Diminished human immunodeficiency virus type 1 DNA yield from dried blood spots after storage in a humid incubator at 37 degrees C compared to -20 degrees C

Collecting whole blood on filter paper simplifies the processing, transport, and storage of specimens used for the diagnosis of human immunodeficiency virus type 1 (HIV-1) and other tests. Specimens may be collected in tropical or rural areas with minimal facilities for handling specimens. To compare simulated tropical conditions with freezer storage, we examined the stability of HIV-1 DNA in dried blood spots (DBS) stored in humid heat and at −20°C. DBS were created by spotting 50-μl aliquots of whole blood on 903 filter paper. DNA was extracted from DBS at baseline and after 2, 6, or 12 months of storage at −20°C or at 37°C with ~85% humidity. The DNA was tested undiluted or diluted using the Amplicor HIV-1 DNA PCR (Roche), version 1.5. Each reaction was scored positive, negative, or indeterminate based on optical density. Results were compared between storage conditions and over time. A total of 1,832 reactions from 916 DBS were analyzed, including 100 DBS at baseline, 418 stored at −20°C, and 398 stored at 37°C. A chi-square test showed fewer positive reactions for DBS stored at 37°C (55%) than for those stored at −20°C (78%) (P < 0.0001). Samples stored at −20°C showed little change in the probability of detection of HIV-1 DNA over time; the odds ratio (OR) was 0.93 after storage for 1 year. Samples stored at 37°C demonstrated a significant change in detection at 1 year (OR, 0.29). We conclude that exposure of DBS to 37°C and high humidity impaired the recovery of HIV-1 DNA from DBS, whereas DNA recovery was preserved when DBS were stored frozen.     

Lymphocyte subpopulations do not alter during blood storage at 4 degrees C

Recent articles have suggested that lymphocyte subpopulations, as determined by monoclonal antibodies, alter during overnight storage of whole blood at 4 degrees C. In contrast, we report here that the expression of lymphocyte subpopulation markers does not alter, but that aberrant results may be obtained due to a large amount of contaminating granulocytes in the lymphocyte preparation, thus indicating that the 'apparent loss' of lymphocyte markers is a technical rather than a biological problem.     

Preservation of Neisseria gonorrhoeae at -20 degrees C.

To explore the feasibility of preserving Neisseria gonorrhoeae at -20 degrees C, we studied its viability quantitatively and qualitatively for 12 and 18 months, respectively, in the following media: a gelatin-based medium used mainly to prepare dried gelatin discs (S. Yamai, Y. Obara, T. Nikkawa, Y Shimoda, and Y. Miyamoto, Br. J. Vener. Dis. 55:90-93, 1979), a simplified version (LSPQ preservation medium), and Trypticase soy broth with 10% (vol/vol) glycerol, a medium commonly used for preservation at -70 degrees C. The latter was studied for 4 months only. Four reference strains and two clinical isolates of N. gonorrhoeae were used. The storage temperature was rigorously preadjusted and monitored at -20 +/- 1 degree C during the entire project. After 12 months of storage, all strains remained viable in both gelatin-based media, whereas a significant loss of viability was observed in Trypticase soy broth-10% glycerol after only 4 months. After 18 months, five strains were still viable in both gelatin-based media and no significant difference was observed between antimicrobial susceptibility results and those of the original strains preserved at -70 degrees C. On the basis of these results, we believe that LSPQ preservation medium represents a good alternative for the storage of N. gonorrhoeae at -20 degrees C for at least a year. Furthermore, it is easy to prepare and use and can by stored at 4 to 8 degrees C for a year prior to use.     

Viability of fungal cultures maintained at -70 degrees C.

One thousand four hundred forty-seven clinical and environmental isolates of molds, yeasts, aerobic actinomycetes, and algae belonging to 164 genera (382 taxa) maintained on potato dextrose agar at -70 degrees C for periods ranging from 6 months to 13 years were subcultured and then incubated at 25 degrees C to determine their viabilities. Thirty-three isolates, Alternaria alternata (n = 1), Apophysomyces elegans (n = 1), Bipolaris spicifera (n = 1), Blastomyces dermatitidis (n = 4), Cokeromyces recurvatus (n = 1), Coremiella cubispora (n = 1), Cryptococcus ater (n = 1), Curvularia sp. (n = 1), Exserohilum monoceras (n = 1), Exserohilum pedicillatum (n = 1), Exserohilum rostratum (n = 1), Filobasidium floriforme (n = 1), Madurella mycetomatis (n = 1), Oedocephalum spp. (n = 2), Penicillium marneffei (n = 1), Pseudomicrodochium spp. (n = 4), Saksenaea vasiformis (n = 1), Sporothrix sp. (n = 1), and Mycelia Sterilia (n = 8), did not grow after repeated attempts at subculturing. Neither time in storage nor taxonomic classification was associated with a lack of viability. Storage at low temperature for either short or long periods of time is an excellent method for maintaining most medically important fungi.     

Intra- and extraerythrocyte pH at 37 degrees C and during long term storage at 4 degrees C: 31P NMR measurements and an electrochemical model of the system

A 31P NMR method based on pH dependent variation of the chemical-shift-difference between the resonances of orthophosphate and methylphosphonate was used to measure simultaneously intracellular pH (pHi) and extracellular pH (pHc) during long term storage of erythrocytes; pH was determined at both 4 degrees C and 37 degrees C. An equation describing the equilibrium distribution of membrane-permeant ions was derived by consideration of the electrochemical and osmotic constraints in the RBC suspension. Calculations using the model-equation and the measured pHi yielded the Donnan ratio and therefore pHo; the relationship between experimentally determined pHi and pHo values was accurately predicted by the model. Sensitivity analysis of the model-equation revealed that the observed increase in transmembrane pH gradient during storage is principally due to the alteration of the total net charge of intracellular (poly)-anions.     

Storage of platete concentrates at +4 degrees C and +22 degrees C

The relationship between the conditions of platelet preservation and their ultrastructure, metabolic activity, and the ability to carry out physiological functions was studied. It was found that storage of platelet concentrates at +4 degrees C for 72 hours reduced the number of platelets, changed their shape decreased the ATP level and the ability of release of adenine nucleotides in response to thrombin. The platelet at +22 degrees C for 72 hours preserved the initial number, normal ultrastructure, intact metabolic activity and the ability of participation in heamostasis processes.     

Thawing of fresh-frozen plasma at 45 degrees C versus 37 degrees C. Comparison using satellite packs of the same donor units

Packs of fresh-frozen plasma (FFP) from the same donor units were thawed at 37 degrees C and at 45 degrees C in order to determine the suitability of the higher temperature for thawing FFP. Measurements of prothrombin time, activated partial thromboplastin time, fibrinogen concentration, and Factor VIII activity showed no significant differences between the temperatures. Thawing time at 45 degrees C was approximately half that at 37 degrees C. The protocol for manipulation and inspection of the FFP was an important determinant of the thawing time, but did not appear to affect coagulation. FFP can safely be thawed in a 45 degrees C water bath if it is removed before thawing is complete.     

Effect of storage time at -20 degrees C on the determination of serum concentrations of tobramycin in the presence of 6 beta-lactams

We assessed the role of six beta-lactam antibiotics and of storage time at - 20 degrees C, in inactivation of tobramycin in patients' sera. Several ranges of concentrations were used for the combined antibiotics. The tobramycin concentrations were measured both by microbiological assay (plate diffusion) and an enzyme mediated immunoassay technique (EMIT). Using the bioassay method, the results after 8 days of frozen storage were as follows: carbenicillin and ticarcillin (256 mg/l) induced a 40-50% reduction of tobramycin activity, whereas mezlocillin (256 mg/l) had less effect: a 15-20% reduction. After 15 days at -20 degrees C the results were nearly the same except for azlocillin (256 mg/l), increasing its percentage reduction to 20%. The results obtained by EMIT procedure were significantly better after 8 days of frozen storage: only mezlocillin (5% reduction) and azlocillin (10%) were effective. Nevertheless after 15 days at -20 degrees C, the inactivating action of the pre-cited antibiotics were similar to those obtained at the same time with the bioassay method. Piperacillin and cefsulodin never induced any reduction of tobramycin levels, whatever the time of storage or quantification procedure used. So, our opinion is that patients' sera containing a beta-lactam antibiotic in combination with tobramycin should be assayed immediately upon receipt. Keeping it at -20 degrees C is not sufficient to prevent in vitro tobramycin inactivation.     

Pilot study for the development of a new campylobacter selective medium at 37 degrees C using aztreonam

AIMS: To overcome contamination and temperature inhibition by isolating campylobacter at 37 degrees C. METHODS: The beta lactam antibiotic aztreonam was included in a selective medium because of its inhibitory activity against Gram negative organisms but not against Campylobacter jejuni. Vancomycin and amphotericin were added to inhibit Gram positive bacteria and yeasts. RESULTS: The aztreonam amphotericin vancomycin (AAV) experimental campylobacter selective medium showed growth microaerobically at 37 degrees C of C jejuni, C coli, C lari, C hyointestinalis, C fetus subsp. fetus, and C jejuni subsp. doylei after 24 to 48 hours of incubation. Six campylobacter NCTC strains demonstrated a minimum inhibitory concentration (MIC) > or = 256 mg/litre for vancomycin and aztreonam, whereas C upsaliensis and two "campylobacter-like" strains now reclassified under genus helicobacter--H cinaedi and H fennelliae--had a MIC of 4 mg/litre for vancomycin and aztreonam. In the pilot study (150 samples), AAV medium (37 degrees C) had a higher sensitivity for isolating campylobacters: 14 were isolated on AAV compared with 10 on modified CDA (43 degrees C) over three days, and nine were isolated on AAV medium compared with five on modified CDA (43 degrees C) after 24 hours of incubation. Contamination rates remained low. CONCLUSION: The medium was devised in a pilot study performed between 1990 and 1993; however, this is the first report of AAV medium used as a selective medium capable of growing six campylobacters of pathogenic importance at 37 degrees C. Further studies are indicated.     

Conversion of Blastomyces dermatitidis to the yeast form at 37 degrees C and 26 degrees C

A partially defined agar medium, KT, has been developed and compared with brain heart infusion agar for the conversion of Blastomyces dermatitidis to the yeast form. On the KT medium, the mold form converted to a yeast form within 72 h of incubation at 37 degrees C or after 3 weeks at 26 degrees C. A nutritionally dependent dimorphism in B. dermatitidis was observed.