W. Göhde

Analysis of PCP-Data to Determine the Fraction of Cells in the Various Phases of Cell Cycle*

SummaryMathematical models for the analysis of pulse-cytophotometric (PCP) data are described. With computer programs based on these models the fractions of cells in G1-, S- and(G2 + M)-phases are obtained. The methods are applied to PCP measurements of Ehrlich ascites tumor cells, human bone marrow cells and L-929-cells in culture. The results of the L-cell experiment are compared with autoradiographic results; for both methods the duration of the various phases has been calculated. Two different mathematical models for PCP-data evaluation and the autoradiographic method yielded results agreeing within statistical error. The application of the two models on different types of DNA-histograms is discussed: One model is suitable for asynchronous cell populations with a low fraction of S-phase cells, the other can be applied for partially synchronized cells and high S-phase fractions as well.

Cellular DNA content as a marker of neoplasia in man

Abstract Cellular DNA content was determined by means of flow cytometry with the use of DNA specific fluorochromes (ethidium bromide and mithramycin) in 516 human tissue samples from 440 subjects. Compared to human granulocytes as diploid reference standard, there was a 91 percent incidence of DNA content abnormality difference in DNA content of tumor G10 cells indicating aneuploidy in 118 patients with neoplastic disease (including nine patients who lacked histopathologic evidence of malignancy at the time of study). Ninety-four percent of aneuploid tumors were hyperdiploid. Except for six solid tumors with biclonal abnormalities in DNA content, the remainder of neoplasms were characterized by uniform DNA content with little dispersion (small coefficient of variation of tumor G10 populations). For the entire group of patients with malignant disease, three modal values of DNA content were recognized at low-degree hyperdiploidy, near triploidy and tetraploidy. Except for the prevalence of high-degree hyperdiploidy in melanomas and low-degree hyper- and hypodiploid abnormalities in malignant lymphomas, significant disease-specific patterns of abnormal DNA content were not apparent. The magnitude of ploidy abnormality was further influenced by patient age and proliferative activity of the tumor. Female patients displayed a preponderance of small-degree hyperdiploid and tetraploid tumors, whereas near-triploid abnormalities prevailed among male patients, who also harbored five of six biclonal tumors. Tumor cell ploidy did not vary among different sites of disease and upon sequential long-term follow-up examination. All 121 benign tumors had a diploid DNA content. Among the group of 209 patients with normal histology or reactive changes were seven patients with a previously established diagnosis of cancer with ploidy abnormality. This discrepancy indicates that monodispersal of the entire tissue aliquot for DNA flow cytometry is superior to histologic examination of focal neoplasia. There were two patients, one with recurrent benign pleural effusions and one with reactive lymphadenopathy, with ploidy abnormality by DNA content in whom malignant lymphoma developed. We conclude that flow cytometry of cellular DNA content is a rapid, objective, quantitative and sensitive method to determine a highly specific and stable tumor cell marker.

Differential pattern of DNA-Aneuploidy in human malignancies

The differential pattern of DNA-aneuploidy, detected by flow cytometry (FCM) regarding its frequency, grade and multiclonality, was investigated and correlated to tumor type, malignancy grade, tumor stage and prognosis in a multi-institutional study at the University of Münster. High resolution measurements using admixed normal blood reference cells were undertaken in 2413 cases of 13 different malignant diseases and in 776 benign lesions or samples. The incidence of DNA-aneuploidy was highest in melanomas, carcinomas, testicular tumors, sarcomas (75%-95%) and myelomas (65%). Acute leukemias showed an intermediate DNA-aneuploidy rate of 40% with special subgroups represented by common ALL (44%), p less than 0.05) and myelomonocytic/monocytic AML (47%, p less than 0.01). The lowest DNA-aneuploidy-rate was found in basal cell skin carcinomas (19%) and congenital melanocytic nevi (9%). No case of DNA-aneuploidy was observed in the 776 benign lesions or samples.--DNA-indices giving the grade of DNA-aneuploidy with 1.0 for normal diploid G1/0 cells were found distributed predominantly between 1.0 and 2.0 in the solid tumors, except testicular tumors, clustering around a triploid maximum at 1.5. DNA-indices of myelomas and acute leukemias generally ranged below 1.25 with lower DNA-aneuploidy grades in AML than in ALL (p less than 0.01).--In melanomas the aneuploidy rate was higher (86%) in metastases than in the primary tumors (54%, p = 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Affordable CD4(+) T-cell counts on 'single-platform' flow cytometers I. Primary CD4 gating.

Here, we demonstrate the flow cytometric concept of ‘primary CD4 gating’ utilizing three different CD4 monoclonal antibodies (mAbs) conjugated with five different fluorochromes. CD4+ lymphocytes were defined by an autogate in a single histogram of CD4 fluorescence intensity (FI) (y‐axis) vs. side light scatter (x‐axis). A wide range of absolute counts for > 600 individuals, including HIV+ patients, were compared with those obtained by ‘state‐of‐the‐art’ single‐platform flow cytometers such as the volumetric Ortho CytoronAbsolute and the Becton Dickinson FACSCalibur using TruCount beads. The correlation between CD4 counts obtained with primary CD4 gating and the full test panel on the Ortho Cytoron was excellent (R2 = 0·999). Bland–Altman statistics showed a mean difference of −2 cells/mm3[confidence interval (CI) 95% = −3 to −1; limits of agreement −27 to +23]. In addition to absolute CD4 counts, CD4% values and CD4/CD8 ratios are also frequently requested. To obtain these, lymphocytes need to be counted using scatter gates, and a second tube stained with a CD8 mAb to count CD8++ lymphocytes can be incorporated. We conclude that primary CD4 gating on single‐platform volumetric flow cytometers is one of the most economical and flexible technologies for routine cost‐conscious service work, particularly during the follow‐up of patients undergoing anti‐HIV therapy and/or vaccination in the developing world.

DNA-fluorometry of mammalian sperm

SummaryThe DNA-content of sperm and testicular cells was measured by pulse-cytophotometry with high resolution. From flat sperm symmetric and narrow fluorescence distributions were obtained. Enzymatic treatment with papain or pronase and staining with an ethidiumbromide-mithramycin dye solution generate stoichiometric DNA-staining including that of mature sperm with a coefficient of variation below 2%.

Measurement of mammalian sperm deoxyribonucleic acid by flow cytometry. Problems and approaches.

Measurement of mammalian sperm deoxyribonucleic acid content is of importance in several areas of biomedical research. When measured in flow systems with orthogonal axes of illumination, flow and detection, an unexpected, distorted distribution consisting of a narrow peak with a lateral extension to the right is observed. Several lines of evidence lead to the conclusion that this effect is an optical-geometric artifact attributable to the flat shape and high index of refraction of mammalian sperm heads. This artifact disappears when an epiillumination flow system is used in which the optic axes for illumination and detection and the flow axis are all coincident. Other approaches also eliminate the artifact. The resulting coefficients of variation observed after acriflavine-Feulgen staining are 4-5%, short of the goal of 1.5% required to distinguish between human sperm bearing X and Y chromosomes and to develop a mutagen test system using mice.

Flow cytometry of human spermatozoa

SummaryMethods are given for the preparation and staining of human spermatozoa for flow cytometric DNA measurements. Using agents for the reductive cleavage of disulfide crosslinks and suitable proteolytic enzymes and effective decondensation of the sperm chromatin and a DNA-proportional uptake of fluorochromes is achieved. Thus reliable and precise measurements of the relative DNA content of human spermatozoa are possible and the two subpopulations of haploid spermatozoa can be distinguished according to the difference in their DNA content.

Flow cytometry as a tool for the prognostic assessment of human neoplasia

Flow cytometry permits the quantitative description of neoplastic cell populations from the point of view of their cytogenetic and cytokinetic features. The advances in preparation of cellular monodispersed samples allow the examination not only of in vitro and hematological, but also of surgical, biopsy, endoscopic, and lavage specimens. The analysis of cytometric DNA content has evidenced the importance of (aneu)ploidy as a remarkable tumor marker. Tumors of different sites and, in some cases, stages and/or grades are characterized by a differential occurrence of diploid vs. aneuploid cell subpopulations and by the eventual presence of different stem cell lines within the same tumor. For certain classes of neoplasms, these parameters can be used for the early recognition of neoplasia and related to disease evolution and dissemination and to the results of therapy. Flow cytometry can also be used to evaluate the fraction of (cycling) cells in the S-phase and of proliferating cells (growth fraction). The percent of S cells can be extracted from cytometric DNA content histograms. Furthermore, the method of Bromodeoxyuridine (BrdUrd) incorporation has been recently introduced into flow cytometry. BrdUrd labeling in cycling cells can be detected either by the induction of quenching or enhancement of specific DNA-dye fluorescence or by fluorescent anti-BrdUrd monoclonal antibodies. This approach has been confirmed by preliminary comparative tests on cultured cells, normal and malignant bone marrow, and human solid tumor specimens. These parameters, together with other cytometric parameters of potential importance for the cellular characterization of malignancy, offer a reliable and real time-saving tool for the prognostic assessment of human tumors and the predicting and monitoring of the results of therapy.

A New No-Lyse, No-Wash Flow-Cytometric Method for the Determination of CD4 T Cells in Blood Samples

Background: Commonly used flow-cytometric methods for immunophenotyping are based on erythrocyte lysing reagents. It is known from the literature that these reagents result in a significant loss of leucocytes caused by membrane destruction. Although the dual-platform method should compensate this phenomenon, the subset-specific individual differences in sensitivity to lysing reagents lead to incorrect values. In order to overcome this problem we introduce a no-lyse, no-wash procedure in combination with absolute true volumetric counting (TVC) for the enumeration of CD4 T cells. Material and Methods: Whole-blood samples of 50 blood donors and 20 samples of patients with acquired immunodeficiency syndrome (AIDS) were treated with both a lyse, no-wash and a nolyse, no-wash procedure. Then, CD4 T cells were counted with a TVC flow cytometer (CyFlow Counter). 30 blood samples of blood donors were treated with a lyse and wash protocol and measured by the CyFlow Counter and FACS-Calibur system (reference method). A new gating strategy was used for the data analysis for both, the lyse and no-lyse method and compared to the traditional gating procedure. Results: The TVC method showed a good reproducibility for both, the lyse (CV 1.9%) and the no-lyse procedure (CV 1.5%). CD4 counts measured by the no-lyse procedure are on average 10% higher than by using the lysing protocol. The comparison of the CyFlow Counter and FACS-Calibur results showed a correlation of r = 0.961. The simplified gating strategy shows a good correlation to the traditional gating procedure (r = 0.998). Conclusion: Erythrocyte lysing procedures cause substantial cell loss with individual values for every single subclass and patient. Therefore, the use of a no-lyse procedure is recommended. The new gating strategy is fully comparable to the traditional one and simplifies enumeration of CD4 T cells. Using the no-lyse procedure in combination with the new gating strategy, it is possible to reduce the costs per sample from EUR 30.– to below EUR 2.–.

“Cytogenetic” studies of spermatids of mice carrying Cattanach's translocation by flow cytometry

The DNA content of spermatids of mice carrying Cattanachs translocation has been measured with high precision by flow cytometry. The observation that the two peaks of DNA content in the haploid region of the DNA histograms represent X-and Y-bearing spermatids was tested and confirmed. Using flow cytometry, the difference in DNA content between the X and Y chromosomes in these mice was measured to be 5.2±0.1% of the total haploid genome as compared to 3.4±0.1% in normal mice. These results demonstrated the precision of flow cytometry for cytogenetic studies. Additional information on spermatogenesis in mice bearing Cattanachs translocation was obtained and showed a gradual loss of cells during spermatogenesis in those bearing the balanced form of the translocation.