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Biomedical subjects

R C Leif

Publications and source records attributed to R C Leif.

At least 37 records · Page 2Linked to original sources
PubMed1977

A procedure for dissociating Ayre scrape samples.

The dissociation of cervical cell suspensions after various chemical and enzymatic treatments was monitored by using the Centrifugal Cytology rotor to produce glutaraldehyde-fixed dispersions on conventional microscope slides and subsequent Pap staining. A special program was written in RPG II to record and analyze the results of the dissociation experiments in terms of white blood cells and the true cervical cells ("other cells"), and the degree of dissociation and recovery of both classes of cells. Since accurate differential counts on the untreated Ayre scrapes were difficult, the samples were syringed gently to break up the large or adventitious clumps. Cumulated results from control preparations indicate that the white blood cells and "other cells" are composed respectively of 92 and 63% single cells. The cells were further dissociated by: dissolving the cervical mucin sequentially with dithiothreitol and iodoacetic acid; depolymerizing the nucleohistone gel with ribonuclease; solubilizing the desmosomes with EDTA; removing the remaining cellular agglutinins with Varidase; and finally mechanical dispersion by hypertonic shock. The optimum procedure for dissociation involves the use of ribonuclease, dithiothreitol, iodoacetic acid EDTA, Varidase and sucrose shock. The white blood cells are now monodisperse and 81% of the "other cells" are found as single cells. If nuclear separation by two diameters is considered sufficient 98% of the "other cells" are single. The slide preparations are now sufficiently good that a scanning system is feasible.

Autoanalysis↗
PubMed1977

Optimization of the binding of dissociated exfoliated cervico-vaginal cells to glass microscope slides.

In order to monitor the development of a cell dissociation technique, it was essential to utilize the Centrifugal Cytology rotor to produce glutaraldehyde-fixed even cellular dispersions. The Cytology rotor has been improved to insure rapid alignment with the centrifugal field during both acceleration and deceleration, and the fixative is now delivered to the surface of the slide. The dissociation of the cells results in a loss of their adhesion to glass slides. Three bonding agents were tested: (a) Poly-L-Lysine; (b) Mayer's albumin fixative; (c) positively charging the slides with a silicone coating. The results with 65% albumin-coated slides were clearly superior to the other two. The addition of a postfixation step of 95% ethanol/4% polyethylene glycol did not significantly affect the recovery of the cells, but did eliminate some unevenness in the Centrifugal Cytology preparations, flattened the cells and expedited the procedure.

Albumins↗
PubMed1977

Development of instrumentation and fluorochromes for automated multiparameter analysis of cells.

We have developed and interfaced to a computer an automated instrument (the AMAC III) which is designed to observe simultaneously several physical parameters of cells. Typical parameters include electronic cell volume (Coulter effect), RF amplitude (opacity), multiwavelength fluorescence of cytological stains, and cell light-scattering. The use of a new ultraviolet laser combined with a holographic grating spectrograph promises to increase the number of fluorescing species that can be detected simultaneously. This number can be further increased by use of special rare-earth-based fluorochromes, that emit well-defined, spectrally distinct peaks.

Autoanalysis↗
PubMed1976

Deoxyribonucleic acid replication in single cells and chromosomes by immunologic techniques.

Antibodies to 5-bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH-Sepharose 4B to which had been coupled BrU. Anti-BrU cross-reacts with iodeodeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti-BrdU treatment was followed by goat anti-rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H-BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cell which had been pulse labled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluoresence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.

Antibodies↗
PubMed1975

Centrifugal cytology. III. The utilization of centrifugal cytology for the preparation of fixed stained dispersions of cells separated by bovine serum albumin bouyant density centrifugation.

This paper describes the modification of Centrifugal Cytology for the preparation of permanent, fixed, stained dispensions for both light and scanning electron microscopy of cells which have been isolated on bovine serum albumin (BSA) boyant density gradients. The principal problem with BSA gradient fractions is that the albumin which is present even after dilution is precipitated by the glutaraldehyde fixative. This problem has been solved by the layering of an intermediate D2O solution under the BSA and subsequent removal of the BSA solution and the underlaying with D2O containing glutaraldehyde. A special layering machine facilitates and expedites these operations. This technique has also been applied to BSA-seperated guinea pig and chicken bone marrow cells, as well as Ehrlich ascites tumor cells, hen and human blood cells. The number of celll present in each area of the slide is maintained at a constant value by utlizing a table of dilution factors. This table was generated by a computer program which calculates the concentration of cells present in the rractions and divides it by the number of celll desired.

Animals↗
PubMed1975

Buoyant density separation of cells. I. The buoyant distribution of guinea pig bone marrow cells.

Guinea pig bone marrow cells were separated by buoyant density utilizing linear gradients of bovine serum albumin (BSA). It has finally become possible to characterize the cells present in the density fractions in terms of classical morphology. The development of the Cell Type computer program which calculates the percentages of the individual types of cells present in the fractions and their buoyant density distributions and plots the data has greatly facilitated and improved the accuracy of these studies. Approximately 40 cell types were observed in guinea pig bone marrow. Cells with definitive morphologies such as erythrocytes, the neutrophilic series, the binucleate blast megakaryocyte precursor and cells in mitosis band as virtually single peaks. Cells which are parts of continua or can easily be wrongly classified are found in multiple peaks. The small lymphocytes which are known to be polydisperse are found as five peaks. Because of the very strong benzidine staining by the glutaraldehyde-fixed hemoglobin, some of the erythroblasts were wrongly staged, resulting in a multimodal distribution. The presence of macrocytes further complicated these distributions. The rule that the younger cells are always less dense than the mature cells was adhered to in those cases where the cells could be definitively characterized, such as the neutrophilic series and the blasts. These results indicate that morphology is a good first approximation of reality.

Animals↗
PubMed1975

The plaque cytogram assay. II. Correlation between morphology and density of linear bovine serum albumin buoyant density gradient-separated immunocompetent cells.

The plaque cytogram assay has been combined with pH 6.8, linear bovine serum albumin (BSA) gradients to demonstrate the correlation between the morphology and buoyant density of immunocompetent cells. Ten types of plaque-forming cells (PFC) and four types of plaques have been identified and enriched in the density gradients. Reproducible density profiles of mouse erythrocytes, lymphoid cells, and PFC have been obtained with BSA gradients. The density peak for mouse erythrocytes was about 1.080 g/ml, while the lymphoid cell density profile had several peaks with a maximum concentration at 1.064 g/ml. Three distinct peaks or shoulders were found in the density profile of hemolytic PFC. Most of the density distributions of the individual morphologic classes of PFC were considerably narrower than that of the entire PFC distribution and were discrete from each other. The combined result that the PFC could be subdivided by morphology and density, and that the two phenomena were correlated establishes that the PFC are truly a heterogeneous population. Whether this heterogeneity is due to different stages in development or due to the existence of differing classes of cells will have to await future experimentation.

Animals↗
PubMed1973

A wet chemical method for rendering scanning electron microscopy samples conductive and observations on the surface morphology of human erythrocytes and Ehrlich ascites cells.

An alternate method to the common technique of evaporating a metallic coating on cells to render them conductive for scanning electron microscopy is described. This wet chemical technique is less expensive and easier to use, but is not as widely applicable as the evaporative technique. It should prove invaluable in the identification of artifacts by comparison of micrographs of material prepared in both manners. The first step in our application of this wet chemical method is to use the Centrifugal Cytology bucket to deposit the cells onto conductive polyethylene. After centrifugation and fixation with 4% glutaraldehyde, the sample is placed in a 50% glutaraldehyde solution. After a rinse in distilled water it is treated with ammoniacal silver nitrate. The reduced silver renders the sample conductive and, thus, for erythrocytes, eliminates artifacts due to charging and reduces these artifacts to virtually acceptable levels for larger cells. The surfaces of erythrocytes appear smooth with this technique while those coated by conventional vapor deposition of Au-Pd alloys often appear slightly wrinkled. Ehrlich ascites cells apparently can be divided into two classes by surface morphology. The surface structure of Ehrlich ascites cells rendered conductive by the wet method appears to be finer than conventionally prepared ones.

Animals↗