Simplified immunophenotypic analysis by laser scanning cytometry.

Immunophenotypic analysis of hematologic specimens is a useful laboratory adjunct to surgical pathology and cytology to confirm or further characterize diagnoses of leukemia or lymphoma. Laser scanning cytometry is a new laboratory technology that has been adapted to perform immunophenotypic analysis of hematologic specimens, with numerous advantages as compared with flow cytometry. In order to make full use of the laser scanning cytometer's capabilities, a new method of specimen preparation and means of performing the immunofluorescent reactions was developed. The technique described in this report, specific only to laser scanning cytometry, enables panels of up to 36 different antibodies to be used on specimens as small as 50,000 total cells. The laboratory methodology is simple, requires 85% less antibody than flow cytometric methods, and allows individual cell cytologic morphology to be correlated with objective physical and fluorescent measurements on a cell-by-cell basis. Other advantages are described in the text. Over the course of nine months in our community hospital, we have used this technique clinically to analyze 172 cases of suspected leukemia or lymphoma. The method has proven remarkably useful, particularly for extremely small specimens such as fine needle aspiration biopsies.

Five-color immunophenotyping plus DNA content analysis by laser scanning cytometry.

Laser scanning cytometry is a new laboratory technology similar to flow cytometry but with advantages for certain clinical and research applications. To date, laser scanning cytometry has been successfully used to perform three-color immunophenotypic analysis of hematologic specimens, single-color immunophenotyping plus DNA content analysis of numerous specimen types, and automated analysis of fluorescence in situ hybridization specimens. Several other interesting applications are also in development. In general, advantages of laser scanning cytometry include reduced specimen size requirements, simplified methodologies, and the ability to microscopically examine individual cells-allowing for the direct correlation of cytologic morphology with objective fluorescence measurements. In this report, we describe a method which more fully takes advantage of the laser scanning cytometer's capabilities for immunophenotypic analysis of hematologic specimens. Specifically, we have devised a method to increase the number of fluorescent parameters from three to a total of six, five representing binding of immunofluorescent antibodies and one for stoichiometric measurements of DNA content. As with most laser scanning cytometric applications, this technique can be utilized on extremely small specimens and enables direct correlation of all of the measured fluorescent parameters with light microscopic cytologic morphology.

Multiparameter analysis of DNA content and cytokeratin expression in breast carcinoma by laser scanning cytometry.

OBJECTIVE: The objective of this study was to test a new laboratory technology, laser scanning cytometry, for the purpose of performing multiparameter DNA content analysis of breast carcinomas. DESIGN: We developed a simplified method of multiparameter DNA content analysis using cytokeratin expression to positively gate epithelial cells. Over 300 consecutive cases of breast carcinoma were analyzed by multiparameter laser scanning cytometry. The first 73 cases were analyzed in parallel by single parameter flow cytometry. SETTING: The Department of Pathology, Christ Hospital and Medical Center, Oak Lawn, Ill. SPECIMENS: Three hundred eighteen consecutive cases of breast carcinoma presenting between March 1994 and December 1995. MAIN OUTCOME MEASURES: Outcome measures included the percentage of cases for which DNA content analysis could be successfully performed given the limitations of specimen size. Additionally, for the first 73 cases, laser scanning cytometry results were compared with flow cytometry results. RESULTS: All of the first 73 cases were successfully analyzed by laser scanning cytometry, but for 8 cases (11%) there was insufficient material for flow cytometry. Correlation of DNA content for the remaining 65 cases analyzed in parallel by the two methods was nearly perfect (p = .994). Five seemingly discrepant cases highlighted the importance of cytokeratin gating of epithelial cells by any technique, as well as other advantages specific to laser scanning cytometry, such as the ability to examine individual cells microscopically and correlate cytologic morphology with DNA content results. CONCLUSIONS: Laser scanning cytometry is a promising new technology for DNA content analysis of solid tissue tumors. Further work needs to be performed to validate the prognostic potential of the laser scanning cytometric assay results and to generate methodologies aimed at providing highly objective determinations of tumor cell S-phase fraction.

Fractionation of human serum lipoproteins by single-spin gradient ultracentrifugation: quantification of apolipoproteins B and A-1 and lipid components.

A sensitive and reproducible method has been developed for separation of the major serum lipoproteins from 1 ml or less of human serum by isopycnic density gradient ultracentrifugation. The serum, applied to a step gradient (total volume 12.8 ml), was spun for 48 hr at 38,000 rpm at 10 degrees C and, in each of the fractions, apolipoproteins B and A-I were quantified by the respective radioimmunoassays. The markers for lipid distribution used were [4-(14)C]cholesterol and [U-(14)C]lecithin, each incubated with an aliquot of serum at 20 degrees C for 75 min prior to ultracentrifugation. In control sera, three main fractions, very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins were clearly separated from a bottom fraction. Their flotational, electrophoretic, and chemical properties were in good agreement with those reported for the corresponding lipoproteins separated by conventional ultracentrifugation. Both apo B and apo A-I were fully recovered. Essentially all of the apo B was found in VLDL (9.3 +/- 3.5%) and LDL (87 +/- 4.6%); of the apo A-I, 81.0 +/- 5.7% was in HDL and the remainder (17.0 +/- 5.8%) was in the bottom fraction. The peak activities of [(14)C]cholesterol coincided with the peak of apo B in both LDL and VLDL, and with the peak of apo A-I in HDL. The results with the radiolabeled cholesterol were in good agreement with those obtained by chemical analyses. Carbon 14-labeled lecithin, although fully recovered, was not an accurate marker of phospholipid distribution because, under our experimental conditions, a significant amount of the lecithin was converted into its lyso derivative. The mechanism of the conversion was not established; it appeared to be unrelated to the activities of either lecithin-cholesterol acyl transferase or a Ca(2+)-dependent phospholipase. Besides its validity in the study of control sera, our method also proved successful in the separation of the serum lipoproteins of the few patients with dyslipoproteinemia (abetalipoproteinemia and familial hypercholesterolemia) who were examined. However, the applicability of the method to all dyslipoproteinemias was not assessed. Taken together, the results indicate that the single-spin method could be useful in clinical studies as a complement to other established techniques.

Vasectomy clinic.

PIP: Data from a questionnaire survey of 38 of 53 physicians in the St. Paul, Minnesota, area on vasectomy and clinic support resulted in the establishment of a low-cost vasectomy clinic by the St. Paul Bureau of Health. The survey results confirmed that many potential vasectomy patients could not afford private medical care and many local physicians were uninterested in performing the surgery or refused to perform it. After initial telephone screening, eligible couples attend a group counseling session. Patients hesitant about the operation are urged to reconsider. Clinics are held on Thursday evenings and Friday mornings to insure minimal loss of work time. Fees are on a sliding scale from zero to 125 dollars. In 8 months, 129 vasectomies have been performed at the clinic, with the majority (60%) desiring vasectomy to limit family size.^ieng