A nonelectrical incubator for developing countries

A nonelectrical human incubator for premature infants has been designed and built for use in rural areas of developing nations where electricity is not readily available. This incubator may be operated using kerosine or gas as the source of energy. The unit uses an automatic self-activating regulator which controls the flow of hot water through a simple heat exchanger; the air in the incubator is heated by natural convection, and the humidity is adjusted by water evaporation. The temperature inside the incubator can be maintained to within±0·3°C of the desired level. The unit operates for extended periods of time with little or no supervision.     

Direct binding enzyme-linked immunosorbent assay (ELISA) for serum amyloid A (SAA)

A solid-phase, direct binding ELISA for serum amyloid A (SAA) proteins is described, in which noncovalent interactions of SAA with other plasma constituents are disrupted to permit direct coating of the wells of flexible polyvinyl chloride microtitration plates with an amount of SAA antigen proportional to its concentration in plasma. The wells are coated overnight at 60 degrees C with plasma diluted in 3 M potassium bromide and 0.1 M sodium bicarbonate. pH 9.6. The next day, any remaining sites on the wells are blocked by incubation for 1 h at ambient temperature with a 5% solution of dry milk solids and 0.05% Tween 20 in 0.02 M phosphate buffer, pH 7.4. The wells are rinsed and incubated for 90 min at 37 degrees C with polyclonal rabbit or rat anti-human SAA antiserum. Then, the wells are rinsed and incubated with goat anti-rabbit or rat IgG antiserum to which has been conjugated horseradish peroxidase. o-phenylenediamine and hydrogen peroxide substrates are added to the wells, color is allowed to develop, and sulfuric acid is added to stop the enzyme-catalyzed reaction. The amount of SAA coated to wells is quantified by absorbance at 490 nm. Four or more serial three-fold dilutions of plasma samples are assayed simultaneously on separate plates. Each plate contains a set of wells with identical concentrations of SAA standard protein diluted in decreasing concentrations of plasma proteins corresponding to the dilution of sample. The method can detect SAA concentrations in plasma samples ranging from 1 microgram/ml to greater than 1000 micrograms/ml. The method is suited to serial monitoring of SAA concentration in patients undergoing treatment for inflammatory conditions and to the quantitative analysis of large numbers of samples.     

Microfluidic gradient PCR (MG-PCR): a new method for microfluidic DNA amplification

This study develops a new microfluidic DNA amplification strategy for executing parallel DNA amplification in the microfluidic gradient polymerase chain reaction (MG-PCR) device. The developed temperature gradient microfluidic system is generated by using an innovative fin design. The device mainly consists of modular thermally conductive copper flake which is attached onto a finned aluminum heat sink with a small fan. In our microfluidic temperature gradient prototype, a non-linear temperature gradient is produced along the gradient direction. On the copper flake of length 45 mm, width 40 mm and thickness 4 mm, the temperature gradient easily spans the range from 97 to 52°C. By making full use of the hot (90–97°C) and cold (60–70°C) regions on the temperature gradient device, the parallel, two-temperature MG-PCR amplification is feasible. As a demonstration, the MG-PCR from three parallel reactions of 112-bp Escherichia coli DNA fragment is performed in a continuous-flow format, in which the flow of the PCR reagent in the closed loop is induced by the buoyancy-driven nature convection. Although the prototype is not optimized, the MG-PCR amplification can be completed in less than 45 min. However, the MG-PCR thermocycler presented herein can be further scaled-down, and thus the amplification times and reagent consumption can be further reduced. In addition, the currently developed temperature gradient technology can be applied onto other continuous-flow MG-PCR systems or used for other analytical purposes such as parallel and combination measurements, and fluorescent melting curve analysis.     

Development of a novel explant culture method for the isolation of mesenchymal stem cells from human breast tumor

Background: Mesenchymal stem cells (MSCs) were isolated from various sources, including various types of tumors. However choosing an appropriate isolation method is an important step in obtaining cells with optimal quality and yield in companion with economical considerations. The purpose of this study was to isolate more pure MSCs from human breast tumor tissue by a modified explant culture method.

Methods and Materials: The tumor tissues (n = 8) were cut into 1 to 3-mm cube-like pieces (explant). Each explant was placed in a well of 24-well format plates, cultured in Dulbecco’s Modified Eagle’s medium (DMEM), and maintained at 37°C with 5% humidified incubator. Morphological phenotypes of the cells were surveyed by an inverted microscope and wells with rather homogenous fibroblast-like morphology cell were considered as positive and selected for more expansion and characterization.

Results: A total of 185 wells, 63.7% of wells were positive that were chosen for expansion. Flowcytometry analysis demonstrated that isolated cells were positive for CD73, CD44, CD29, CD105, and CD90 but negative for CD11b, CD45, CD34, and HLA?DR. In addition, cells possessed the capability of multipotential differentiation into osteoblasts and adipocytes.     

Rapid microassays for the measurement of superoxide and hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader

Two simple semiautomated microassays for the measurement of superoxide (O-2) and hydrogen peroxide (H2O2) production by cultured macrophages (MPs) are described. The measurement of O-2 is based on the reduction of ferricytochrome c as assayed by the increase in its absorbance at 550 nm. Quantitation of H2O2 is based on the horseradish peroxidase (HRPO)-dependent oxidation of phenol red which is assayed by its increased absorbance at 600 nm. MPs are cultured in monolayers in 96-well flat-bottom tissue culture plates and covered with 100 mul amounts per well of either a ferricytochrome c solution containing phenol red and HRPO. Following the addition of an agent eliciting an oxidative burst (OB) and incubation of the plates at 37 degrees C for various time intervals, the changes in the absorbance of ferricytochrome c and phenol red, respectively, are measured directly in the wells of the tissue culture plates with the cells in situ, by using an automatic 8-channel photometer which reads absorbances vertically through individual wells. This instrument, which was originally designed for reading enzyme immunoassays in microtitration plates, can be easily adapted for use in the above test, when fitted with interference filters with wave lengths of 550 nm (for the assay of O-2) and 600 nm (for the assay of H2O2). The principal advantages of this techniques are: the ability to perform the assays directly in the culture plates with cells in situ; the small amounts of cells and reagents needed; its sensitivity and reproducibility; the ease with which kinetic experiments can be done; the large number of samples which can be tested in parallel, and especially the speed and convenience offered by the automated reading and printout of absorbance values.     

The use of beeswax as heating element in non-electric infant incubator

Non-electric infant incubators are needed in remote areas that have no access to electricity to reduce infant mortality nationwide. In previous studies, non-electric infant incubators have been developed using phase change material of beeswax as the heating element. This study aims to improve the performance of beeswax non-electric infant incubator to obtain a more reliable and practical one. The design of the original beeswax cartridge in the form of copper boxes was modified into tubes of stainless steel. The geometry and location of the air holes were also modified. Wood that was previously used as the body material was replaced with polyurethane to reduce the weight of the incubator. The beeswax cartridges were heated using boiling water until the beeswax melted. For temperature measurement, five 0.5?mm k-type thermocouples were placed inside of the incubator according to the National Industrial Standard of SNI 16-4221. The beeswax cartridge arrangement was varied to obtain the best performance. The results showed that polyurethane provides infant incubator lighter and more practical to use. The new design of non-electric infant incubator was capable of providing a temperature of 32–36?°C for 2?h.     

The Ames miniscreen assay: Volatility of sodium azide can cause an increase in the reversion frequencies of adjacent,untreated wells

Sodium azide, when added to wells adjacent to untreated wells, caused an increase in the reversion rate of Salmonella typhimurium TA100 in a 12-well plate format. Increases in the reversion frequency in adjacent, untreated wells were observed when a single well on the plate was treated with as little as 1 μg of sodium azide. This effect is probably caused by the hydrolysis of sodium azide to form hydrazoic acid. Hydrazoic acid has a boiling point of 37°C and, thus, would become a diffusible gas duringthe incubation of the plates. Our findings suggest that a diffusible gas is present and that this gas has the ability to contaminate nearby wells when using the multiwell version of the Ames assay. Furthermore, it may be prudent to isolate all positive controls and negative controls on separate plates with no test material since a volatile test material could produce spurious results in the Ames miniscreen. Environ. Mol. Mutagen. 29:217–219, 1997. © 1997 Wiley-Liss, Inc.     

Application of indium tin oxide (ITO)-based microheater chip with uniform thermal distribution for perfusion cell culture outside a cell incubator

This study reports a transparent indium tin oxide (ITO)-based microheater chip and its applicability for perfusion cell culture outside a cell incubator. The attempt of the proposed ITO microheater is to take the role of conventional bulky incubator for cell culture in order to improve integratability with the experimental setup for continuous/perfusion cell culture, to facilitate microscopic observation or other online monitoring activities during cell culture, or even to provide portability of cell culture operation. In this work, numerical simulation and experimental evaluation have been conducted to justify that the presented device is capable of providing a spatially uniform thermal environment and precise temperature control with a mild deviation of ±0.2°C, which is suitable for a general cell culture practice. Besides, to testify that the thermal environment generated by the presented device is well compatible with conventional cell incubator, chondrocyte perfusion culture was carried out. Results demonstrated that the physiology of the cultured chondrocytes on the developed ITO microheater chip was consistent with that of an incubator. All these not only demonstrate the feasibility of using the presented ITO microheater as a thermal control system for cell culture outside a cell incubator but also reveal its potential for other applications in which excellent thermal control is required.     

A simple and rapid flow cytometric method for detection of porcine cell surface markers

The objective of this study was to develop a rapid and reliable method for flow cytometric analysis of porcine whole blood cells. Fifty-microliters of heparin- or EDTA-treated whole blood was added to wells of a round-bottom 96-well microtitration plate. Each well contained 10 microl of an appropriate dilution of four different antibodies (40 microl total; two primary monoclonal antibodies and two fluorescent-labeled secondary antibodies). For convenience, the antibody mixture could be added to plates 1-2 days prior to assay and stored at 4 degrees C. Once whole blood was added to wells, plates were mixed gently, placed in a sealed bag and incubated in the dark at room temperature for 20 min. Contents of wells were then transferred to polystyrene tubes containing 2 ml of 1.5% formalin in distilled water and mixed gently. Cells were fixed for a minimum of 30 min and then stored in the dark at 4 degrees C until analysis by flow cytometry. Analysis of cell samples may be done up to 3 days after fixation. Results indicate that the percentages of Class I, Class II, CD3, CD8, CD4, CD45, monocyte, gamma-delta T-cell populations, and total number of granulocytes identified using this method were comparable to standard values or to values obtained following separation of white blood cells from red blood cells. The percentage of labeled B-cells was lower than standard values. Total assay time from receipt of blood to acquisition of data by flow cytometry required less than 2 h. This modified assay was shown to be simple, reliable, and useful for screening large numbers of porcine samples in a minimal period of time.     

Rapid screening of monoclonal antibodies: new ''microstick'' radioimmunoassay

A semi-automation of fluid phase double antibody radioimmunoassay has been developed. The immune precipitate that was formed in 96-well microtitration plates was harvested and washed on microfibre filters using a Titertek cell harvester. A disc transfer system originally designed for use with the harvester was used as a quick and easy method of transferring the filter discs containing immune precipitate into vials for counting. The results of radioimmunoassay using the microtitration plate-filtration and conventional tube-centrifugation method are essentially identical. The microtitration plate-filtration radioimmunoassay has the following advantages over the conventional tube-centrifugation method: (1) there is no centrifugation required; (2) handling of microtitration plate is easier than the tubes in racks; and (3) it requires much less time to perform the assay.     

Experimental and numerical studies on convective heat transfer in a neonatal incubator

Thermo-neutrality is one of the major environmental factors affecting a premature or low-birth-weight neonate inside an incubator. Severe temperature differences inside an incubator lead to neonate heat loss, hypothermia and apnoea, which are closely related to air flow and air velocity. In the study, flow visualisations, hot-wire velocity measurements and computational fluid dynamics simulate the airflow inside a neonatal incubator. An anatomically correct neonate model is designed using a three-dimensional laser scanner system and a rapid prototyping machine. Flow visualisations demonstrate that large-scale rotating airflow is produced inside the chamber, and a number of small, stationary eddies are found in regions between the air inlet and the neonate. Hot-wire measurements show that air velocities along the long inlets are not uniform. Computational fluid dynamics show relatively uniform temperatures of about 34°C on the neonate's anterior aspect and the highest temperature of 36.1°C at the right armpit and the crotch. Flow fields from airflow visualisations, hot-wire measurements and computational fluid dynamics are very similar, both qualitatively and quantitatively. The small eddies produced between the neonate and the mattress could interfere with convective and evaporative heat transfers from the neonate. Therefore it is important to eliminate eddies around the neonate in future designs of neonatal incubators.     

A simple and inexpensive method for assessing in vitro candidacidal activity of leukocytes

Candidacidal activity of mouse neutrophils and macrophages was determined directly in microtiter plates. After a suitable period of interaction between phagocytic cells and C. albicans in the wells, the mouse cells were lysed with distilled water and corn meal agar was added to each well. Following incubation at 37°C, viability was assessed using an inverted microscope and counting the number of germ tubes or microcolonies which developed. This method does not use radioisotopes or vital stains and should be applicable to other genera of yeasts.     

Direct counting of tritium by dissolving Microelisa wells in scintillation fluid

Radioassays with low-energy beta-emitting nuclides (e.g., 3H, 14C, 35S) in 96-well plastic plates are tedious and frequently inaccurate because of the necessity of quantitatively removing and transferring the contents of each well to scintillation fluid. We therefore investigated the possibility of counting these nuclides by directly placing the entire break-apart well (Microelisa) along with the sample into one of four scintillation counting fluids: toluene-PPO-POPOP with or without Protosol, ACSR, and EcoLite. Although some of these scintillation fluids fully dissolved the plastic wells and other did not, we found that the presence of the wells did not appreciably interfere with the efficiency of tritium counting. This technique saves considerable time and reduces possible errors in liquid scintillation counting of samples from plastic microtitration plates.     

Contents to volume 44 (1981)

The use of Terasaki (10 μl samples) and microtitration (100 μl samples) plates as the solid phase in enzyme immunoassays was compared. Various antigens were used for coating the plates and antibodies present in human sera were evaluated using the same anti-human Ig antibody labelled with either β-galactosidase, alkaline phosphatase, peroxidase or glucose oxidase. The results obtained, either by scoring with the naked eye or by absorbance reading with appropriate densitometers, showed that both plates were equally suitable and that the 4 enzymes were equally effective in detecting the same lowest quantity of antibody. A comparative evaluation using either Terasaki or microtitration plates for the quantitation of human anti-Echinococus granulosus antibody in 50 sera demonstrated that there was a good correlation between the two procedures (r=0.8097). Finally, the use of glucose oxidase as the enzyme marked allowed a clear-cut distinction to be made between positive and negative samples with the naked eye alone.     

Theoretical simulation of temperature distribution in the brain during mild hypothermia treatment for brain injury

Mild or moderate hypothermia (>30°C) has been proposed for clinical use as a therapeutic option for achieving protection from cerebral ischaemia in brain injury patients. In this research, a theoretical model was developed to examine the brain temperature gradients during selective cooling of the brain surface after head injury. The head was modelled as a hemisphere consisting of several layers, representing the scalp, skull and brain tissue, respectively. The dimensions, physical properties and physiological characteristics for each layer, as well as the arterial blood temperature, were used as the input to the Pennes bioheat transfer equation to simulate the steady-state temperature distribution within the brain. Depending on the head surface temperature, a temperature gradient of up to 13°C exists in the brain tissue. The results have shown that the volumetric-averaged brain tissue temperature T_{bt, avg} for adults and infants can be 1.7 and 4.3°C, respectively, lower than the temperature of the arterial blood supplied to the brain tissue. The location where the probe should be placed to measure T_{bt, avg} was also determined by the simulation. The calculation suggests that the temperature sensor should be placed 7.5mm and 5.9 mm beneath the brain tissue surface for adults and infants, respectively, to monitor T_{bt, avg} continuously.     

Additional double-wall roof in single-wall,closed, convective incubators: Impact on body heat loss from premature infants and optimal adjustment of the incubator air temperature

Radiant heat loss is high in low-birth-weight (LBW) neonates. Double-wall or single-wall incubators with an additional double-wall roof panel that can be removed during phototherapy are used to reduce Radiant heat loss. There are no data on how the incubators should be used when this second roof panel is removed. The aim of the study was to assess the heat exchanges in LBW neonates in a single-wall incubator with and without an additional roof panel. To determine the optimal thermoneutral incubator air temperature.Influence of the additional double-wall roof was assessed by using a thermal mannequin simulating a LBW neonate. Then, we calculated the optimal incubator air temperature from a cohort of human LBW neonate in the absence of the additional roof panel.Twenty-three LBW neonates (birth weight: 750–1800 g; gestational age: 28–32 weeks) were included. With the additional roof panel, R was lower but convective and evaporative skin heat losses were greater. This difference can be overcome by increasing the incubator air temperature by 0.15–0.20 °C.The benefit of an additional roof panel was cancelled out by greater body heat losses through other routes. Understanding the heat transfers between the neonate and the environment is essential for optimizing incubators.     

Thermoregulatory behavior in rat pups from birth to weaning

One–19 day old rat pups were placed individually in a thermal gradient (floor temperatures 17–45°C) at either 45°, 30°, or 20°C. From the first day of life, pups placed at 45° or 30°C and given sufficient time (up to 2 hrs) oriented and moved along the gradient to an area of moderate temperatures (35°–40°C). The pups regulated their body temperatures by remaining in appropriate positions in the gradient. Most pups under a week old were immobilized when placed in the cold area. During the next two weeks, pups placed at 20°C moved up the gradient to the 35°–40°C area. Response latency was related to both starting temperature and age. Observations of the pups' behavior in different temperatures are discussed.     

Temperature distribution effects on micro-CFPCR performance

Continuous flow polymerase chain reactors (CFPCRs) are BioMEMS devices that offer unique capabilities for the ultra-fast amplification of target DNA fragments using repeated thermal cycling, typically over the following temperature ranges: 90°C–95°C for denaturation, 50°C–70°C for renaturation, and 70°C–75°C for extension. In CFPCR, DNA cocktail is pumped through the constant temperature zones and reaches thermal equilibrium with the channel walls quickly due to its low thermal capacitance. In previous work, a polycarbonate CFPCR was designed with microchannels 150 μm deep, 50 μm wide, and 1.78 m long—including preheating and post-heating zones, fabricated with LIGA, and demonstrated. The high thermal resistance of the polycarbonate led to a high temperature gradient in the micro-device at steady-state and was partly responsible for the low amplification yield. Several steps were taken to ensure that there were three discrete, uniform temperature zones on the polycarbonate CFPCR device including: reducing the thickness of the CFPCR substrate to decrease thermal capacitance, using copper plates as heating elements to ensure a uniform temperature input, and making grooves between temperature zones to increase the resistance to lateral heat conduction between zones. Finite element analyses (FEA) were used to evaluate the macro temperature distribution in the CFPCR device and the micro temperature distribution along a single microchannel. At steady-state, the simulated CFPCR device had three discrete temperature zones, each with a uniform temperature distribution with a variation of ±0.3°C. An infrared (IR) camera was used to measure the steady-state temperature distribution in the prototype CFPCR and validated the simulation results. The temperature distributions along a microchannel at flow velocities from 0 mm/s to 6 mm/s were used to estimate the resulting temperatures of the DNA reagents in a single microchannel. A 500 bp DNA fragment was generated from a bacteriophage λ-DNA target using 20 cycles of PCR. The amplification efficiencies compared to a commercial thermal cycler were 72.7% (2 mm/s), 44% (3 mm/s), and 29.4% (4 mm/s). The amplification efficiency with the modified CFPCR device increased by 363% at 2 mm/s and 440% at 3 mm/s compared to amplification obtained using a CFPCR device with the same fluidic layout, (Hashimoto et al., Lab Chip 4:638, 2004) strictly due to the improved temperature distribution.     

Use of stable, sensitized cells in an indirect microhemagglutination test for melioidosis.

Two evaluations were carried out in this study. The first was a comparison of the standard tube test with the automated microtitration test for the detection of antibodies to Pseudomonas pseudomallei by the indirect hemagglutination method. Data from this comparison indicated that the tests were equivalent. The second evaluation consisted of reproducibility studies on two lots of pyruvic aldehyde-stabilized sensitized erythrocytes in comparison with freshly prepared sensitized erythrocytes in the automated microtitration test. The influence of different types of the microtitration plates used was also examined. Results indicated that the use of stabilized antigens is feasible, and these antigens offer the advantage of being ready for immediate use.     

Comparative clinical evaluation of a prototype non-electric transport incubator and an electrical infant incubator in a neonatal unit

A new non-electric transport incubator has been developed for transferring babies between health facilities in developing countries. The temperature performance of this prototype was compared with a commercial electric incubator. The warm-up time for the prototype was 51.8 min, compared with 48.1 min for the electric incubator. Forty-five non-distressed premature babies, aged 24–72 h, with a gestational age of less than 37 weeks, were continuously evaluated for a 2 h period. Twenty-five babies, with a mean weight of 2073 g (range 1500–2500 g), were studied in the prototype, and 20 babies, with a mean weight of 2076 g (range 1550–2500 g), were studied in the electrical incubator. The rectal and abdominal skin temperature, heart rate, oxygen saturation and respiratory rate of the babies were recorded. The temperature, oxygen and humidity level of the canopy and the room temperature were also measured. The SaO₂, heart rate and respiratory rate were within the normal range (in the prototype: 96.5%, 130.5 beats min⁻¹ and 43 breaths min⁻¹, respectively; and, in the electric incubator: 96.5%, 128.5 beats min⁻¹ and 40 breaths min⁻¹, respectively). No evidence of carbon dioxide narcosis, hypoxia, acidosis or adverse thermoregulatory behaviour were observed in the two groups. The mean rectal temperature for both groups was within the range 36.5°C–37.5°C. There was no significant difference between the measurements of the two groups. The level of oxygen inside the canopy was 21%, and no decrease was observed. The new non-electric transport incubator confirmed its safety and efficiency in providing a warm environment for non-distressed premature babies over a 2 h period.