Measuring percent lymphocytes by flow cytometry to calculate absolute lymphocyte subset counts for HIV+ specimens.

A comparison was made of lymphocyte percentages from an automated hematology analyzer (ELT 15) vs. a fluorescence flow cytometer (Cytofluorograf). The hematology values were consistently higher than the flow (by greater than 10% for 13 of 50 HIV+ specimens). The findings were similar to three other pairings: H*1 vs. Cytofluorograf, Cell Dyn 3000 vs. Cytofluorograf, and Cell Dyn 3000 vs. EPICS Profile. In another comparison manual percent lymphocytes matched much better with flow values. Factors of sample preparation and instrument analysis as they relate to nonrandom cell loss and lymphocyte resolution are examined.     

Membrane fluidity measured by fluorescence polarization using an EPICS V cell sorter

We have characterized the measurement of fluorescence polarization on single cells using an EPICS V cell sorter. A critical analysis is made of the balancing and calibration of the system. The system is highly linear for polarization measurements. Cellular membranes were labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) to measure membrane fluidity. Fluorescence polarization histograms had coefficients of variation as low as 7%. Cells labeled with DPH after 24 hr incubation in medium lacking serum showed a significantly higher fluorescence polarization than cells in medium containing serum. The fluorescence polarization measured at 15 degrees C was 0.311 compared to 0.270 at 25 degrees C for cells labeled with DPH, verifying that temperature affects the membrane fluidity as measured by flow cytometry.     

Macintosh graphics for the EPICS flow cytometer user

Graphic options for the EPICS flow cytometer user have been restricted in the past to software written specifically for the analysis and graphics of flow cytometric data. The software is limited to only a few graphic presentation styles. The technique described will allow the EPICS user to translate histogram files into text files that can be used in an alternative computer format.     

How the confocal laser scanning microscope entered biological research

A history of the early development of the confocal laser scanning microscope in the MRC Laboratory of Molecular Biology in Cambridge is presented. The rapid uptake of this technology is explained by the wide use of fluorescence in the 80s. The key innovations were the scanning of the light beam over the specimen rather than vice-versa and a high magnification at the level of the detector, allowing the use of a macroscopic iris. These were followed by an achromatic all-reflective relay system, a non-confocal transmission detector and novel software for control and basic image processing. This design was commercialized successfully and has been produced and developed over 17 years, surviving challenges from alternative technologies, including solid-state scanning systems. Lessons are pointed out from the unusual nature of the original funding and research environment. Attention is drawn to the slow adoption of the instrument in diagnostic medicine, despite promising applications.     

Comparison of lymphocyte immunophenotypes obtained simultaneously from two different data acquisition and analysis systems on the same flow cytometer.

Immunophenotyping of different lymphocyte populations was carried out in parallel on 113 consecutively received specimens of human peripheral blood using 2 different data acquisition and analysis systems (EPICS C and 4Cyte-Acmecyte) on the same flow cytometer (EPICS C). The phenotypes analyzed were CD3+, CD4+, CD8+ CD56+ CD16+ CD3-, TCR-gamma delta+ CD8-, and TCR-gamma delta+ CD8+. Both HIV- and HIV+ specimens were used for this study, including some with CD4 levels as low as 2% of all lymphocytes. Despite differences in gating procedures and shapes of bitmap (rectilinear vs. "amorphous"), the 2 methods agreed to within 2% positive cells in 97% of the cases. Although some statistically significant biases in the methods were observed, these were small and not biologically important. We conclude that both methods of data acquisition and analysis, as employed by experienced operators on the EPICS C flow cytometer, gave essentially equivalent results for lymphocyte sub-populations in peripheral blood preparations.     

Integration of a barcode reader with a commercial flow cytometer.

This report describes the application and installation of a barcode reader on a standard EPICS Elite flow cytometer. The barcode reader system eliminates keyboard entry of sample information on the cytometer. The system automates the transfer of sample information already present in our laboratory database to the cytometer at run time. The system uses a standard "off-the-shelf" bar code wand with a personal computer keyboard interface and requires no additional software at run time. No typing of sample information is required by the operator at any stage of normal sample operation at the cytometer. All operations are automatically coded into the cytometry software using the macro functions of the software. Tubes are inserted into the tube reader and sample information is transferred automatically into the cytometer. We have found that the system allows rapid and continuous operation of routine clinical and research samples. This automated data entry also reduces the possibility of data input errors.     

A New Ultrasensitive Scanning Calorimeter

A new ultrasensitive differential scanning calorimeter is described, having a number of novel features arising from integration between hardware and software. It is capable of high performance in either a scanning or isothermal mode of operation. Upscanning is carried out adiabatically while downscanning is nonadiabatic. By using software-controlled signals sent continuously to appropriate hardware devices, it is possible to improve adiabaticity and constancy of scan rate through use of empirical prerun information stored in memory rather than by using feedback systems which respond in real time and generate thermal noise. Also, instrument response time is software-selectable, maximizing performance for both slow- and fast-transient systems. While these and other sophisticated functionalities have been introduced into the instrument to improve performance and data analysis, they are virtually invisible and add no additional complexities into operation of the instrument. Noise and baseline repeatability are an order of magnitude better than published raw data from other instruments so that high-quality results can be obtained on protein solutions, for example, using as little as 50 μg of protein in the sample cell.     

An adapter for defined sample volumes makes it possible to count absolute particle numbers in flow cytometry.

A device is described which makes it possible to count absolute particle (cell) numbers per volume by flow cytometry. It can easily by adapted to several types of flow cytometers, especially to the Coulter EPICS V and EPICS 750 series. A volume adapter has been installed in place of the normal sample handling system without any further modifications of the instrument or the data acquisition program. The adapter consists of a special pipette with two opto-electronic detectors for the beginning and end of the measuring period. These switch on/off a shutter for the illuminating laser beam so that acquisition of the data is controlled indirectly. Sample volumes of 50 microliters were measured at flow rates up to 10(3) particles/s. Calibration beads as well as blood cells were enumerated according to FALS (forward angle light scatter), to SSC (90 degrees light scatter), and to fluorescence parameters. The results were compared to the evaluation made on a Coulter counter or in a Neubauer chamber of a light microscope. Using a concentration of 1 x 10(5)-5 x 10(5) particles/ml, the absolute numbers of particles were determined with a high reproducibility and an estimated error rate of 2-5%.     

Flow cytometric discrimination of various phycobilin-containing phytoplankton groups in a hypertrophic reservoir

BACKGROUND: Knowledge of phytoplankton structure is important information in water quality control. Lake restoration and sanitation measures in particular must be evaluated on the organismic level to valuate biological effects and assess the risk of potentially toxic Cyanobacteria blooms. We used and comparatively tested three independent methods for phytoplankton analysis in a hypertrophic reservoir under restoration. METHODS: Nine unialgal cultures and outdoor samples were examined by high-performance liquid chromatography pigment analysis, microscopical cell counting, and flow cytometric (FCM) light scatter and fluorescence analysis to measure the percentage contribution of the major algal groups to chlorophyll a and biovolume. The FCM instrument settings and identification criteria were developed using a single excitation wavelength at 514 nm to differentiate nine algal species representing the major groups of algae. Fluorescence was detected at 585, 620, 650, and 680 nm. RESULTS: The results show that FCM is the only method for determining changes in the phytoplankton composition on both a chlorophyll a and biovolume basis. CONCLUSIONS: Each of the three methods has specific advantages and disadvantages, and should be chosen depending on the experimental problem. FCM sorting allows the combination of all three and offers further new perspectives.     

Comparison of nucleic acid extraction platforms for detection of select biothreat agents for use in clinical resource limited settings

High-quality nucleic acids are critical for optimal PCR-based diagnostics and pathogen detection. Rapid sample processing time is important for the earliest administration of therapeutic and containment measures, especially in the case of biothreat agents. In this context, we compared the Fujifilm QuickGene-Mini80 to Qiagen's QIAamp Mini Purification kits for extraction of DNA and RNA for potential use in austere settings. Qiagen (QIAamp) column-based extraction is the currently recommended purification platform by United States Army Medical Research Institute for Infectious Diseases for both DNA and RNA extraction. However, this sample processing system requires dedicated laboratory equipment including a centrifuge. In this study, we investigated the QuickGene-Mini80, which does not require centrifugation, as a suitable platform for nucleic acid extraction for use in resource-limited locations. Quality of the sample extraction was evaluated using pathogen-specific, real-time PCR assays for nucleic acids extracted from viable and γ-irradiated Bacillus anthracis, Yersinia pestis, vaccinia virus, Venezuelan equine encephalitis virus, or B. anthracis spores in buffer or human whole blood. QuickGene-Mini80 and QIAamp performed similarly for DNA extraction regardless of organism viability. It was noteworthy that γ-irradiation did not have a significant impact on real-time PCR for organism detection. Comparison with QIAamp showed a less than adequate performance of the Fujifilm instrument for RNA extraction. However, QuickGene-Mini80 remains a viable alternative to QIAamp for DNA extraction for use in remote settings due to extraction quality, time efficiency, reduced instrument requirements, and ease of use.     

Assessment of the viability and fertilizing potential of cryopreserved bovine spermatozoa using dual fluorescent staining and two-flow cytometric systems

A dual fluorescent staining system utilizing 5 (and-6)-carboxy-4′,5′-dimethyl fluorescein diacetate (CDMFDA) and Hydroethidine (HED) was developed to provide quantifiable information reflective of spermatozoal viability and fertilizing potential. Cryopreserved spermatozoa from ten bulls on which there was fertilizing capacity information were incubated for 1.5, and 3 hr at 39°C prior to fluorogenic staining. Spermatozoa were analyzed using both a FACS Analyzer and an EPICS V flow cytometer to determine if a particular fluorescence pattern was due to an instrumental artifact or cellular processes. Five fluorescent cellular populations were identified by the FACS Analyzer and three populations by the EPICS V. Spermatozoa were quantified after each incubation time for red (HED) and green (CDMFDA) fluorescence. Viable spermatozoa retained the greatest amount of both green and red fluorescence. Dead or moribund spermatozoa had a decrease in over-all fluorescence. The number of viable cells at 0 hr plus the number of dead or morbid cells at any time period were identified by the FACS Analyzer as important in estimating the potential fertility of a bull. The EPICS V identified the number of dead or moribund cells as being related to nonreturn rates. Incubation of samples decreased cellular viability, which resulted in reduced levels of both green and red fluorescence. Similarities between data obtained with both flow cytometers illustrated that cellular processes, not instrumental artifacts, were responsible for the decrease in over-all fluorescence when viability declined, the relationship between the number of cells with specific fluorescence levels and nonreturn rates, and the incubative-induced changes in fluorescence patterns.     

Flow cytometric analysis of bacteria- and virus-like particles in lake sediments

Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (x1/20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or -22 degrees C. For FCM analysis, the samples were diluted in Tris-EDTA buffer (pH 8) and heated for 10 min at 75 degrees C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.     

Vectorcardiographic significance of three-dimensional devices ended and nevertheless without boundaries

The circular boundary of a disk may be suppressed by connecting it to that of an other disk presenting the same characteristics (isomorph). In such a condition, energizing the first element indirectly feeds the second one by the same amount than it would be delivered to infinite surroundings. In mathematical terms, this means that summing up the potentials arising at the same points of the two connected disks leads to the potential identically located in the disk activated as before but disconnected from its isomorph: the statement being true except for a constant. In three dimensions, the problem is somewhat more difficult because a single isomorph cannot drain all the current that would be transmitted by a sphere to the infinite medium in which it is immersed. So, connecting two spheres results in a partial confinement which, however, does not rule out the aforementioned statement. On the other hand, the potential differences around the isomorph centre are well-suited to a vectorcardiographic use like Rijlant's one; this method is consequently justified as to its principle. Finally, we have studied the radial alinearity which is introduced by Rijlant to improve his instrument. We have shown the changes related to Rijlant's way of building his network and we conclude that the use of many equivalent layers bears on grounds similar to that which are developed for the linear isomorphs.     

System for acquisition and real-time processing of multidimensional slit-scan flow cytometric data

The high-speed sampling requirements of multidimensional slit-scan signals (cell contours) have typically required custom hardware. This specialized hardware has often lacked the flexibility to adapt to varying instrument setups and experimental requirements. A hardware and software system capable of sampling multiple slit-scan cell contours at rates of up to 40 MHz with 10-bit resolution is described. It utilizes commercially available CAMAC transient recorders, a Digital Equipment Corp. PDP-11/83 computer, and custom hardware for signal conditioning and trigger generation. The modular design of the software system allows various hardware options with minimal additional coding. Real-time digital processing checks each cell contour for multiple peaks; extracts morphological features such as width, height, and area; accumulates gated histograms of these data; and optionally saves the derived data, selected contours, or both into list mode files on disk.     

Reducing the man - machine barrier: the Sequence Analysis Workbench

The conventional software paradigm in which the machine actsas an unintelligent but patient servant often increases theperceived distance between scientist and data. Direct manipulationoffers an alternative paradigm in which the scientist uses thecomputer as a scientific instrument for examining and modifyingthe data directly, rather than by issuing instructions to anagent. The Sequence Analysis Workbench provides several experimentaltools for direct manipulation of sequence data; object-orientedprogramming makes it possible to construct sophisticated toolsquickly, and facilitates critical examination and review ofscientific software. Received on December 30, 1986; accepted on June 27, 1987     

An inexpensive microcomputer-based stopped-flow data acquisition system.

A low-cost (less than $2,500) microcomputer-controlled data acquisition system for use with a stopped-flow instrument is described. Data acquisition, reduction, signal averaging, kinetic modeling, and plotting are performed under software control. Applications to biological and inorganic systems are presented.     

Use of the Brown-Roberts-Wells stereotactic frame for functional neurosurgery

The Brown-Roberts-Wells (BRW) stereotactic unit has proven itself to be a highly accurate instrument for biopsying or locating pathologic intracranial lesions based on CT scan information. We utilized the BRW frame to select 18 target sites in 12 patients undergoing functional stereotactic procedures. Two patients had bilateral cingulumotomies, 5 had thalamotomies for movement disorders, and 5 underwent electrode implantations for the treatment of chronic pain. Stereotactic frame settings were determined using a positive contrast ventriculogram, orthogonal radiographs, and a computer program provided with the BRW system. In addition, attempts were made to select targets based on CT scan landmarks alone, and these were compared to those derived using ventriculography. We found the BRW frame to be a satisfactory device for performing functional neurosurgical procedures based on ventriculographic landmarks. Coordinates derived from CT scans were similar to those obtained with ventriculography, but were not accurate enough to permit the use of CT scanning as the sole means of target identification. Although future improvements in imaging techniques and computer software are likely to occur, our experience supports ventriculography as the current method of choice for the precise localization of functional targets with the BRW stereotactic system.     

CHASM and SNVBox: toolkit for detecting biologically important single nucleotide mutations in cancer

SUMMARY: Thousands of cancer exomes are currently being sequenced, yielding millions of non-synonymous single nucleotide variants (SNVs) of possible relevance to disease etiology. Here, we provide a software toolkit to prioritize SNVs based on their predicted contribution to tumorigenesis. It includes a database of precomputed, predictive features covering all positions in the annotated human exome and can be used either stand-alone or as part of a larger variant discovery pipeline. Availability and Implementation: MySQL database, source code and binaries freely available for academic/government use at http://wiki.chasmsoftware.org, Source in Python and C++. Requires 32 or 64-bit Linux system (tested on Fedora Core 8,10,11 and Ubuntu 10), 2.5*≤ Python <3.0*, MySQL server >5.0, 60 GB available hard disk space (50 MB for software and data files, 40 GB for MySQL database dump when uncompressed), 2 GB of RAM.     

Reference master: A microcomputer-based storage and retrieval system for bibliographic references

A complete system for housekeeping and retrieval of bibliographic references managing individual reprint collections is described. By the use of special hardware and individual data base software even large reprint collections in the range up to 65 000 papers are handled economically. A fast 8-bit microprocessor (HD 64180) in combination with a Winchester hard disk drive serves as the basis for rapid access to the desired information. An efficient string search algorithm written in assembly language guarantees a fast operation with a search speed of more than 6,000 entries/ minute. The system cannot only prepare reference lists and reference files, but also incorporates an editor and maintains the control whether reprints are already on file or requested. The implementation of back-up schemes assure against data losses. Using a state of the art design single board computer and the most recent mass storage device technology, the system is as well small and cost effective, and thus suitable for personal use. In addition, some general questions and pitfalls concerning the management of scientific literature collections are touched upon.     

Rapid and sensitive detection of an intracellular pathogen in human peripheral leukocytes with hybridizing magnetic relaxation nanosensors

Bacterial infections are still a major global healthcare problem. The quick and sensitive detection of pathogens responsible for these infections would facilitate correct diagnosis of the disease and expedite treatment. Of major importance are intracellular slow-growing pathogens that reside within peripheral leukocytes, evading recognition by the immune system and detection by traditional culture methods. Herein, we report the use of hybridizing magnetic nanosensors (hMRS) for the detection of an intracellular pathogen, Mycobacterium avium spp. paratuberculosis (MAP). The hMRS are designed to bind to a unique genomic sequence found in the MAP genome, causing significant changes in the sample's magnetic resonance signal. Clinically relevant samples, including tissue and blood, were screened with hMRS and results were compared with traditional PCR analysis. Within less than an hour, the hMRS identified MAP-positive samples in a library of laboratory cultures, clinical isolates, blood and homogenized tissues. Comparison of the hMRS with culture methods in terms of prediction of disease state revealed that the hMRS outperformed established culture methods, while being significantly faster (1 hour vs 12 weeks). Additionally, using a single instrument and one nanoparticle preparation we were able to detect the intracellular bacterial target in clinical samples at the genomic and epitope levels. Overall, since the nanoparticles are robust in diverse environmental settings and substantially more affordable than PCR enzymes, the potential clinical and field-based use of hMRS in the multiplexed identification of microbial pathogens and other disease-related biomarkers via a single, deployable instrument in clinical and complex environmental samples is foreseen.