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Cells from a group of 185 patients suffering from malignant tumours (160 non-small-cell lung carcinoma, 13 small-cell lung carcinoma, and 12 non-epithelial tumours) and 6 with benign lung tumours were studied by flow cytometry in order to detect the prognostic value of DNA content. A total of 144 (90%) non-small-cell lung carcinomas (NSCLC) and 8 (62%) small-cell lung carcinomas (SCLC) exhibited aneuploidy. Furthermore 52% (83 patients) NSCLC, 24% (3 patients) SCLC and 50% (6 patients) non-epithelial tumours demonstrated multiclonality. Benign cases showed diploid DNA content. For actuarial survival analysis using the Bergesson and Gage method and the Greenwood variance, 142 patients were selected. Statistical comparisons were made by the use of the t-test for unpaired data between fixed times. No correlation was observed between ploidy and stage, histological grading or treatment modality. A statistically significantly better survival was observed after 12, 18 and 24 months of follow-up for diploid and monoclonal (with the exclusion of hypo- and hypertetraploid) patients. Thus, flow-cytometric DNA analysis may be useful in prognostic assessment of human lung tumours.

A previously described technique from the author's laboratories for purification of pancreatic islets by fluorescence-activated cell sorting used the dye neutral red (NR) to obtain specific fluorescence of islets sufficient to give a sorting signal. A major drawback with this technique was the need to inject the dye intravascularly before excision of the pancreas. Preliminary investigations showed that NR would produce selective staining of islets by topical application in vitro but only at low concentrations that were insufficient to give fluorescence strong enough for sorting. The chelating agent dithizone (DTZ) produces bright red staining of islets by topical application in vitro. Further studies showed that dithizone-stained islets exhibited moderately strong fluorescence that faded too quickly for reliable sorting. By combining both NR and DTZ staining in vitro, selective fluorescence of islets was obtained that was sufficient to allow efficient sorting. Using the combined DTZ/NR stain the yield of islets obtained by sorting from a single rat pancreas was 569 +/- 72 (n = 16), corresponding to 83% of the islets present in the digest. The mean purity of the preparation, confirmed by histologic examination, was 80%. The viability of the islets was shown to be good both by supravital staining and by the successful correction of streptozotocin diabetes in syngeneic rats following transplantation of sorted islets.

Using flow cytometric DNA measurement and sorting combined with morphometric light microscopy, different groups of cells were studied in a human melanoma pleural effusion, a human melanoma lymph node metastasis and a mouse tumor, as well as in normal reference tissues. Beside cells of the predominant tumor cell population, three types of rare tumor cells were studied after enrichment by sorting: a) giant cells from the greater than 8c region, comprising about 5% of the tumor cells, b) binucleated and multinucleated cells with unequal nuclear sizes within the same cell, found at frequencies of about 1.5%, and c) less than 2c cells which were derived from the so-called "debris"-region of the DNA histogram, found at frequencies of about 1 to 6%. All these rare cells were found only in the malignant tumors and not in the benign reference tissues. Morphometry showed that the increase in the cellular DNA content in the different fractions of tumor cells was combined with an increase in the cellular and nuclear sizes. However, the n/c-ratio was constant in the whole range of tumor cell fractions, including the fractions from the the less than 2c and the greater than 8c regions. The n/c-ratio of the less than 2c cells and giant cells differed from that of corresponding normal cells underlining their origin from the predominant tumor cell population. The possible linkage between the occurrence of the three rare cell types and genetic instability of tumors related to faulty nucleus and cell division is discussed.

Correlated DNA- and protein-determinations were performed using flow cytometry and DAPI-sulforhodamine 101 (SR101) staining. Two-wavelength UV-green excitation using a single HBO lamp was found to be superior to single wavelength UV excitation, since it resulted in a higher specificity in the SR101 measurements. The effects of overlapping in the emission spectra and of energy transfer from DAPI to SR101 were minimised. Different staining conditions and the specificity of SR101 staining were examined. No binding of SR101 to DNA and RNA could be detected. Good reproducibility was achieved in the protein measurements using human lymphocytes as an internal reference. Examples are given of the analysis of mixed cell populations with different cellular protein contents and different growth and metabolic activities. Correlated measurements of DNA and protein appear to be useful for further analysis of tumor cell populations.

Kinetics-directed drug treatment schedules were tested in Chinese hamster ovary cells. Ten hr after treatment with 1,2:5,6-dianhydrogalactitol (DAG) (at a dose lethal to less than 5% of the cells), a 150% enrichment of cells into the S phase of the cell cycle was observed. This blockade in S phase was reversible and was followed at 18 hr after an exposure to DAG by a 200% increase in the fraction of cells in the G2-M phases of the cell cycle. Bleomycin, known to be most effective against G2 + M cells, had the greatest effect on cell killing when administered at that time. Rapid analysis by flow microfluorometry techniques was used to determine the DAG-induced kinetics changes, thus allowing treatment with the second drugs at the most opportune time. The DAG-induced kinetics changes were also demonstrated in a line of human adenocarcinoma of the stomach in vitro and in Ehrlich ascites tumor cells in vivo. In all cases, the enrichment of cells into S phase was reversible at the doses used and was followed by a reversible blockade in G2-M.

Flow fluorometry and Coulter type sizing analysis of sperm have been applied separately in order to improve human semen analysis. Different methods of sample preparation were evaluated and a protocol involving prestaining pepsin treatment of sperm samples is proposed for fluorometric analysis. The data obtained with fluorometry and sizing analysis result in different kinds of information: Coulter counting allows to automate sperm counting and fluorometry yields more detailed information about normozoospermia and oligozoospermia by determining the proportion of mature spermatozoa and immature germ-cells. These two methods, together with light microscopy, may help to explore the correlation of fertility and pathology of spermatozoa. The aim of these investigations is to yield the preconditions for simultaneous two-parameter analysis of DNA content and cellular size distributions.

Tumour biopsies of 93 patients with head and neck malignancy were analysed by cytophotometry. Ploidy and the proportion of cells in S-phase were measured. Tumours were classified as proliferative if the S-phase fraction was greater than 8%; the rest were classified as being less proliferative. Proliferative tumours produce local and distant metastases earlier and more frequently, so that the proportion of cells in S-phase is of prognostic importance. The response to chemotherapy of irradiation was no different in either group.

We have analysed spermatogenetic cells by flow cytometry to quantify effects of ionizing radiation. The radiation-induced reductions of testicular DNA-synthesizing cells, primary spermatocytes, haploid round and elongated spermatids as well as the increases of numerical chromosome aberrations (abnormal diploid spermatids and aneuploidies) in NMRI inbred mice are described. Testicular weights were determined as a parameter of germ cell decrease, and histologic cross sections of the testes were analysed. Since even an exposure of 0.05 Gy (= 5 rad) may be detected by a reduction of DNA-synthesizing cells (Acta Radiol. Oncol. Radiat. Phys. Biol. 21, 349-351 (1982) [1]), the use of the in vivo system "spermatogenesis" as a biological dosimeter to monitor low dose effects and to determine RBE values of different radiation qualities is suggested.

Flow cytometric and histological analysis, measurements of testicular weight and sperm head counts were performed to analyze the effects of doxorubicin (DX) and 4'-epi-doxorubicin (4'-epi-DX), two closely related antineoplastic agents, on mouse spermatogenesis. The DNA distribution patterns obtained by flow cytometry indicate the frequency of different germ cell types: elongated and round spermatids, primary spermatocytes with a 4 c DNA content, and S-phase spermatogonia and spermatocytes. Following the injection of different doses of DX, characteristic changes of the frequencies of those germ cell types are observed with time, indicating selective inactivation of spermatogonia followed by sequential depletion of spermatocytes, round spermatids and elongated spermatids, and then recovery of these cell types. Similar changes were observed with 4'-epi-DX; the dose-response curves indicated that 4'-epi-DX might be slightly, although not significantly, less effective than DX. The mutagenic potential of DX and 4'-epi-DX is reflected by an increase of the coefficient of variation in the DNA histogram as a measure of aneuploidy, and an increase of diploid spermatids. Flow cytometric analysis of spermatogenesis offers a sensitive in vivo system to monitor mutagenic agents.

The cytokinetic response of a human colon carcinoma cell line to cis-dichlorodiammineplatinum(II) was investigated using flow cytometry of DNA content, autoradiography after pulse and continuous tritiated thymidine exposure, and mitotic accumulation after continuous Colcemid treatment. With increasing concentration and exposure time, cis-dichlorodiammineplatinum(II) delayed and then blocked cycle traverse in S and G2 phases. After prolonged treatment with high concentrations of cis-dichlorodiammineplatinum(II), an additional block in G1 or at the G1-S boundary was established. Irreversibility of cell cycle distribution changes after prolonged observation periods suggests cell death in G2, S, and G1 compartments.

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.

Mathematical 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)-phase 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.

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)

The DNA content of spermatids of mice carrying Cattanach's 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 Cattanach's translocation was obtained and showed a gradual loss of cells during spermatogenesis in those bearing the balanced form of the translocation.

Because cytokinetic studies of the human bone marrow aspirate as a prognostic factor and as a monitor of drug perturbation are frequently inconsistent, we investigated reproducibility of DNA distribution measured by flow cytometry of DNA content in patients with morphologically normal bone marrow. In 15 patients, correlation was noted between DNA distributions simultaneously obtained on right and left iliac crest bone marrow aspirates (r = .588), although considerable variation in individuals was encountered. Much better reproducibility (r = .879) was achieved using bilateral core biopsy of bone marrow in these same patients. In 60 samples, comparison of DNA distribution between bone marrow aspirate and simultaneously obtained biopsy revealed higher relative proportions of S and G2 + M phase cells in biopsies (p less than 0.001), suggesting peripheral blood contamination of aspirate material. Brisk shaking of biopsy specimens in saline expelled a representative sample in the supernatant that could be subjected to simultaneous cytomorphological and cytokinetic analysis. To improve reproducibility of DNA content determinations in normal human bone marrow, bone marrow biopsy should be utilized.

Flow cytometric measurements of the cellular DNA contents of 16 testicular biopsies of 15 patients with maldescended testes were performed. 2 patients were children. 11 patients of pubescent or adult age had no haploid germ cells in the tissue of their maldescended testis. One patient with a testis mobilis had about a normal percentage of haploid germ cells. In one patient a malignant tumor was detected unexpectedly by an aneuploid stem line at 2.2 c. Flow cytometric analysis of human testicular biopsy material is an easy and a fast way to quantify the function of spermatogenesis and detect malignancies.

To provide a rapid method for examining cell cycle dynamics, we utilized continuous exposure of Chinese hamster ovary cells and human colon cancer cells to colcemid to block cycling cells in metaphase, suppressing re-entry into G1. Changes in cell cycle compartment distribution were monitored by DNA flow cytometry. Analysis of the rate of G2 + M compartment accumulation after addition of colcemid permitted calculation of all cycle transit parameters. These compared favorably with data in the same cell lines determined by the fraction of labeled mitoses technique. Serial assessment of DNA flow cytometry after addition of colcemid permits rapid quantitation of cycle traverse rates.

Methods 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 an 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.

The cellular DNA-content of 75 malignant testicular germ-cell tumours was determined by flow cytometry. The results are: Aneuploidy is a certain tumour marker in 95% of germ-cell tumours. The DNA-indices range from 2 c (diploid) to 6 c with a modal value around 3 c. 23% of germ-cell tumours are multiclonal. The S-phase percentage is higher than those of the most other solid tumours investigated so far and is 25.5% in seminomas and 35% in non-seminomatous germ-cell tumours. Further investigations of a greater number of tissues will show the importance of DNA-contents measurement by flow cytometry for the prognosis of testicular tumours, representing an addition to histopathology.

A case of prolymphocytic leukemia (PL) is reported, which showed a good response to a new antiblastic schedule (4-epidoxorubicin-asparaginase-dexamethasone) in spite of the resistance to other chemotherapy regimens. However during the course of the disease it was possible to observe the terminal appearance of a small aneuploid cell population in the peripheral blood of the patient and, in the same time, the clinical condition deteriorated considerably. The significance of this neoplastic progression and the pros and cons of aggressive chemotherapy regimens remain to be carefully evaluated in PL and related disorders.

The Dna-content of sperm and testicular cells was measured by pulse-cytophotometry with high resolution. From flat sperm symmetric and narrow fluorescene 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%.

After testing the BrdUrd technique on experimental tumour cell lines, we applied the technique to human renal cell carcinoma in vitro. We compared the results with the data acquired after FCM analysis and 3H-thymidine treatment. In contrast to BrdUrd the 3H-thymidine uptake seemed to be limited in suspended cells. FCM data represented the DNA distribution of cells. BrdUrd labelling on the other hand detected DNA synthesizing cells. Only both methods in parallel were able to discriminate between proliferating cells and resting cells with an S-phase equivalent DNA content.