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Objectives:

This study evaluated the effect of refrigeration at 4°C and post-refrigeration times (immediate, 5, 10, 15, or 20 min) on the viscosity and conversion kinetics of adhesive bonding resins.

Methods:

Scotchbond Dual-Cure (3M ESPE) and Clearfil SE Bond (Kuraray) were tested. Control samples were kept at 25°C for 24 h. At each post-refrigeration time, the temperature was checked with a K-type thermocouple. Viscosity measurements as a function of temperature were performed using a cone-plate viscometer. Real-time polymerization was monitored by infrared spectroscopy. Degree of conversion (DC) was calculated for each second during polymerization, and the rate of polymerization analyzed. Data were separately submitted to two-way ANOVA and Tukey’s test (P<.05).

Results:

Clearfil presented faster increase in temperature after exposure to room temperature than Scotchbond. A continuous decrease in viscosity (Pa.s) was observed for both Scotchbond (0.49, 0.34, 0.30, 0.26, 0.23, 0.23) and Clearfil (0.38, 0.37, 0.34, 0.25, 0.24, 0.22). For Scotchbond, higher final DC was detected for the control (62.7%) compared with the immediate (53.3%) and 5 min (54.7%) groups. For Clearfil, the control sample (81.4%) showed higher DC than all refrigerated groups (68.8–69.5%). Clearfil always showed significantly higher DC than Scotchbond.

Conclusions:

Refrigeration presented a significant time- and material-dependent effect on the viscosity and polymerization kinetics of the bonding resins. Under clinical conditions, adhesive agents should be removed from the refrigerator at least 20 min before being used.

Three methods of preserving simulated specimens of urine were studied with six test strains of bacteria. Viable counts were measured by a surface viable count and by the filter-paper-strip method during a holding period of 72 hours. Refrigeration at approximately 4 degrees C was effective and reliable. Boric acid (1-8%) at room temperature was toxic for the strain of Escherichia coli at a density of 10(7) cfu/ml but this may not be significant at the higher concentration of bacterial cells often found in clinical specimens. NaCl-polyvinylpyrrolidone (PVP) solutions containing PVP of mol. wt 44 000 or 700 000 were not effective; they were toxic for the Gram-negative strains and did not retard the growth of Micrococcus subgroup 3. The two methods of measuring viable counts were compared for specimens held under different conditions; the specificity of the filter-paper-strip method was high but the sensitivity was low when many of the specimens contained approximately 10(5) cfu/ml.

Purpose

Thiosulfate has been shown to reduce the risk of cyanide toxicity during nitroprusside administration. Admixtures containing both agents may provide a safe and effective alternative to more expensive agents used to reduce blood pressure in the critically ill patient. This study determined the physical and chemical stability of a 1:10 nitroprusside:thiosulfate admixture, stored up to 48 hours. The economic consequences of a shift toward using thiosulfate and nitroprusside, and away from higher cost alternatives, are considered.

Methods

Seven samples of 50 mg nitroprusside and 500 mg thiosulfate were prepared and stored away from light, at room temperature, and in a refrigerator prepared in D5W and NS. Each sample was analyzed via a novel high-performance liquid chromatographic (HPLC) method at time 0, 8, 24, and 48 hours. The method was tested and passed specifications for linearity, reproducibility, and accuracy. A visual inspection by 9 licensed pharmacists was used to demonstrate physical stability. A cost evaluation comparing nitroprusside and thiosulfate to alternative agents was completed.

Results

The concentration of both nitroprusside and thiosulfate remain greater than 95% of the initial concentration through 48 hours. Physical compatibility was confirmed in all samples tested through 72 hours.

Conclusion

The combination of nitroprusside and thiosulfate is chemically and physically stable as a single compounded dose for up to 48 hours when stored at room temperature and protected from light. The admixture represents an inexpensive option to other higher cost alternatives such as nicardipine or clevidipine.

OBJECTIVES

To formulate a liquid preparation of ziprasidone in a convenient concentration to allow dosing of less than 20 mg and of sufficient chemical and physical stability to enable an entire prescription or course of treatment to be prepared in a single batch.

METHODS

Geodon for injection (ziprasidone mesylate), 20 mg/mL, was diluted to 2.5 mg/mL in a commercially available sugar-free and alcohol-free, flavored syrup and stored at room temperature under ambient fluorescent light illumination, at room temperature in darkness, and under refrigeration. The ziprasidone content was measured in samples at various time intervals using a stability-indicating high-performance liquid chromatographic method.

RESULTS

When refrigerated, the ziprasidone syrup that was compounded in a commercially available, sugar-free and alcohol-free vehicle maintained at least 90% of stated potency for at least 6 weeks. Samples stored under other conditions were less stable, underscoring the manufacturer's labeling regarding refrigerated storage of the reconstituted injection.

CONCLUSIONS

The findings suggest that chemical and physical stability are maintained for 2 weeks under refrigeration, allowing the convenience of compounding for the long-term needs of a particular patient, rather than daily compounding. The only storage condition we recommend is refrigeration at 5°C.

In the context of a larger clinical study in Nouna, Burkina Faso, we evaluated a simplified dual-platform (DP) flow cytometric (FCM) method that allows the determination of major lymphocyte subsets in a single test tube. We compared the phenotyping of lymphocytes with DP FCM and simultaneous measurements with standard single-platform (SP) FCM for samples from 177 individuals. Analysis of the comparative measurements revealed that DP FCM systematically underestimates the proportion of NK cells, overestimates the percentage of CD3+ CD8+ lymphocytes, and yields proportions of B cells and CD4+ T cells comparable with the results from SP FCM. Bland-Altman analysis showed a low bias between both methods and an acceptable precision for percent values of CD4+ T cells (bias ± precision, −1% ± 6%) and CD8+ T cells (−3% ± 6%). The absolute cell numbers of all lymphocyte subpopulations, however, were systematically biased towards lower values being obtained by DP FCM. Reference values for the distribution of T-cell maturation phenotypes in 177 healthy adults were calculated using DP FCM. The mean ± standard deviation (SD) CD4+-to-CD8+ T-cell ratio was 1.61 ± 0.61, the mean percentage ± SD of CD4+ T cells was 42% ± 7%, and that of CD8+ T cells 29% ± 7%. Among CD4+ lymphocytes, 28% ± 7% were classified as central memory (CD45RAlow CCR7+), 22% ± 10% as naïve (CD45RAhigh CCR7+), 45% ± 12% as effector memory (CD45RAlow CCR7−); and 5% ± 3% as terminally differentiated effector memory expressing CD45RA (CD45RAhigh CCR7−). Among CD8bright lymphocytes, 3% ± 2% had a central memory phenotype, 27% ± 13% were naïve, 37% ± 13% had an effector memory phenotype, and 34% ± 12% were terminally differentiated effector memory cells expressing CD45RA.

Background:

Solutions of vancomycin for oral administration are not available commercially in Canada or the United States but are needed for patients who cannot swallow capsules.

Objective:

To evaluate the stability of vancomycin solutions stored in unit-dose cups and plastic bottles under refrigeration (4°C) and at room temperature (25°C) for up to 75 days.

Methods:

Vancomycin 25 mg/mL in Ora-Sweet vehicle and water (1:1 ratio by volume) was dispensed into opaque blue polyethylene unit-dose cups with aluminum seal (14 replicates) or amber plastic prescription bottles (6 replicates). Seven cups and 3 bottles were refrigerated (4°C), and the remainder of the containers were stored at room temperature (25°C). At the time of preparation and at 15, 30, 40, 50, 63, and 75 days, 3 aliquots were collected from one of the cups and from every bottle and were stored frozen (−85°C) until the time of analysis. Physical characteristics were evaluated at each time point, including measurement of pH and visual assessment of colour and precipitation. After thawing, the samples were analyzed in triplicate by a validated stability-indicating high-performance liquid chromatography method. A solution was considered stable if 90% of the initial concentration of vancomycin was maintained.

Results:

No notable changes in colour, taste, or pH were observed in vancomycin solutions stored in the unit-dose cups at 4°C or 25°C or in the plastic bottles stored at 4°C over the 75-day study period. Starting on day 63, a white precipitate was observed in the solutions stored in plastic bottles at 25°C, but there were no notable changes in taste or pH during the 75-day period. The 95% confidence interval of the slope of the curve relating concentration to time, determined by linear regression, indicated that vancomycin solutions stored in cups or bottles at 4°C would maintain at least 93.6% of the initial vancomycin concentration for 75 days and that solutions stored at 25°C would maintain at least 90.0% of the initial concentration for 30 days (cups) or 26 days (bottles), with 95% confidence.

Conclusions:

Vancomycin 25 mg/mL stored in unit-dose cups or plastic bottles at 4°C was stable for at least 75 days, whereas solutions stored in cups or bottles at 25°C are expected to be stable for 30 or 26 days, respectively.

The influence of preconditioning temperature, length of the preconditioning period, host and age of the infection on the survival of Trichinella spiralis nativa larvae in musculature to low temperature refrigeration was investigated. Dogs, foxes, ferrets, mink and guinea pigs were infected with a T. spiralis nativa isolate, killed at various times postinfection, preconditioned at temperature of -10 degrees C, -15 degrees C or -20 degrees C for varying periods of time prior to low temperature refrigeration and subsequent pepsin digestion to determine survival of larvae. The preconditioning temperature played an important role in the subsequent survival of larvae in musculature at low refrigeration temperatures. Under the conditions of this study, survival of larvae was greater as the preconditioning temperature became lower. The minimum period of preconditioning required had an inverse relationship with the refrigeration temperature. Preconditioning of the T. spiralis nativa isolate used occurred in the musculature of guinea pigs, foxes, ferrets, mink and dogs with larvae surviving longer in vulpine and canine musculature than in the other hosts studied. Age of the infection was not a major factor in the survival of preconditioned larvae in musculature at low refrigeration temperatures although survival was slightly longer in older infections.

Swab transport systems are used for a variety of specimen types and must maintain organism viability throughout the transport process. The Copan ESwab is a new nylon-flocked swab designed to optimize specimen collection and to minimize entrapment of the specimen. We used the quantitative elution method with recommended strains, as described in CLSI document M40-A, to evaluate the ESwab for maintenance of viability of aerobic and anaerobic microorganisms for 0, 6, 24, and 48 h during room temperature and refrigerated temperature storage. The Becton Dickinson CultureSwab MaxV swab and the Remel BactiSwab were used as comparators. The ESwab met CLSI acceptance criteria for all aerobic isolates stored at both temperatures and for all anaerobic isolates stored at refrigerated temperature. The ESwab also met CLSI criteria for four of five anaerobic strains at room temperature. Prevotella melaninogenica was not recovered after 24 or 48 h of room temperature storage with any of the three swab transport systems tested. Overall, the ESwab was equivalent to the Becton Dickinson CultureSwab MaxV swab in organism recovery but recovered more isolates than Remel BactiSwab.

The clinical application and reliability of the leukocyte culture method for the diagnosis of freemartinism were examined and the length of time that blood samples could be held at room temperature and in the refrigerator prior to culturing, was investigated.

The chromosome findings by the leukocyte culture method in 14 freemartins and 9 non-freemartin females belonging to heterosexual twins or triplets revealed that XX-XY cell chimerism exists only in the former, whereas the latter were exclusively of normal female complement.

The mitotic index in bovine blood after preservation for varying periods was studied on samples from two animals. Blood samples from these two animals stored at 5°C for 6 hours in a refrigerator showed the mitotic index to be 3.8 and 5.3 per cent which gradually decreased in samples stored for longer than 12 hours. After 72 hours, a very rapid decrease in mitotic index occurred in both cases, reaching zero in samples stored for 96 and 108 hours. Samples kept at room temperature followed a similar pattern as under refrigeration but with slightly lower values throughout.

Images

Simplification of cell enumeration technologies is necessary, especially for resource-poor countries, where reliable and affordable enumeration systems are greatly needed. In this paper, an immunomagnetic single-platform image cytometer (SP ICM) for cell enumeration based on antibody specificity is reported. A chamber/magnet assembly was designed such that the immunomagnetically labeled, acridine orange-stained cells in a blood sample moved to the surface of the chamber, where a fluorescent image was captured and analyzed for cell enumeration. The system was evaluated by applying one kind of antibody to count leukocytes and one kind for each leukocyte subpopulation: CD45 for leukocytes, CD3 for T lymphocytes, and CD19 for B lymphocytes. Excellent precision and linearity were achieved. Moreover, these cell counts, each from blood specimens of 42 to 52 randomly selected patients, were compared with those obtained by SP (TruCount) and dual-platform (DP) flow cytometry (FCM) technologies. The cell counts obtained by our system were in between those obtained from the TruCount and DP FCM methods; and good correlations were achieved (R ≥ 0.95). For CD4+ counts, as we expected, the cell count by our system was significantly higher than the CD4+ T-lymphocyte counts obtained by SP and DP FCM methods. Immunophenotyping of the immunomagnetically selected CD4+ cells showed that, besides CD4+ T lymphocytes, a proportion of the CD4+ dim monocytes was also selected. Our system is a simple immunomagnetic SP ICM, which can potentially be used for enumeration of CD3+ CD4+ T lymphocytes in resource-poor countries if an additional CD3 immunofluorescent label is applied.

Delayed offline measurement of exhaled nitric oxide (eNO), although useful in environmental and clinical research, is limited by the instability of stored breath samples. The authors characterized sources of instability with the goal of minimizing them. Breath and other air samples were stored under various conditions, and NO levels were measured repeatedly over 1–7 d. Concentration change rates varied positively with temperature and negatively with initial NO level, thus “stable” levels reflected a balance of NO-adding and NO-removing processes. Storage under refrigeration for a standardized period of time can optimize offline eNO measurement, although samples at room temperature are effectively stable for several hours.

Background:

CD4+ T lymphocyte enumeration plays a critical role in the initiation and monitoring of HIV-infected patients on antiretroviral therapy. There is an urgent need for low-cost CD4+ enumeration technologies, particularly for use in dry, dusty climates characteristic of many small cities in Sub-Saharan Africa.

Design:

Cross-sectional study.

Methods:

Blood samples from 98 HIV-infected patients followed in a community HIV clinic in Ouahigouya, Burkina Faso were obtained for routine CD4+ T lymphocyte count monitoring. The blood samples were divided into two aliquots, on which parallel CD4+ measurements were performed using microcapillary (Guava EasyCD4) and dedicated (Becton Dickinson FACSCount) CD4+ enumeration systems. Spearman rank correlation coefficient was calculated, and the sensitivity, specificity and positive predictive value (PPV) for EasyCD4 <200 cells/µL were determined compared to the reference standard FACSCount CD4 <200 cells/µL.

Results:

Mean CD4 counts for the EasyCD4 and FACSCount were 313.75 cells/µL and 303.47 cells/µL, respectively. The Spearman rank correlation coefficient was 0.92 (p<0.001). Median values using EasyCD4 were higher than those with the FACSCount (p=0.004). For a CD4<350 cells/uL, sensitivity of the EasyCD4 was 93.9% (95%CI 85.2-98.3%), specificity was 90.6% (95% CI 75.0-98.0%), and PPV was 95.4% (95%CI 87.1-99.0%).

Conclusion:

Use of the EasyCD4 system was feasible and highly accurate in the harsh conditions of this remote city in Sub-Saharan Africa, demonstrating acceptable sensitivity and specificity compared to a standard operating system. Microcapillary flow cytometry offers a cost-effective alternative for community-based, point-of-care CD4+ testing and could play a substantial role in scaling up HIV care in remote, resource-limited settings.

Fresh milk samples and potassium dichromate preserved milk samples were stored at both ambient, approximately 21 degree C, and refrigerator temperatures, 3-5 degree C, for varying lengths of time before somatic cell counts were performed on an electronic particle counter. Fresh milk samples stored at ambient temperatures became unacceptable for somatic cell counting by 16 hours while those stored in the refrigerator were acceptable for up to three days. Once dichromate had been added to the milk no difference in cell counts attributable to temperature of storage were detected and there was very little change with time up to 14 days. On the average the addition of the dichromate elevated the cell counts/mL. As well a method of rapid fixation of milk involving the addition of glutaraldehyde prior to counting was evaluated. In fresh milk samples the use of glutaraldehyde as a fixative required adjustment of the threshold setting on the cell counter in order to produce results comparable to those obtained from formalin fixed samples. With dichromate preserved milk samples, glutaraldehyde fixation generally elevated the cell counts but the results were variable.

The present preliminary study was performed to find out stability of total prostate specific antigen (PSA) and free prostate specific antigen (FPSA) in serum of healthy males as well as in patients of benign and malignant disorders of prostate at various freezing and nonfreezing temperatures and at different duration of time.

The results of our study indicated long-term stability of both the analytes in frozen serum. Serum total and free PSA were stable only for three to four days in regular refrigerators in unfrozen states. Clotted blood kept at room temperature (25°C–30°C) did not cause change in concentrations of both the analytes for twenty four hours.

A single-platform volumetric flow cytometer, the Partec Cyflow SL_3, was evaluated against a BD FACSCalibur/Sysmex XT1800i dual platform for measuring CD4+ lymphocytes, total lymphocytes, and the percentage of CD4 lymphocytes in whole-blood samples for monitoring the immune systems of human immunodeficiency virus (HIV)/AIDS patients. Statistical analyses for precision, correlation, and agreement were performed. Coefficients of variation (CV) of 5.8, 4.6, and 3.9% were obtained for low, medium, and high CD4+ cell counts, respectively, using the SL_3, and CV of 3.7, 4.0, and 0.94 were obtained for the same categories, using the BD FACSCalibur. Significant correlations (P < 0.005) between the two assays for CD4 counts, total lymphocyte counts, and percentages of CD4 were obtained, with correlation coefficients of 0.99, 0.96, and 0.99, respectively (n = 229). Using the Bland-Altman plot, mean biases of −18 cell/μl (95% confidence interval (CI); −91 to 54 cells/μl), −0.8% (95% CI; −3.6 to 2%), and −36.8 cells/μl (95% CI; −477 to 404 cells/μl) were obtained for comparisons of CD4 counts, percentages of CD4 cells, and total lymphocyte counts, respectively. The effects of the age of the samples on the three parameters were also analyzed by comparing results from the same samples analyzed at 6, 24, and 48 h after collection. The correlation coefficients for comparisons among different time points for the same machine and among all the time points for the two different machines were greater than 0.90. These data showed that the Partec Cyflow SL_3 assay is comparable to the BD FACSCalibur/Sysmex XT1800i dual-platform method for measuring the amount of CD4+ cells and total lymphocytes and the percentages of CD4 cells in blood samples for the purpose of monitoring HIV/AIDS patients.

Although mycophenolate is widely prescribed in India, therapeutic drug monitoring of mycophenolic acid is not performed in most centers. This could be due to many factors such as the large investment and expertise required for high performance liquid chromatography, or the high costs involved as specialized refrigeration is required when transporting patient specimens to the laboratories with the facility to analyze MPA. The Clinical Pharmacology unit of the Christian Medical College Hospital routinely monitors the area under the curve of MPA. In order to determine if this unit could act as a central laboratory for MPA monitoring, the stability of MPA in plasma under a series of storage and transport conditions was assessed. The procedures involved the analysis of plasma specimens from patients on mycophenolate mofetil and blank plasma spiked with MPA reference standard. A range of low and high concentrations were separately analyzed to confirm long term and short term stability. The measured concentrations of MPA showed no significant change over 5 months when stored at −20° or over five days under conditions encountered during transport.

Background

HIV testing on sputum using the QraQuick HIV1/2® assay has high sensitivity and specificity, and holds promise for application in tuberculosis surveys. Its performance under conditions that may occur during surveys in resource-poor countries is however, unknown. We assessed, in a blinded comparison with HIV serum testing, the sensitivity and specificity of the OraQuick® assay for detecting HIV antibody in sputum specimens kept at ambient temperature for up to 7 days, with and without decontaminant.

Methods

Paired sputum and blood specimens from consecutively diagnosed smear-positive tuberculosis patients were tested with OraQuick® and 2 HIV-1/2 ELISA's. Sputum was tested within 24 hours of collection, split into 2 aliquots with and without addition of cetylpyridium chloride, and tested again after 4 and 7 days.

Results

Complete data was available for 377/435 (87%) enrolled patients; 132 (35%) tested HIV positive on serum. The sensitivity of the sputum test was 94.7% (95% CI 89.4–97.8) on day 1, 93.2% on day 4 and 92.9% on day 7. The specificity was 92.9% (95% CI 88.9–95.8) on day 1, and declined to 76.7% on day 4 (p < 0.001) and to 62.7% on day 7 (p < 0.001). Adding cetylpyridium chloride further decreased the specificity to 67.8% on day 4 (p = 0.04) and to 49.6% on day 7 (p = 0.004).

Conclusion

Transportation of sputum specimens at ambient temperatures for 4 days or more, and addition of decontaminant, strongly affect the specificity of the OraQuick® assay. Unless applied within one day, this assay is not suitable for estimation of HIV-prevalence among tuberculosis patients in survey settings.

Culture results of urine specimens transported conventionally (sterile cup) and in a commercial liquid or an investigational lyophilized preservative were compared in a hospital that experiences substantial delays in specimen transport to the laboratory (greater than 40% of specimens received after a delay of greater than or equal to 2 h). At the time of initial plating in the laboratory, 106 of 111 (95.5%) specimens that were positive (greater than or equal to 10(5) CFU of a single organism per ml in pure culture) after conventional transport were also positive in liquid preservative. After a 24-h holding period (cup refrigerated, preserved urine at room temperature), agreement was 91.4% (96 of 105). At the time of initial plating, agreement between results obtained by the conventional method and those obtained by using lyophilized preservative was 96.9% (63 of 65); after 24 h, agreement was 92.4% (61 of 67). Complete inhibition of growth of three Klebsiella pneumoniae isolates was observed in liquid preservative; however, urine processed in the lyophilized preservative did not show inhibition. The proportion of urine cultures showing no change in quantitative growth between the time of initial plating and repeat plating at 24 h was virtually identical for all three processing methods (83.6 +/- 0.9%). After the 24-h holding period, specimens processed in lyophilized preservative were less likely to show diminished quantitative growth than were specimens processed conventionally or in liquid preservative but were more likely to show an increase in growth of greater than or equal to 1 log. Nonetheless, the apparent lack of toxicity of lyophilized preservative may make it preferable to the currently available liquid preservative.

Large field studies of travelers' diarrhea for multiple destinations are limited by the need to perform stool cultures on site in a timely manner. A method for the collection, transport, and storage of fecal specimens that does not require immediate processing and refrigeration and that is stable for months would be advantageous. This study was designed to determine if enterotoxigenic Escherichia coli (ETEC) and enteroaggregative E. coli (EAEC) DNA could be identified from cards that were processed for the evaluation of fecal occult blood. U.S. students traveling to Mexico during 2005 to 2007 were monitored for the occurrence of diarrheal illness. When ill, students provided a stool specimen for culture and occult blood by the standard methods. Cards then were stored at room temperature prior to DNA extraction. Fecal PCR was performed to identify ETEC and EAEC in DNA extracted from stools and from occult blood cards. Significantly more EAEC cases were identified by PCR that was performed on DNA that was extracted from cards (49%) or from frozen feces (40%) than from culture methods that used HEp-2 adherence assays (13%) (P < 0.001). Similarly, more ETEC cases were detected from card DNA (38%) than from fecal DNA (30%) or by culture that was followed by hybridization (10%) (P < 0.001). The sensitivity and specificity of the card test were 75 and 62%, respectively, compared to those for EAEC by culture and were 50 and 63%, respectively, compared to those for ETEC. DNA extracted from fecal cards that was used for the detection of occult blood is of use in identifying diarrheagenic E. coli.

The objectives of this study were to determine the cause of the crystallization in a large volume creatine supplement solution made from effervescent powders containing di-creatine citrate, and to characterize these crystals using thermal analyses and x-ray diffractometry. Creatine effervescent powders were dissolved in deionized water (pH 6.2) and stored both at room temperature (RT) (25°C) and refrigerated condition (4°C) over a period of 45 days. Creatine concentration was determined using high-performance liquid chromatography (HPLC). Intrinsic dissolution and saturated solubility of creatine, creatine monohydrate, and di-creatine citrate in water were determined and compared. Crystal growth was detected only in the refrigerated samples on the seventh day of storage. Differential Scanning Calorimetry (DSC) and x-ray diffraction (XRD) studies revealed that the crystals formed were of creatine monohydrate. Ninety percent creatine degradation was observed within 45 days for RT samples. However, at refrigerated condition this degradation was 80% within the same time period. The pH of the RT samples also increased from 3.6 to 4.5 during storage. No such increase was observed in the case of refrigerated samples. The intrinsic dissolution rate constants of the compounds decreased in the following order: dicreatine citrate>creatine>creatine monohydrate. In conclusion, di-creatine citrate used in effervescent formulation dissociates to creatine in aqueous solution and eventually crystallizes out as creatine monohydrate. Significant decrease in solubility and effect of pH contribute to this crystallization process.

Introduction

C-peptide and insulin measurements in blood provide useful information regarding endogenous insulin secretion. Conflicting evidence on sample stability and handling procedures continue to limit the widespread clinical use of these tests. We assessed the factors that altered the stability of insulin and C-peptide in blood.

Methods

We investigated the impact of preservative type, time to centrifugation, storage conditions and duration of storage on the stability of C-peptide and insulin on three different analytical platforms.

Results

C-peptide was stable for at least 24 hours at room temperature in both centrifuged and whole blood collected in K+-EDTA and serum gel tubes, with the exception of whole blood serum gel, which decreased to 78% of baseline at 24 hours, (p = 0.008). Insulin was stable at room temperature for 24 hours in both centrifuged and whole blood collected in K+-EDTA tubes. In contrast insulin levels decreased in serum gel tubes both centrifuged and whole blood (66% of baseline, p = 0.01 and 76% of baseline p = 0.01, by 24 hours respectively). C-peptide and insulin remained stable after 6 freeze-thaw cycles.

Conclusions

The stability of C-peptide and insulin in whole blood K+-EDTA tubes negates the need to conform to strict sample handling procedures for these assays, greatly increasing their clinical utility.

The balance between free radical production and antioxidant defenses in the body has important systemic and oral health implications. There is convincing evidence that breastmilk containing antioxidants is important in the prevention of diseases in infancy. This study compared the total antioxidant concentration of human breastmilk expressed at different stages of lactation, stored at various temperatures and durations. Expressed breastmilk (EBM) samples of the third, seventh and 30th day were collected from women who had term and preterm deliveries (n=20). Another cohort of women (n=20) was also assessed; these women were more than five months postpartum and lactating. The total antioxidant capacity (TAC) of EBM was assessed at zero hours at room temperature, at 48 hours, one week post-refrigeration (4 °C), and freezing (-8 °C) respectively using the phosphomolybdenum method. The highest antioxidant levels were found in colostrum. The TAC of EBM reduced with time and at post-refrigeration and after freezing (p<0.0005). No significant difference in the mean TAC was observed between the EBM samples obtained from women with either term or preterm deliveries. The progressive loss of antioxidant content of EBM emphasizes the need of awareness and curtailment of the practice of storing and later use of EBM.

Background

Information concerning the effects of HIV-1 infection, disease progression and antiretroviral therapy (ART) on male genital white blood cell (WBC) profiles could provide important insight into genital immune defense in HIV-infected men and seminal HIV transmission mechanisms.

Objective

To compare concentrations of WBC populations in semen from HIV-1-seronegative (HIV−) and seropositive (HIV+) men, and determine whether HIV disease stage and ART are associated with alterations in seminal WBC profiles.

Subjects and Methods

Subjects were 102 HIV− men, 98 ART-naive (ART−) HIV+ men, and 22 HIV+ men on dual nucleoside ART, before and six months after addition of indinavir. Seminal WBCs, macrophages (MØ), and T lymphocyte subpopulations were enumerated by immunohistology technique.

Results

Seminal CD4+ and CD8+ T cell populations were severely depleted in ART− HIV+ men regardless of peripheral blood CD4+ cell count; seminal MØ counts were also reduced. HIV+ men on dual nucleoside ART had significantly higher seminal MØ, CD4+ and CD8+ T cell counts than ART− HIV+ men; addition of indinavir led to a dramatic (>25-fold, p<0.001) increase in seminal CD4+ T cell counts which paralleled an increase in blood CD4+ cell counts. Two ART− HIV+ men with notably elevated seminal WBC profiles (>20 × 106 WBCs/ml) and infectious cell-associated HIV in semen are described.

Conclusions

HIV infection severely depletes CD4+ T cells in the male genital tract as it does at other mucosal sites. This provides evidence that ART− HIV+ men have depressed T cell-dependent genital immune defense functions, and are vulnerable to other genital infections that could promote HIV transmission. Seminal CD4+ T cell counts rebounded following treatment with a viral-suppressing ART regimen, indicating that ART may reverse HIV-associated genital immunosuppression. The relative abundance of seminal MØ in HIV+ men suggests that these cells are important HIV host cells in the male genital tract and vectors of HIV transmission. A subgroup of HIV+ men with exceptionally elevated seminal MØ and CD4+ T cell counts and HIV titers may be highly infectious and contribute disproportionately to HIV transmission.

Background:

Prescribing of diclofenac for children usually involves a dose different from commercially available strengths. This drug is available only as tablets, which can be divided only so many times before the dose obtained becomes inaccurate. In addition, children may have difficulty swallowing tablets. For these reasons, a compounding formula for a liquid dosage form is essential to ensure effective delivery of the drug to pediatric patients.

Objectives:

To develop a compounding formula for diclofenac sodium and to determine the extended physical and chemical stability of this compound when stored in amber polyvinyl chloride (PVC) prescription bottles under refrigeration and at room temperature.

Methods:

A suspension of diclofenac sodium (10 mg/mL) was prepared from commercially available diclofenac sodium tablets, with Ora-Blend as the suspending and flavouring agent. The suspension was packaged in 60-mL amber PVC prescription bottles and stored at either room temperature (23°C) or under refrigeration (5°C). Samples were collected on days 0, 7, 14, 21, 27, 56, and 93. Chemical stability was determined using a validated stability-indicating high-performance liquid chromatography method. At each sampling time, the suspensions were checked for changes in appearance (i.e., colour, layering, caking, ease of resuspension), odour, and pH.

Results:

The diclofenac sodium suspensions were very stable, retaining at least 99.5% of the original concentration for up to 93 days, regardless of storage temperature. There were no apparent changes in the physical appearance of the suspensions, nor were there any substantial changes in odour or pH.

Conclusions:

Suspensions of diclofenac sodium (10 mg/mL) were quantitatively stable but difficult to prepare because of the enteric coating of the tablets. Therefore, it is recommended that diclofenac powder be used for the preparation of suspensions. For pediatric use, palatability is a consideration, and a masking agent should be added before administration. An expiry date of up to 93 days is suggested.

Use of the standard dual-platform flow cytometric method for determination of CD4+ T-lymphocyte counts, which needs both a flow cytometer (FCM) and hematological analyzer, would inevitably lead to increased variability. The development of new single-platform (SP) FCMs that provide direct CD4+ T-lymphocyte counts for improved assay precision and accuracy have recently attracted attention. This study evaluated one of those systems, CyFlowgreen (Partec), a single-parameter SP volumetric FCM. The performance of CyFlowgreen was compared with those of two reference standard SP microbead-based technologies of the three-color TruCOUNT tube with the FACScan FCM and a two-color FACSCount system (Becton Dickinson Biosciences). Absolute CD4+ and CD8+ T-lymphocyte counts in 200 human immunodeficiency virus type 1-seropositive blood specimens were determined. Statistical analysis for correlation and agreement were performed. A high correlation of absolute CD4 counts was shown when those obtained with CyFlowgreen were compared with those obtained with the bead-based three-color TruCOUNT system (R2 = 0.96; mean bias, −69.1 cells/μl; 95% confidence interval [CI], −225.7 to +87.5 cells/μl) and the FACSCount system (R2 = 0.97; mean bias, −40.0 cells/μl; 95% CI, −165.1 to +85.1 cells/μl). The correlation of the CD4+ T-lymphocyte counts obtained by the two bead-based systems was high (R2 = 0.98). Interestingly, CyFlowgreen yielded CD4+ T-lymphocyte counts that were 21.8 and 7.2 cells/μl lower than those obtained with the TruCOUNT and the FACSCount systems, respectively, when CD4+ T-lymphocyte counts were <250 CD4+ T-lymphocyte counts/μl range or 17.3 and 5.8 cells/μl less, respectively, when CD4+ T-lymphocyte counts were <200 cells/μl. The single-parameter CyFlowgreen volumetric technology performed well in comparison with the performance of the standard SP bead-based FCM system. However, a multicenter comparative study is needed before this FCM machine is implemented in resource-limited settings.