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PubMed1999

A combined solution exchange/plunge-freezing device for skinned muscle fibers.

For many contractility studies, defined functional states of skinned muscle fiber preparations can be introduced by application of standardized perfusion protocols with large varieties of experimental solutions. Functionally important subcellular element distributions in the myoplasm and in the sarcoplasmic reticulum can be measured with high spatial resolution by electron microscopic microanalysis. Capturing these subcellular ion distributions requires their rapid immobilization by quick-freezing. We therefore combined a plunge-freezing device with a semiautomatic solution exchanger to reproducibly perfuse skinned muscle fiber bundles with multiple solutions. The isometric tension produced is simultaneously recorded as an indicator for the functional state. The samples can be quick-frozen at any chosen time of the tension transient. A cryoglueing technique finally delivers specimens suitable for cryoultramicrotomy.

Animals↗
PubMed2006

Influence of hemolysis on routine clinical chemistry testing.

BACKGROUND: Preanalytical factors are the main source of variation in clinical chemistry testing and among the major determinants of preanalytical variability, sample hemolysis can exert a strong influence on result reliability. Hemolytic samples are a rather common and unfavorable occurrence in laboratory practice, as they are often considered unsuitable for routine testing due to biological and analytical interference. However, definitive indications on the analytical and clinical management of hemolyzed specimens are currently lacking. Therefore, the present investigation evaluated the influence of in vitro blood cell lysis on routine clinical chemistry testing. METHODS: Nine aliquots, prepared by serial dilutions of homologous hemolyzed samples collected from 12 different subjects and containing a final concentration of serum hemoglobin ranging from 0 to 20.6 g/L, were tested for the most common clinical chemistry analytes. Lysis was achieved by subjecting whole blood to an overnight freeze-thaw cycle. RESULTS: Hemolysis interference appeared to be approximately linearly dependent on the final concentration of blood-cell lysate in the specimen. This generated a consistent trend towards overestimation of alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine, creatine kinase (CK), iron, lactate dehydrogenase (LDH), lipase, magnesium, phosphorus, potassium and urea, whereas mean values of albumin, alkaline phosphatase (ALP), chloride, gamma-glutamyltransferase (GGT), glucose and sodium were substantially decreased. Clinically meaningful variations of AST, chloride, LDH, potassium and sodium were observed in specimens displaying mild or almost undetectable hemolysis by visual inspection (serum hemoglobin < 0.6 g/L). The rather heterogeneous and unpredictable response to hemolysis observed for several parameters prevented the adoption of reliable statistic corrective measures for results on the basis of the degree of hemolysis. CONCLUSION: If hemolysis and blood cell lysis result from an in vitro cause, we suggest that the most convenient corrective solution might be quantification of free hemoglobin, alerting the clinicians and sample recollection.

Artifacts↗
PubMedDate not supplied

Two urinary catecholamine measurement indices for applied stress research: effects of time and temperature until freezing.

Research on stress at work often involves the analysis of urinary adrenaline and noradrenaline. It is usually assumed that samples have to be cooled quickly and stored at refrigerator temperature before freezing. This is often difficult to achieve in field studies. This experimental study therefore tests the robustness of results when samples are not cooled immediately. Samples of 9 men and women, collected at 3 points in time, were immediately frozen, kept for a variable delay in a warm room, or stored in a refrigerator before freezing. Two indices were calculated: (a) the ratio of hormones to liquid volume, period of excretion, and body weight; and (b) the ratio of hormones to urinary creatinine. The reliability of high performance liquid chromatography analysis was satisfactory, as was the comparability of the 2 indices. Unfavorable storage up to 24 hr did not cause bacteria-driven decreases of catecholamines, regardless of storage temperature or sampling time. Results suggest high stability for at least 24 hr without cooling, provided the samples are immediately acidified. Cooling may therefore be handled less restrictively than has been assumed. The application of this research is to facilitate research in settings where samples are collected at different places, such as participants' homes or different workplaces.

Adult↗
PubMed1999

The feasibility of using blood bank-stored (4 degrees C) cord blood, unmatched for HLA for marrow transplantation.

The main purpose for storing large numbers of umbilical cord blood (CB) units by cryopreservation is to obtain a close HLA match for use in bone marrow transplantation. The use of partially matched or unmatched CB has been suggested, and publications about the success of 3 antigen mismatches have given some credence to this suggestion. Graft vs host disease still is considered a major barrier for successful CB transplantation. The cost per frozen CB unit of approximately $15,000 considerably limits its availability in developing countries. Eleven human umbilical cord specimens were stored in gas-permeable bags at 4 degrees C for up to 3 weeks. Clonal growth, replating efficiency in methylcellulose cultures, differential count, and flow cytometric immunophenotyping results were examined at intervals up to 21 days. Mixed lymphocyte cultures were evaluated on 13 similarly stored specimens at intervals up to 14 days. When plated at 1, 10, and 21 days, the combined percentage of the more primitive colonies increased on days 10 and 21. Replating efficiency of blast cell colonies when stem cell factor was added was 81.2% and 67.8% on days 10 and 21, respectively. When mononuclear cells were immunophenotyped, the mean percentage of CD34+ and CD117+ cells, considered primitive stem cell markers, increased significantly from day 1 to day 21. The ability of stored CB cells to respond to phytohemagglutinin or alloantigens decreased progressively from day 1 to day 14. By day 14, the reactivity of CB responder cells, in mixed lymphocyte cultures, to fresh allogeneic CB stimulator cells declined significantly. These findings suggest that CB can be stored in existing blood bank facilities and retain its hematopoietic potential for transplantation. Furthermore, it may be feasible to combine individual CB samples to provide a sufficient number of viable stem cells for transplantation, substantially expanding the number of potential recipients.

Blood Banks↗
PubMed1996

Comparison of the different strategies for cryopreserving and storage of the bone marrow CD34+ cells. Possibility of unprogrammed rate freezing and storage at -80 degrees C mechanical freezer.

We have compared the efficiency of the different strategies allowing for a long term storage of a human CD34+ bone marrow cells. Accordingly, the aliquots of CD34+ cells isolated from bone marrow were frozen using: controlled rate freezing equipment, or freezer unprogrammed in a -80 degrees C mechanical freezer. After freezing, CD34+ cells were subsequently stored for one month in a liquid nitrogen tank at -196 degrees C or in mechanical freezer at -80 degrees C. We have found that both the viability and the recovery of clonogeneic progenitors of CD34+ cells samples stored at different temperature were similar. Therefore, regarding the costs and simplicity, we recommend the unprogrammed freezing and storage of human CD34+ cells at -80 degrees C in a mechanical freezer as a convenient, inexpensive, and reliable method for storing marrow for transplantation. This data also indirectly indicate that the aliquots of the CD34+ cells can be shipped frozen on dry ice (-80 degrees C), and that these cells will maintain viability under this conditions. Furthermore, in this study we have confirmed validity of our earlier observation that human CFU-Meg progenitors are more sensitive to cryopreservation.

Antigens, CD↗
PubMed1993

Invited commentary: cryopreservation as a tool for the study of selectively bred lines in rodent behavioral genetics.

Cryopreserved embryos should survive centuries. Upon rederivation, in vitro survival is high. Upon reimplantation, in vivo survival is substantial. Genetic stability of the embryos at -196 degrees C is theoretically absolute for several decades and likely high for centuries. Cryopreservation and rederivation services are available at a reasonable cost from commercial vendors. Preservation of a sufficient number of embryos can serve as an insurance policy against loss of the lines from several causes. In addition to the protective utility of a single banked sample, repeated preservation during the course of selection offers several advantages. Correlated responses to selection which appear late in selection may be verified by use of a cross-sectional experimental design to take a "snapshot" of the gradually developing response. After attainment of the selection limit, a generation may be chosen retrospectively which represents the best compromise between maximizing response and minimizing inbreeding irrelevant to the selected trait. Estimates of changes in gene frequencies could be derived from molecular biological analyses. An example of such an experiment in progress employing embryos from the COLD and HOT selected mouse lines is discussed.

Animals↗
PubMed2001

Long-term outcomes of elective human sperm cryostorage.

BACKGROUND: Sperm cryopreservation allows men with threatened fertility to preserve their progenitive potential, but there is little data on long-term outcomes of elective sperm cryostorage programmes. METHODS AND RESULTS: Over 22 years, 930 men sought semen cryostorage in a single academic hospital, of which 833 (90%) had spermatozoa cryostored. Among 692 (74%) men surviving their illness, sperm samples were discarded for 193 (21% of all applicants, 28% of survivors) and cryostored spermatozoa were used for 64 men (7% of all applicants, 9% of survivors) in 85 treatment cycles commencing at a median of 36 months post-storage (range 12-180 months) with nearly 90% of usage started within 10 years of storage and none after 15 years. Pregnancy was most efficiently produced by intracytoplasmic sperm injection (median three cycles) compared with conventional IVF (median eight cycles) or artificial insemination (median more than six cycles; P < 0.05). A total of 141 (15%) of men had died and of these, 120 (85% of those dying) had their spermatozoa discarded; requests to prolong cryostorage were received from relatives of 21 men (2% of all applicants, 15% of deceased) of which three cases had spermatozoa transferred for use with no pregnancies reported. Sperm concentration was lower for all cryostorage groups compared with healthy sperm donor controls (P < 0.05). Following orchidectomy, men with testicular cancer had sperm density approximately half that of all other groups of men seeking cryostorage (P < 0.05), the lowering attributable to removal of one testis rather than in defects in spermatogenesis. CONCLUSION: Elective sperm cryopreservation is an effective, if sparsely used, form of fertility insurance for men whose fertility is threatened by medical treatment and is an essential part of any comprehensive cancer care programme.

Adult↗
PubMed1993

Validation of the hemizona assay (HZA) in a monkey model. II. Kinetics of binding and influence of cryopreserved-thawed spermatozoa.

We compared fresh and frozen-thawed cynomolgus monkey spermatozoa tight binding to the zona pellucida under hemizona assay (HZA) conditions. Monkey oocytes were recovered after superovulation and stored in salt solution. Matching hemizonae were obtained by micromanipulation. Semen, obtained by electroejaculation, was used fresh or was cyropreserved, thawed, and washed by swim-up separation. At the standard initial dilution of 500,000 motile sperm/ml (or 5 x 10(4) motile sperm/hemizona), binding was significantly higher for fresh sperm (P = 0.00004). For frozen-thawed samples, there was a linear increase in the number of tightly bound sperm with increasing sperm concentration (r = 0.95). At 1.5 x 10(6) motile sperm/hemizona, binding of frozen-thawed spermatozoa was similar to that of fresh at a standard concentration. Kinetic studies showed peak binding at 1 hr of gametes coincubation. We conclude that, in this monkey model, the HZA is a valuable bioassay for evaluation of sperm binding to the zona pellucida, the initial requisite for fertilization and embryo development.

Animals↗
PubMed2006

Influence of various permeating cryoprotectants on freezability of Iberian red deer (Cervus elaphus hispanicus) epididymal spermatozoa: effects of concentration and temperature of addition.

With the aim of finding an ideal cryoprotectant (CPA) in a suitable concentration for red deer epididymal spermatozoa cryopreservation, we evaluated the effects of the 3 most commonly used CPAs, glycerol (GLY), ethylene glycol (EG), and propylene glycol (PG), on sperm cryoresistance. The aim of Experiment 1 was to evaluate the influence of 3 different final concentrations (3%, 6%, and 12%) of each CPA on sperm freezability. Sperm samples were diluted to a final sperm concentration of approximately 400 x 10(6) spermatozoa/mL with a Tris-citrate-fructose-EY extender (TCF) prior to freezing. Sperm cryosurvival was judged in vitro by microscopic assessments of individual sperm motility (SMI), viability, and plasma membrane (by means of the HOS test) and acrosome (NAR) integrities. Thawed samples were incubated at 37 degrees C for 2 hours in the freezing medium. At the end of this incubation period, sperm suspensions were again assessed. Our results showed that 12% of any CPA was toxic to red deer epididymal spermatozoa membrane integrity (P < .05). Moreover, regardless of the level of CPA, results indicated that the cryoprotective effects on red deer epididymal spermatozoa of the 3 CPAs after thawing are in the following sequence: GLY > EG > PG (higher symbols mean P < .001). Furthermore, our results also showed an improvement in sperm parameters when the TCF diluent contained 6% of GLY. In Experiment 2 extenders were prepared using GLY 6%. This experiment was designed to investigate the effect of 2 different temperatures of GLY addition -22 degrees C (ambient temperature) and 5 degrees C- on sperm freezability. Our results showed a differential response (P < .05) of motility (SMI) to temperature of GLY addition before freezing, the best being 22 degrees C (81.94 +/- 2.4% vs 72.38 +/- 2.4%). Although there were no statistically significant differences (P > .05) between the 2 temperatures of GLY addition after thawing in terms of sperm quality, after 2 hours of incubation, results tended to be better when CPAs were added at 22 degrees C. In conclusion, our work showed the efficacy of a TCF diluent with 6% of GLY and its addition at 22 degrees C, as an alternative to the more common 3%-4% of GLY and addition at 5 degrees C, in red deer semen freezing protocols.

Acrosome↗
PubMed1987

Effects of exposing bovine sperm to bovine serum albumin, or freeze-thawing, on sperm-bound amidase activity.

Isolation of a self-selected population of motile spermatozoa is possible by using a gradient of bovine serum albumin (BSA). We determined if exposure to BSA altered the sperm or if isolated sperm differed from nonisolated cells in terms of motility or activity of sperm-bound amidase, either before or after subsequent cryopreservation. Exposure of sperm to 6% BSA in egg yolk Tris extender induced changes in the plasma and acrosomal membranes of sperm that resulted in exposure and activation of sperm-bound amidase (P less than .01). In experiment 2, semen extended in egg yolk Tris was cooled to 5 degrees C or layered onto a solution of 6% BSA in extender at 37 degrees C, from which the sperm that had swum into the BSA solution were recovered 2 h later and cooled to 5 degrees C. Sperm in both treatments were cryopreserved. The percentage of progressively motile sperm was determined visually and by track motility. Activity of sperm-bound amidase exposed to substrate was evaluated. After recovery of sperm from the 6% BSA solution, 81% were progressively motile as compared to 59% in the starting samples (P less than .01). However, the amount of exposed sperm-bound amidase also was greater (P less than .05); this was a deleterious change. Immediately after thawing, more (P less than .01) sperm were motile in samples of isolated sperm than for nonisolated cells (43 vs 24%), but after incubating the thawed sperm for 1 h at 37 degrees C there was no difference.(ABSTRACT TRUNCATED AT 250 WORDS)

Amidohydrolases↗
PubMed2006

Suprazero cooling conditions significantly influence subzero permeability parameters of mammalian ovarian tissue.

To model the cryobiological responses of cells and tissues, permeability characteristics are often measured at suprazero temperatures and the measured values are used to predict the responses at subzero temperatures. The purpose of the present study was to determine whether the rate of cooling from +25 to +4 degrees C influenced the measured water transport response of ovarian tissue at subzero temperatures in the presence or absence of cryoprotective agents (CPAs). Sections of freshly collected equine ovarian tissue were first cooled either at 40 degrees C/min or at 0.5 degrees C/min from 25 to 4 degrees C, and then cooled to subzero temperatures. A shape-independent differential scanning calorimeter (DSC) technique was used to measure the volumetric shrinkage during freezing of equine ovarian tissue sections. After ice was induced to form in the extracellular fluid within the specimen, the sample was frozen from the phase change temperature to -50 degrees C at 5 degrees C/min. Replicate samples were frozen in isotonic medium alone or in medium containing 0.85 M glycerol or 0.85 M dimethylsulfoxide. The water transport response of ovarian tissue samples cooled at 40 degrees C/min from 25 to 4 degrees C was significantly different (confidence level >95%) from that of tissue samples cooled at 0.5 degrees C/min, whether in the presence or absence of CPAs. We fitted a model of water transport to the experimentally-derived volumetric shrinkage data and determined the best-fit membrane permeability parameters (L(pg) and E(Lp)) of equine ovarian tissue during freezing. Subzero water transport parameters of ovarian tissue samples cooled at 0.5 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.06 to 0.73 microm/min.atm and E(Lp) = 6.1 to 20.5 kcal/mol. The corresponding parameters of samples cooled at 40 degrees C/min from 25 to 4 degrees C ranged from: L(pg) = 0.04 to 0.61 microm/min.atm and E(Lp) = 8.2 to 54.2 kcal/mol. Calculations made of the theoretical response of tissue at subzero temperatures suggest that the optimal cooling rates to cryopreserve ovarian tissue are significantly dependent upon suprazero cooling conditions.

Animals↗
PubMed2005

Changes of viability and composition of the Escherichia coli flora in faecal samples during long time storage.

Long-time storage of faecal samples is necessary for investigations of intestinal microfloras. The aim of the present study was to evaluate how the viability and the composition of the Escherichia coli flora are affected in faecal samples during different storage conditions. Four fresh faecal samples (two from calves and two from infants) were divided into sub-samples and stored in four different ways: with and without addition of glycerol broth at -20 degrees C and at -70 degrees C. The viability and the phenotypic diversity of the E. coli flora in the sub-samples were evaluated after repeated thawings and after storage during 1 year. The samples stored for 1 year without thawing were also kept at room temperature for 5 days and subsequently analysed. According to phenotyping (PhP analysis) of 32 isolates per sample on day 0, all four samples contained two dominating strains of E. coli each, and between one and eight less common strains. Samples that were stored at -70 degrees C in glycerol broth showed equal or even higher bacterial numbers as the original samples, even after repeated thawings, whereas samples stored at -20 degrees C showed a considerably lower survival rate, also with addition of glycerol. Sub-samples containing glycerol broth that were kept at room temperature after storage for 1 year showed a clear increase in the number of viable cells as well as in diversity. The diversities in each sub-sample showed a tendency to decrease after several thawings as well as after storage. Generally, the E. coli populations in samples stored at -20 degrees C were less similar to the population of the original sample than that in samples stored at -70 degrees C. Samples that had been mixed with glycerol broth had an E. coli flora more similar to that in the original sample than those without glycerol broth. Furthermore, the sub-samples that were kept at room temperature after storage for 1 year generally were more similar to the original samples than if they were processed directly. We conclude that for long time storage of faecal samples, storage at -70 degrees C is preferable. If samples have to be thawed repeatedly, addition of glycerol is preferable both for samples stored at -70 degrees C and for samples stored at -20 degrees C. Our data also have indicated that when E. coli isolates from faecal samples are selected for, e.g. analysis of virulence factors, it is necessary to pick several isolates per sample in order to obtain at least one isolate representing the dominating strain(s).

Animals↗
PubMed2005

New assays for detection and localization of endogenous lipid peroxidation products in living boar sperm after BTS dilution or after freeze-thawing.

Reactive oxygen species have been implicated in sperm aberrations causing multiple pathologies including sub- and infertility. Freeze/thawing of sperm samples is routinely performed in the cattle breeding industries for semen storage prior to artificial insemination but unusual in porcine breeding industries as semen dilution and storage at 17 degrees C is sufficient for artificial insemination within 2-3 days. However, longer semen storage requires cryopreservation of boar semen. Freeze/thawing procedures induce sperm damage and induce reactive oxygen species in mammalian sperm and boar sperm seems to be more vulnerable for this than bull sperm. We developed a new method to detect reactive oxygen species induced damage at the level of the sperm plasma membrane in bull sperm. Lipid peroxidation in freshly stored and frozen/thawed sperm cells was assessed by mass spectrometric analysis of the main endogenous lipid classes, phosphatidylcholine and cholesterol and by fluorescence techniques using the lipid peroxidation reporter probe C11-BODIPY(581/591). Peroxidation as reported by the fluorescent probe, clearly corresponded with the presence of hydroxy- and hydroperoxyphosphatidylcholine in the sperm membranes, which are early stage products of lipid peroxidation. This allowed us, for the first time, to correlate endogenous lipid peroxidation with localization of this process in the living sperm cells. Cytoplasmatic droplets in incompletely matured sperm cells were intensely peroxidized. Furthermore, lipid peroxidation was particularly strong in the mid-piece and tail of frozen/thawed spermatozoa and significantly less intense in the sperm head. Induction of peroxidation in fresh sperm cells with the lipid soluble reactive oxygen species tert-butylhydroperoxide gave an even more pronounced effect, demonstrating antioxidant activity in the head of fresh sperm cells. Furthermore, we were able to show using the flow cytometer that spontaneous peroxidation was not a result of cell death, as only a pronounced subpopulation of living cells showed peroxidation after freeze-thawing. Although the method was established on bovine sperm, we discuss the importance of these assays for detecting lipid peroxidation in boar sperm cells.

Animals↗
PubMed1998

Two-year stability of free and total PSA in frozen serum samples.

OBJECTIVES: To investigate the stability of free and total prostate-specific antigen (PSA) in serum stored at -20 degrees C and -70 degrees C for 2 years. METHODS: Fresh blood from 29 patients was drawn and allowed to clot at room temperature. Serum was prepared by centrifugation 1 hour later and tested to determine the free and total PSA levels. Samples were aliquoted, frozen at -20 degrees C or -70 degrees C, and monitored for long-term stability over the course of 2 years. RESULTS: On average, 91% of the free PSA, 92% of the total PSA, and 89% of the percent free PSA values were recovered after 2 years of storage at -20 degrees C; and 96% of the free PSA, 101% of the total PSA, and 95% of the percent free PSA values were recovered after 2 years of storage at -70 degrees C. Seventy percent of the samples stored at -20 degrees C and 100% of the samples stored at -70 degrees C showed no statistically significant decline in free PSA values over the 2-year period. Only one total PSA sample (-20 degrees C) showed a statistically significant loss after 2 years of storage. CONCLUSIONS: Our results suggest that samples stored frozen for up to 2 years at -70 degrees C should give acceptable percent free PSA results.

Aged↗
PubMed2003

Role of incidental and/or cured intestinal parasitic infections on profile of CD4+ and CD8+ T cell subsets and activation status in HIV-1 infected and uninfected adult Ethiopians.

Intestinal parasitic infections have been suggested to cause persistent immune activation leading to an unbalanced immune state. Such a state has been proposed to be a major factor in the pathogenesis of AIDS in an African context. The present study investigated the effect of incidental parasitic infection and treatment on the profile of T cell differentiation and activation markers on CD4+ and CD8+ T cells from HIV-1 infected and uninfected adult Ethiopians. Cryopreserved PBMCs from 64 subjects (41 HIV-negative and 23 HIV-positive) with follow-up visits at 6-monthly intervals were used to compare the effect of incidental intestinal parasites and their treatment upon T cell subset profiles and activation status. The samples were stained with antibodies to various T cell differentiation and activation markers allowing naive, memory, effector, memory/effector, activated and resting CD4+ and CD8+ T cell subsets to be quantified by triple-colour FACScan. Incidental intestinal parasitic infections resulted in a significant increase in memory CD4+ T cell numbers both in HIV-negative and HIV-positive subjects (P < 0.05). There was also a significant increase in the percentage of CD8+ HLA-DR+ T cells (P < 0.05) in HIV-positive subjects co-infected with parasites. In HIV-negative subjects, a significant decline in activated cells and a significant increase in resting CD8+ T cells (P < 0.05) was observed after treatment for parasites. These data suggest that intestinal parasitic infections could result in the alteration of T cell subset counts and also in the up-regulation of T cell activation markers in peripheral blood. Treatment of parasitic infections showed a tendency to reduce the activation suggesting that, together with other community based intervention strategies, such treatment could be used to down-regulate immune activation and hence protect the host from being easily attacked by HIV.

Adult↗
PubMed1989

Human immunodeficiency virus type 1 infection in homosexual men who remain seronegative for prolonged periods.

Infection with the human immunodeficiency virus type 1 (HIV-1), as demonstrated by viral cultures, has been described in some patients before antibodies to HIV-1 can be detected, but the duration and frequency of such latent infections are uncertain. We selected prospectively a cohort of 133 seronegative homosexual men who continued to be involved in high-risk sexual activity, and we cultured 225 samples of their peripheral-blood lymphocytes, using mitogen stimulation to activate the integrated HIV-1 genome. HIV-1 was isolated in blood samples from 31 of the 133 men (23 percent), 27 of whom have remained seronegative for up to 36 months after the positive culture. The other four men seroconverted 11 to 17 months after the isolation of HIV-1. In three of them, we studied cryopreserved lymphocytes obtained earlier, using the polymerase chain reaction to amplify small amounts of viral DNA, and we demonstrated that HIV-1 provirus had been present 23, 35, and 35 months before seroconversion. We conclude that HIV-1 infection in homosexual men at high risk may occur at least 35 months before antibodies to HIV-1 can be detected. A prolonged period of latency in such infections may be more common than previously recognized; the degree of infectiousness during such periods is unknown.

Acquired Immunodeficiency Syndrome↗
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↗
PubMed2003

Measurement of plasma viscosity in stored frozen samples: a general population study.

To establish whether plasma viscosity could be accurately measured from frozen-stored (instead of fresh) plasma samples in epidemiological studies, we prospectively compared fresh and frozen-stored samples from 1361 men and women aged 25-74 years participating in the Glasgow MONICA survey. Mean plasma viscosity (37 degrees C) was significantly higher (P < 0.0001) in fresh samples (mean = 1.310 mPa/s, standard deviation = 0.079 mPa/s) compared with frozen samples (mean = 1.278 mPa/s, standard deviation = 0.075 mPa/s). However, this difference (mean = 0.030 mPa/s, 95% confidence interval = 0.029-0.031) did not vary significantly with increasing viscosity (kappa statistic for consistency of categorization by thirds = 0.824, standard error = 0.021). We conclude that frozen samples are acceptable for epidemiological studies of plasma viscosity, which should increase their utilization.

Adult↗