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PubMed2004

Intramatrix events during cryopreservation of porcine articular cartilage using rapid cooling.

Cryopreservation of articular cartilage may improve long-term transplantation results if cell and matrix integrity can be maintained. This study examined intramatrix events in intact porcine articular cartilage that occurred during a rapid-cooling technique with various concentrations of dimethyl sulfoxide (DMSO) (1, 3, 5, 6 and 7 M). Thermocouples were inserted into the solution and in the cartilage matrix to record the temperature during rapid cooling. In addition, scanning electron microscopy of freeze-substituted samples was performed and quantitatively evaluated for the areas representing ice in the matrix. The results of this study showed that low concentrations of DMSO resulted in the largest temperature gradient between the matrix and the surrounding solution, which occurred near the freezing point of the cryoprotectant solution. At higher concentrations of DMSO, the peak temperature gradient occurred near the glass transition temperature. The temperature measurements suggested that a significant amount of ice formed within the matrix at lower DMSO concentrations. At higher DMSO concentrations that resulted in vitrification of the external solution, there was evidence of some ice in the matrix. The scanning electron micrographs demonstrated significantly more matrix disruption (likely due to ice formation) (P<0.02) in the lower DMSO concentrations (1 and 5 M) while the 6 M DMSO concentration demonstrated minimal matrix disruption. Cryopreservation of articular cartilage with a rapid-cooling technique and high concentrations of DMSO resulted in partial vitrification of the matrix and significantly less matrix disruption. It appears that successful cryopreservation of viability and function in articular cartilage will require high concentrations of cryoprotectants and rapid cooling.

Animals↗
PubMed2003

Seasonal changes in semen quality and freezability in the Warmblood stallion.

The objective of this study was to investigate seasonal changes in stallion semen quality and to determine the best time for semen cryopreservation. Experiments were performed using 10 Warmblood stallions from the National Stud Farm in Avenches (Switzerland). Ejaculates were collected and frozen every other week during 1 year from January to December 1999. Volume, concentration, and motility, and the number of morphologically normal sperm and sperm with major defects (abnormal heads, acrosome defects, nuclear vacuoles, proximal droplets, abnormal midpieces) were evaluated. For all frozen-thawed semen samples motility as well as viability (SYBR-14/PI) was tested, and the hypoosmotic swelling test (HOS) was performed. To analyze seasonal differences 4 periods of 3 months each were defined: autumn (September, October, November), winter (December, January, February), spring (March, April, May) and summer (June, July, August). During the 1 year experiment all semen quality parameters showed a clear seasonal pattern. The volume, total sperm count and motility in fresh semen were significantly higher (P<0.05) in summer than in winter, while sperm concentration was significantly lower in summer compared to the other seasons. Regarding morphology, normal sperm was significantly lower (P<0.05) in summer than at any other time of the year and higher values (P<0.05) were found for major defects in summer than in spring and autumn. In frozen-thawed semen motility was significantly (P<0.05) improved in autumn when compared to spring and summer. Viability was lowest in summer and differed significantly (P<0.05) from other seasons. The HOS test revealed significantly more (P<0.05) membrane damaged spermatozoa in winter than in spring, summer and autumn. Our results demonstrate that in our climatic conditions clear seasonal differences occur in semen quality of fresh and frozen-thawed semen and that cryopreservation of stallion semen should preferably be performed in autumn.

Acrosome↗
PubMed1996

Sources and sequelae of bacterial contamination of hematopoietic stem cell components: implications for the safety of hematotherapy and graft engineering.

BACKGROUND: It is important to compare the incidence of bacterial contamination of components collected from the peripheral blood or bone marrow (BM), as well as of components processed with or without cell selection or depletion, and to evaluate the sequelae of such contamination. STUDY DESIGN AND METHODS: Bacterial contamination rates were compared in 1380 untreated autologous peripheral blood progenitor cells (PBPCs), 291 untreated autologous BM samples, 916 monoclonal antibody (MoAb)-treated autologous and allogeneic BM samples, and in 45 autologous PBPC components from which the CD34+ cells were selected. Bacterial cultures were performed at sequential time points during the processing of MoAb-treated BM. RESULTS: Bacterial contamination was documented in 44 of 2632 components from 1593 patients (1.67% of components, 2.76% of patients) before cryopreservation. Although only 0.65% of untreated PBPCs were contaminated before cryopreservation, each patient was more likely to have given a contaminated PBPC component than a contaminated BM component (2.41% vs. 0%, p < 0.01). Bacterial contamination of MoAb-treated BM was greater during or after manipulation than it was before (2.33% vs. 0.77%, p < 0.05). At thawing, contamination was documented in 42 (1.97%) of 2136 components cultured. Ten (13.7%) of 73 patients who received hematopoietic progenitor cells that were contaminated before cryopreservation or at thawing developed fever or positive blood cultures within 48 hours of transfusion. Fever was associated with bacteremia in two cases, but no irreversible clinical sequelae were noted. CONCLUSION: These studies suggest that, despite careful attention to sterile procedures, low-level contamination of hematopoietic stem cell components can be introduced before or during manipulation as well as at thawing, and that standards for monitoring of the procedures for collection, processing, cryopreservation, thawing, and transfusion of hematopoietic progenitor cells are necessary.

Antibodies, Monoclonal↗
PubMed2000

Correlation of in vivo and in vitro functions of fresh and stored human platelets.

BACKGROUND: Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. STUDY DESIGN AND METHODS: Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. RESULTS: Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. CONCLUSION: It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.

Animals↗
PubMed1993

Centrifugation of human spermatozoa induces sublethal damage; separation of human spermatozoa from seminal plasma by a dextran swim-up procedure without centrifugation extends their motile lifetime.

While washing of human sperm cells by centrifugation and resuspension is a procedure in widespread use, there have been indications that this procedure per se may be harmful to the cells. The objective of this study was to investigate this question. To this end, a method for the clean separation of motile human spermatozoa from seminal plasma in the absence of centrifugation was developed, using a modified swim-up procedure, in which liquefied semen was mixed with an equal volume of 30 mg/ml dextran in medium, and the mixture overlaid with medium containing 5 mg/ml bovine serum albumin, forming two discreet layers with stable interface. The percentage of motile cells in a given sample was consistently > 80% immediately after recovery. Damage to the cells was assessed by loss of motile cells during incubation up to 96 h post-recovery. Comparison of aliquots of spermatozoa obtained by the dextran swim-up procedure showed that the aliquot subjected to centrifugation had 4 +/- 3% motile cells after 48 h, while the untreated aliquot had 52 +/- 12%. The aliquots showed no difference 1 h post-recovery. Similar results were obtained with spermatozoa that had been centrifuged in seminal plasma and resuspended in fresh plasma, then recovered by dextran swim-up. The delayed onset of motility loss in the centrifuged samples implies that this treatment induces sublethal damage in the cells. Comparison of the standard swim-up and Percoll gradient methods for sperm recovery, both of which involve centrifugation steps, showed decline in motility of the samples similar to that seen with dextran swim-up of centrifuged cells. We conclude that centrifugation per se induces sublethal damage in human spermatozoa, independently of treatment method, and suggest that recovery methods for human spermatozoa which avoid centrifugation might partially alleviate the damage incurred by these cells during cryopreservation.

Adult↗
PubMed2003

Mineralization and organic phase modifications as contributory factors of accelerated degeneration in homograft aortic valves.

BACKGROUND AND AIM OF THE STUDY: The study aim was to assess histological and mineralogical properties of leaflets in human, antibiotic-preserved aortic homograft valves (recovered during surgical replacement) in order to identify factors accounting for pathological changes leading to accelerated graft dysfunction. A comparison was made with aortic valves prepared for grafting, with a view to assessing morphological and mineralogical characteristics as a potential preimplantation risk factor. METHODS: Valve leaflets were examined with light and scanning electron microscopy. Mineralization was assessed histochemically, and also physicochemically by atomic absorption spectroscopy (AAS). RESULTS: All explanted homograft valve leaflets revealed prominent degenerative changes seen as decreased surface area, fibrosis, mineralization and focal thrombosis. Substantial loss of endothelium and fibroblasts, reduced collagen bundles crimping, inflammation (81%) and disappearance of layered structure (59%) was identified. The elastic elements were relatively stable, though a gradual age-dependent loss was observed in both groups. Accelerated mineralization was seen in all explanted homografts; inorganic deposits were composed mainly of hydroxyapatite. Two types of mineralization were identified: large limited nodular structures, and diffuse mineral deposits. Homografts with moderately elevated mineralization, well-preserved layered structure and sporadic infective changes proved to have the greatest durability. Homograft durability was also affected by the difference in host and donor age. CONCLUSION: Preimplantation factors affecting pathological changes determining homograft durability included morphological status of the graft itself and donor age; host-related factors included recipient age, endocarditis, native valve calcification, and host-donor matching (age difference between host and donor). A limited molecular mineralization may increase valvular durability, provided that no focal nodular calcifications exist that might adversely affect overall homograft integrity.

Adult↗
PubMed2006

Histologic analyses of human mineralized bone grafting material in sinus elevation procedures: a case series.

Human mineralized bone allograft is a solvent-preserved allograft and is therefore composed of a unique bone substitute that differs from other forms of bone allograft processed via standard cryopreservation. During solvent preservation, the mineral and collagen structures appear to remain intact, thus possibly facilitating bony ingrowth when the mineral is used as a bone graft. This case series illustrates the histologic and clinical effects of this bone graft material for sinus elevation procedures. Histology from bone biopsy core samples of four patients revealed newly formed bone with a well-organized lamellar bone structure in general, and in some cases, remaining particles were observed in contact with surrounding newly formed bone. Histomorphometric analysis demonstrated a mean of 73.3% of bone formation (range, 66.1% to 85.0%) in the grafted sites, resulting in bone density that was similar to that of the original host bone (mean, 73.2%; range, 64.4% to 84.8%). Clinically, no complications were observed, and all implants were considered clinically osseointegrated after 6 months (range, 4 to 8 months). The results of this study suggest that human mineralized bone allograft could be successfully used in sinus lifting procedures and encourage further research of this solvent-preserved bone allograft material in oral reconstruction for future implant placement.

Adult↗
PubMed1983

Transplantation and in vitro perifusion of rat islets of Langerhans after slow cooling and warming in the presence of either glycerol or dimethyl sulfoxide.

The cryoprotectants dimethyl sulfoxide (Me2SO) and glycerol have been used for the cryopreservation of fetal rat pancreases but only Me2SO has been reported for the cryopreservation of adult rat islets. Since glycerol may be preferred to Me2SO for clinical use, this study was undertaken to compare the effectiveness of these cryoprotectants during the slow cooling of isolated adult rat islets. Islets of Langerhans prepared from the pancreases of WAG rats by collagenase digestion were stored at -196 degrees C after slow cooling (0.3 degrees C/min) to -70 degrees C in the presence of multimolar concentrations of either Me2SO or glycerol. Samples were rewarmed slowly (approximately 10 degrees C/min) and dilution of the cryoprotectant was achieved using medium containing sucrose. Function was assessed by determination of the time course of the glucose-induced insulin release during in vitro perifusion at 37 degrees C and also by isograft transplantation. Transplants were carried out by intraportal injection of a minimum of 1700 frozen and thawed islets into streptozotocin-induced diabetic recipients and tissue function was assessed by monitoring blood glucose levels and body weight changes. Without exception the islets frozen and thawed in the presence of glycerol failed to reduce high serum glucose levels of recipient rats and in vitro dynamic release curves showed to demonstrate a glucose-sensitive insulin release pattern. Reversal of the diabetic conditions was achieved in two of five animals receiving islets which had been frozen and thawed with 2 M Me2SO; and in one of three animals receiving islets cryopreserved with 3 M Me2SO. Nevertheless, perifusion studies showed that the pattern of insulin secretion from groups of cryopreserved islets which did show an ability to secrete insulin was atypical compared with that of untreated controls, suggesting that the tissue was altered or damaged in some way.

Animals↗
PubMed1996

Viability analysis of cryopreserved rat pancreatic islets using laser scanning confocal microscopy.

We have developed a digital image analysis technique to assay the viability of frozen-thawed pancreatic islets by using laser scanning confocal microscopy (LSCM) in conjunction with double fluorescent staining [acridine orange/propidium iodide (AO/PI)]. Freshly isolated rat pancreatic islets were cultured for 18-24 h and then brought to a 2 M concentration of dimethyl sulfoxide (Me2SO) by serial addition at decreasing temperatures. Ice was nucleated in the islet suspension at a defined temperature (-10 degrees C), followed by a controlled period for equilibration and then cooling in a programmable bulk freezer at rates of 0.3, 1, 3, 10 and 30 degrees C/min to -70 degrees C. Samples were then stored in liquid nitrogen. Subsequent to rapid thawing and serial dilution with sucrose solution to remove Me2SO, AO/PI-stained individual islets were prepared for imaging on the LSCM. A series of optical sections through individual stained islets were obtained and processed to obtain high-contrast images at two different wavelengths; 488 nm and 514 nm for viable and damaged tissue, respectively. Image analysis algorithms consisted of template masking, generation of histograms of the pixel intensity profile, and gray level thresholding to obtain binary images. The total percentages of both types of tissue in the islet were computed by summing the two populations in each serial section. The spatial distributions of viable and damaged tissue were calculated from the three-dimensional (3-D) data base for both cultured (control) and cryopreserved islets. The 3-D spatial distributions of damaged and viable tissue in the islets were computed by determining the normalized distance of each viable/damaged voxel from the centroid of the islet volume to a mathematically estimated 3-D superquadric surface used to estimate the outer boundary of the islet. Further, each isolated damaged cell was identified and its volume determined. These studies indicate that cryopreserved islets exhibit shape distortion and a decrease in the numerical density of cells in comparison to unfrozen controls. Maximal survival was observed at the slower cooling rates. Accordingly, damage was found to occur throughout the 3-D islet volume in distinct spatial distributions for islets frozen at the slower and the faster cooling rates. Further, it was found that the volume of the majority of damaged cells identified was consistent with that of cells ranging in diameter from 5 to 9 micrometers.

Algorithms↗
PubMed1998

Use of stored pig eggs to assess boar sperm fertilizing functions in vitro.

Methodology for studying sperm-zona interaction in the pig was established using cryopreserved cumulus-free immature eggs obtained in large numbers from gilts' ovaries. Boar sperm were preincubated in a medium known to support in vitro fertilization (IVF) and coincubated briefly with groups of eggs, and the resultant sperm-egg complexes were transferred to fresh sperm-free medium so that the behavior of the egg-associated sperm sample could be assessed during further incubation ("postincubation"). Complexes were passaged repeatedly through a wide-bore pipette tip to rinse off loosely bound sperm and leave tightly bound sperm; alternatively, they were passaged repeatedly through a narrow-bore pipette tip to strip off sperm bound to the zona surface, whence residual zona-penetrated sperm could be counted. Zona-binding ability was present in the sperm populations very soon after the start of preincubation, although it increased during the following 3 h; considerable binding was also noted in a medium that did not support IVF. Zona-penetrating ability was absent at the start of preincubation, increased slowly to a maximum after 3 h, and declined thereafter; penetration was insignificant in the medium that did not support IVF. Associated sperm numbers remained constant during postincubation of sperm-egg complexes. However, numbers of penetrated sperm rose slowly in a curvilinear fashion to maximize after some 3 h of postincubation, when they constituted less than about 15% of the bound sperm. No rapidly penetrating cohort was detectable, and the proportion of sperm that became tightly bound to the zona was unaffected by either preincubation or postincubation. It was concluded that 1) the strength of sperm-zona attachment reflected the area of the sperm head in contact with the zona rather than any physiologically specific binding and 2) zona attachment was not a functional or temporal indicator of zona penetration.

Acrosome↗
PubMed1990

Development of bioartificial liver: bilirubin conjugation in Gunn rats.

We developed a novel bioartificial liver using microcarrier-attached hepatocytes and a bioreactor. The bioreactor consists of an intracapillary chamber made up of porous (0.2 micron) cellulose acetate hollow fibers enclosed in a polycarbonate module forming an extracapillary chamber. Cryopreserved (-80 degrees C, 3 weeks) microcarrier-attached hepatocytes (3-4 X 10(7] were inoculated into the extracapillary chamber. The bioartificial liver, containing either microcarrier hepatocytes (n = 6) or microcarriers alone (n = 5); was attached to adult Gunn rats via cannulas in the aorta and inferior vena cava. Bile samples were collected before and at hourly intervals after attachment to the bioartificial liver. The same animal model was used to demonstrate de novo conjugation of free bilirubin by the bioartificial liver (n = 3) following systemic administration of unconjugated [3H]bilirubin. Bile was analyzed for bilirubin mono- and diglucuronides by high-performance liquid chromatography and confirmed by digestion with beta-glucuronidase and cochromatography with normal rat bile. A progressive increase in the concentration of bilirubin monoglucuronide and bilirubin diglucuronide in bile was seen as early as 30 min and lasted up to 4 hr following attachment to a bioartificial liver containing microcarrier hepatocytes (3.53 +/- 0.68 to 8.07 +/- 0.85 microM; P less than 0.01). Approximately 22% of the radiolabeled bilirubin excreted in bile was seen in the form of bilirubin conjugates, no radioactivity was seen in the free bilirubin fraction. In conclusion, we developed a bioartificial liver using microcarrier hepatocytes which carry out an important differentiated liver function which results in partial correction of a specific metabolic liver defect, i.e., conjugation of bilirubin.

Animals↗
PubMed2006

RNA quality and gene expression analysis of ovarian tumor tissue undergoing repeated thaw-freezing.

Gene expression profiles evaluated by microarray-based quantization of RNA are used in studies of differential diagnosis and prognosis in cancer. RNA of good quality is mandatory for this evaluation. The RNA most often comes from tumor banks with limited amount of tissue, and the tissue often undergoes repeated thawing and freezing. We evaluated the influence of repeated division of tumor samples at room temperature, on RNA quality and quantity, in addition to the gene expression profile. Sixteen ovarian tumor samples were divided in three aliquots each, undergoing respectively one, two, and three thaw-freeze cycles. RNA from each aliquot was extracted on the day of division, and quantity and quality were evaluated. RNA from all three aliquots of four tumor samples underwent microarray analysis on Affymetrix Human Genome U133A 2.0 arrays. Microarray data were evaluated using both unsupervised, and supervised multivariate statistical methods, reliability analysis, as well as verification using published gene lists in ovarian cancer studies. RNA quality and quantity did not change during the division procedure and microarray data showed insignificant difference in gene expression. Tumor samples from tumor banks can be frozen and thawed at least three times without compromising the RNA integrity and genetic expression profile.

Cryopreservation↗
PubMedDate not supplied

LC-mS analysis of human urine specimens for 2-oxo-3-hydroxy LSD: method validation for potential interferants and stability study of 2-oxo-3-hydroxy LSD under various storage conditions.

2-Oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD), a major LSD metabolite, has previously been demonstrated to be a superior marker for identifying LSD use compared with the parent drug, LSD. Specifically, O-H-LSD analyzed using liquid chromatography-mass spectrometry has been reported to be present in urine at concentrations 16 to 43 times greater than LSD. To further support forensic application of this procedure, the specificity of the assay was assessed using compounds that have structural and chemical properties similar to O-H-LSD, common over-the-counter products, prescription drugs and some of their metabolites, and other drugs of abuse. Of the wide range of compounds studied, none were found to interfere with the detection of O-H-LSD or the internal standard 2-oxo-3-hydroxy lysergic acid methyl propylamide. The stability of O-H-LSD was investigated from 0 to 9 days at various temperatures, pH conditions, and exposures to fluorescent light. Additionally, the effect of long-term frozen storage and pH was investigated from 0 to 60 days. There was no significant loss of O-H-LSD under both refrigerated and frozen conditions within the normal human physiological pH range of urine (4.6-8.4). However, significant loss of O-H-LSD was observed in samples prepared at pH 4.6-8.4 and stored at room temperature or higher (24-50 degrees C).

Biomarkers↗
PubMed1983

Establishment of a method for preservation of human cervical mucus for in vitro penetration tests.

Midcycle cervical mucus was obtained from 56 women. Each mucus sample was divided into four portions collected into a capillary tube and kept either at room temperature or at 4 degrees C, -20 degrees C or in liquid nitrogen for 1 h, afterwards thawed at 37 degrees C and used in the in vitro penetration device for 1 h. 56 semen samples of good quality were used, the same sample for all four types of the same cervical mucus. Very high significant correlations were calculated between the penetration value (PV) rates in all four methods of cervical mucus preservation. Paired t test revealed a significant decrease (p less than 0.025) in the PV rates of sperm in the cervical mucus cryopreserved in liquid nitrogen. The results of this study recommend the establishment of a cervical mucus bank using the 4 degrees C and -20 degrees C as possible methods for preservation of cervical mucus.

Cervix Mucus↗
PubMedDate not supplied

Evaluation of a container for collection and shipment of semen with potential uses in population-based, clinical, and occupational settings.

Large, population-based studies of semen quality are encumbered by the logistics and expense of obtaining semen samples from men who live in a variety of locations. A prototype semen collection and transportation kit, the TRANSEM100, can be distributed to study participants and then directly shipped to a central laboratory for analysis. This study was designed to evaluate the ability of male volunteers to correctly use the kit. Thirty volunteers aged 20 to 44 years with no history of diabetes, recent chemotherapy, fertility problems, or vasectomy were recruited through a newspaper advertisement, interviewed to obtain demographic information, and instructed on the use of the kit. Twenty-six of the initial subjects provided at least 1 semen specimen using the kit and returned the specimens by overnight delivery to the laboratory for analysis, 25 completed a follow-up interview on the use of the collection kit, and 20 submitted a second semen sample using the same method. The average volunteer was white, 27.8 years old, and held at least a college degree. Forty percent of the volunteers were married. In general, participants correctly followed the instructions for collecting, packaging, and shipping the semen samples. Volunteers were instructed to collect samples after at least 2, but no more than 7 days of abstinence. For the first and second samples submitted, participants collected semen samples after an average of 3.3 and 3.9 days of abstinence, respectively. Seventeen (65%) of the samples from the first sampling period and 16 (80%) of the samples from the second period were received in the laboratory the day after they had been collected. In summary, the TRANSEM100 may prove to be useful for collecting human semen in field studies. Further testing of this method is warranted to evaluate preservation of sample quality and use of the kit by men among diverse socioeconomic groups.

Adult↗
PubMed2001

Stability of ketone bodies in serum in dependence on storage time and storage temperature.

The serum concentrations of beta-hydroxybutyrate and acetoacetate as well as the beta-hydroxybutyrate/acetoacetate ratio are important parameters for the differential diagnosis of certain inborn errors of metabolism. Acetoacetate, however, is an unstable compound which becomes rapidly decarboxylated. At a storage temperature of -20 degrees C about 40% of the acetoacetate is lost within 7 days and after 40 days storage at this temperature virtually all of the acetoacetate has become degraded. At -80 degrees C the decomposition of acetoacetate occurs with a much slower rate and only 15% of the initial acetoacetate is lost after 40 days storage. The rate constants for the decarboxylation reaction were found to be (6.4 +/- 2.9) * 10(-5) [min(-1)] at -20 degrees C and (0.4 +/- 0.3) * 10(-5) [min(-1)] at -80 degrees C. In contrast, beta-hydroxybutyrate is very stable during storage and hence should be used as main parameter for the evaluation of ketonemia. If determination of acetoacetate and/or of the beta-hydroxybutyrate/acetoacetate ratio is necessary, an assay immediately after collecting the serum samples is recommended. Otherwise, the serum samples should be frozen as soon as possible and stored at -80 degrees C during transport and storage.

3-Hydroxybutyric Acid↗
PubMed2002

[The gynecology-obstetrics department].

The scientific and clinical activities of the Department of Obstetrics and Gynaecology have involved the three main subdivisions: the gynecological surgery, the obstetrics and fetal medicine, the endocrinology and the reproductive medicine. Minimal invasive surgery including laser assisted laparoscopy or robotic assisted surgery has been particularly developed. Endometriosis, a frequent and sometimes particularly invasive disease, and oncologic surgeries have been developed in collaboration with the digestive surgery department. The department has also contributed to the comprehension and treatment of prenatal pathologies such as premature labor and deliveries or the gestational diabetes. The department has supported the development of techniques to study the fetal well-being in utero: the prenatal echography, the chorionic villous sampling, the amniotic puncture or the cordocentesis for prenatal genetic diagnosis or fetal infectious contaminations, the CMV transmission more specifically. In endocrinology and reproductive medicine, the department has mainly developed the in vitro fertilization techniques. The prolonged embryo culture, the study of preimplantation embryo metabolism, the preimplantation genetic diagnosis and the cryopreservation of ovarian fragments to preserve fertility in women undergoing oncologic treatments represent the more recent developed topics. Finally, the security of viral transmission in assisted procreation and the treatment of these patients with chronic viral diseases (Hepatitis C or HIV) are another domain with important scientific activity.

Belgium↗
PubMed1996

Positive selection of CD34+ cells from cryopreserved peripheral blood stem cells after thawing: technical aspects and clinical use.

Autologous stem cell transplantation has become an important therapy in lymphoma, multiple myeloma and solid tumors. The rationale for the selection of CD34+ cells from peripheral blood or bone marrow progenitor cell collections is based on the observation that contaminating tumor cells can be depleted approximately 3 to 6 logs. This procedure may be limited because of lack of sufficient numbers of progenitor cells in the leukapheresis concentrates. The use of frozen/thawed peripheral blood mononuclear cell (PBMC) samples makes it possible to pool two or even more stem cell harvests collected at different time points to increase the total number of CD34+ progenitor cells. We report in this work the feasibility of frozen/thawed peripheral blood CD34+ positive cell selection, using the large-scale (Ceprate SC) and the lab-scale avidin-biotin immunoadsorption system (Ceprate LC). This procedure consists of a washing step and a positive selection step. Our results show that frozen/thawed CD34+ cells were obtained with a purity of 86.68 +/- 3.62%, a viability of 97.94 +/- 0.97% and a recovery of 91.85 +/- 10.84% (range 80 to 112%). The CFU-GM assays were performed in a methylcellulose based medium; 89.13 +/- 19.63 colonies were obtained for 10(3) cells plated. Two patients were grafted with peripheral blood CD34+ cells selected after freezing. Our clinical data show that these cells are capable of rapidly reconstituting hematopoiesis after high-dose chemotherapy.

Antigens, CD34↗