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PubMed1991

The use of an electric freezer in human semen banking.

The use of an electric freezer for cryostorage of human semen is described. A simple method for semen freezing in liquid nitrogen after dilution with 7.5% glycerol was used. Thawing for quality analysis revealed only a small decrease (10%) in post-thaw sperm motility (59 +/- 1.4 vs. 53 +/- 1.8%; mean +/- SE, n = 12, P less than 10(-5)) and 14% in post-thaw sperm vitality (85.0 +/- 1.3 vs. 72.9 +/- 1.8%). After the freezing process, the samples, three of each donor, were cryopreserved in a regular electric freezer which maintained temperatures in the range of -85 degrees C (+/- 2 degrees C). The samples were stored for 1 week, 2 and 6 months, thawed and then assayed for motility and vitality. No effect of storage was found for a period up to 2 months. An additional decrease of 17.2% in sperm motility and 18% in sperm vitality were noted only after 6 months of preservation. The final motility and vitality rates of these sperm samples were 44 +/- 2.4 and 60 +/- 3.0%, respectively. According to these results, in cases of sperm storage for limited periods, it is recommended to cryopreserve human semen by the use of a combination of freezing in liquid nitrogen and storage of the samples in an electric freezer at -85 degrees C.

Analysis of Variance↗
PubMed2005

Cryopreservation of hematopoietic progenitor cells from apheresis at high cell concentrations does not impair the hematopoietic recovery after transplantation.

BACKGROUND: The high number of nuclear cells (NCs) from hematopoietic progenitor cells-apheresis (HPC-A) requires cryopreservation in large volumes or at high NC concentrations. The effect of NC concentration during cryopreservation has yet to be examined. STUDY DESIGN AND METHODS: In the experimental arm (n = 610, Protocol B), the first HPC-A sample from the patient was cryopreserved in two cryobags and subsequent collections in one cryobag, resulting in high NC concentrations (>100 x 10(6) NCs/mL) in most cases. The effect of NC concentrations at freezing in NC recovery after thawing and engraftment kinetics was analyzed and compared with a group of HPC-A cryopreserved at standard NC concentrations (n = 455, Protocol A). RESULTS: The mean (SD) NC concentration at freezing was 78 (28) x 10(6) per mL (median, 82 x 10(6)/mL; range, 12 x 10(6)-156 x 10(6)/mL) and 183 (108) x 10(6) per mL (median, 156 x 10(6)/mL; range, 16 x 10(6)-678 x 10(6)/mL), for HPC-A cryopreserved according to Protocols A and B, respectively. The NC viabilities of the test vials and HPC-A components after thawing were 88 percent versus 85 percent and 85 percent versus 82 percent, and the cloning efficiency was 49 percent versus 33 percent for Protocols A and B, respectively (p < 0.001). Significant differences were not observed in the recovery of NCs. Days to neutrophil and platelet engraftment were not different between patients transplanted in the standard- (n = 143) or high-cell-concentration group (n = 238). CONCLUSION: The cryopreservation of HPC-A at higher than standard NC concentrations has no adverse impact on hematopoietic reconstitution after transplantation.

Adolescent↗
PubMed1990

The hypoosmotic swelling test and cryosurvival of human spermatozoa.

The hypoosmotic swelling test is a simple laboratory test to evaluate the functional integrity of the membrane of human spermatozoa. This test was performed on 83 samples of human semen before cryopreservation to determine whether it has any predictive value for the cryosurvival of human spermatozoa. Stepwise regression analysis demonstrated that conventional sperm characteristics, including the concentration, motility, normal morphology and viability, of pre-freeze semen samples were of limited value in predicting the cryosurvival of human spermatozoa. Further, the hypoosmotic swelling test results from pre-freeze semen samples did not correlate with the post-thaw motility or the survival rate of spermatozoa after cryopreservation.

Cell Survival↗
PubMed2006

Imatinib activity in vitro in tumor cells from patients with chronic myeloid leukemia in chronic phase and blast crisis.

The aims of this study were to evaluate the feasibility of using the non-clonogenic fluorometric microculture cytotoxicity assay in drug sensitivity testing of tumor cells from patients with chronic myeloid leukemia. In nine samples (six chronic phase, three blast crisis), the drug sensitivities in tumor cells from blood versus from bone marrow and fresh tumor cells versus cryopreserved were compared. In 26 samples obtained in chronic phase (pretreatment), in six samples from patients in blast crisis and in the K 562 cell line, the activity of imatinib alone and in combination with cytarabine, vincristine, daunorubicin, interferon, arsenic trioxide and homoharringtonine was evaluated. All chronic myeloid leukemia chronic phase samples were sensitive to imatinib, with a mean IC50 at 10.3 mumol/l. The chronic myeloid leukemia samples from blast crisis (n=6) were significantly more sensitive to imatinib than the samples from chronic phase (n=26) (P<0.05), with an IC50 mean at 0.4 mumol/l. In blast crisis samples, significant positive interaction effects were observed between imatinib and all other tested drugs except for interferon. In chronic phase samples, interferon, daunorubicin and arsenic trioxide were the drugs with the highest frequency of positive interactions with imatinib (P<0.05). We conclude that the fluorometric microculture cytotoxicity assay may be a useful method for drug sensitivity testing in chronic myeloid leukemia patient samples from both chronic phase and blast crisis, and that testing primary tumor cells may have advantages over cell line studies. Imatinib shows a higher in vitro activity and more positive drug interactions in cells from blast crisis than chronic phase chronic myeloid leukemia patients. Combinations between imatinib and interferon, daunorubicin and arsenic trioxide may be interesting for future clinical trials in patients with chronic myeloid leukemia chronic phase.

Antineoplastic Combined Chemotherapy Protocols↗
PubMedDate not supplied

Cryopreservation of axillary buds of grape (Vitis vinifera) in vitro plantlets.

Axillary buds sampled from in vitro plants of four grape varieties could withstand cryopreservation using the encapsulation-dehydration technique. Regrowth percentages ranged between 15 and 40%. Buds were encapsulated in alginate beads containing 0.5M sucrose, precultured at 5 degrees C on media containing daily increasing sucrose concentrations from 0.1 to 1.0M, then desiccated to 21% moisture content (fresh weight basis) and frozen slowly (pre-freezing to -40 degrees C at 0.2 degrees C/min followed by immersion in liquid nitrogen). The physiological state of the in vitro mother-plants and the freezing procedure employed dramatically influenced the results. Regrowth after cryopreservation was achieved only when buds were sampled from mother-plants that had been kept without subculture for 3 to 4 months. An additional one-month period of cold-acclimation of mother-plants at 5 degrees C and a two-step freezing procedure improved growth recovery. The multiplication rate of shoots produced from cryopreserved buds was lower than that of controls during the first subcultures after thawing. However, the rooting ability of control and cryopreserved plantlets became similar from the fourth subculture onwards.

Cryopreservation↗
PubMed2006

Sperm cryopreservation in oncological patients: a 14-year follow-up study.

OBJECTIVE: Oncologic treatments can destroy spermatogenic dividing cells and cause azoospermia which could be irreversible. Sperm banking is the best option to preserve male fertility after these treatments. It is easy, inexpensive, and safe. To date, few clinical data are available about large series of cancer patients. Our objective was to determine the usefulness of these preventive sperm freezing protocols. DESIGN: Prospective study. SETTING: University-affiliated private fertility center. PATIENT(S): One hundred eighty-six cancer patients who banked sperm samples at our center before surgery or chemo- or radiotherapy treatments from 1991 to 2004. INTERVENTION(S): Conjugal status, age, type of cancer, treatment, and future use (if any) of the cryopreserved sperm samples for assisted reproduction technology (ART), and cycle results were recorded, analyzed, and compared with a control group. MAIN OUTCOME MEASURE(S): Basic sperm analysis of semen samples from cancer patients prior to freezing, after thawing, and after capacitation for ART. RESULT(S): A total of 320 semen samples were frozen before antineoplasic treatment. Six months later, 27% of the males recovered normal sperm production. From all frozen samples, 8.7% were discarded; the reasons were pregnancy achievement (55%), normal sperm production (28%), and patient death (18%). Finally, 5 IUI cycles and 30 ICSI cycles were done from frozen samples, with 1 and 15 pregnancies, respectively; results were comparable with those obtained in a control group. CONCLUSION(S): A significant number of males who cryopreserved semen samples before receiving antitumoral treatments have employed them. The results obtained showed that this is the strategy of choice, aiming to preserve fertility for the future, because the cost/benefit ratio is favorable. Patients should be counseled accordingly.

Adult↗
PubMed1991

A simplified method for cryopreservation of peripheral blood stem cells at -80 degrees C without rate-controlled freezing.

A simplified method was established for cryopreservation of peripheral blood stem cells (PBSCs) using hydroxyethyl starch (HES) and dimethylsulfoxide (DMSO) as a cryoprotective agent at -80 degrees C without rate-controlled freezing. The data indicate that a cryoprotective solution consisting of 6% HES and 5% DMSO produced the highest recovery rates for nucleated cells (92.0 +/- 3.5%), CFU-GM (73.8 +/- 4.1%) and BFU-E (82.2 +/- 6.9%), and the highest trypan blue viability (88.4 +/- 3.6%). For long-term cryopreservation of PBSCs, CFU-GM recovery rates remained almost unchanged during 5-18 months; the mean CFU-GM recovery rate after 18 months of cryopreservation was 70 +/- 11%. When large-scale samples of PBSCs in 100-ml freezing bags were cryopreserved for clinical use, cell and CFU-GM recoveries were similar. Using this method, 10 patients with hematological malignancy received PBSC transplants after marrow-ablative chemotherapy. All demonstrated early engraftment; seven are now alive in unmaintained complete remission for 3.5-15 months after PBSC transplant. This simple and inexpensive method will be useful for the wider application of PBSC transplant as a therapeutic alternative in the treatment of malignant diseases.

Acute Disease↗
PubMed2005

Long-term functionality of cryopreserved parathyroid autografts: a 13-year prospective analysis.

BACKGROUND: The functional results of cryopreserved heterotopic parathyroid autotransplantation (CHPA) are not well defined. The authors evaluated the outcomes of delayed CHPA for the treatment of surgically induced hypoparathyroidism. METHODS: Since November 1991, 448 parathyroid samples from 436 patients were cryopreserved at our institution. Of these, 29 patients underwent 34 CHPA procedures, with placement of 20 to 25 pieces of parathyroid tissue (approximately 50 to 75 mg) into the forearm. Outcomes were determined based on peripheral parathyroid hormone (PTH) levels and, where available, PTH gradients between grafted and nongrafted arms. Graft function results were defined as completely functional (patients with normal PTH and calcium levels off all calcium/vitamin D supplementation), partially functional (normal PTH levels and mild hypocalcemia on calcium supplementation), or nonfunctional (low PTH levels and dependent on calcium/vitamin D supplementation). RESULTS: Of the 29 patients with CHPA, prospective data were available for 26 patients undergoing 30 CHPA procedures (9 patients with MEN 1, 4 with MEN 2A, 1 with MEN 2B, and 12 with sporadic hyperparathyroidism). The mean follow-up interval was 2 years. Twelve of 26 patients (46%) had completely functional grafts, 6 patients (23%) had partially functional grafts, and the remaining 8 patients (31%) had nonfunctional grafts. No patient with CHPA had graft-dependent recurrent hyperparathyroidism. Of the 14 patients (15 autografts) with MEN, 7 patients (50%) had fully functional grafts, and 2 patients (14%) had partially functional grafts. The mean cryopreservation period was 7.9 months (range, 1 week to 22 months) for functional autografts and 15.3 months (range, 2 weeks to 106 months) for nonfunctional autografts (P < .01). CONCLUSIONS: Based on these data and those in previous studies, approximately 60% of delayed, cryopreserved parathyroid autografts are functional. In this study 40% autografts (46% of patients) achieved full competency off supplements. Some patients have evidence of graft function with normal PTH levels but are not normocalcemic. Results were similar for patients with MEN and nonhereditary hyperparathyroidism. The duration of cryopreservation was a significant indicator of graft failure, and no functional autograft was observed beyond 22 months of preservation. CHPA is a useful treatment modality for patients with postoperative hypocalcemia after thyroid or parathyroid surgery, who do not respond to immediate parathyroid autotransplantation.

Adolescent↗
PubMedDate not supplied

[Biological specimen banks: epidemiological tools and scientific enterprises].

In the last twenty years many large epidemiological studies have made efforts to collect material for biological specimen banks. These banks contain samples collected for short or long-term cryopreservation at low (e.g. -70 degrees C) or very low (-192 degrees C). This report describes general guidelines and methodological issues to take into consideration in designing and developing a biological specimen bank and is based on experience of three large scale studies. The author describes quality, quantity, methods to collect samples, and condition of cryopreservation that ensure both testing of scientific hypotheses and integrity of the stored samples. Feasibility and long-term storage effect studies should always be part of the design and the basic organization of a biological specimen bank. All operative choices a researcher makes in setting up a biological specimen bank have a direct impact on the future use of the biological samples and should be carefully considered.

Biological Specimen Banks↗
PubMed2006

Apoptotic lymphocytes and CD34+ cells in cryopreserved cord blood detected by the fluorescent vital dye SYTO 16 and correlation with loss of L-selectin (CD62L) expression.

Discrimination between live and apoptotic cells is important for accurate determination of viable CD34(+) cells in hematopoietic stem cell transplant products. SYTO16 is a sensitive fluorescent dye for discriminating live from apoptotic leukocytes. The incidence of apoptotic leukocytes in paired samples of fresh and cryopreserved-thawed cord blood (CB) was determined by the SYTO16/7-AAD flow cytometric assay. Cell migration and expression of the cell homing molecule L-selectin (CD62L) was determined in relation to SYTO16 staining. SYTO16 detected significant proportions of apoptotic lymphocytes and CD34(+) cells in fresh and thawed CB buffy-coat samples that were not detected by 7-AAD. Compared to fresh CB, the proportion of apoptotic lymphocytes and CD34(+) cells significantly increased following thawing. Significantly higher proportions of live SYTO16(bright) lymphocytes and CD34(+) cells were found in the migrated cell population compared to the non-migrated population. Significantly fewer lymphocytes and CD34(+) cells expressed CD62L following thawing. Absence of CD62L expression was strongly correlated with apoptotic/SYTO16(dim) lymphocytes and CD34(+) cells. Cryopreserved-thawed CB contains significant proportions of apoptotic lymphocytes and CD34(+) cells that are not detected by 7-AAD. SYTO16 offers a sensitive method for discrimination of live from apoptotic leukocytes and assists in accurate assessment of CB quality and suitability for use in clinical transplantation.

Antigens, CD34↗
PubMed2000

Congenital bilateral absence of the vas deferens, cystic fibrosis mutation analysis and intracytoplasmic sperm injection.

The aim of this study was to assess the outcome of intracytoplasmic sperm injection (ICSI) with fresh and frozen-thawed surgically retrieved spermatozoa from men diagnosed with congenital bilateral absence of the vas deferens (CBAVD). Twenty-seven azoospermic men with their partners were treated [25 with CBAVD and two with clinical cystic fibrosis (CF)]. CF gene mutation analysis and genetic counselling was provided. Spermatozoa were aspirated by microsurgical epididymal sperm aspiration (MESA), percutaneous epididymal sperm aspiration (PESA) or open testis biopsy. Of the men with CBAVD, 60% carried a single mutation, 20% were compound heterozygotes, and 20% had no CF mutation identified. Of the 28 sperm aspiration procedures, 86% had supplementary spermatozoa for cryopreservation with 83% of those samples assessed as satisfactory when thawed. Of 29 cycles with fresh spermatozoa a fertilization rate of 76% of oocytes injected and 17% embryo implantation rate occurred. Twenty-four cycles in which cryopreserved spermatozoa were used resulted in an oocyte fertilization rate of 69% and embryo implantation rate of 20%. Eighteen clinical pregnancies occurred with 14 live births without congenital anomaly. Two pregnancies were achieved following pre-implantation genetic diagnosis. It is concluded that the presence of CF mutations in the male partner does not compromise in-vitro fertilization treatment outcomes or the opportunity for healthy live births.

Adult↗
PubMed2002

Cryopreserved versus freshly isolated lymphocytes in human biomonitoring: endogenous and induced DNA damage, antioxidant status and repair capability.

Lymphocytes are routinely used in human biomonitoring to assess the potential toxic and cytoprotective effects of diet on both DNA damage and repair and, by implication, health. Logistically, samples may require to be cryopreserved and stored. How this affects cells used in human biomonitoring is often not considered. In this study we have evaluated the influence of cryopreservation on endogenous and induced DNA strand breakage, altered bases (oxidized purines, oxidized pyrimidines and misincorporated uracil), antioxidant capacity and DNA repair capability in human peripheral blood lymphocytes. Neither isolation nor freezing increased DNA strand breakage above endogenous levels found in freshly isolated human lymphocytes. Oxidized bases (both pyrimidines and purines) and misincorporated uracil, were similar for fresh and frozen lymphocytes. Fresh and frozen lymphocytes responded almost identically to hydrogen peroxide. Quercetin-mediated cytoprotection against hydrogen peroxide-induced strand breakage was maintained in cryopreserved lymphocytes after short-term (24 h) and longer term (2 months) storage compared with freshly isolated and treated cells. Hydrogen peroxide-induced DNA strand breakage was repaired in fresh lymphocytes. Cryopreserved lymphocytes were unable to repair oxidant-induced DNA strand breaks. Frozen human lymphocytes can therefore be successfully used for most aspects of DNA damage biomonitoring, but not for repair.

Antioxidants↗
PubMed1995

Density adjustment of software settings minimizes bias in automated sperm motility estimation.

To minimize overestimation of motility, it is recommended that fresh semen be diluted with seminal plasma prior to automated analysis. However, for glycerolated or cryopreserved semen this is impractical, and alternative methods are needed to minimize automated motility bias. In the present study, the proportion of motile spermatozoa was determined in fresh, diluted and cryopreserved semen (n = 25 ejaculates) using visual and automated methods. The effect of software settings on motility was investigated by assessing samples at a range of modified settings. At standard settings, automated motility was biased in fresh semen (+7.2%) after dilution with cryopreservative (-2.9%) and after cryopreservation (-7.8%) (P < 0.0001 versus visual). Automated motility was inversely related to the minimum number of frames for motility sampling (P < 0.0001), with mean estimates of 41.0, 46.1, 52.0 and 58.2% generated at settings of 8, 4, 2 and 1 frame(s) respectively (n = 15 fresh, diluted and cryopreserved samples). Based on an arbitrary ordinal scale, a method was developed whereby motility sampling was adjusted prior to analysis according to sperm density. Analysis of an independent set of semen samples with density-adjusted software settings reduced bias in automated estimates (n = 30) before and after freezing (P < 0.0001). In addition, bias was no longer related to sperm density. In conclusion, modification of software settings is an effective alternative to dilution to minimize bias in automated motility estimates in fresh, diluted and cryopreserved human semen.

Autoanalysis↗
PubMedDate not supplied

The emergence of specialized procedures for the acquisition, processing, and cryopreservation of epididymal and testicular sperm in connection with intracytoplasmic sperm injection.

Epididymal and testicular spermatozoa have been used widely for ICSI among men with obstructive azoospermia and NOA. New aspiration and biopsy techniques are available, and several sperm preparation methods have been described to isolate sperm. Most samples have sufficient sperm for cryopreservation. Accordingly, clinicians must sort through these new procedures to develop treatment plans that best suit their practices. In the future, other methods will be developed, and clinicians will have to critically review them prior to use.

Cryopreservation↗
PubMed1994

Analysis of human antiviral cytotoxic T-lymphocyte responses for vaccine trials using cryopreserved mononuclear leukocytes: demonstration of feasibility with influenza virus-specific responses.

The feasibility of measuring virus-specific human cytotoxic T-lymphocyte (CTL) activity by using cryopreserved mononuclear leukocytes to support clinical vaccine trials was addressed. Autologous fresh and cryopreserved cells from the same sample of peripheral blood were used as sources of CTL precursors and were tested for influenza virus-specific activity. The data indicated that virus-specific CTL activity could be measured by using cryopreserved cells; this could also be done in assays that are designed to characterize the responsible effector cell population.

Cryopreservation↗
PubMed2000

Cryopreservation of epididymal sperm obtained at necropsy from goats.

In the field of transgenic production, the ability to carry a male's genetic contribution beyond its natural life span is remarkably important. The ability to successfully collect and cryopreserve sperm from the epididymis at necropsy may prove to be a useful technique for preserving valuable genes. Thirty-two bucks ranging in age from 13 days to 7 years were examined in this study and 25 had epididymal sperm extracted at necropsy. Seven bucks yielded clear fluid with no spermatozoa; all were under four months of age. Testes were removed from the scrotal sac, small lateral incisions made across the convoluted tubules, pressure applied to the tail of the epididymis and small droplets of sperm pipetted into equilibrated extender. The average initial analysis of wave motion (0 to 5, 5 being rapid wave motion), live/dead sperm percentage and acrosomal integrity of 25 fresh epididymal samples were 5.0, 92%, and 100%, respectively. By comparison, the same parameters obtained from 206 fresh ejaculated samples were 3.0, 86%, and 95%, respectively. After being cryopreserved in liquid nitrogen, one straw from each sample was thawed after 3 to 60 days of cryostorage. Results of post-thaw analysis of 25 cryopreserved epididymal sperm samples for live/dead percentage and acrosomal integrity were 82% and 84%, respectively. By comparison, results of post-thaw analysis of 206 cryopreserved ejaculated sperm samples for live/dead percentage and acrosomal integrity were 60% and 89%, respectively. To assess the competence of the frozen epididymal sperm, IVF and AI were performed. In parallel IVF experiments, 40% of the oocytes showed cleavage patterns, with 6% developing to the blastocyst stage using frozen epididymal sperm, while 37% of the oocytes showed cleavage patterns and 4% developed into blastocysts using frozen ejaculated sperm. One artificial insemination out of 20 resulted in a pregnancy using frozen epididymal sperm, while 7 of 18 artificial inseminations resulted in a pregnancy using frozen ejaculated sperm. This data documents the successful collection and cryopreservation of epididymal sperm from the goat and its use for in vitro fertilization and artificial insemination.

Acrosome↗
PubMed2003

[The effect of the period of cryopreservative storage on the cryosurvival of human spermatozoa].

OBJECTIVES: To study the effect of cryopreservative period on the cryosurvival of human spermatozoa and find out the optimal recovery time of cryopreservation. METHODS: Eighty-eight semen samples were collected from normal donors and divided randomly into 5 groups according to the period of cryopreservative storage (1 d, 7 d, 30 d, 180 d, 300 d) in liquid nitrogen after being frozen by the programized, three-step freezing method. Fresh and frozen-thawed semen were examined by the routine analysis of semen and then the sperm recovery rate were calculated. RESULTS: There were no significant differences in sperm recovery rate between group I and the others (P > 0.05). The period of cryopreservative storage in liquid nitrogen had no correlation with the cryosurvival of human spermatozoa (r = 0.05, P > 0.05). CONCLUSIONS: It was indicated that freezing-thawing after 24 h would be helpful to the screening of semen donors in batches for donor insemination of human sperm bank.

Cryopreservation↗
PubMed1998

Control and surveillance of African trypanosomiasis. Report of a WHO Expert Committee.

Current estimates indicate that 60 million people are at risk of infection with human African trypanosomiasis or sleeping sickness, with about 300,000 new cases each year. However, less than 4 million people are under surveillance and only 10% of new cases are diagnosed and treated. This report of a WHO Expert Committee reviews current epidemiological information on African trypanosomiasis and its vectors, and evaluates recent advances in drug treatment and the development of tools for the control and surveillance of the disease. The report provides examples of treatment schedules, vector control operations, indicators for monitoring control and surveillance activities and sample calculations for analysing the cost-effectiveness of different strategies, as well as details of methods for cryopreservation of trypanosome-infected blood samples and a description of traps and screens for the control of the insect vector, Glossina. Although primarily addressed to health policy-makers in countries endemic for sleeping sickness, this report will also be a useful reference source for health care staff at all levels, including physicians, nurses, auxiliary staff and students, as well as those engaged in research on the disease.

Africa South of the Sahara↗