Analytical methods and stability assessment of liquid yeast derived sucrase

Two independent analytical methods for determining the activity and stability profile of liquid yeast derived sucrase (YS) were established and validated in order to conduct preliminary stability studies as a function of temperature. The methods included a hexokinase-based (HK) enzymatic assay for determining the formation of glucose upon hydrolysis of sucrose by YS, and a direct polarimetric procedure to quantitate YS hydrolysis of sucrose. Both assays were validated with respect to YS dilution, incubation time, sucrose or glucose concentration, linearity of response and within- and between-day variability. A preliminary stability study was conducted over a 24 week period with liquid YS samples stored at -20, 4, 30, 40 and 50 degrees C. Enzymatic activity was monitored as a function of time using both the HK and polarimetric assays. Liquid YS samples stored at -20, 4 and 30 degrees C retained 100% activity after 24 weeks storage, while the samples stored at 40 degrees C lost approximately 70% activity over the same storage period and samples stored at 50 degrees C lost approximately 95% activity after 12 weeks storage. The two methods of analysis gave consistent results over the course of the study.     

Measurement of fibrinogen in frozen plasma

Several large studies have compared fibrinogen measurements determined over a particular time interval. These assays are subject to difficulties encountered by all laboratories on tests carried out over a period of time such as assay drift. To avoid this problem, plasma can be stored frozen and fibrinogen determined in a large number of samples simultaneously. However, a thorough comparison of measurements carried out in fresh and frozen plasma has not yet been performed. Fibrinogen concentration was therefore determined in fresh plasma samples and then at a later date in the same samples after storage at -70 degrees C. A good correlation was observed between the two measurements, however, bias increased at the higher fibrinogen levels which are most critical in the determination of thrombotic risk. An increase in measurement error as a result of freezing was also observed. These effects may, therefore, be important considerations in future studies of this nature.     

Degradation of histamine solutions used for bronchoprovocation

STUDY OBJECTIVES: To determine optimal storage conditions for histamine diphosphate (HDP) solutions used for bronchoprovocation. DESIGN: HDP was dissolved in buffered saline solution to concentrations of 0.125 to 16 mg/mL and stored in 3-mL unit dose syringes at different temperatures for varying lengths of time, with and without protection from fluorescent light. SETTING: Dark freezer (-20 degrees C), dark refrigerator (4 degrees C), and laboratory counter top (20 degrees C) illuminated by fluorescent light (375 foot-candles). MEASUREMENTS: HDP concentrations were measured after the solutions were prepared and during storage by a high-performance liquid chromatographic assay that differentiates histamine from its break down products. RESULTS: All dilutions were sterile after preparation and contained 97 to 110% of the labeled amount of HDP. Solutions constantly exposed to fluorescent light (375 foot-candles) and room temperature (20 degrees C) contained only 20 to 37% of the initial concentrations after 7 days. The same dilutions stored at room temperature, but protected from light, contained 83 to 94% of the initial concentrations. Dilutions stored in the dark in a refrigerator (4 degrees C) retained 97% of the initial concentrations after 8 weeks, while dilutions stored in the dark freezer (-20 degrees C) were stable for 12 months. CONCLUSIONS: Exposure to fluorescent light at room temperature results in degradation of histamine solutions used for bronchoprovocation. Dilutions stored in unit dose syringes and protected from light are stable for at least 8 weeks in the refrigerator and up to 12 months frozen. Once removed from the refrigerator or freezer, the solutions should be used within 6 h or discarded.     

Hospital controls versus community controls: differences in inferences regarding risk factors for hip fracture

In cranioplasty complexity is proportional to the size of the detect, particularly if greater than 50 cm2. If the patient's own bone flap is not available, allogenic frozen bone graft can be used instead. Between June 1990 and June 1995 twenty cranioplasties with allogenic frozen bone grafts were performed. Age of patients ranged between 23 and 63 years (average 38.4 years). Male/female ratio was 2:1.7. Size of craniectomy ranged between 65 and 150 cm2 (average 83.3 cm2). Follow-up ranged between 10 and 58 months (average 41 months). Donors were tested to rule out transmissible diseases, infections, sepsis and/or cancer. Bone grafts were removed under aseptic conditions, microbiological cultures were taken, wrapped in a gauze soaked with Gentamicin sulphate and Bacitracin, sealed in three sterilised vinyl plastic bags, and stored in a deep freezer for a minimum of 30 days (range 36-93 days, average 67 days), at a temperature of -80 degrees C. Grafts were placed in the defect after a step was carved on its borders to facilitate the contact between host and graft. Vancomycin 1 g. IV/12 hours and Ceftriaxone 1 g. IV/12 hours were administered for five days. Grafts were covered by means of scalp flaps. Only one required a musculocutaneous free flap. None was exposed, extruded or had to be removed. Plain skull X-ray studies showed progressive remodelling of the grafts. Partial resorption was observed in two (2/20, 10%) and loss of thickness in another 3/20 (15%), but with no changes in the contour. Biopsies were taken in 3/20 (15%) cases at a second surgical procedure. Areas of osteoclastic resorptive activity mixed with others of osteoblastic bone apposition, showed replacement with new bone. We conclude that cranial vault frozen allografts are a good alternative to autologous bone when the latter is absent or not present in sufficient amount.     

Retrospective diagnosis of bluetongue virus in stored frozen and fixed tissue samples using PCR

Stored frozen (-70 degrees C) and formalin-fixed tissue samples constitute a valuable resource for retrospective studies of infectious diseases, or for diagnostic investigations. The polymerase chain reaction (PCR) affords an accurate and rapid method for detection of viral nucleic acids. It was applied to stored tissue samples collected from sheep inoculated with two Australian serotypes of bluetongue virus, BTV 1 and 23, and two North American serotypes, BTV 11 and 17. Specific nested PCR products were detected in both frozen and formalin-fixed samples from the Australian sheep after storage for 3.5 years. The tissues from sheep inoculated with the North American serotypes yielded specific nested PCR products after storage at -70 degrees C for 14 years. No specific primary PCR products were detected in any frozen or formalin-fixed samples. The PCR assay offers a potential benefit for epidemiological studies, and for screening of stored semen, embryos and tissue banks.     

Storage temperature of the unbuffered rapid urease test

OBJECTIVES: The unbuffered rapid urease test (RUT) is an accurate, rapid, and inexpensive method for detecting Helicobacter pylori. However, it is generally recommended that the reagent be prepared daily. This prospective study was undertaken to evaluate the shelf life of our unbuffered RUT when stored at 4 and -20 degrees C. METHODS: Ninety-five patients were studied. Three sets of antral (X2) and body (X1) biopsy samples were taken from each patient. The samples were subjected to histological examination, with the RUTs stored at 4 and -20 degrees C. The RUT tubes were examined at 1 and 15 min. RESULTS: Fifty-six patients (59%) were infected with H. pylori as defined by histological examination. The reagent was classified according to storage time (group I, < or = 5 days; group II, > 5 days). The mean (SD) storage time of group I (n = 59) and group II (n = 36) was 3.2 (1.4) and 9.9 (5.0) days, respectively. At 15 min, the sensitivity of our RUT stored at 4 degrees C was significantly higher in group I than in group II (92 vs 47%). On the other hand, the sensitivity of our RUT stored at -20 degrees C remained consistently high in both groups (15 min: group I, 92%; group II, 100%). Our RUTs stored at 4 and -20 degrees C were highly specific in both groups. CONCLUSIONS: Our RUT remains highly sensitive and specific when it is stored at 4 degrees C for up to 5 days. When the RUT is expected to be stored for a longer period of time, the bottles should be frozen at -20 degrees C.     

Suggestibility in hypochondriacal patients and healthy control subjects. An experimental case-control study

The effects of slow freezing and thawing on enzyme compartmentalization and ultrastructure were studied in rat liver slices frozen in dry ice, isopentane/ethanol-dry ice, or liquid nitrogen, and stored at -80 degrees C for 1-14 days. Non-frozen slices served as controls. Frozen liver slices were thawed in a Karnovsky fixative and processed for transmission electron microscopy (TEM). After all freezing protocols, the outer zone of frozen-thawed tissue was ultrastructurally very similar to that of non-frozen liver. Towards the center of the tissue, the ultrastructure progressively deteriorated. Comparison with 50-microm cryostat sections prepared for TEM showed that thawing and not freezing is the detrimental step for fair preservation of ultrastructure. After thawing, homogenization, and differential centrifugation, distribution patterns of soluble marker enzymes were analyzed (cytosol, lactate dehydrogenase; mitochondrial matrix, glutamate dehydrogenase; lysosomes, acid phosphatase). The enzyme activities were not affected by storage for 2 weeks and the activity distributions showed that protein leakage from compartments was only minimally increased in frozen-thawed tissue compared with that from non-frozen tissue, irrespective of the method of freezing. In conclusion, fairly large tissue slices (20x5x3 mm) may be frozen and stored at -80 degrees C for biochemical, ultrahistochemical or ultrastructural studies. For ultrastructural analysis, only the periphery of the tissue slice should be used.     

Gonadotrophins and prolactin rise after bilateral oophorectomy for benign conditions

The aim of this study was to analyse the changes in follicle stimulating hormone (FSH), luteinizing hormone (LH) and prolactin concentrations in the 3 months following oophorectomy in pre-menopausal women operated on for benign gynaecological conditions. Included in this analysis were 21 women (mean age 47 years, range 46-52) who underwent bilateral oophorectomy plus hysterectomy for fibroids or ovarian cysts. Plasma concentrations of FSH, LH and prolactin were measured before and on days 2, 4, 6, 14 and 30 after surgery; in 10 cases measurements were made on day 60, and in five cases on day 90 after surgery. Hormone concentrations were measured in duplicate daily samples, and immunoenzymatic assay kits were used for all the immunoassays. The FSH and LH concentrations increased constantly after surgery. Mean prolactin concentrations also increased from 12.1 ng/ml before surgery to 31.5 ng/ml on day 14 after bilateral oophorectomy, but decreased thereafter to 18.2 ng/ml on day 30, 10.9 ng/ml on day 60 and 6 ng/ml on day 90. In conclusion, transient (2-3 weeks) increased prolactin concentrations are observed after surgical castration.     

Effect of different storage temperatures, sample collection procedures and immunoassay methods on osteocalcin measurement

The apparent instability of measured osteocalcin has been reported as method-dependent and related to preanalytical variables such as storage temperature, and the use of anticoagulants and protease inhibitors. The aim of this study was to determine a sample collection procedure which minimised osteocalcin degradation. Blood samples from five normal individuals were collected with or without anticoagulants and protease inhibitors (heparin, EDTA, or heparin and aprotinin) and stored at 4 degrees C, -20 degrees C or -70 degrees C for up to 7 days, 28 days and 90 days respectively. Osteocalcin was measured by both a monoclonal EIA specific for intact osteocalcin and a bovine polyclonal RIA. Osteocalcin concentrations in serum and EDTA-treated samples significantly decreased by 40% (P < 0.001) with the ELISA and 72% (P < 0.001) with the RIA after 7 days storage at 4 degrees C. Similar falls were documented in these samples when stored at -20 degrees C and -70 degrees C and measured by the ELISA. Minimal changes in osteocalcin immunoreactivity were observed in either assay when heparin-treated plasma with or without aprotinin was stored at -20 degrees C or -70 degrees C for up to 90 days. The apparent instability of measured osteocalcin can be minimised using these conditions.     

Cytotoxic agents active against mucinous adenocarcinoma of the ovary

OBJECTIVE: To determine the stability of immunoglobulin E levels in obstetric sera. METHODS: AlaSTAT(R) and AlaTOP(R) (Diagnostic Products) were used to assay total and specific IgE levels in obstetric sera collected in Memphis, TN and Portland, OR. The samples were collected from the Collaborative Perinatal Project (CPP) between 1959 and 1965 and stored at -20 degrees C. The assay results were compared with IgE levels found in sera collected at the same locations for the Calcium for Pre-eclampsia Prevention Study (CPEP) and stored since 1992 at -70 degrees C. The samples were also assayed for cockroach (CR) and mouse urine specific IgE using the AlaSTAT(R) assay (Diagnostic Products). RESULTS: Total IgE and specific IgE to CR and mouse urine were detectable in older and recent samples. The median total IgE for the recent and older Portland samples was 26 IU/ml and 65 IU/ml, respectively. The median total IgE was identical (40 IU/ml) in the recent and older Memphis samples. CONCLUSION: Long-term storage does not diminish the ability to measure serum IgE. Levels of IgE in sera stored 32-37 years were equal to or greater than levels in sera stored for 5 years. reserved.     

Hepatitis C virus RNA in dried serum spotted onto filter paper is stable at room temperature

To overcome the instability of viral RNA, we carried out hepatitis C virus (HCV) RNA detection in dried serum spotted onto filter paper. The spotted serum samples were stored at room temperature and then processed for PCR assay at intervals of 1, 2, 3, and 4 weeks. The results showed that serum HCV RNA is stable in a dried condition, as it was detectable in spotted serum samples stored for 4 weeks at room temperature. Furthermore, although the HCV RNA titer showed an approximately 10-fold reduction in virus yield in dried serum stored at room temperature for 4 weeks, the PCR results of frozen serum samples and dried serum samples matched completely. This storage method facilitates transport and analysis by nucleic acid amplification techniques even when freezing conditions are not available.     

Solid-phase assay for luteinizing hormone in mouse plasma

A solid-phase tube assay for measuring LH levels in mouse plasma is described. The assay utilizes an antiserum to ovine LH and ovine LH standards and it measures LH levels in 20 mul of plasma with a sensitivity of less than 0.6 ng/ml. Various parameters affecting the sensitivity and specificity of the assay were investigated. Serial dilutions of plasma from pregnant mice, a pituitary homogenate from mice and plasma from hypophysectomized mice, injected subcutaneously with ovine LH, ran parallel with ovine LH standards in plasma from hypophysectomized mice and plasma with low LH levels from intact mice. Ovine TSH showed about 12% cross reaction in the assay system, whilst rat FSH and prolactin and also ovine FSH, prolactin and GH showed practically no cross reaction. Measurements of plasma LH levels have been made in hypophysectomzied mice after injection with different vehicles containing 10 or 50 mug LH or 50 mug FSH per animal. Daily measurements of LH levels throughout pregnancy in the mouse show a rise in LH level prior to implantationand a further rise around mid-pregnancy which drops off sharply to levels which remain fairly constant until parturition when there is another rise.     

Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk

An ELISA containing lipoarabinomannan (LAM) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to LAM ELISA testing immediately, and after 1 year of storage at -70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in LAM ELISA results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk LAM ELISA and was 0.15 for serum LAM ELISA, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to LAM ELISA testing. Results were compared, using the area under the receiver operating characteristic curves. The milk LAM ELISA had specificity (+/- 95% confidence limits) of 87 +/- 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 +/- 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 +/- 0.03 for the milk ELISA and 0.75 +/- 0.02 for the serum ELISA. In this population of cattle, the milk LAM ELISA had comparable accuracy to serum LAM ELISA, although the milk LAM ELISA was slightly less reproducible (higher coefficient of variation).     

Impact of extreme donor age on the outcome of living-related donor kidney transplantation

Eosinophil cationic protein (ECP) in sputum may be used to estimate the severity of bronchial inflammation and obstruction in asthmatics as well as to monitor asthma drug therapy. For this purpose, standardized processing of sputum is important. The aim of our study was to determine whether time and temperature influence the ECP concentration in the sputum of asthmatics. The samples of induced sputum obtained from 12 patients with stable asthma were homogenized using ultrasonification, and centrifuged. Supernatants were evenly divided and stored for 1, 6, 24 or 72 h at either 4 or 25 degrees C, then frozen at -80 degrees C. The ECP concentrations were determined using fluoroimmunoassay and compared with the immediately frozen samples. After storing at 4 degrees C, the ECP levels at the four time points were 101.2, 96.0, 98.2 and 90.6% of the initial concentration, respectively. When sputum specimens were stored at 25 degrees C, ECP levels decreased to 96.1, 94.4, 90.7 and 87.7%, respectively. The influence of time on ECP concentrations in sputa was statistically significant (p=0.02). A significant temperature effect was found when comparing the specimens stored at 4 degrees C with those at 25 degrees C (p=0.03). Looking at individual time points, a significant decrease in ECP concentration was only seen at 25 degrees C after 24 and 72 h. We conclude that eosinophilic cationic protein in the sputum of asthmatics decreases in a time- and temperature-dependent process. If sputa cannot be processed after obtaining the specimens, they should be stored in a refrigerator at 4 degrees C, until eosinophilic cationic protein is measured.     

Reproducibility of electronic cell counts in milk; a study of 5 further factors

This paper is a sequel to a previous one in which a number of factors likely to influence the accuracy of counting somatic cells in milk was assessed; in the present work the effects of 5 other factors are investigated. In a study of storage time and temperature of milk samples fixed in formalin, a significant increase in cell count occurred after 5-7 d when samples were stored at room temperature (17-23 degrees C), compared with those maintained at 4 degrees C. When manual and mechanical mixing of fixed samples were compared only marginal differences in cell counts were observed. An increase in cell counts followed manual dilution of milk samples in comparison with automatic dilution. The temperature of samples prepared for counting was also studied and no significant variations occurred between mean temperatures of 12-7 and 32-9 degrees C. The final factor evaluated was that of holding time before counting; using 4 cell-count levels it was observed that counts were acceptable up to 1 1/2 h.     

Readability of self-report clinical outcome measures

Since plasma is generally employed for amino acid analysis, we compared amino acid levels in plasma with those in serum for healthy individuals and examined the influence of separation and storage conditions on the stability of the samples. Then, we determined the amino acid levels of frozen serum samples obtained from sarin poisoned patients. A. Comparison of Amino Acid Levels in Plasma and Those in Serum Blood was collected from 5 healthy individuals. Then, heparinated plasma and serum were separated by centrifugation immediately after blood collection. Serum was also separated by centrifugation after standing whole blood at room temperature for 1 hour. Frozen plasma and serum were store at -40 degrees C for 5 months. All were subjected to analysis in an amino acid analyzer. It was found that the cystine (Cys) and 3-methyl-histidine (3-M-His) levels in serum and plasma were affected when stored in a frozen state, that the aspartate (Asp) level was changed according to the method of collecting serum, and that the taurine (Tau) and ornithine (Orn) levels were affected by standing blood. B. Analysis of Blood Taken from Sarin Poisoned Patients Twelve sarin poisoned patients were selected as subjects, and serum cholinesterase (Ch-E) and serum albumin (Alb) levels were determined. Amino acid analysis was conducted using an amino acid analyzer. Serum samples which had been obtained from the 6 patients and frozen and stored at -40 degrees C from 5 months were used for amino acid analysis. As a result, the serum Ch-E level decreased and the Alb level tended to rise. Since the Ch-E/Alb ratio was reduced in the sarin poisoned patients, it is considered useful for discrimination from liver cirrhosis in which both Ch-E and Alb levels decreased. Amino acid levels in the serum obtained from the sarin poisoned patients were compared with those of healthy individuals, both of which had been stored under the same conditions. There were significant differences in Asp, glutamate (Glu), phenylalanine (Phe), 3-M-His, glutamine (Gln), and Cys levels. The Glu, Phe, and Gln levels were not affected by storage of serum in a frozen state, while the Glu and Phe levels were elevated and the Gln level was reduced. Although Cys exhibited lower values in frozen serum samples, the Cys level was elevated with a rise in the serum Ch-E levels. Therefore, we deduced that Cys metabolism disorders also occur in sarin poisoning. As stated above, the Glu and Phe levels were elevated and the Gln and Cys levels were reduced, suggesting the presence of abnormal amino acid metabolism, in patients with sarin-poisoning.     

Improving survival of culture bacteria in frozen desserts by microentrapment

Lactobacillus bulgaricus cells were entrapped in beads of calcium alginate and evaluated for their ability to survive freezing processes. Cells survived freezing (without agitation) in ice milk mix much better than in distilled water, and more entrapped cells survived than did cells that were not entrapped. Glycerol and mannitol were cryoprotective, but glucose was not, when each was added (6%) separately to the beads. Entrapment protected the lactobacilli in batch frozen and continuously frozen ice milk mixes. The percentage of survival for entrapped and unentrapped cells in continuously frozen ice milk approximated 90 and 40%, respectively. Lactobacilli survived better in beads with mean diameters > 30 microns than in those averaging 15 microns. Addition of entrapped lactobacilli had no measurable effect on the sensory characteristics of the ice milk.     

Plasma concentrations and pharmacokinetics of idebenone and its metabolites following single and repeated doses in young patients with mitochondrial encephalomyopathy

The stability of enoxaparin sodium in 0.9% sodium chloride injection in polyvinyl chloride (PVC) containers was studied. Triplicate solutions of 120 mg (1.2 mL) of enoxaparin (as the sodium salt) and 98.8 mL of 0.9% sodium chloride injection were prepared in 250-mL PVC containers and stored at room temperature (20-22 degrees C). Samples were taken immediately after preparation and at 0.25, 0.5, 0.75, 1, 4, 12, 16, 24, and 48 hours. Inspections for color change and precipitation were performed with a clarity inspection station and a magnifying glass. Samples of the three admixtures were evaluated in duplicate for pharmacologic activity by an automated coagulation heparin assay. Throughout the 48-hour study period, the enoxaparin admixtures were free of color change, evolution of gas, and precipitates. The pharmacologic activity of enoxaparin in the PVC containers remained > 94% of the initial measured activity for 48 hours. Enoxaparin 1.2 mg/mL (as the sodium salt) in 0.9% sodium chloride injection in PVC containers was stable for up to 48 hours at 20-22 degrees C.     

Clinical experience with transfusion of cryopreserved platelets

Multiple units of platelet concentrate obtained by plateletpheresis of normal, 'random' or HL-A matched donors were pooled and frozen in polyolefin bags using 5% dimethysulphoxide (DMSO) as a cryoprotective agent and a controlled freezing rate of I degrees C/min. The platelets were stored at approximately-I20 degrees C for as long as 20I days, thawed rapidly at 37 degrees C, washed once and resuspended in ACD plasma prior to transfusion. Two different final concentrations of platelets (approximately 2.7 and 9.0 X 10(12)/1.) were studied. Twenty-three thrombocytopenic patients have received a total of 40 frozen platelet transfusions. The mean freeze-thaw loss was 2I% and was similar for both platelet concentrations. All transfusions were well tolerated and there were no side effects attributable to the small amounts of DMSO infused. Increments in platelet counts I h after transfusion ranged from 0 to 102 X 10(9)/1. with an overall mean corrected increase in evaluable patients of 12 800 (increase x surface area (m2)/number of platelets transfused x 10(11)). Corrected increases tended to be greater with the low concentration of platelets. Overall, the increase in count for the frozen platelet transfusions was 65% of the increments obtained with fresh platelet transfusions administered within 1 week of the frozen platelets. Bleeding times were partially corrected after four out of six transfusions with post-transfusion counts greater than 50 X 10(9)/1., and active haemorrhage was controlled in some patients by frozen platelet transfusions. These results indicate that pooled platelets can be frozen, thawed and transfused with reasonable efficiency. The frozen platelets can circulate and function haemostatically and may eventually play an important role in supportive care.