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PubMed1987

Flow cytometry of X and Y chromosome-bearing sperm for DNA using an improved preparation method and staining with Hoechst 33342.

A new and improved method of preparing mammalian spermatozoa for high resolution flow cytometric DNA analysis and flow sorting is described. Ejaculated or cryopreserved sperm were briefly sonicated to remove tails and then stained with Hoechst 33342. This simple procedure was found superior to more severe treatments of dimethylsulfoxide washes, fixation in 80% ethanol, and protease digestion of the sperm membranes and tails by papain. Flow cytometric DNA analyses of sperm samples subjected to varying sonication times indicated that X and Y chromosome-bearing sperm populations could be well resolved with as little as 15-sec sonication. In addition, a comparison of sonicated samples stained with four concentrations of bisbenzimide (Hoechst 33342) or 4',6-diamidino-2-phenylindole (DAPI) indicated that 2.5 or 5.0 micrograms/ml of Hoechst was sufficient to resolve the X and Y sperm populations. In order to quantitatively describe the flow cytometric data, several indices (sample quality, orientation and splitting) were developed.

Animals↗
PubMed1994

Quantitative micro P30 and reverse transcriptase assays for Moloney murine leukemia virus.

An anti-P30 immunohistochemical and a reverse transcriptase assay for Moloney murine leukemia virus (MoMLV) are adapted to 96-well plates. The assay results are shown to be directly proportional to the number of infectious particles, and can therefore be used to estimate the infective titers of a virus preparation. The micro P30 assay yields a direct estimate of infectious centers, and the reverse transcriptase assay quantitates progeny from a single cycle of replication. The semi-automated nature of these assays is well suited to the analysis of a large number of samples and therefore permits the examination of the efficiency of the process of retroviral/MoMLV infection under varied times or conditions.

3T3 Cells↗
PubMed2000

Increased sperm motility after in-vitro culture of testicular biopsies from obstructive azoospermic patients results in better post-thaw recovery rate.

The objective of this study was to optimize the use of testicular biopsies in 14 patients with obstructive azoospermia. Testicular specimens were retrieved from six patients (group I) and cultured at 32 and 37 degrees C for up to 20 days; changes in percentage motile spermatozoa were compared. In four men of group I, one portion of the specimen was frozen at retrieval, and changes in post-thaw motility after 24 h of culture at 37 degrees C were recorded. In the other eight patients (group II), testicular specimens were frozen at retrieval and after 72 h culture at 37 degrees C. Pre and post-freezing motility and post-thaw recovery rate were compared. No significant differences were observed until day 8 in the improvement of motility between 32 and 37 degrees C in-vitro culture. Maximum motility was reached, under both conditions, between 48 h and 72 h. Post-thaw 24 h culture at 37 degrees C of specimens frozen at retrieval did not improve motility; however, 72 h pre-freezing culture significantly improved initial motility (P: < 0.01), post-thaw motility (P: < 0.01) and post-thaw recovery rate (P: < 0. 001). The higher recovery rate of samples frozen 3 days after retrieval allows more economical use of the tissue that is available.

Biopsy↗
PubMedDate not supplied

Evaluation of sperm cryoprotective media in respect to fertilizing capacity tested in vitro.

Nine different cryoprotectant buffers were tested to measure their protective ability towards main sperm seminological parameters. These were: maintained sperm motility, progressive motility and sperm viability. Out of the nine tested buffers, medium E (TES-Tris without glycerol) and H (glycerol only) showed significantly lower (P < 0.001) values than the rest of the studied buffers in respect to all tested seminological features. The other media did not differ significantly in their cryoprotective abilities to sperm. Richardson's medium (A) preserved sperm viability significantly better (P < 0.001) than the other tested buffers, reaching 63.1% of viable spermatozoa in proportion to the fresh sperm sample before freezing. Three cryoprotectants, A (with egg yolk, no TEST buffer system), D (neither egg yolk nor TEST buffer system), F (TEST-egg yolk buffer system) were further studied for their ability to preserve sperm function in sperm-cervical mucus penetration (Penetrak) and sperm penetration assay (SPA). In our hands, neither supplementation of the buffer with egg yolk nor TEST-egg yolk buffer system promoted sperm capacity in functional tests. A,D,F buffers did not significantly differ among each other in applied functional assays, however, they all diminished (P < 0.001) sperm penetration ratios when compared with fresh sperm samples. Therefore enhancement of sperm capacity to fertilize after equilibration with TEST-egg yolk buffer system should be contested.

Adult↗
PubMed2002

Analysis of differential messenger RNA expression between bovine blastocysts produced in different culture systems: implications for blastocyst quality.

Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

Animals↗
PubMed2002

New trends in gamete's cryopreservation.

We developed new techniques to improve freezing and vitrification of sperm, oocytes and embryos. Our novel freezing technology is based on 'Multi-Thermal-Gradient' (MTG) freezing that is used for sperm. The freezing apparatus has the ability to control ice crystals propagation by changing thermal gradient or the liquid-ice interface velocity which optimizes ice crystals morphology during freezing of cells and tissue. Using this apparatus we were able to freeze bull, stallion, boar, ram, fowl and human sperm with normal post-thaw motility/pre-freezing motility of 70-100%. The vitrification method includes the cooling of nanoliter sample (the 'Minimum Drop Size' technique) in 'super-cooled' liquid nitrogen (-210 degrees C), which maximized cooling rate to the highest physically possible (24-130000 degrees C/min). Using this method we achieved very high survival of bovine oocytes and embryos. Vitrification of oocytes at the MII stage resulted with cleavage and blastocyst rate of 50 and 20%, respectively. The vitrification of in-vitro production (IVP) of bovine embryos allowed the production of a healthy calf after embryo-transfer carrying the name 'Zegugit' (in Hebrew: made from glass).

Animals↗
PubMed2005

Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation?

Previous studies have shown sperm quality post-cryopreservation differs depending on the fraction of the seminal plasma boar spermatozoa are fortuitously contained in. As such, spermatozoa contained in the first 10 mL of the sperm-rich fraction (portion I) have better sustained handling procedures (extension, handling and freezing/thawing) than those contained in the ulterior part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction, portion II). However, those studies were performed using pooled samples. In the present study, individual ejaculates were used. Split ejaculates (portions I and II) from five boars were frozen and thawed using a conventional freezing protocol, followed by computer-assisted motility and morphology analysis (CASA and ASMA, respectively), as well as an Annexin-V assay for spermatozoa from each boar and ejaculate portion. Significant differences between portions were observed in all ASMA-derived variables, except in one boar. Also significant differences were observed between boars and ejaculate portions in sperm quality post-thaw. We identified, however, boars showing best results of motility and sperm membrane integrity post-thaw in portion I, while in other boar the best results was observed in portion II. It is concluded that the identification of the ejaculate portion more suitable to sustain cryopreservation in each individual boar may be a readily applicable and easy technique to diminish variation in sperm freezability among boars.

Animals↗
PubMed1995

Use of polymerase chain reaction and quantitative antibody tests in children born to human immunodeficiency virus-1-infected mothers.

The diagnosis of human immunodeficiency virus (HIV) infection in children born to HIV-infected mothers is complicated by the presence of passively acquired maternal antibodies, and exclusion of infection in these infants remains problematic. The use of genome detection by polymerase chain reaction (PCR) amplification and the quantification of anti-HIV-1 antibodies were examined as methods for early diagnosis. Blood samples were taken from 84 non-breast-fed infants of HIV-infected mothers in five Italian and Spanish centres, a subgroup of children enrolled in the European Collaborative Study (ECS) for whom clinical and immunological information has been documented from birth. Whole blood was added to glycigel cryopreservative, stored, and tested in the United Kingdom by a nested PCR method. Antibody to HIV-1 was detected and quantified by titration using a gelatin particle agglutination test. PCR sensitivity and specificity were assessed. Twenty-one of the 84 children tested were infected. The estimated PCR sensitivity ranged from 0% (95% CI 0-26%) on day 1, 57% (19-85) on day 7, to 63% (33-92) on day 30. The negative predictive value of PCR ranged from 85% (83-88) on day 0 to 98% (94-100) at 3 months of age. On average, the level of maternal antibody halved every 33 days (31-36.5) in uninfected children. Between 6 and 9 months of age, increases in antibody titres in infected children were not more informative than absolute levels. These findings suggest that antibody measurement may supplement genomic diagnosis and that this collection method provides an alternative to the use of dried blood spots.

Age Factors↗
PubMed1993

Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. I. Enzyme activity and calorimetric studies.

Stabilization of labile proteins during lyophilization requires protection of the protein against both freezing and dehydration stresses. Solutions of 1-10% (wt/vol) polyethylene glycol (PEG) fully protected both lactate dehydrogenase and phosphofructokinase during freezing and thawing, but did not stabilize the proteins during freeze-drying. Thus, with this lyophilization system a second compound could be tested for its capacity to stabilize dried proteins, independent of its ability to provide cryopreservation. In the presence of low concentrations of glucose or trehalose (which alone provided minimal protection) and 1% PEG (wt/vol), almost full enzyme activity was recovered after freeze-drying and rehydration. Differential scanning calorimetry indicated that the PEG was crystalline and the sugars were amorphous in the dried samples. Experiments with lactose and mannitol demonstrated that if these compounds also crystallized during freeze-drying, protein stabilization was reduced or abolished. PEG stabilizes the proteins during freezing, due to preferential exclusion of PEG from the protein's surface. The sugars protect the proteins during dehydration by hydrogen bonding to the dried protein, thus serving as water substitutes. This report provides the first example of stabilization of proteins during lyophilization through separate, specific treatments of the fundamentally different stresses of freezing and dehydration.

Animals↗
PubMed1997

Detection of a dormant 20q- leukemia clone in bone marrow cultures with hematopoietic growth factors: implications for secondary leukemia post-transplant.

A patient developed secondary acute myelogenous leukemia with a 20q- marker chromosome abnormality six years following chemotherapy and radiation for Hodgkins disease (HD). Routine cytogenetics on the bone marrow which had been harvested and cryopreserved immediately following completion of initial therapy for HD showed no cytogenetic abnormality. However, a 20q- clonal marker was detected after culturing bone marrow with hematopoietic growth factors (HGF). The marrow was used successfully for an autotransplant. Post-transplant, the 20q- marker was again detected in HGF cultured samples. The patient relapsed at 165 days post-transplant with the 20q- marker and trisomy 21. These data suggest that standard cytogenetic assays may not detect abnormal clones which can cause leukemia post-transplant.

Acute Disease↗
PubMed1993

Automated processing of human bone marrow grafts for transplantation.

Prior to purging or cryopreservation, we concentrated 21 bone marrow (BM) harvests using a modification of the 'grancollect-protocol' of the Fresenius AS 104 cell separator with the P1-Y set. Within 40-70 min, the initial marrow volume of 1,265 ml (+/- 537 ml) was processed two to three times. A mean of 47% (+/- 21%) of the initial mononuclear cells was recovered in a mean volume of 128 ml (+36 ml). The recovery of clonogenic cells, measured by CFU-GM assays, was 68% (+/- 47%). Red blood cells in the BM concentrates were reduced to 7% (+/- 4%) of the initial number. The procedure was efficient and yielded a BM cell fraction suitable for purging, cryopreservation and transplantation. At this time, 10 of the 21 patients whose BM was processed using this technique have been transplanted. Seven of these 10 patients have been grafted using the BM alone. Three of the 10 patients showed reduced cell viability and colony growth in the thawed BM samples, and therefore obtained BM and peripheral blood-derived stem cells. All transplanted patients showed an evaluable engraftment, achieving 1,000 granulocytes per microliter of peripheral blood in a mean of 18 days.

Automation↗
PubMedDate not supplied

Isolation procedures for blood lymphocytes produce artifacts. Detection by changes of electrophoretic mobilities of lymphocytes.

The changes induced in the distribution of the electrophoretic mobility (EPM) of human peripheral blood lymphocytes (HPBL), by various methods used to prepare the lymphocyte suspensions and eliminate platelets from them, were investigated on blood samples collected from healthy individuals and thrombopenic patients. Data showed that the distribution of the lymphocyte EPMs, i.e., the "lymphocyte electrophoregram," was dependent on the method chosen to enrich the suspension in the cell type of interest. The relative percentages of the low and high mobility cells, the two main subpopulations defined by lymphocyte electrophoresis, were different. The most striking artifactual differences in the lymphocyte electrophoregram were induced by the method of elimination of platelets; the distribution was unimodal and asymmetric when thrombin was used and bimodal when the blood sample, or the lymphocyte suspension, was placed on ice for 30 min (as is the practice in some laboratories). The "split" of the lymphocyte electrophoregram was found to be reversible within 90 min. Similar changes were observed on lymphocyte suspensions and blood samples of thrombopenic patients when the step for the elimination of platelets was not involved.

Adult↗
PubMed2004

A procedure for isolating and freezing metacyclic Trypanosoma brucei gambiense forms in the field.

An adaptation of a modified procedure using anion-exchange centrifugation was evaluated to isolate and freeze in field conditions a high number of bloodstream forms of Trypanosoma brucei gambiense for direct in vitro culture. Pellets of trypanosomes were obtained from 62 patients in Central Africa. The presence of trypanosomes was detected in all 62 eluates. The isolation system was efficient. Preliminary results of in vitro culture of 10 thawed samples of trypanosomes were highly promising because no fungal or bacterial contamination was noticed even after 2 weeks of culture.

Animals↗
PubMed2002

Spot urine analysis: acidification does not increase calcium recovery.

INTRODUCTION: We investigated the effects of acidification and storage on calcium recovery in spot urine samples. METHODS: In a retrospective study, 43 spot urine samples from patients with metabolic bone disease, which had been previously analysed fresh for calcium and creatinine, were acidified and incubated for 2 h at room temperature and re-analysed. In a prospective study, fresh spot urine samples from 15 volunteers were split into two aliquots--one left unacidified and the other acidified with 6 mol/L HCl to pH < 2. Both aliquots were analysed immediately and after storage at 4 degrees C for 24 h. RESULTS: In both studies, the calcium creatinine ratios obtained on acidified and non-acidified spot urine samples were not different, both when measured immediately as well as after storage at 4 degrees C for 24 h. CONCLUSIONS: The acidification of spot urine samples would thus appear to be unnecessary for calcium/creatinine measurements in metabolic bone disease if they were undertaken within at least 24 h.

Bone Diseases, Metabolic↗
PubMed1989

Sex and storage affect cholinesterase activity in blood plasma of Japanese quail.

Blood plasma cholinesterase (ChE) activity is a sensitive indicator of exposure to organophosphorus and carbamate insecticides. Effects of sex and storage of samples were studied as sources of variability by treating breeding Japanese quail (Coturnix japonica) with 3 mg of dicrotophos or carbofuran per kg of body weight and comparing blood plasma ChE activities for samples collected at 1 hr postdosage and assayed fresh, after 1 and 2 days of refrigeration (4 C), and after 1, 7 and 28 days of freezing (-25 C). ChE activity of fresh control plasma was 34% (P less than 0.01) higher in males than females. Male ChE activity remained essentially unchanged during storage while female ChE activity increased (P less than 0.05) gradually over time under both storage conditions. In contrast, when plasma ChE activity was inhibited by either antiChE, male plasma ChE activity was depressed further than female ChE (P less than 0.01) and remained constant during storage while female ChE activity continued to decrease (P less than 0.05). These divergent effects of exposure to antiChE compounds and sample storage indicate extreme care should be exercised in use of blood plasma for evaluation of antiChE exposure in wild birds.

Animals↗
PubMed1991

[Management of fresh urine].

To obtain an accurate urinalysis result, the process from the sampling to completion of the examination should be carried out as quickly as possible. Management of fresh urine is important for this purpose. While urinalysis should basically be performed immediately after voiding, the sample should be maintained in a refrigerator (4 degrees C) if this is not possible. Adequate preservation of the urine according to the purpose of the test is important.

Cryopreservation↗
PubMed1995

An evaluation of cold chain in Maharashtra & Karnataka states by potency testing of field samples of oral poliovirus vaccine.

The cold chain for oral poliovirus vaccine was monitored in Maharashtra and Karnataka by potency testing of vaccine vials collected from various stages of the delivery system. Results showed that cold chain maintenance improved in the state of Maharashtra within a period of three years as the monitoring began in 1987. Of the 6289 samples of trivalent OPV collected from all 30 districts of the state during 1990 to 1992, 5834 (92.8%) had retained virus titre of at least 10(5.81) TCID50/dose. In comparison, 72 per cent of the 1660 samples collected from the state of Karnataka during the same period were found to contain this minimum required virus titre. Defects in cold chain maintenance in Karnataka could be demonstrated by plotting virus titre of samples of individual batches collected from different outlets. It was concluded that potency retesting of OPV samples for cold chain monitoring will ensure proper storage, transport and use of potent vaccine in the field.

Cryopreservation↗
PubMed1996

Enzymes protective of oxidative damage present in all decades of life in the trabecular meshwork, as detected by two-dimensional gel electrophoresis protein maps.

Our purpose was to determine whether there are age-related differences in the presence of enzymes protective against oxidative damage in normal human trabecular meshwork (TM). The study was also done to determine whether there are significant changes in proteins related to age, gender, or position around the circumference of the meshwork. Human TM was dissected from both eyes of 16 normal donors ranging in age from 9 to 91 years. Proteins were mapped with two-dimensional (2D) gel electrophoresis. The microanalytical method used allowed analysis of samples as small as one fourth of a single meshwork. Selected proteins were identified by Western blot. Superoxide dismutase (SOD) and glutathione reductase were present in all ages on the 2D maps. Age-related changes in the TM were not apparent, although slight differences may exist between juvenile and adult TM, since differences in the relative intensities of some polypeptides were evident. In any single quadrant of the TM, > or = 160 protein spots could be counted visually. The 2D maps of the various quadrants were very similar, and the protein patterns were analogous between fellow eyes. Distinct differences were noted between maps from TM and adjacent cornea and sclera. Other protein spots identified included actin, albumin, transferrin, and vimentin. The TM samples had a mean cellular protein content of 66.3 +/- 15.4 micrograms.

Adolescent↗