Larry Seamer

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Papers by Larry Seamer

Biosafety guidelines for sorting of unfixed cells

Cytometry, 1997

The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelin... more The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflecteddroplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples.

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Thermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): Critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram

Cytometry, 1989

Significant inter- and intraexperimental variations of the relative antibromodeoxyuridine fluores... more Significant inter- and intraexperimental variations of the relative antibromodeoxyuridine fluorescence were found during measurement of DNA synthesis rates using flow cytometric analysis of 5-bromodeoxyuridine (BrdUrd)-labeled cells with an anti-BrdUrd antibody. Fluctuations in other endpoints associated with levels of denaturation (integrity of DNA and cell size) were also observed to vary widely among samples that were otherwise thought to have been treated identically. Therefore, the denaturation step has been carefully re-examined, and several critical factors were identified that influence the denaturation and subsequent binding of the anti-BrdUrd to the labeled DNA. These factors include cell density, volume of water, and pH of the sample during heating. Appropriate adjustments are now included in the protocol, resulting in more consistent anti-BrdUrd measurements in the face of routine (and sometimes necessary) experimental variations.

Sheath fluid control to permit stable flow in rapid mix flow cytometry

Cytometry, 1999

Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell res... more Flow cytometry is a potentially powerful tool to analyze the kinetics of ligand binding, cell response and molecular assembly. The difficulty in adding reactant to cells, achieving adequate mixing, delivering those cells to the laser focal point and establishing stable flow, has historically limited flow cytometry to systems with reactions times longer than 5 s. With the advent of automated syringes and flow injection methods, sample injection times shorter than 1 s have become routine. However, an inherent problem in acquiring time courses starting under 1 s is that rapid sample introduction through the flow tip to the detection point perturbs laminar flow. The purpose of this work was to determine if stable flow could be reestablished more quickly if the sheath flow was reduced during sample introduction, returning to normal sheath and sample rates afterward. We used programmable syringes and valves to control sample mixing as well as sheath and sample delivery through the flow tip to the detection point for stream-in-air detection. Stable flow was monitored by mean particle fluorescence during sample introduction. With no sheath reduction, stable flow recovered after more than 1 s. By reducing sheath flow during the short period (300 msec) of sample mixing and delivery, stable laminar flow recovered within 200 msec. This use of automated syringes to control both sheath and sample flow provides a potential for robust sample handling applicable to kinetic as well as high throughput flow cytometric analysis.

Characterization by flow cytometry of fluorescein-methotrexate transport in chinese hamster ovary cells

Cytometry, 1989

We have studied by flow cytometry the transport of fluorescein-methotrexate in Chinese hamster ov... more We have studied by flow cytometry the transport of fluorescein-methotrexate in Chinese hamster ovary cells. Fluorescein-methotrexate appears to enter cells via a mechanism different from the carrier-mediated system for methotrexate. This conclusion is supported by the following observations: (1) Fluorescein-methotrexate is transported equally well into normal and mutant cells defective in the in ward methotrexate uptake. (2) Folic acid and its reduced states, which competitively inhibit methotrexate uptake, do not alter fluorescein-methotrexate transport. (3) Fluorescein-methotrexate accumulation exhibits a low temperature coefficient (Q10 = 1.6) compared with the influx of methotrexate (Q10 = 6–8). (4) Initial rates of fluorescein-methotrexate uptake are concentration dependent but are not saturable. (5) Fluorescein-methotrexate uptake is very slow and reaches steady state after 8 h, whereas at an equimolar concentration methotrexate reaches saturation after 20 min. (6) Initial influx rates of fluorescein-methotrexate are not affected by the presence of methotrexate. (7) Sulfhydryl-reactive mercurials, which block methotrexate transport, do not reduce fluorescein-methotrexate influx, but rather stimulate it. Thus, based on the nonsaturability of fluorescein-methotrexate inward transport, its low temperature coefficient, and lack of inhibition with structural analogs, we conclude that fluorescein-methotrexate is accumulated in hamster cells by a passive diffusion process.

Sample handling for kinetics and molecular assembly in flow cytometry

Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrou... more Flow cytometry discriminates particle associated fluorescence from the fluorescence of the surrounding medium. It permits assemblies of macromolecular complexes on beads or cells to be detected in real-time with precision and specificity. We have investigated two types of robust sample handling systems which provide sub-second resolution and high throughput: (1) mixers which use stepper-motor driven syringes to initiate chemical reactions in msec time frames; and (2) flow injection controllers with valves and automated syringes used in chemical process control. In the former system, we used fast valves to overcome the disparity between mixing 100 (mu) ls of sample in 100 msecs and delivering sample to a flow cytometer at 1 (mu) l/sec. Particles were detected within 100 msec after mixing, but unstable flow was created which lasted for 1 sec after injection of the sample into the flow cytometer. We used optical criteria to discriminate particles which were out of alignment due to the unstable flow. Complex sample handling protocols involving multiple mixing steps and sample dilution have also been achieved. With the latter system we were able to automate sample handling and delivery with intervals of a few seconds. We used a fluidic approach to defeat the instability caused by sample introduction. By controlling both sheath and sample with individual syringes, the period of instability was reduced to approximately 200 msecs. Automated sample handling and sub-second resolution should permit broad analytical and diagnostic applications of flow cytometry.

Special Reports Biosafety Guidelines for Sorting of Unfixed Cells

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Method to improve the sensitivity of flow cytometric membrane potential measurements in mouse spinal cord cells

Cytometry, 1992

We have developers a technique to improve the sensitivity of relative membrane potential measurem... more We have developers a technique to improve the sensitivity of relative membrane potential measurements in mouse spinal cord cells using the fluorescent, anionic, voltage sensitive dye, DiBa-C4(3) (Oxonol) and flow cytometry. In order to attribute cellular fluorescence primarily to membrane potential, signal variability due to cell size and shape was reduced by dividing the log fluorescence signal from each cell by either its log forward angle light scatter or log side scatter signals. The use of these ratios in place of log oxonol fluorescence reduced the coefficient of variation of the distributions while leaving the changes in mean fluorescence largely unaffected. Kolmogorov-Smirnov analysis of pre- vs. postkainate stimulation (an excitatory amino acid) showed improved sensitivity of the assay with the use of this ratio technique.

Proposed new data file standard for flow cytometry, version FCS 3.0

Cytometry, 1997

In 1984, the first flow cytometry data file format was proposed as Flow Cytometry Standard 1.0 (F... more In 1984, the first flow cytometry data file format was proposed as Flow Cytometry Standard 1.0 (FCS1.0). FCS 1.0 provided a uniform file format allowing data acquired on one computer to be correctly read and interpreted on other computers running a variety of operating systems. That standard was modified in 1990 and adopted by the Society of Analytical Cytology as FCS 2.0. Here, we report on an update of the FCS 2.0 standard which we propose to designate FCS 3.0. We have retained the basic four segment structure of earlier versions (HEADER, TEXT, DATA and ANALYSIS) in order to maintain analysis software compatibility, where possible. The changes described in this proposal include a method to collect files larger than 100 megabytes (not possible in earlier versions of the standard), the inclusion of international characters in the TEXT portions of the file, a method of verifying data integrity using a 16-bit cyclic redundancy check, and increased keyword support for cluster analysis and time acquisition. This report summarizes the work of the ISAC Data File Standards Committee. The complete and detailed FCS 3.0 standard is available through the ISAC office [Sherwood Group, 60 Revere Drive, Ste 500, Northbrook, IL 60062, phone: (847) 480-9080 ext. 231, fax: (847) 480-9282, E-mail: isac@sherwood-group.com] or through the internet at the ISAC WWW site, http:// nucleus.immunol.washington.edu/ISAC.html. Cytometry 28:118-122, 1997. r 1997 Wiley-Liss, Inc.

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DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens

Cancer, 1988

Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along wit... more Breast cancer proliferative capacity as determined by the DNA thymidine labeling index, along with estrogen and progesterone receptor status, is highly predictive for risk of relapse and overall survival. Recently, DNA ploidy and proliferative capacity (S-phase fraction [SPF]) as determined by flow cytometry have also shown significant prognostic value. The authors have developed a technique which allows a 50 to 100 mg aliquot of the same frozen breast tumor specimen routinely employed in steroid receptor assays, to be assayed for both DNA ploidy and SPF by flow cytometry. Of the 1331 tumors examined, DNA histograms were evaluable for ploidy in 89% (1184) of specimens examined; 57% of these were aneuploid. Adapting a trapezoidal model to estimate SPF in both diploid and aneuploid tumors, the authors found 81% (1084) to be evaluable for SPF, with a median SPF of 5.8% for the entire population. The median SPF was significantly lower in diploid tumors (2.6%) than in aneuploid tumors (10.3%, P less than 0.0001). Both aneuploidy and high SPF were strongly associated with absence of steroid receptors. Aneuploid tumors showed more striking differences in the frequency of high S-phase values with respect to receptor status and age or menopausal status, whereas diploid but not aneuploid tumors showed lower SPF in node-negative versus node-positive patients. Because it is particularly important to identify the high-risk minority of node-negative patients, the authors examined the node-negative group separately. High SPF subgroups appeared in each category of receptor status and age or menopausal status within the node-negative group, suggesting that SPF will be an independent prognostic factor. With the DNA flow cytometric methods used here, it is now practical to determine ploidy and SPF for nearly every breast cancer patient. These factors, which show associations with established prognostic factors, such as receptor status can now be fully evaluated for their prognostic significance in broad patient populations.

Fluorescence drift detection as a novel QC procedure for DNA cell-cycle analysis

Cytometry, 1995

The goal of this work was to develop an objective, quantitative, and reproducible method of detec... more The goal of this work was to develop an objective, quantitative, and reproducible method of detecting fluorescence drift which may have occurred during DNA cell-cycle data acquisition. Quality control software, “TruPloid©” is described that analyzes list-mode files to detect and quantify fluorescence drift using three separate statistical tests. We show that fluorescence drift may lead to a variety of measurement artifacts including high coefficients of variation, obscuring of small populations and creation of distinct artificial peaks. Forty percent of 50 archived list-mode files displayed fluorescence drift, which demonstrates the need for detection methods to deal with this source of DNA cell-cycle histogram artifacts. © 1995 Wiley-Liss, Inc.