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1.  Time-Dependent Effect of Refrigeration on Viscosity and Conversion Kinetics of Dental Adhesive Resins 
European Journal of Dentistry  2010;4(2):150-155.
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.
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).
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.
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.
PMCID: PMC2853827  PMID: 20396445
Bonding resins; Degree of conversion; Polymerization kinetics; Refrigeration; Viscosity
2.  Laboratory assessment of physical and chemical methods of preserving urine specimens. 
Journal of Clinical Pathology  1977;30(6):532-536.
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.
PMCID: PMC476463  PMID: 326817
3.  Stability of Sodium Nitroprusside and Sodium Thiosulfate 1:10 Intravenous Admixture 
Hospital pharmacy  2010;45(10):779-784.
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.
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.
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.
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.
PMCID: PMC3102563  PMID: 21625332
drug stability; high-pressure liquid chromatography; pharmacoeconomics
4.  Effect of a Common Diet and Regular Beverage on Enamel Erosion in Various Temperatures: An In-Vitro Study 
This study compared diet and regular Coca-Cola on enamel erosion in cold and room temperatures.
Materials and Methods:
Seventy five enamel specimens were prepared and divided into 5 equal groups (N=15) as follows: Group 1: regular beverage at room temperature, Group 2: regular beverage at refri-gerator temperature, Group 3: diet beverage at room and Group 4: diet beverage at refrige-rator temperature. The specimens were immersed in the regular or diet beverage (Coca-Cola, trade mark regd. Khoshgovar Co., Tehran, Iran) at room (20°C) or refrigerator (2°C) temperatures for 20 minutes, 3 times per day for 7 days. Specimens in the control subjects (group 5) were placed in synthetic saliva at room temperature for 7 days. The hardness of specimens was tested using Vickers test under 500 gr loads for 5 seconds. The data were analyzed using two-way ANOVA and Tukey tests.
The mean and standard deviations of micro-hardness values of the studied groups were as follow: G1: 304.26±29.71, G2: 285.53±42.14, G3: 279.06±39.52, G4: 266.80±23.98 and G5: 319± 30.79. There was a significant difference in the beverage type as the main factor (p<0.05), but temperature factor and their interaction effect on enamel hardness showed no significant difference (p>0.05). Tukey tests showed that there were significant differences between control and diet groups as well as regular and diet groups.
Diet Coca-Cola is more erosive than the regular type and the temperature of the beverages used had no significant influence on enamel erosion.
PMCID: PMC4025425  PMID: 24910648
Beverages; Cola; Tooth Erosions; Enamel
5.  Stability of Extemporaneously Prepared Lansoprazole Suspension at Two Temperatures 
The purpose of this study was to examine the stability of a generic lansoprazole product in a 3 mg/mL sodium bicarbonate suspension under room temperature and refrigerated conditions.
Lansoprazole suspensions (3 mg/mL) were prepared in triplicate using an 8.4% sodium bicarbonate vehicle for each storage condition (room temperature and refrigerated). During 1 month, samples from each replicate were periodically removed and analyzed for lansoprazole concentration by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Each sample was spiked with 10 mg/L omeprazole to serve as the internal standard. A positive electrospray LC-MS/MS method was validated over the calibration range of 5 to 25 mg/L using Food and Drug Administration Guidance. The identities of the analyte and internal standard in the samples were verified by monitoring the MS/MS transitions of m/z 370 to m/z 252 and m/z 346 to m/z 198 for lansoprazole and omeprazole, respectively. Additionally, the pH of the suspensions was monitored throughout the study.
The stability of lansoprazole in the oral sodium bicarbonate suspension under refrigeration is compromised prior to what has been previously reported in the literature. Samples kept at room temperature lost >10% of the lansoprazole after 48 hours compared with the refrigerated samples, which maintained integrity up to 7 days. No statistically significant difference was found between the pH of the room temperature and refrigerated suspension samples, indicating that this factor is not the cause for the differences in stability at these two conditions.
This study suggests that the extemporaneously compounded lansoprazole oral suspension prepared in 8.4% sodium bicarbonate should not be stored in plastic oral syringes longer than 48 hours at room temperature and no longer than 7 days when refrigerated. These data indicate an expiration time earlier than that previously reported for the refrigerated product (14 days).
PMCID: PMC3668940  PMID: 23798906
drug stability; lansoprazole; suspensions
6.  Stability of Ziprasidone Mesylate in an Extemporaneously Compounded Oral Solution 
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.
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.
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.
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.
PMCID: PMC3018175  PMID: 22477804
extemporaneous compounding; high performance liquid chromatography; oral solution; stability; ziprasidone
7.  Stability of Vancomycin 25 mg/mL in Ora-Sweet and Water in Unit-Dose Cups and Plastic Bottles at 4°C and 25°C 
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.
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.
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.
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.
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.
PMCID: PMC2999368  PMID: 22479004
vancomycin; high-pressure liquid chromatography; stability; vancomycine; chromatographie liquide haute performance; stabilité
8.  Evaluation of a Simplified Dual-Platform Flow Cytometric Method for Measurement of Lymphocyte Subsets and T-Cell Maturation Phenotypes in the Population of Nouna, Burkina Faso▿  
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.
PMCID: PMC1951080  PMID: 17442847
9.  Factors affecting preconditioning of Trichinella spiralis nativa larvae in musculature to low temperatures. 
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.
PMCID: PMC1255297  PMID: 3607648
10.  An Immunomagnetic Single-Platform Image Cytometer for Cell Enumeration Based on Antibody Specificity▿  
Clinical and Vaccine Immunology  2007;14(4):412-419.
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.
PMCID: PMC1865616  PMID: 17287315
11.  Validation of Microcapillary Flow Cytometry for Community-Based CD4+ T Lymphocyte Enumeration in Remote Burkina Faso  
The Open AIDS Journal  2010;4:171-175.
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.
Cross-sectional study.
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.
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%).
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.
PMCID: PMC3023948  PMID: 21253463
Low-cost CD4; CD4+ count; EasyCD4 assay; Guava Technologies, Inc.,; microcapillary flow cytometry; resource-limited setting.
12.  Comparison of the Copan ESwab System with Two Amies Agar Swab Transport Systems for Maintenance of Microorganism Viability▿  
Journal of Clinical Microbiology  2008;46(5):1655-1658.
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.
PMCID: PMC2395074  PMID: 18353935
13.  Chiral Stability of an Extemporaneously Prepared Clopidogrel Bisulfate Oral Suspension 
The purpose of this study was to evaluate the chiral stability of clopidogrel bisulfate in an extemporaneously compounded oral suspension for a period of 60 days.
A 5 mg/mL oral suspension of clopidogrel bisulfate was prepared from commercially available Plavix tablets. The clopidogrel suspension was then evenly divided between two light-resistant prescription bottles and stored either under refrigeration (4°C) or at room temperature (25°C). Samples were drawn from the stored suspensions immediately after preparation and on days 7, 14, 28, and 60. Samples were subsequently analyzed at each time point by high-performance liquid chromatography using a reversed-phase column, with chemical stability defined as the retention of at least 90% of the initial intact clopidogrel concentration measured. To determine the chiral stability of the suspension, samples were also analyzed by high-performance liquid chromatography using a chiral column to investigate possible enantiomeric inversion. Chiral stability was defined as the retention of at least 90% of the initial concentration of the suspension as the S-enantiomer, the active moiety of Plavix.
Regardless of storage conditions, the oral suspension of clopidogrel retained at least 98% of the active S-enantiomer for 60 days after preparation. Compared with the clopidogrel suspension stored in the refrigerator, more chiral inversion was noted in the clopidogrel suspension stored at room temperature.
Our investigation of chiral stability indicates that a 5 mg/mL clopidogrel oral suspension stored under refrigeration and at room temperature maintains chiral stability as the active S-enantiomer.
PMCID: PMC3998964  PMID: 24782688
chiral; clopidogrel bisulfate; compounded oral suspension; enantiomers; high-performance liquid chromatography
14.  The Leukocyte Culture Method in the Diagnosis of Free-martinism 
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.
PMCID: PMC1319303  PMID: 4234791
15.  Exhaled Nitric Oxide: Sources of Error in Offline Measurement 
Archives of environmental health  2004;59(8):385-391.
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.
PMCID: PMC1866170  PMID: 16268114
air samples; breath samples; exhaled nitric oxide; NO
16.  Effect of Pre-treatment and Storage Conditions on the Quality Characteristics of Ginger Paste 
This study was performed to investigate the effects of pre-treatment and storage temperature and periods on the quality characteristics of ginger paste. The pH of the ginger paste remained constant during room temperature storage but increased with prolonged refrigerated storage periods. During five months of frozen storage, regardless of pre-treatment, the pH of most of the samples decreased slightly and then remained constant. In the color value of ginger paste stored at room temperature, the samples with and without chemical additives changed in color more prominently than fermented or pasteurized samples. Intriguingly, the color value for samples containing chemical additives changed more dramatically when stored under refrigerated conditions. However, the L, a, and b values of samples stored under frozen storage conditions did not change even after ten months. Most of the samples contained glucose and fructose, except for the fermented samples. The free sugar content of samples slowly decreased with increasing storage periods, while the organic acid content generally decreased also, regardless of sample type. Depending on pre-treatment and storage temperature, the gingerol content of the samples was either retained or decreased with prolonged storage time.
PMCID: PMC3866759  PMID: 24471062
ginger paste; pre-treatment; storage condition; quality characteristics
17.  The effects of storage and method of fixation on somatic cell counts in bovine milk. 
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.
PMCID: PMC1320159  PMID: 7337865
18.  Field evaluation in Chad of community usage of CD4 T lymphocyte counting by alternative single-platform flow cytometry 
Field and community evaluation of the routine usage of CD4 T counting platforms is essential in resource-poor countries for efficient and cost-effective monitoring of HIV-infected adults and children attending health care centers.
We herein addressed the principal issues raised by the implementation of the single-platform, volumetric Auto40 flow cytometer (Apogee Flow Systems Ltd, Hemel Hempstead, UK) in 8 community HIV monitoring laboratories of different levels throughout Chad. This is a country with particularly difficult conditions, both in terms of climate and vast geographical territory, making the decentralization of the therapeutic management of HIV-infected patients challenging.
The routine usage of the Auto40 flow cytometers for a period of 5 years (2008–2013) confirms the reliability and robustness of the analyzer for community-based CD4 T cell enumeration in terms of both absolute numbers and percentages to enable accurate monitoring of HIV-infected adults and children. However, our observations suggest that the Auto40 mini flow cytometer is not suitable for all laboratories as it is oversized and ultimately very expensive.
The Chad experience with the Auto40 flow cytometer suggests that its usage in resource-limited settings should be mainly reserved to reference (level 1) or district (level 2) laboratories, rather than to laboratories of health care centres (level 3).
PMCID: PMC3849920  PMID: 24083615
Flow cytometry; CD4 T cell count; CD45; Decentralization; Sub-Saharan Africa; Chad
19.  Stability of total and free prostate specific antigen in serum samples at different storage conditions 
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.
PMCID: PMC3454192  PMID: 23105449
Free PSA and Total PSA; BPH; Cancer Prostate
20.  Extended Stability of Intravenous 0.9% Sodium Chloride Solution After Prolonged Heating or Cooling 
Hospital Pharmacy  2014;49(3):269-272.
The primary objective of this study was to evaluate the stability and sterility of an intravenous 0.9% sodium chloride solution that had been cooled or heated for an extended period of time.
Fifteen sterile 1 L bags of 0.9% sodium chloride solution were randomly selected for this experiment. Five bags were refrigerated at an average temperature of 5.2°C, 5 bags were heated at an average temperature of 39.2°C, and 5 bags were stored at an average room temperature of 21.8°C to serve as controls. All samples were protected from light and stored for a period of 199 days prior to being assayed and analyzed for microbial and fungal growth.
There was no clinically significant difference in the mean sodium values between the refrigerated samples, the heated samples, and the control group. There were no signs of microbial or fungal growth for the duration of the study.
A sterile intravenous solution of 0.9% sodium chloride that was heated or cooled remained stable and showed no signs of microbial or fungal growth for a period of 199 days. This finding will allow hospitals and emergency medical technicians to significantly extend the expiration date assigned to these fluids and therefore obviate the need to change out these fluids every 28 days as recommended by the manufacturer.
PMCID: PMC3971113  PMID: 24715747
refrigerated fluids; sodium chloride; stability; sterility; warmed fluids
21.  Evaluation of the Partec Flow Cytometer against the BD FACSCalibur System for Monitoring Immune Responses of Human Immunodeficiency Virus-Infected Patients in Zimbabwe▿  
Clinical and Vaccine Immunology  2007;14(3):293-298.
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.
PMCID: PMC1828850  PMID: 17267593
22.  Stability Issues of Mycophenolic Acid in Plasma: From Patient to Laboratory 
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.
PMCID: PMC2866351  PMID: 20502578
Mycophenolate; stability; transport; temperature
23.  Low specificity of HIV-testing on sputum specimens kept at ambient temperatures for 4 to 7 days: a blinded comparison 
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.
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.
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).
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.
PMCID: PMC2072942  PMID: 17880720
24.  Evaluation of PIMA™® Point of Care Technology for CD4 T Cell Enumeration in Kenya 
PLoS ONE  2013;8(6):e67612.
CD4+ T cell enumeration is used to determine eligibility for antiretroviral therapy (ART) and to monitor the immune status of HIV-positive patients; however, many patients do not have access to this essential diagnostic test. Introducing point of care (POC) testing may improve access. We have evaluated Alere’s PIMA™, one such POC device, against conventional CD4+ testing platforms to determine its performance and validity for use in Kenya. In our hands, Alere PIMA™ had a coefficient of variability of 10.3% and of repeatability of 175.6 cells/µl. It differed from both the BD FACSCalibur™ (r2 = 0.762, mean bias −64.8 cells/µl), and the BD FACSCount™ (r2 = 0.874, mean bias 7.8 cells/µl). When compared to the FACSCalibur™ at a cutoff of 350 cells/µl, it had a sensitivity of 89.6% and a specificity of 86.7% in those aged 5 years and over (Kw = 0.7566). With the BD FACSCount™, it had a sensitivity of 79.4% and a specificity of 83.4% in those aged 5 years and over (Kw = 0.7790). The device also differed from PARTEC Cyflow™ (r2 = 0.781, mean bias −24.2 cells/µl) and GUAVA™ (r2 = 0.658, mean bias −0.3 cells/µl) platforms, which are used in some facilities in Kenya. We conclude that with refinement, Alere PIMA™ technology has potential benefits for HIV-positive patients. This study highlights the difficulty in selecting the most appropriate reference technology for technical evaluations.
PMCID: PMC3692483  PMID: 23825674
Transfusion  2012;52(9):2043-2048.
The FDA requires that red cells must be refrigerated within 8 hours of whole blood collection. Longer storage of whole blood at 22°C before component preparation would have many advantages.
Study Design And Methods
Two methods of holding whole blood for 20 to 24 hours at room temperature were evaluated; i.e., refrigerated plates or a 23°C incubator. After extended whole blood storage, platelet concentrates were prepared from platelet-rich-plasma on day 1 post-donation, and the platelets were stored for 6 more days. On day 7 of platelet storage, blood was drawn from each subject to prepare fresh platelets. The stored and fresh platelets were radiolabeled and transfused into their donor.
Eleven subjects’ whole blood was stored using refrigerated Compocool plates and 10 using an incubator. Post-storage platelet recoveries averaged 47 ± 13% versus 53 ± 11% and survivals averaged 4.6 ± 1.7 days versus 4.7 ± 0.9 days for Compocool versus incubator storage, respectively (p=N.S.). Using all results, post-storage platelet recoveries averaged 75 ± 10% of fresh and survivals 57 ± 13% of fresh; platelet recoveries met FDA guidelines for post-storage platelet viability but not survivals.
Seven-day post-storage platelet viability is comparable when whole blood is stored for 22 ± 2 hour at 22°C using either refrigerated plates or an incubator to maintain temperature prior to preparing platelet concentrates.
PMCID: PMC3366010  PMID: 22320682
Platelets; Platelet Concentrates; Platelet Storage

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