Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “sample cryopreservation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 487 records · Page 27Linked to original sources
PubMed2004

Single sperm cryopreservation on cryoloops: an alternative to hamster zona for freezing individual spermatozoa.

Methodology enabling cryopreservation of individual spermatozoa in extreme cases of oligozoospermia would tremendously benefit patients. This study explores the use of a nylon loop for cryopreservation of small quantities of spermatozoa, and also describes a novel technique for freezing individual spermatozoa with this cryoloop. Experiments were conducted to compare sperm recovery and viability after cryopreservation in conventional vials versus on the cryoloops. Discarded human sperm specimens with varying parameters were utilized. The study also examines two different glycerolbased cryoprotectants, with and without test yolk buffer. For single sperm cryopreservation, 5-10 spermatozoa were selected and loaded onto cryoloops with the aid of a microscope and micromanipulation equipment. Sperm function testing was performed on both human and bovine spermatozoa frozen on cryoloops. Microquantities of spermatozoa frozen on cryoloops exhibited overall motility and viability parameters similar to control samples frozen in cryovials. Individually selected spermatozoa cryopreserved on loops were easily recovered and post-thaw motility was generally good. Sperm function testing demonstrated that both human and bovine spermatozoa cryopreserved on loops were able to undergo sperm head decondensation when injected into oocytes. Cryoloops may be an excellent alternative to hamster zonae for cryopreserving small numbers of human spermatozoa.

Animals↗
PubMed2001

Effect of freezing-thawing on the expression of mannose-ligand receptors on human spermatozoa: the impact on sperm capacitation and acrosome reaction.

The aim of this study was to evaluate the change in the expression of mannose-ligand receptors following a freezing-thawing procedure, in order to assess its impact on sperm capacitation and acrosome reaction. Twenty semen samples were obtained from fertile donors. Sperm samples were divided into two equal volumes. One aliquot was cryopreserved and the other aliquot was incubated at 32 degrees C. After 2 h the frozen sample was thawed and both samples were further incubated at 32 degrees C to allow capacitation. Mannose receptors were examined following 4 and 22 h of incubation using a mannosylated-BSA-FITC probe. The expression of mannose-ligand receptors on the sperm plasma membrane was determined according to the fluorescence pattern: pattern I represents pre-capacitation, pattern II represents capacitated spermatozoa and pattern III represents acrosome-reacted spermatozoa. After 4 h incubation in capacitating medium, the percentages of patterns I, II and III were 90, 7 and 3% for fresh spermatozoa and 89, 8 and 3% for frozen-thawed spermatozoa, respectively (P > 0.05). Following 22 h of incubation, the percentages of patterns I, II and III were 84, 11 and 5 for fresh spermatozoa and 83, 11 and 6% for frozen-thawed spermatozoa, respectively (not significant at P > 0.05). The percentages of patterns II and III in fresh and frozen-thawed spermatozoa were increased by the same magnitude with longer incubation in the capacitating conditions. It was concluded that the freezing-thawing procedure for human spermatozoa does not affect the expression of mannose-ligand receptors and the dynamics of sperm pre-fertilization processes.

Acrosome Reaction↗
PubMed1984

Antibody-dependent cell-mediated cytotoxicity of cryopreserved human monocytes.

Human peripheral blood monocytes (PBM) modulate and participate in a variety of host defences. Cryopreservation of PBM has facilitated studies of their function. Peripheral blood samples cleared of red cells and granulocytes by centrifugation over Ficoll-Hypaque were cryopreserved at 1 degree C/min in 10% Me2SO and stored at -150 degrees C. Cryopreserved cells were thawed rapidly, diluted at a constant rate over 10 min with 9 vol of media, and washed twice prior to study. Antibody-dependent cell-mediated cytotoxicity (ADCC) activity against anti-D-coated Rh-positive erythrocytes of both fresh and cryopreserved PBM was tested and found to be equal (52.5 vs 51%). The myeloperoxidase positive, EA-rosette-positive population in cryopreserved cells was 39% compared with 17% for fresh cells (P less than 0.0001). This difference is due to preferential recovery of cryopreserved monocytes among mononuclear cells. The proportion of cells expressing Fc receptors among the myeloperoxidase-positive mononuclear cell population increased after freezing, suggesting an alteration in membrane structure induced by cryopreservation. It is concluded that PBM can be cryopreserved in Me2SO and that ADCC function is fully retained in the cryopreserved cells. This study along with a previous study (R.S. Weiner and S.J. Norman, J. Natl. Cancer Inst. 66, 255-260, 1981) demonstrate the feasibility of using cryopreserved human PBM for functional studies.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1999

Effect of cryopreservation of zona-binding capacity of canine spermatozoa in vitro.

The hemizona assay (HZA) was used as a functional test for zona pellucida binding capacity of fresh and frozen-thawed canine spermatozoa. We investigated 30 ejaculates from 3 dogs with sperm motility > 70% and sperm concentration > 5.10(8) cells per ejaculate with up to 20% abnormal and dead spermatozoa. Fifteen ejaculates were each divided into 2 portions: one portion was used for analysis of fresh semen, the other for cryopreserved semen. On the day of the experiments, in vitro-matured canine oocytes were bisected into 2 equal hemizonae. One half of the hemizonae were coincubated with fresh capacitated (control) spermatozoa, the other half of the hemizonae were coincubated with frozen-thawed (tested) spermatozoa at final concentration of 1 to 2 x 10(6) cells/mL in 200 microL droplets of BSA-supplemented Toyoda, Yokojama and Hoshi (TYH) medium at 37 degrees C, 5%, CO2 for 1 h. Sperm suspensions were examined kinesigraphically for post capacitation type of movement. The Student's t-test was used to compare differences between semen parameters. The data on HZA binding activity of fresh and frozen-thawed canine semen were analyzed by ANOVA and then by the Newman-Keuls multiple range method. The results showed no differences in the initial semen quality parameters among the 3 dogs. After thawing, the semen from Dog 1 and Dog 2 demonstrated relatively uniform sperm parameters, while in Dog 3 sperm motility, and viability and the percentage of morphologically normal spermatozoa were significantly decreased. The binding activity of frozen-thawed spermatozoa from the 3 dogs was significantly reduced (29.40 +/- 9.02, 18.60 +/- 3.30, 8.20 +/- 4.49) compared with that (107.20 +/- 19.22, 109.80 +/- 20.75, 78.20 +/- 12.47; P < 0.01) of fresh spermatozoa. The results showed that semen samples with similar sperm parameters prior to cryopreservation displayed different sperm zona-binding capacity after freezing. The HZI (value of sperm binding capacity of frozen-thawed vs fresh semen samples) was higher in Dog 1 (27.43) than in Dog 2 (16.90) or Dog 3 (10.40), and thus confirmed the variation of zona binding activity after thawing between dogs. The freezability of individual dog semen is discussed. In conclusion HZA may be a valuable tool for evaluating the post-thaw fertilizing ability of canine spermatozoa.

Animals↗
PubMed2003

Effects of vascular endothelial growth factor released from cultured dermal substitute on proliferation of vascular endothelial cells in vitro.

Allogeneic cultured dermal substitute (CDS) was prepared by culturing fibroblasts on a two-layered spongy matrix of hyaluronic acid and atelocollagen. CDS can be cryopreserved and transported to other hospitals in a frozen state. The present study was designed to analyze the amounts of vascular endothelial growth factor (VEGF) released from fibroblasts in fresh and cryopreserved CDS and to investigate the effects of this VEGF on proliferation of vascular endothelial cells in vitro. The culture medium used in preparing CDS (fresh CDS culture medium sample) was collected and stored at -30 degrees C for the quantitative analysis of VEGF. After thawing cryopreserved CDS, it was recultured in a culture medium for 1 week. The culture medium used was collected and stored at -30 degrees C for quantitative analysis of VEGF. The amounts of VEGF released from the fresh and cryopreserved CDS into the culture medium were about 610 pg/ml and 640 pg/ml, respectively. This finding suggests that the cryopreserved CDS retains its ability to release VEGF. Immunohistological analysis indicated that some of the VEGF adhered to the matrix. Human vascular endothelial cells were cultured in medium mixed with the fresh or cryopreserved CDS culture medium sample. Proliferation of vascular endothelial cells was enhanced by increasing the concentration of both CDS culture medium samples. When antihuman VEGF antibody was added to the culture medium, the proliferative activity of vascular endothelial cells was reduced. These findings confirm that VEGF released from CDS promotes proliferation of vascular endothelial cells.

Cell Division↗
PubMed2004

Problems in isolation of Campylobacter jejuni from frozen-stored raw milk and bovine fecal samples: genetic confirmation using multiplex PCR.

The objectives of this study were to evaluate the use of various protocols for the isolation of Campylobacter jejuni from bulk tank milk and bovine fecal samples that were stored frozen for varying times, and to develop a rapid DNA-based protocol that distinguishes C. jejuni from other thermophilic Campylobacter spp. The pathogen was recovered from fecal samples that had been stored for 96-251 days at -20 degrees C with glycerol as the cryopreservative. In a separate study, C. jejuni-positive bovine fecal samples were stored at 5 degrees C for up to 70 days without compromising subsequent recovery of the pathogen. However, the pathogen could not be recovered from pathogenpositive fecal samples stored with or without glycerol (5 mL/11 g sample) for 21 days at -20 degrees C. Bolton broth (BB) and Bolton broth with 5% blood (BBB), containing BB supplement, were used for enrichment. Bacterial isolation was achieved by streaking from BB and BBB, and filtering from BBB onto blood-free charcoal cefoperazone deoxycholate agar (CCDA). The use of BB for the recovery of Campylobacter was more sensitive than BBB, and streaking achieved better isolation rates than filtration. Multiplex PCR incorporating thermophilic Campylobacter-specific 23S rRNA and C. jejuni-specific hippuricase gene sequences was used to confirm C. jejuni. All 265 bulk tank milk samples analyzed were negative for C. jejuni, whereas five of 411 (1.2%) fecal samples tested positive. This is the first report that has used a combination of sequences of the two genes in a multiplex format to identify C. jejuni to the species level. The method described has potential for routine use in the detection of thermophilic Campylobacter in farm environmental samples as well as other samples.

Animals↗
PubMed2003

Free radical scavenger effect of rebamipide in sperm processing and cryopreservation.

AIM: To study the effect of rebamipide added to semen samples and cryoprotectant on reactive oxygen species (ROS) production. METHODS: Semen samples from 30 fertile and healthy volunteers were collected by masturbation after 2 days approximately 3 days of abstinence. After liquefaction, the specimens were diluted with sperm wash media to a uniform density of 20 x 10(6)/mL. Rebamipide was added to semen samples and cryoprotectant to a final concentration of 10 micromol/L, 30 micromol/L, 100 micromol/L or 300 micromol/L. Specimens were incubated at 37 degree C in a 0.5 % CO(2) incubator for 1 h or cryopreserved at -196 degree C LN(2) for 3 days. The sperm motility and viability and the levels of ROS and lipid peroxidation of sperm membrance were assessed before and after incubation and cryopreservation by means of computer assisted semen analyzer, eosin-nigrosin stain, chemiluminescence and thiobarbituric acid assay, respectively. RESULTS: The sperm motility was significantly increased after incubation with 100 micromol/L and 300 micromol/L rebamipide (P<0.05). After cryopreservation, the sperm motility was significantly decreased in all concentrations (P<0.05), but the decrease was less with 100 micromol/L and 300 micromol/L rebamipide than that with other concentrations. The sperm viability showed no significant difference before and after incubation (P>0.05). The levels of ROS and lipid peroxidation in semen were significantly decreased in proportion to the concentrations of rebamipide both after incubation and cryopreservation (P<0.05). CONCLUSION: Rebamipide is an effective free radical scavenger in semen in vitro.

Alanine↗
PubMed1980

Cryopreservation of buffalo semen.

Twenty-one split semen samples from five bulls were diluted in five diluents. The diluents comprised lactose-egg yolk-glycerol, fructose-egg yolk-glycerol and three different combinations of lactose and fructose with equal proportions of egg yolk and glycerol. The straw-method of deep freezing was used. Frozen semen was evaluated on the basis of post thawing motility percentage, survival of spermatozoa at 37 degrees C and absolute index of survival. Statistical analysis of data revealed highly significant differences between diluents for the three characteristics tested. The diluent comprising 11 per cent lactose 56.25 ml + 6 per cent fructose 18.75 ml + egg yolk 20 ml + glycerol 5 ml achieved the best results.

Animals↗
PubMed2006

The effects of cancer and cancer treatments on male reproductive function.

In male cancer patients, surgery, radiotherapy and chemotherapy can be followed by transient or permanent infertility by affecting ejaculatory or erectile function, or by impairing spermatogenesis. Cancer specialists should, therefore, discuss the impact of different therapies on fertility with their patients prior to treatment, and consider fertility-preserving measures before and during treatment, such as nerve-sparing operative techniques, adequate testicular shielding during radiotherapy and the avoidance of unnecessary gonadotoxic chemotherapy. Pretreatment sperm-cell cryopreservation should be offered, even in cases where the individual's risk of post-treatment infertility might seem minimal or if it might require testicular sperm-cell extraction. Samples that are severely oligospermic should also be cryopreserved. Post-treatment ejaculatory or erectile dysfunction can be reversed pharmacologically, but the success rate varies with the extent of neurologic sequelae of the treatments used. At present there is no established method to stimulate post-treatment impaired spermatogenesis, although currently available assisted reproductive techniques overcome some of the existing problems of infertility in cancer survivors, and ongoing research will hopefully increase these possibilities. A multidisciplinary approach that depends on close cooperation between relevant medical specialists is central to achieving such advances.

Combined Modality Therapy↗
PubMedDate not supplied

Effect of semen dilution on bovine sperm viability as determined by dual-DNA staining and flow cytometry.

Living and dead spermatozoa were examined for the effects of sperm concentration level on sperm viability. Semen was collected from two different bulls on each of four collection dates. A ninth bull was collected on all four collection dates as a control for effects of collection date. The ejaculates from these nine bulls were diluted to 30 x 10(6) spermatozoa/0.5 ml and then serially diluted to 20, 10, 5, or 1 x 10(6) spermatozoa/0.5 ml French straw. One-half of the straws for each dilution series was stored 24 hours at 5 degrees C, while the other half was cryopreserved. Spermatozoa were stained with SYBR-14 and propidium iodide (PI) to assess viability. Flow cytometry yielded dot plots showing three distinct sperm populations: dead red-stained spermatozoa (PI), viable green-stained spermatozoa (SYBR-14), and moribund spermatozoa that stained both red and green (doubly-stained). Populations were expressed and analyzed in terms of mean percentage of viable spermatozoa and by actual numbers of viable spermatozoa per insemination dose. The mean percentage of living spermatozoa decreased linearly with decreasing sperm concentration; whereas the decrease was parabolic when those same samples were expressed as the mean number of living spermatozoa per insemination dose. The percentage of SYBR-14-stained spermatozoa differed among concentration levels and among bulls (P < 0.01). There were no differences among straws from the same ejaculate. The total volume of ejaculated semen and the concentration of spermatozoa in that ejaculate were both significantly positively correlated with the percentage of SYBR-14-stained spermatozoa in that semen when it was cryopreserved and diluted to < 10 x 10(6) spermatozoa/0.5 ml. In contrast, there were no significant correlations between the initial ejaculate characteristics and the proportion of SYBR-14-stained spermatozoa in the 24-hour-stored samples at any concentration. In conclusion, the percentage of viable spermatozoa in an ejaculate significantly decreased with increasing dilution. Further, in cryopreserved samples, the percentage of living spermatozoa < 10 x 10(6) spermatozoa/0.5 ml depended on the original volume and the sperm concentration of that particular ejaculate.

Analysis of Variance↗
PubMed2000

Etiologic and early marker studies in the prostate, lung, colorectal and ovarian (PLCO) cancer screening trial.

The Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial, which is randomizing 74,000 screening arm participants (37,000 men, 37,000 women; ages 55-74) and an equal number of nonscreened controls, is a unique setting for the investigation of the etiology of cancer and other diseases and for the evaluation of potential molecular markers of early disease. At entry, baseline information is collected by questionnaire on dietary intake, tobacco and alcohol use, reproductive history (for women), family history of cancer, use of selected drugs, and other selected risk factors. Blood samples collected at the baseline screening exam are aliquoted to serum, plasma, red blood cell, and buffy coat fractions. At the next two annual screening visits, serum samples are collected. At the third annual reexamination, cryopreserved whole blood is obtained, in addition to serum, plasma, red blood cell, and buffy coat fractions. At the fourth and fifth years, serum, plasma, and buffy coat are collected. All blood samples are shipped to a central repository for long-term storage at -70 degrees C. Dietary questionnaires and buccal cells for DNA analysis are obtained from nonscreened controls. Cancer cases are identified through annual follow-up questionnaires, and all deaths are identified through vital status tracing mechanisms. Procedures are being developed to obtain archival pathologic material for selected cases of cancer and related diseases. Initial investigations are focusing on the etiology of colorectal cancer and on the operative characteristics of tests for the early detection of colorectal and prostate cancer.

Aged↗
PubMed2004

Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa.

We investigated the impact of cryopreservation and thawing on levels of caspases-3, -8, and -9 activity, intact mitochondrial membrane potential (Deltapsim), and DNA fragmentation in human spermatozoa. Eleven pools of cryopreserved and eight pools of fresh semen samples were examined. Mature and immature fractions were separated on a two-layer density gradient (47% and 90%) and further subdivided based on the externalization of phosphatidylserine and its binding to annexin V-labeled superparamagnetic microbeads (ANMB). Levels of activated caspases were assessed using fluorescein-labeled inhibitors of caspases (FLICA), Deltapsim using a lipophilic cationic dye, and DNA fragmentation by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Cryopreservation was significantly associated with activation of caspases-3, -8, and -9, as well as disruption of the mitochondrial membrane potential but no significant changes were observed in DNA fragmentation. In mature sperm, caspase activation was only detected in the ANMB+ fraction, whereas in immature sperm, both ANMB+ and ANMB- fractions showed activated caspase levels. In ANMB+ immature sperm, apoptosis seemed to be triggered by a surface ligand-receptor mechanism as well as by disruption of mitochondria, whereas in ANMB- immature sperm, apoptosis was induced by activation of caspase-9 following loss of intact Deltapsim. These results demonstrate that selection of annexin V-negative mature spermatozoa might be of clinical relevance for fertility preservation, as this sperm fraction shows no activated apoptosis during the cryopreservation process.

Annexin A5↗
PubMedDate not supplied

Association of human sperm nuclear decondensation and in vitro penetration ability.

Sperm nuclear decondensation is an integral step in fertilization which leads to the formation of the male pronucleus. The association between the in vitro spontaneous nuclear decondensation of human sperm and its fertilizing ability was studied in infertile male patients. The ability of sperm to fertilize an egg using the discontinuous two-layer Percoll method was significantly correlated to the percentage of decondensed swollen head (r = 0.43; P less than 0.005). The fertilizing ability of sperm processed with Test-Yolk buffer was correlated with the percentage of sperm at the fully decondensed stalk stage (r = 0.51; P less than 0.05). There were insignificant correlations for the whole-wash centrifugation, cryopreserved-thawed and swim-up methods. Samples of sperm that were positive (greater than 0% fertilization) in the sperm penetration assay had a higher percentage of decondensed sperm heads (66.7% vs. 20.6%) after Percoll wash or whole-wash centrifugation (60.5% vs. 44.3%) treatments compared with samples with no fertilization. Treatments that included Test-Yolk resulted in high percentages of decondensed swollen heads. The results suggest a positive association between sperm nuclear decondensation and the fertilizing ability of sperm, and affirm the importance of nuclear decondensation to the study of fertilization events.

Animals↗
PubMedDate not supplied

Transplantation performance of human skin cryopreserved by programmed or stepwise freezing and stored at -80 degrees C or -180 degrees C.

We developed a mouse recipient model that was used to evaluate and compare four cryopreservation procedures for human cadaveric skin stored for two time periods. Skin specimens were identically processed and preserved by programmed (1 degree C/min), or stepwise freezing, and stored at -180 degrees C or -80 degrees C for periods of 1 month and 6 to 10 months. Samples were grafted on Balb/c mice, and primary take was evaluated after 7 days. The results indicate that although all grafted specimens were initially accepted, as indicated by gross observations, histologic differences were evident and significant. The study groups were analyzed for the effect of method and skin sample variety; the effect of freezing procedure and temperature level; time effect (storage period); and advantage of method 1 (programmed freezing at -180 degrees C) over the other methods. The significance (p value) was determined for separate histologic criteria and average skin score or quality. The overall results indicate that average score of skin preserved by method 1 is highest for both storage periods. This method has an almost significant advantage (p = 0.057) over the others on quality of skin stored for 1 month, and a highly significant advantage (p = 0.007) on graft adherence of skin stored for 6 to 10 months. The effect of method and samples variety on the separate histologic criteria and average score of skin is not always significant. However, an interaction factor (between method and samples) has a highly significant effect (p < 0.001) on almost all of the histologic criteria and average skin score. The effects of freezing method is significant only on average skin score, for 1 month of storage; whereas temperature effect is seldom significant. Evaluating the effects of time, samples, and the interaction factor (between time and samples) indicated that the interaction factor is highly significant (p < 0.001). Time and samples effects are rarely significant. Thus the quality of the final product-the cryopreserved skin-is determined by many factors, and quite often they interact. Highly significant is the combined effect, or interaction factor, of sample variability with method of cryopreservation or with storage period.

Animals↗
PubMed2002

Cryopreserved chronic lymphocytic leukemia cells analyzed by multicolor fluorescence in situ hybridization after optimized mitogen stimulation.

We investigated the utility of multicolor in situ fluorescence hybridization (mFISH) on cryopreserved blood cells from 11 chronic lymphocytic leukemia (CLL) patients. The results demonstrate that an individually chosen optimized mitogen combination induces proliferation of neoplastic B-cells after cryopreservation. Abnormal cells were detected in eight samples by mFISH, and, in six samples, the abnormality could be verified by comparative genomic hybridization or interphase FISH. In addition to typical CLL abnormalities, such as del(11q) or +12, several balanced translocations and single-cell abnormalities were found. Thus, mFISH can reveal new prognostically relevant chromosome aberrations in CLL.

Cell Division↗
PubMed1979

Studies on the presence and possible oscillations of granulocytic progenitor cells (CFU-C) in human blood.

The presence of CFU-C in the peripheral blood of 3 healthy volunteers was monitored over a period of 70 d. Blood samples were taken 3 times a week. Mononuclear cells (MNC) were separated from blood leucocytes by dextran sedimentation and the iron ingestion method. After cryopreservation the cells were cultured in soft agar. One sample of mononuclear cells was cultured using a leucocyte feeder layer as a source of colony stimulating activity (CSA) and another sample using human placental conditioned medium as CSA. In cultures using a leucocyte feeder layer, the average number of CFU-C was 29, 43, 118 per ml blood. No systematic oscillations were observed. When human placental conditioned medium was used the average number of CFU-C was 255, 125, 222 per ml blood, respectively. Significant oscillations with periods of 23, 19 and 25 d were observed.

Adult↗
PubMed2005

Standardization of cytokine flow cytometry assays.

BACKGROUND: Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online). RESULTS: Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells. CONCLUSION: ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.

Blood Preservation↗
PubMedDate not supplied

Cryopreservation of Salix species using sections from winter vegetative scions.

Twigs of Salix species are candidates for cryopreservation procedures because they become tolerant of freezing temperatures during mid-winter. We examined several variables in developing a two-step cryopreservation procedure for sections from these twigs. Samples of Salix triandra cooled to -30 or -35 degree C and then transferred to the vapor phase over liquid nitrogen gave the greatest percent shoot formation. Cooling rate to -35 degree C had a major influence on shoot formation. Samples cooled at rates greater than 10 degree C/hour showed no shoot formation. The highest percent of shoot formation was achieved by cooling at 0.21 degree C/hour. Cooling rate from -35 degree C to liquid nitrogen did not influence shoot formation. Warming procedures affected shoot formation. Transferring samples from -160 degree C to either a +2 degree C cold room or to -3 degree C methanol gave similar levels of shoot formation. No shoot formation occurred either with warming in +40 degree C water or very slowly in a Styrofoam box. Eight of eleven Salix taxa tested using the established protocol had significant levels of shoot formation after cryogenic treatment.

Cryopreservation↗