning one; ii) CS and context freezing were measured at increased delays after the training session (days 3 and 4, 14 and 15, 28 and 29). The results show that there are significant differences between CS and context freezing retention, which are clearly related to delay after the initial session and to US intensity. In particular: 1) conditioned freezing to a discrete tone is better retained than conditioned freezing to context (irrespective of US intensity); 2) context freezing is directly related to US intensity much more than to tone freezing; 3) context freezing is easier to extinguish than tone freezing. The results are discussed in relation to previous ones and to their relevance to freezing genesis neural correlates.

Acoustic Stimulation↗
PubMed1994

Protein purification process engineering. Freeze drying: A practical overview.

Freeze drying provides a valuable tool to the formulation scientist by permitting dehydration of heat-sensitive drugs and biologicals at low temperature. The final product is quickly and easily reconstituted, and the process is compatible with aseptic operations. Freezing is a critical step, since the microstructure established by the freezing process usually represents the microstructure of the dried product. The product must be frozen to a low enough temperature to be completely solidified. If the solute crystallizes during freezing, this temperature is the eutectic temperature. If the solute remains substantially amorphous with freezing, the relevant temperature is the collapse temperature. Understanding the physical form of the solute--crystalline or amorphous--after freezing can be important from the standpoint of drying characteristics, appearance of the final product, and even product stability during storage. Supercooling is a significant factor in freezing of formulations intended for freeze drying--prior to both primary and secondary (eutectic) crystallization. The driving force for freeze drying is the difference in vapor pressure of ice between the sublimation zone and the condenser. Because the vapor pressure of ice increases sharply with increased product temperature, it is important from the standpoint of process efficiency to maintain product temperature as high as possible during primary drying without damaging the product. The upper limit of product temperature during primary drying again depends on the physical form of the solute. Exceeding either the eutectic temperature (crystalline solute) or the collapse temperature (amorphous solute) results in loss of the desirable properties of a freeze dried product. Freeze drying is a coupled heat and mass transfer process, where either heat transfer or mass transfer may be rate limiting with respect to the overall drying rate. Heat transfer is often the rate-limiting transfer operation because of the high heat of sublimation of ice and the inefficiency of heat transfer. Conduction is the primary mechanism of heat transfer, as opposed to convection or thermal radiation. The rate-limiting resistance to heat transfer is usually the interfacial, or contact, resistance caused by poor contact between materials--the heated shelf, metal trays, and the bottom surface of glass vials. Since the thermal conductivity of a gas is directly proportional to pressure in the free molecular flow regime, the chamber pressure during primary drying is an important determinant of the overall heat transfer rate. As a result, the drying rate for a heat transfer-limited process increases sharply with chamber pressure up to a pressure where free molecular flow conditions no longer apply.(ABSTRACT TRUNCATED AT 400 WORDS)

Biotechnology↗
PubMed2001

Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying.

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.

Animals↗
PubMed2000

A freezing-sensitive mutant of Arabidopsis, frs1, is a new aba3 allele.

To investigate the molecular mechanisms controlling the process of cold acclimation and to identify genes involved in plant freezing tolerance, mutations that impaired the cold acclimation capability of Arabidopsis thaliana (L.) Heynh were screened for. A new mutation, frs1 (freezing sensitive 1), that reduced both the constitutive freezing tolerance as well as the freezing tolerance of Arabidopsis after cold acclimation was characterized. This mutation also produced a wilty phenotype and excessive water loss. Plants with the frs1 mutation recovered their wild-type phenotype, their capability to tolerate freezing temperatures and their capability to retain water after an exogenous abscisic acid (ABA) treatment. Measurements of ABA revealed that frs1 mutants were ABA deficient, and complementation tests indicated that frs1 mutation was a new allele of the ABA3 locus showing that a mutation in this locus leads to an impairment of freezing tolerance. These results constitute the first report showing that a mutation in ABA3 leads to an impairment of freezing tolerance, and not only strengthen the conclusion that ABA is required for full development of freezing tolerance in cold-acclimated plants, but also demonstrate that ABA mediates the constitutive freezing tolerance of Arabidopsis. Gene expression in frs1 mutants was altered in response to dehydration, suggesting that freezing tolerance in Arabidopsis depends on ABA-regulated proteins that allow plants to survive the challenges imposed by subzero temperatures, mainly freeze-induced cellular dehydration.

Acclimatization↗
PubMed2005

Effect of freeze-drying and gamma irradiation on biomechanical properties of bovine pericardium.

Freeze-drying and gamma irradiation are the techniques widely use in tissue banking for preservation and sterilization of tissue grafts respectively. However, the effect of these techniques on biomechanical properties of bovine pericardium is poorly known. A total of 300 strips of bovine pericardium each measured 4 cm x 1 cm were used in this study to evaluate the effect of freeze-drying on biomechanical properties of fresh bovine pericardium and the effect of gamma irradiation on biomechanical properties of freeze-dried bovine pericardium. The strips were divided into three equal groups, which consist of 100 strips each group. The three groups were fresh bovine pericardium, freeze-dried bovine pericardium and irradiated freeze-dried bovine pericardium. The biomechanical properties of the pericardial strips were measured by a computer controlled instron tensiometer while the strips thickness was measured by Mitutoyo thickness gauge. The results of the study revealed that freeze-drying has no significant (p > 0.05) effect on the tensile strength, Young's modulus (stiffness) and elongation rate of fresh bovine pericardium. Irradiation with 25 kGy gamma rays caused significant decreased in the tensile strength, Young's modulus and elongation rate of the freeze-dried pericardium. However, gamma irradiation has no significant effect on the thickness of freeze-dried bovine pericardium, while freeze-drying caused significant decreased in the thickness of the fresh bovine pericardium. The outcome of this study demonstrated that freeze-drying has no significant effect on the biomechanical properties of fresh bovine pericardium, and gamma irradiation caused significant effect on the biomechanical properties of freeze-dried bovine pericardium.

Animals↗
PubMed1986

Comparison of methods for freezing interleukin-dependent murine cell lines.

Various methods for freezing several interleukin-dependent murine cell lines have been compared and were shown to differ in their efficacy from line to line. An interleukin-2-dependent cell line, T572.C5, which was more sensitive to dimethylsulfoxide (DMSO) than the other cell lines, was the most difficult to freeze in a viable state. In order to define better conditions for freezing, the effects of the changes in freezing rates and concentrations of DMSO and of the addition of Ficoll were determined. The optimal freezing rates and DMSO concentrations varied from line to line. The addition of Ficoll increased the viability in some, but not all, cell lines, depending on the freezing conditions used. Thus, the optimal conditions for freezing interleukin-dependent cell lines varied from line to line. In general, the direct freezing procedure was better than the styrofoam-box freezing method. Although programmed freezing under direct temperature control was the most suitable among the freezing procedures examined, unprogrammed procedures also were of practical use.

Animals↗
PubMed2004

Exposure of hepatitis B vaccine to freezing temperatures during transport to rural health centers in Mongolia.

BACKGROUND: A previous evaluation of the Mongolian hepatitis B virus (HBV) infant vaccination program showed that only 70% of rural subjects developed protective antibody levels compared to 94% of urban subjects. The difference is likely due to damaged vaccine being administered to rural infants. HBV vaccine is heat-stable, but freezing destroys immunogenicity. The current study was designed to ascertain if HBV is subject to freezing temperatures during transport to rural health centers. METHODS: During several time periods from 2001 to 2003, HBV vaccine transported from the national vaccine store to provincial stores and from two provincial stores to rural health centers was monitored for freezing using 3M Freeze Watch indicators. RESULTS: Of 181 provincial-to-rural transports, 19% (95% CI 13-25%) resulted in freezing. One of 59 or 1.6% (95% CI 0-5.6%) of national-to-provincial store transports indicated freezing. In the second half of each substudy, freezing events diminished. Duration of transport was associated with freezing. CONCLUSION: Identification of freezing temperatures during provincial-to-rural vaccine transport provides evidence that HBV vaccine is being damaged. This is the likely cause of the poor vaccine response in the Mongolian countryside. The authors speculate that packing vaccines for transport with ice taken directly from storage deep freezes at -20 degrees C is the cause of the freezing.

Freezing↗
PubMed2003

Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis.

The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6+/-0.2%) and sucrose the highest (6.4+/-1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1 x 10(7) to 5 x 10(10) CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium. The highest freeze-drying survival values, 15-25%, were obtained for initial cell concentrations between 1 x 10(9) and 1 x 10(10) CFU/ml. For cell concentrations outside this window more than 10 times lower survival values were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26+/-6%.

Animals↗
PubMed2001

Effect of dissolved organic material and cations on freeze-thaw conditioning of activated and alum sludges.

Freeze-thaw conditioning effectively dewaters alum and activated sludges, but it works better on alum sludge than it does on activated sludge. The main difference between alum sludge and activated sludge is that activated sludge has high concentrations of both dissolved organic material and ions. Dissolved organic material and ions may possibly alter the freezing process and decrease the effectiveness of freeze-thaw conditioning on activated sludge. The objective of this study is to investigate the effect of dissolved organic material and cations on freeze-thaw conditioning of sludges, and to improve the effectiveness of freeze-thaw conditioning on activated sludge. The results of this study show that although protein, carbohydrate and cation concentrations in activated sludge supernatant are initially high, they dramatically increase after freeze-thaw conditioning. The increase is likely to come from the release of extracellular and intracellular material to sludge supernatant. The observed increase in the DNA concentration in activated sludge supernatant after freeze-thaw conditioning indicates that freeze-thaw causes cell disruption. Alum sludge supernatant, on the other hand, initially contains low concentrations of proteins, carbohydrates and cations which do not noticeably change after freeze-thaw conditioning. When ECPs (extracellular polymers) and cations are extracted from activated sludge before freeze-thaw conditioning. the sludge settles and dewaters better after the freeze-thaw. The resulting aggregates are smaller and denser resembling the "coffee ground" aggregates of alum sludge.

Adjuvants, Immunologic↗
PubMed1998

Effects of freeze-dry processing conditions on the crystallization of pentamidine isethionate.

The results of this study show that pentamidine isethionate (PI) can exist in at least four crystalline forms-three anhydrates designated as forms A, B, and C, and a trihydrate. Form C is the high-temperature modification, produced by heating forms A, B, and the trihydrate above 130 degrees C and cannot be produced under actual lyophilization conditions. The crystal forms of PI present after freeze-drying depend on the initial solution concentration and the thermal history of freezing. At low concentrations of PI (4% and less), form A is observed regardless of freezing method. At a higher concentration (10%), the crystal forms observed are a function of the freezing method. Three freezing methods were used to effect different cooling rates: (1) cooling on the shelf to 2 degrees C and holding for 3 h prior to decreasing the temperature to -45 degrees C, (2) directly cooling on the shelf from room temperature to -45 degrees C, and (3) dipping the vials in liquid nitrogen. The results show that form A, form B, or a mixture of both forms are present in the freeze-dried solid depending upon whether the trihydrate crystallizes during freezing or not. Since form B can only be produced by dehydration of the trihydrate at low temperature, the presence of this form in the freeze-dried powders depends on the nucleation and growth of the trihydrate during freezing. Photostability studies have demonstrated marked differences between freeze-dried solids frozen under different conditions. The results underscore the importance of recognizing that seemingly subtle differences in processing conditions can have a significant impact on critical quality attributes of freeze-dried products.

Antiprotozoal Agents↗
PubMed2007

A new scaleable freeze-thaw technology for bulk protein solutions.

A new method of freeze-thaw is described using experimental data obtained from freezing of purified rhGH (recombinant human growth hormone). The method is based on freezing protein solutions in rectangular rather than cylindrical containers. It is hypothesized that the change in container geometry allows for linear scale-up of the freeze-thaw operation based on equivalency of temperature-time profile. The hypothesis is tested using freeze-thaw data from a miniature (30 ml) and a 2.4 litre container. Computational fluid dynamics techniques are used to simulate the freeze process and the simulations are compared with experimental results. Protein quality is assessed as a function of freeze conditions using dynamic light scattering, circular CD, size-exclusion and reverse-phase HPLC measurements. The results demonstrate the applicability of the new approach. Freezing of rhGH solution at concentrations of approx. 30 mg/ml is shown to be possible with no damage to the molecule for up to five cycles of freeze-thaw. A nitrogen blast chest-freezer is designed and evaluated as part of the process. The refrigeration system and the freeze-thaw method can be used to freeze-thaw bulk protein solutions for development work and has the potential for transfer to manufacturing.

Chromatography, Gel↗
PubMedDate not supplied

Freezing of plasma and recovery of factor VIII.

The freezing procedure and its influence on the plasma quality was studied with a new plasma container and freezing bath and compared with the results from those obtained with commonly used equipment. Cryoprecipitation was performed on plasma pools frozen at three different rates, and quality and recovery of the cryoprecipitates were determined by analysis of factor VIII, factor VIII-related antigen (vWF:Ag), and fibrinogen. The plasma freezing time was 95 and 65 percent shorter with a new -40 degrees C freezing bath as compared to -25 and -80 degrees C freezing boxes, respectively. A further 30 percent reduction of the plasma freezing time was gained by the introduction of a new flat 750-ml plasma container. Rapid plasma freezing prevented substantial loss of factor VIII in frozen plasma. Cryoprecipitate purity measured as factor VIII-to-fibrinogen ratio increased from 0.50 to 0.82 (IU/mg) when the freezing time was decreased from 10 hours to 45 minutes, although the recovery of factor VIII increased less. In summary, freezing of plasma in small flat containers in an effective ethanol bath resulted in rapid freezing with high recovery of factor VIII in plasma, and increased purity and recovery of subsequently processed cryoprecipitate. This freezing concept, adapted at Swedish blood banks, has contributed to higher source plasma quality and increased self-sufficiency.

Blood Preservation↗
PubMed2003

Freezing tolerance of conifer seeds and germinants.

Survival after freezing was measured for seeds and germinants of four seedlots each of interior spruce (Picea glauca x engelmannii complex), lodgepole pine (Pinus contorta Dougl. ex Loud.), Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and western red cedar (Thuja plicata Donn ex D. Donn). Effects of eight seed treatments on post-freezing survival of seeds and germinants were tested: dry, imbibed and stratified seed, and seed placed in a growth chamber for 2, 5, 10, 15, 20 or 30 days in a 16-h photoperiod and a 22/17 degrees C thermoperiod. Survival was related to the water content of seeds and germinants, germination rate and seedlot origin. After freezing for 3 h at -196 degrees C, dry seed of most seedlots of interior spruce, Douglas-fir and western red cedar had 84-96% germination, whereas lodgepole pine seedlots had 53-82% germination. Freezing tolerance declined significantly after imbibition in lodgepole pine, Douglas-fir and interior spruce seed (western red cedar was not tested), and mean LT50 of imbibed seed of these species was -30, -24.5 and -20 degrees C, respectively. Freezing tolerance continued to decline to a minimum LT50 of -4 to -7 degrees C after 10 days in a growth chamber for interior spruce, Douglas-fir and lodgepole pine, or after 15 days for western red cedar. Minimum freezing tolerance was reached at the stage of rapid hypocotyl elongation. In all species, a slight increase in freezing tolerance of germinants was observed once cotyledons emerged from the seed coat. The decrease in freezing tolerance during the transition from dry to germinating seed correlated with increases in seed water content. Changes in freezing tolerance between 10 and 30 days in the growth chamber were not correlated with seedling water content. Within a species, seedlots differed significantly in freezing tolerance after 2 or 5 days in the growth chamber. Because all seedlots of interior spruce and lodgepole pine germinated quickly, there was no correlation between seedlot hardiness and rate of germination. Germination rate and freezing tolerance of Douglas-fir and western red cedar seedlots was negatively correlated. There was a significant correlation between LT50 after 10 days in the growth chamber and minimum spring temperature at the location of seedlot origin for interior spruce and three seedlots of western red cedar, but no relationship was apparent for lodgepole pine and Douglas-fir.

Freezing↗