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Biomedical subjects

D Pinkel

Publications and source records attributed to D Pinkel.

At least 145 records · Page 8Linked to original sources
PubMed1985

The LLNL high-speed sorter: design features, operational characteristics, and biological utility.

This report describes a dual-beam, high-speed sorter (HiSS) with a droplet production rate of 220,000 s-1 now in routine use at the Lawrence Livermore National Laboratory. The system can process and sort objects at rates in excess of 20,000 s-1. We report here on the development of HiSS, describe its operational characteristics, and evaluate its utility for analysis and purification of human chromosomes, human erythrocytes, and living Chinese hamster ovary cells.

Cell Separation↗
PubMed1985

Flow cytometric determination of the proportions of X- and Y-chromosome-bearing sperm in samples of purportedly separated bull sperm.

A rapid assay for determining the proportions of X- and Y-chromosome-bearing sperm in semen samples would benefit research aimed at sex ratio control through sperm separation. It also would be of value for quality control should a separation technique be developed. Flow cytometric methods capable of measuring sperm DNA content precisely enough to resolve and quantify the X and Y populations in many mammalian species have been developed. They are effective for fresh and cryopreserved sperm of most domestic animals. Results are reported of flow cytometric analyses of bull sperm samples from seven commercial and academic sources after processing with procedures purported to separate the X and Y populations. In no case was enrichment of either sperm population observed. Breeding trials carried out by the sources of two of the sets of samples showed these procedures were ineffective in altering the sex ratio.

Animals↗
PubMed1985

Measurement of sister chromatid exchanges at very low bromodeoxyuridine substitution levels using a monoclonal antibody in Chinese hamster ovary cells.

A monoclonal antibody to bromodeoxyuridine (BrdUrd) incorporated into DNA allowed visualization of sister chromatid exchanges (SCE) when as little as 0.6% of the thymine in a single DNA strand has been substituted. Measurement of the SCE frequency as a function of BrdUrd substitution in a normal Chinese hamster ovary cell line showed a plateau of six SCEs per cell for substitution levels up to at least 20%. A clear elevation in frequencies was noted at 60% substitution. However, in the mutant line EM9, previously shown to have a highly elevated frequency of SCE, the level of exchanges declined continuously as the percentage of BrdUrd substitution decreased. At 0.6% substitution, the frequency of SCE was still 4-fold higher than that of the parental cells. The antibody procedure described here should be useful in evaluating the extent to which SCEs induced by mutagenic agents result from interactions between the DNA damage caused by the agent and the BrdUrd routinely used for measuring SCE.

Animals↗
PubMed1983

Bacterial characterization by flow cytometry.

Bacteria were analyzed in a dual-beam flow cytometer after double staining with the fluorescent dyes chromomycin A3 and Hoechst 33258, which bind preferentially to DNA that is rich in guanine-cytosine and adenine-thymine, respectively. The measurements were indicative of the cellular DNA content and base composition, cell concentration, and proliferative state of the population. The ratio of the chromomycin A3 signal to the Hoechst 33258 signal increased with the guanine-cytosine content of the cellular DNA for the six cultured species measured, following expectation. Bacteria in urine from patients with urinary tract infections were characterized without interference from host cell DNA, debris, or other particulates.

Bacteria↗
PubMed1983

Quantification of the X- and Y-chromosome-bearing spermatozoa of domestic animals by flow cytometry.

The relative content of DNA in spermatozoa presumed to be the X- and Y-chromosome-bearing gametes from bulls, boars, rams and rabbits and the amount of DNA in spermatozoa of cockerels was determined by flow cytometry. Differences in the relative content of DNA and proportions of the presumed X- and Y-sperm populations in cryopreserved semen from Holstein, Jersey, Angus, Hereford and Brahman bulls were also determined. Spermatozoa were washed by centrifugation using a series of dimethyl sulfoxide solutions made in isotonic sodium citrate, fixed in ethanol, treated with papain and dithioerythritol to loosen the chromatin structure and remove cellular organelles, and stained quantitatively for DNA with the fluorochrome 4'-6-diamidino-2-phenylindole (DAPI). Approximately 5000 stained sperm nuclei, which were nonviable due to the removal of other cellular organelles during the washing procedure, were measured for DNA in an epi-illumination flow cytometer. A single distinct peak for cockerel spermatozoa and two symmetrical, overlapping peaks for species with X- and Y-spermatozoa were seen. This and other evidence strongly supports the interpretation that the peaks represent the X- and Y-sperm populations. The content of DNA in sperm nuclei from cockerels, bulls, boars, rams and rabbits, as determined by fluorescence flow cytometry, corresponded to biochemical estimates of DNA per sperm cell. Analyses of the bimodal histograms by computer-fitting two Gaussian distributions to the data showed the means of the peaks differed by 3.9, 3.7, 4.1 and 3.9% for bulls, boars, rams and rabbits, respectively. In four replicate analyses of semen from 25 bulls representing 5 breeds, the average population of sperm nuclei in the Y-peaks ranged from 49.5 to 50.5% for all breeds. The X-Y peak differences did not vary within each breed, but were significantly different when the breeds were compared. Spermatozoa from Jersey bulls had larger X-Y peak differences (P less than 0.001) than spermatozoa from Holstein, Hereford, and Angus bulls; spermatozoa from Brahman bulls had smaller X-Y differences (P less than 0.004). It is suggested from the evidence obtained in these studies that flow cytometry can be used to assess the proportion of X- and Y-spermatozoa in semen of domestic animals and is thereby applicable to verification of the effectiveness of enrichment techniques for X- or Y-spermatozoa.

Animals↗
PubMed1983

Radiation-induced DNA content variability in mouse sperm.

Mouse sperm collected from the cauda epididymidis 35 days after acute testicular X-ray exposure and fluorescently stained for DNA show dose-dependent increases in the coefficient of variation (CV) of flow cytometrically obtained fluorescence distributions. By comparing dose-response curves obtained with three protocols which overcome the optical and cytochemical difficulties of sperm measurement in different ways we conclude the response is due to X-ray-induced DNA content variability. In the range between 0 and 600 rad the dose dependence of the square of CV of the DNA content variability, delta CV2D, is described by delta CV2D = Bx + Cx2, with 0 less than or equal to B less than or equal to 0.23 X 10(-2) and C = (0.44 +/- 0.06) X 10(-4). The dose x is measured in rad and delta CVD is expressed in percent. Computer modeling of the shapes of the fluorescence distributions show that at 600 rad 30 to 40% of the sperm have abnormal DNA content. Some have errors as large as two whole chromosomes, but it is not clear whether they are due to whole chromosome nondisjunction or a finer fragmentation of the genome. Exposures to benzo(a)pyrene and mitomycin C cause no detectable DNA content variability. We conclude mouse sperm DNA content measurements are not sensitive to small amounts of aneuploidy and as such will only be useful in detecting agents that produce substantial DNA content variability. Another animal with a smaller number of chromosomes might be more favorable. These sperm measurement techniques may find additional application in other areas of reproductive biology, such as the determination of the relative numbers of X and Y chromosome-bearing sperm in semen that may be artificially enriched in one population.

Animals↗
PubMed1982

Sex preselection in mammals? Separation of sperm bearing Y and "O" chromosomes in the vole Microtus oregoni.

The two sex determining sperm populations of the vole Microtus oregoni were separated according to DNA content by use of flow sorting instrumentation. Although the sperm were not viable, they should be useful for addressing the question of haploid expression of genes linked to sex chromosomes and for efficiently searching for biochemical markers that differentiate the two populations.

Animals↗
PubMed1982

High resolution DNA content measurements of mammalian sperm.

The high condensation and flat shape of the mammalian sperm nucleus present unique difficulties to flow cytometric measurement of DNA content. Chromatin compactness makes quantitative fluorescent staining for DNA difficult and causes a high index of refraction. The refractive index makes optical measurements sensitive to sperm head orientation. We demonstrate that the optical problems can be overcome using the commercial ICP22 epiillumination flow cytometer (Ortho Instruments, Westwood, MA) or a specially built cell orientating flow cytometer (OFCM). The design and operation of the OFCM are described. Measurements of the angular dependence of fluorescence from acriflavine stained rabbit sperm show that it is capable of orienting flat sperm with a tolerance of +/- 7 degrees. Differences in the angular dependence for the similarly shaped bull and rabbit sperm allow discrimination of these cells. We show that DNA staining with 4-6 diamidino-2-phenylindole (DAPI) or an ethidium bromide mithramycin combination allows resolution of the X and Y populations in mouse sperm. They have also been successful with sperm from the bull, ram, rabbit, and boar. Reliable results with human sperm are not obtained. The accuracy of the staining and measurement techniques are verified by the correct determination of the relative DNA content of these two populations in sperm from normal mice and those with the Cattanach [7 to X] translocation. Among the potential uses of these techniques are measurement of DNA content errors induced in sperm due to mutagen exposure, and assessment of the fractions of X and Y sperm in semen that may have one population artifically enriched.

Acriflavine↗
PubMed1982

Simple methods to determine and compare the sensitivity of flow cytometers.

Knowledge of the sensitivity of a flow cytometer is important both in choosing an instrument for a particular application and in determining whether the resolution obtained on real samples is limited by instrumental factors or sample variability. The coefficient of variation (CV) obtained for measurement is given by CV2 = CVS2 + CVI2 + 1/n + K(delta VB2)/n2, where CVS is the variability due to the sample, CVI that due to variations in illumination of the sample particles, n the number of photoelectrons emitted from the photomultiplier photo cathode per sample particle (which is the signal intensity), delta VB2 the mean square background noise voltage (usually due to background illumination), and K a constant. Measurement of the CV of light flashes from a light emitting diode as a function of their intensity allows development of standard curves which can be used to directly determine the degree to which the resolution obtained on actual samples is limited by signal intensity and background light. The use of standard particles allows relative calibration of the curves for different instruments and which also ranks their sensitivities.

Flow Cytometry↗
PubMed1982

Doxorubicin-vincristine therapy for Wilms' tumor: a pilot study.

Doxorubicin plus vincristine chemotherapy was given to 31 children following nephrectomy for Wilms' tumor. Radiation therapy was used as indicated. Disease-free survival by stage is: eight of nine patients (stage I), eight of nine (stage II), nine of ten (stage III), and two of three (stage IV). Median follow-up of survivors is 28 months (range, 2-67); for all but four patients, follow-up is greater than 12 months. Two of the three stage I-III failures occurred in children with unfavorable histologies; the third failure was due to fatal anthracycline cardiomyopathy. Lowering the maximal cumulative doxorubicin dose from 450 to 240 mg/m2 did not increase failures. Doxorubicin-vincristine appears to be effective chemotherapy for Wilms' tumor.

Adolescent↗
PubMed1982

Doxorubicin cardiomyopathy in children with left-sided Wilms tumor.

Two children with Wilms tumor of the left kidney experienced severe anthracycline cardiomyopathy after irradiation to the tumor bed and conventional dosage of doxorubicin. The cardiomyopathy is attributed 1) to the fact that radiation fields for left Wilms tumor include the lower portion of the heart and 2) to the interaction of doxorubicin and irradiation on cardiac muscle. It is recommended that doxorubicin dosage be sharply restricted in children with Wilms tumor of the left kidney who receive postoperative irradiation.

Cardiomyopathies↗
PubMed1981

A flow system for partial automation of plating efficiency tests.

A flow system that sterilely deposits a known number of cells into a collection vessel has been constructed and evaluated. In this device, the cells from a single-cell suspension flow through a nozzle that constrains them to the center of the emerging jet. The cells in the jet are illuminated by a 0.5-mW helium-neon laser; they are detected, sized and counted on the basis of the light scattered as they pass through the laser beam. The device has been used for plating efficiency tests in a tissue culture laboratory. We found that it saved considerable labor by eliminating the dilution step from the standard plating efficiency procedure. The biologic effect of machine plating was determined by comparing plating efficiencies of KHT, CHO-T5, and CHO-AA8 cells established by hand plating with those established by machine plating. Results indicate that machine plating reduces the plating efficiency of the CHO-T5 cells while the plating efficiencies of the CHO-AA8 and KHT cells are unaffected.

Animals↗
PubMed1981

On the possibility of automated scoring of pollen mutants.

Both flow cytometry and automated image analysis techniques may be useful in relieving the drudgery, increasing the speed, and adding quantitation to the scoring of pollen mutants, but practical problems related to the detection of very rare events have to be overcome. The features of flow and image cytometry instrumentation that may be useful for pollen measurements are discussed and a qualitative framework is presented from which to view the rare event problem.

Cell Count↗