Performance Verification of Alternative Quality Control Materials for Urine Albumin Assessment.

BACKGROUND: Accurate detection of urine albumin is important for evaluating the progression of diabetic kidney disease. However, two levels of daily quality control may not be practically feasible in some small clinical laboratories owing to a small number of patient samples and high costs. We aimed to prepare homemade quality control material (HQM) to measure urine albumin and then verify its performance. METHODS: Normal saline solution and fresh mixed urine samples from five donors with serious kidney disease were used to prepare two levels of HQM (HQM1 and HQM2). Anhydrous ethylene glycol and sodium azide were used as antifreeze and as a preservative, respectively. RESULTS: Before being separated into Eppendorf tubes, 20 tests for HQM1 and HQM2 were performed, resulting in mean ± SD of 19.52 ± 0.91 mg/L and 105.28 ± 3.71 mg/L, respectively. After having been divided, the vial-to-vial variations of HQM1 and HQM2 were small (4.93% and 3.70%, respectively). The stability of HQM1 and HQM2 stored at 2 - 8°C was about 2 months and 80 days, respectively, and when stored at -20°C, remained stable for more than 8 months. After 1 - 8 months of cryopreservation at -20°C, once opened, the HQM in every Eppendorf tube could be kept for at least five days (CV < 6.1%). CONCLUSIONS: Our HQM stored at -20°C remained stable for a long time, and so could be considered as an alternative to standard QMs in the clinical laboratory.

Long-Term Storage of Cryptosporidium parvum for In Vitro Culture.

The long-term storage of Cryptosporidium life-cycle stages is a prerequisite for in vitro culture of the parasite. Cryptosporidium parvum oocysts, sporozoites, and intracellular forms inside infected host cells were stored for 6-12 mo in liquid nitrogen utilizing different cryoprotectants (dimethyl sulfoxide [DMSO], glycerol and fetal calf serum [FCS]), then cultured in vitro. Performance in vitro was quantified by estimating the total Cryptosporidium copy number with quantitative polymerase chain reaction (qPCR) in 3- and 7-day-old cultures. Although few parasites were recovered either from stored oocysts or from infected host cells, sporozoites stored in liquid nitrogen recovered from freezing successfully. More copies of parasite DNA were obtained from culturing those sporozoites than sporozoites excysted from oocysts kept at 4 C for the same period. The best performance was observed for sporozoites stored in Roswell Park Memorial Institute (RPMI) medium with 10% FCS and 5% DMSO, which generated 240% and 330% greater number of parasite DNA copies (on days 3 and 7 post-infection, respectively) compared to controls. Storage of sporozoites in liquid nitrogen is more effective than oocyst storage at 4 C and represents a more consistent approach for storage of viable infective Cryptosporidium aliquots for in vitro culture.

Cryopreservation of Sambar deer semen in Thailand.

Little is known of the different freezing and thawing techniques for post-thaw survival of spermatozoa in Sambar deer. So, this study determined the effect of seminal plasma, egg yolk and glycerol extenders and their concentrations, plus cooling, freezing, and thawing protocols on the post-thaw quality of their semen. Semen samples were collected by electro-ejaculation from four Thai Sambar deer stags (Cervus unicolor equinus). As evaluated by post-thaw progressive motility and acrosome integrity removal of seminal plasma was beneficial; Tris-egg yolk was the most efficient extender; a 20% egg yolk concentration was better than the 0%, 10%, or 30%; and a 3% glycerol concentration was better than 5%, 7%, or 9%. Using the optimum dilution techniques, semen was loaded in 0.5 ml plastic straws. Cooling times from ambient temperature to 5°C in 3 hr resulted in higher post-thaw progressive motility and acrosome integrity than 1, 2, or 4 hr. Suspending the straws 4 cm above the surface for 15 min before plunging into liquid nitrogen was better than suspending at 2 or 6 cm. For thawing frozen semen, an intermediate thawing (50°C, 8 sec) protocol was more effective than the slower (37°C, 10 sec) or faster (70°C, 5 sec) thawing rates. Timed insemination following estrus synchronization of 10 hinds resulted in six confirmed pregnancies at 60 days. Five hinds delivered live fawns. This study provides an effective approach for semen cryopreservation and artificial insemination (AI), which should be valuable to scientists for genetics and reproductive management of Sambar deer in developing countries.

Vitrification of caudal fin explants from zebrafish adult specimens.

No data on vitrification of tissue samples are available in fishes. Three vitrification solutions were compared: V1: 20% ethylene glycol and 20% dimethyl sulphoxide; V2: 25% propylene glycol and 20% dimethyl sulphoxide, and; V3: 20% propylene glycol and 13% methanol, all three prepared in Hanks' buffered salt solution plus 20 percent FBS, following the same one step vitrification procedure developed in mammals. Caudal fin tissue pieces were vitrified into 0.25 ml plastic straws in 30s and stored in liquid nitrogen for 3 days minimum, warmed (10s in nitrogen vapour and 5s in a 25 degree C water bath) and cultured (L-15 plus 20% FBS at 28.5 degree C). At the third day of culture, both attachment and outgrowing rates were recorded. V3 led to the worst results (8% of attachment rate). V1 and V2 allow higher attachment rates (V1: 63% vs V2: 50%. P < 0.05) but not significantly different outgrowing rates (83% to 94%). Vitrification of caudal fin pieces is advantageous in fish biodiversity conservation, particularly in the wild, due to the simplicity of procedure and equipment.

Cryopreservation in assisted reproductive technology: new trends.

During the last few years, cryopreservation has become a relevant addition to therapeutic concepts in reproductive medicine. New data and publications have made it difficult to maintain an overview of all of the new developments and their results. The focus of interest more recently, especially with the cryopreservation of human oocytes and human ovarian tissue, has been vitrification as an interesting alternative to slow freezing methods. Even though studies investigating the slow freezing of human mature oocytes have resulted in very different survival rates, it could be an option for donor oocyte programs, in the case of threatened ovarian loss or when there is an objection to embryo freezing. An optimal freezing protocol and later use of thawed human ovarian tissue is still a point of discussion. There are encouraging results regarding different kinds of autotransplantation, and recently the first birth after orthotopic autotransplantation of cryopreserved/thawed human ovarian tissue was described in the literature. Independent of any objections to cryopreservation in general, vitrification is a potential and effective alternative to conventional slow cryopreservation, especially for oocytes and embryos. Vitrification might be also be an option for human ovarian tissue; however this is only in its infancy and requires much additional investigation. Our article discusses new trends and results of actual studies regarding these issues.

Novel approaches to cryopreservation of human pancreatic islets.

BACKGROUND: Optimized conditions for survival and function of human islets must be defined if sufficient islets are to be recovered from a single human donor pancreas to reverse type-1 diabetes after isolation, cryopreservation, and transplantation. The objective of this study was to compare the cryoprotective effect of ethylene glycol (EG) with the standard cryoprotectant, dimethyl sulfoxide (DMSO), on isolated human islet survival and function. Furthermore, the effect of different addition protocols and equilibrium concentrations of the cryoprotectants were studied. METHODS: Islets were isolated from human pancreata by using standard techniques of collagenase digestion and discontinuous Ficoll gradient purification. Aliquots of freshly isolated human islets were cryopreserved in six groups by using DMSO or EG. Cryoprotectants were added stepwise to produce a final concentration of 1.5 or 2.0 M, or added in a single step to a concentration of 1.5 M. Islets were cryopreserved by using established protocols and cultured for 48 hr at 37 degrees C before assessment of percentage of recovery and in vitro viability. RESULTS: After cryopreservation, percentage of recovery of islets was significantly higher in the group treated with 1.5 M of DMSO added in a stepwise protocol (74+/-3%, mean+/-SEM) compared with the standard 2.0 M of DMSO (62+/-4%) (P<0.05, unpaired t test, n=6). There was no difference between the recovery of islets cryopreserved with either 1.5 M of DMSO stepwise (74+/-3%) or 1.5 M of DMSO one-step (69+/-3%). Islet recovery was higher in groups treated with DMSO compared with EG, regardless of concentration of cryoprotectant or addition protocol, although the difference was significant only when comparing DMSO and EG 1.5 M one-step. Furthermore, islets treated with 1.5 M of DMSO, added either stepwise (6.0+/-0.4) or in one-step (6.5+/-0.8), had significantly higher stimulation indices compared with islets treated with the standard cryoprotectant for human islets, 2.0 M of DMSO (4.5+/-0.5) (P<0.05). CONCLUSIONS: These results demonstrate that a lower concentration of DMSO (1.5 M) allows for the cryopreservation of human islets with superior survival and preservation of function post-culture compared with 2.0 M of DMSO and various concentrations of EG.

Comparison of cryoprotectants in the preservation of Theileria parva sporozoites using an in vitro infectivity assay.

An in vitro infectivity assay was used to examine five cryoprotectants for their suitability for preserving Theileria parva sporozoites. All five were capable of preserving T. parva sporozoites through freezing, the optimal concentrations being 7.5% for glycerol, 5% for dimethyl sulphoxide (DMSO), poly (vinylpyrrolidone) (PVP) and poly(ethylene glycol) (PEG), and 2.5% for hydroxyethyl starch (HES). When the five cryoprotectants were compared at their optimal concentrations, using a modification of the standard method of stabilate preparation, glycerol was significantly better than the others (p < 0.05). Measurement of the effects of each cryoprotectant on the osmolality of the media revealed that glycerol and DMSO elevated the osmolality significantly (p < 0.05). Resuscitation of glycerol-preserved sporozoites required the presence of glycerol in the diluent to maintain infectivity. Studies on the effects of equilibration time in glycerol on the infectivity of sporozoites showed that those frozen immediately after mixing (2 min) were as infective as those frozen after 60 min of equilibration.

Usefulness of polyethylene glycol for cryopreservation by vitrification of in vitro-derived bovine blastocysts.

A series of five experiments measured the high survival of bovine blastocysts produced in vitro after cryopreservation by vitrification. The vitrification solution (designated VS) contained 40% (v/v) ethylene glycol, 6% (w/v) polyethylene glycol and 0.5 M sucrose in phosphate-buffered saline. Embryos developed in vitro at Days 7 and 8 (Day 0 = insemination day) were exposed in one step to VS for 1 min or two steps with 10% ethylene glycol for 5 min and then VS for 1 min. In both cases, the embryos were finally cryopreserved in liquid nitrogen. After the embryos were warmed rapidly and the VS solution diluted, the survival rates were assessed by monitoring hatching rate in vitro. They were 13.0% for the one-step and 72.7% for the two-step procedures (P < 0.001). When embryos were exposed to individual solutions containing 6% (w/v) of each of 4 macromolecules (polyethylene glycol, BSA, polyvinylpyrrolidone or Ficoll) in the two-step protocol and then cryopreserved, the survival rates were 79.3, 34.8, 41.4 and 57.1%, respectively. After embryos had been exposed to the VS in two steps and then cryopreserved, there were no significant differences in survival rates when the solutions were diluted with or without sucrose. These results indicated that a vitrification solution containing polyethylene glycol can be used for cryopreservation of bovine blastocysts produced in vitro, and that a two-step addition of VS improved the in vitro survival of post-warming embryos. It was also shown to be possible to dilute post-warming embryos directly without the use of sucrose solution.

Differential freezing tolerance of rat pancreatic islets depending on their size variation.

We have investigated the freezing tolerance of rat pancreatic islets. Freshly isolated rat pancreatic islets were divided into three groups based on their longest diameter (small; 100 - 200 microns, medium; 201 - 300 microns, large; > 300 microns). They were then cryopreserved at a slow cooling rate (-0.3 degrees C/min) in the presence of dimethyl sulfoxide (Me2SO) or ethylene glycol (EG). After storage at -196 degrees C for 1 - 4 weeks, they were thawed and their ability to secrete insulin in response to fluctuations in glucose concentration was examined during three consecutive static incubations in vitro (1st; 2.8 mM, 2nd; 16.7 mM, 3rd; 2.8 mM). Morphological examination of the beta-granule population was determined by image analysis, and correlation with islets size was analyzed. The amount of insulin released from large-sized islets was significantly suppressed in EG (p < 0.05) and Me2SO (p < 0.01) groups compared to unfrozen islets. However, the mean volume of the large-sized islets isolated from one rat accounted for 43.0% of the total volume. On the other hand, the amount of insulin released from small- and medium-sized islets did not differ from those of unfrozen islets, and their mean volumes were 13.2 and 43.8% respectively. The percentage of cells with beta-granules was significantly correlated with size in both EG (r = -0.52) and Me2SO (r = -0.35) groups, but no significant correlation was observed in the unfrozen islets groups. These findings suggest that large-sized islets are more susceptible to freezing injury than small- or medium-sized islets. Moreover, the volume distribution of isolated islets indicated that it may be important to retain the ability of insulin secretion from the large-sized islets.

Studies on cryopreservation of Toxoplasma gondii and its antigenicity in mice.

Two strains (RH and GC, the latter of which is a Taiwan isolate of porcine origin) of Toxoplasma gondii were kept at -20 degrees C, -60 degrees C, and in liquid nitrogen (-196 degrees C) to follow the time course change in viability and virulence of the parasites by direct count and animal inoculation methods. Changes in antibody titers in some of the mice inoculated with the thawed organisms were assayed by the indirect immunofluorescent antibody test. Viability and virulence of T. gondii were best preserved by storage in liquid nitrogen. Tachyzoites kept in liquid nitrogen for eight years still can lead to the death of the injected mice in 2-3 weeks. Virulence of the tachyzoites could be maintained for eight weeks at most at -20 degrees C and -60 degrees C. Dimethylsulfoxide (DMSO) seemed to be a better cryoprotectant for T. gondii than glycerol, but the DMSO-preserved organisms resulted in fewer tachyzoite-containing peritoneal exudates in inoculated mice than the glycerol-preserved organisms. The local isolate (GC strain) tachyzoites tolerated cryopreservation less well than the RH strain parasites. Low antibody titers (at most 1:64) were produced in mice that survived more than 16 days after inoculation with thawed tachyzoites.

Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human sperm: glycerol and other polyols as sole cryoprotectant.

One effect of cryopreservation on human sperm is sublethal cryodamage, in which cell viability post-thaw is lost more rapidly at later times than in fresh cells. We hypothesized two modes of sublethal cryodamage: one is peroxidation-related involving plasma membrane damage due to lipid peroxidation; the other is membrane stress-related involving membrane embrittlement during phase transitions occurring during freeze-thaw. If the peroxidation-related mode contributed substantially to sublethal cryodamage, the hypothesis predicts that lipid peroxidation inhibitors should reduce this damage. To test this prediction, we examined the effect of the lipid peroxidation inhibitors, hypotaurine, bovine serum albumin (BSA), and alpha-tocopherol (Vit. E) on the time to loss of motility (TLM), taken as a measure of cell viability over time, for sperm samples cryopreserved in glycerol plus egg yolk medium. These agents had no effect on TLM of these samples, indicating that this mode contributes little to sublethal cryodamage. If the membrane stress-related mode contributed, the hypothesis predicts rapid recovery of motility in the presence of egg yolk plus glycerol, but slow recovery in the presence of glycerol alone. It also predicts that an appropriate polyol may be both necessary and sufficient for cryopreservation. In the presence of egg yolk plus glycerol, motility recovery was complete within 5 minutes, but the percent motile cells then decreased linearly with time. With glycerol alone in the range 3-12%, at 5 minutes post-thaw the percent motile cells was 5-10%, but by 40 minutes post-thaw had risen to 60-80%, approaching that in the fresh sample, and was maintained up to 4 hours. In the absence of glycerol, the percentage of motile cells post-thaw was nil and remained nil up to 4 hours. The polyols, erythritol, ribitol, and sorbitol had similar effects to that of glycerol, but the recovery of motility was not as complete. These results indicate that the membrane stress-related mode contributes substantially to sublethal cryodamage. They also indicate that glycerol and other polyols can function alone as cryoprotectants, but that recovery of motility is slow in these systems.

Malaria parasite strain characterization, cryopreservation, and banking of isolates: a WHO memorandum.

There has been considerable progress in the biological characterization of malaria parasites in the past few years. Physiological parameters such as host adaptation, virulence, exoerythrocytic development, in vitro growth of erythrocytic stages, and drug sensitivity are of particular importance to epidemiologists. Advances in enzyme analysis, 2-dimensional protein electrophoresis, and nucleic acid analysis have produced several new techniques that can be applied to the malaria parasite. Similarly, antigenic characterization is expected to progress as a result of technical improvements. Many of the biological parameters are needed for the study of parasite genetics, a field which has expanded greatly through the development of cloning techniques. The latter also hold interest for the production, and the future use in research, of biologically well characterized standard clones. In this connexion, the cryopreservation and banking of malaria parasites deserve attention, in order to ensure the supply of well defined, viable isolates and clones to interested research workers.

The effect of increasing glucose in CPD on the quality of stored blood.

Changes in quality of blood units containing one and a half or double amounts of glucose, stored at +4 degrees C for three weeks were analysed. An experimental preservative containing glucose and fructose (1 : 1) was also used. No other additives (purine or purine-nucleoside) were applied. A standard CPD preservative of the National Inst. of Haematology and Blood Transfusion was used as control. The pH, plasma free haemoglobin, K+ content, red blood cell (RBC) ATP and 2,3-DPG content, and RBC fragility index were determined in each sample. Increase of glucose concentration, the addition of fructose had a beneficial effect on blood pH, and on plasma free haemoglobin and K+ concentration. 150% glucose improved the 2,3-DPG maintenance in stored blood.

Some aspects for the further improvement in cell cryopreservation.

According to theoretical considerations and to results of own experimental investigations in heart muscle fragments of adult rats the authors are of opinion that more attention should be attributed to the cryoprotective medium as a whole, i.e. not only to the proper cryoprotectant, but to the other components of the cryoprotective medium as well. The suppose that the still unsatisfactory results of cell preservation, including blood and bone marrow cells, can be essentially improved by optimizing the composition of the basal medium.