Prognostic classification of malignant melanomas by combining clinical, histological, and immunohistochemical parameters.

In a retrospective study the prognostic relevance of clinical, histopathological, immunohistochemical, and flow-cytometric parameters in primary malignant melanomas was evaluated using both the receiver operating characteristic ROC procedure and the logistic regression model. The proteolytic enzymes collagenase IV, cathepsin B, and cathepsin D proved to be significant prognostic factors. Combining the results obtained with these enzymes with gender, anatomic site, tumour thickness, Clark's level, ulceration, pattern of invasive growth, and presence of large round cells resulted in greatly improved discrimination between metastasized and non-metastasized cases. It is anticipated that this method could allow for precise individual prognostic characterization and in particular for identification of high-risk patients for adjuvant therapy.

Quantification of cathepsin D in plasma of patients with malignant melanoma.

The high mortality rate of melanoma patients who develop metastases prompted us to seek for a prognostic soluble marker to identify high-risk and non-risk patients at the stage of the primary tumour. Therefore, we developed a new ELISA for quantifying plasma concentrations of the proteolytic enzyme cathepsin D (CD) in patients with primary tumours (MM-P) and with metastases (MM-M), respectively, compared to a control group. Whereas healthy probands (n = 56) and MM-P (n = 68) showed similar mean values of CD (0.73 +/- 0.45 ng/ml and 0.82 + 0.80 ng/ml), MM-M (n = 40) yielded significantly reduced plasma levels (0.43 +/- 0.53 ng/ml) revealing a high significant discrimination both between controls and MM-M, and MM-P and MM-M (p < 0.0001). From the beginning of the study (1990) to the present 11 of 68 MM-P developed metastases. In order to test the prognostic efficiency of this enzyme to determine those patients at high-risk and non-risk for developing metastases, the receiver operating characteristic analysis was used showing that CD plasma levels cannot supply reliable prognostic values (W = 0.53, p = 0.66).

Soluble HLA class I antigens in plasma of patients with malignant melanoma.

The high mortality rate of melanoma patients who develop metastases prompted us to look for prognostic markers to determine high-risk and non-risk patients at the primary tumour stage. Therefore, we quantified plasma concentrations of soluble HLA class I antigens (sHLA-I) by ELISA in patients with primary tumours (MM-P) and with metastases (MM-M), respectively, and compared them to a control group. Whereas healthy probands (n = 55) and MM-M (n = 38) showed similar mean values of sHLA-I (1.30 +/- 1.44 micrograms/ml and 1.29 +/- 1.27 micrograms/ml), MM-P (n = 67) revealed significantly reduced levels of this marker (0.84 +/- 0.85 microgram/ml). This result matches with our immunohistological staining of membrane-bound HLA-I in sections of paraffin-embedded melanoma. Further subdivision of the MM-P substantiated the observation that mean values of decreased sHLA-I concentrations are in line with high tumour thickness. Since the beginning of this study (1990) to date, 11 of 67 MM-P have developed metastases. The prognostic efficiency of sHLA-I to identify high-risk and non-risk patients was tested by ROC-analysis (receiver operating characteristic) and did not demonstrate good prognostic relevance for sHLA-I (W = 0.64, p = 0.04).

Flow cytometric detection of unbalanced ram spermatozoa from heterozygous 1;20 translocation carriers.

Flow cytometric analysis of enzymatically decondensed, DAPI-stained spermatozoa was performed to confirm the suspected production of unbalanced spermatozoa in heterozygous rams carrying a 1;20 translocation. High-precision flow cytometry (coefficient of variation, 0.6-0.8%) with a PAS II flow cytometer depicted Y- and X-chromosome-bearing spermatozoa from three cytogenetically normal rams as two distinct peaks. The difference in DNA fluorescence intensity between the gonosomes averaged 4.8%. Analysis of sperm samples from three heterozygous 1;20 translocation carriers yielded histograms with five peak distributions. The individual peaks were attributed to spermatozoa with a normal, balanced, and unbalanced chromosomal status. Peaks within Y- and X-spermatozoa populations were distributed in a ratio of 1:2:1 and were almost completely separated, with a coefficient of variation of 0.5-0.6%. Owing to the relative size of the translocated chromosomal segment (2.4% of the total DNA content, as determined from the flow cytometric data), histograms with five instead of the expected six peaks were observed.

A rapid method for flow cytometric cell counting and cell cycle analysis from monolayer cultures of tumor cells.

A rapid, simple, and reliable flow cytometric method using the histochemical fluorescent stain Hoechst 33342 in presence of the non-ionic detergent Triton X-100 has been reported. The processing of melanoma cell cultures to get nuclei stained with the fluorescent dye was accomplished in one step and within an hour permitted concurrent flow cytometric measurement of cell density and cell cycle analysis. The preparation is stable for more than three weeks at room temperature for flow cytometry. The histograms are reproducible and exhibit a coefficient of variation of less than 2.5% (G1 peak). The cell density measurements varied within +/- 5% limits.

DNA flow cytometry in the prognosis of primary malignant melanoma.

DNA flow cytometry was carried out on 804 primary melanomas. The data were analyzed with a follow-up of 24-96 months. 57% of the cases were diploid, 32% had one abnormal cell population, and 11% were multiclonal. In 8% of the aneuploid tumors there were cell lines in the hypertetraploid range. A reliable S phase determination was possible in 524 cases. Among these 11% had an S phase exceeding 15%. Using an increased tumor thickness, relapse rate and mortality as criteria of tumor progression, aneuploidy and multiclonality, the occurrence of hypertetraploid cell lines and a high S phase (greater than 15%) proved to be correlated with a poor prognosis.

Flow cytometric discrimination of human semen cells.

An improved method for differential staining and high resolution flow cytometric measurement of human semen cells is presented. Using a mild pretreatment with citric acid/detergent and staining with DAPI, the new procedure provides excellent preservation and good discrimination of all cells which are present in normal and pathological semen samples.

[Differentiation of human sperm cells using flow cytometry]. / Differenzierung menschlicher Spermazellen mit Hilfe der Durchflusszytometrie.

An improved method for differential staining and high-resolution flow cytometric measurement of human sperm cells is presented. Involving a mild pretreatment with citric acid/detergent and staining with DAPI, the new procedure provides excellent preservation and good discrimination of all cells present in normal and pathologic semen samples.

Radiation-induced diploid spermatids in mice.

Diploid elongated spermatids of mice were enriched by flow cytometry and cell sorting using a new type of sorter (Partec). The sorted abnormal spermatids were identified morphologically and by nuclear area integration. The radiation-induced increase in the frequency of diploid elongated spermatids was monitored with time following acute X-ray exposure of mice. Dose-response curves for acute 60Co-gamma and 14 MeV neutron irradiations yielded an RBE value of 4.3 for the doubling of the control level.

Assessment of persisting chromosome aberrations by flow karyotyping of cloned Chinese hamster cells.

A preparation, staining and measuring protocol for high resolution flow cytometry of chromosomes was developed. This method allows us to identify all chromosome types and is suited for characterization of permanent cell lines and cell clones by establishing their flow karyotypes. In cell clones this procedure can be used for the detection of chromosomal aberrations which appear spontaneously or are induced by mutagen treatment and persist in the cell population.

Shape and position of the haploid peak in flow cytometric sperm histograms for the assessment of andrological diseases.

Ejaculates from 45 patients with various andrological diseases and from healthy men were analyzed by flow cytometric DNA measurements. The sperm histograms obtained by this method were subjected to mathematical analysis. The height, width and position of the haploid (1 C) peak proved to be a useful criterion for the discrimination of spermatozoa from patients with heterogeneous testicular disorders (SHTD, "mixed atrophy") and homogeneous testicular disorders (SETD) with delivery of immature spermiogenetic cells as compared to healthy persons.

[Pepsin pretreatment of human semen as a standard procedure for flow cytometry]. / Die Pepsinvorbehandlung menschlichen Spermas als Standardmethode in der impulscytophotometrischen Analyse.

The 30 minutes pepsin incubation of human semen has proved to be a standard pretreatment procedure for impuls cytophotometry (ICP). This method, in comparison to papain treatment, shows a better preservation of the material and avoids the phenomenon of "disintegration" and reduced staining property. The CV of 1C peak is small enough. Additionally, the immature spermatozoa, earlier dissociated late elongated spermatids, diploid spermatozoa, dual forms and diploid semen cells (spermatogenetic cells, leukocytes, epithelial cells are demonstrated in small peaks, a fact which can help explain the disturbances in pathological cases.

Flow cytometric measurement of nuclear DNA content variations as a potential in vivo mutagenicity test.

Mutagen-induced variations of the cellular DNA content have been studied in mouse bone marrow cells in vivo using high resolution flow cytometric techniques. During the first days after a single injection of the chemical mutagen cyclophosphamide an increased coefficient of variation in the G1 peak of the flow histograms was observed. The magnitude and duration of this effect were dose-dependent. The reproducibility of the measurements was high, indicating that individual variability between animals and instrumental dispersion is small. The results demonstrate that on the basis of the flow cytometric measurement of cellular DNA content, a short-term in vivo test for mutagenicity can be established which is much faster than conventional cytogenetic methods.

[Disintegration of human spermatozoa in flow cytometry of semen]. / Desintegration von Spermatozoen bei der impulscytophotometrischen Analyse des menschlichen Spermas.

In some cases, the pretreatment of human spermatozoa produced desintegration of various degrees, while in others a reduced staining was observed. For the evaluation of these phenomena the following techniques were applied: papain/DTE solution at pH 5.5 and 6.4, pepsin solution and a staining procedure without any preceeding decondensation of the spermatozoa nuclei. Pathological spermatozoa from patients with severe disorders of spermatogenesis showed an extended resistance to the pretreatment procedures, the spermatozoa being stained after papain/DTE pretreatment at pH 5.5 only. Contrary to this finding, normal spermatozoa and spermatozoa with slight morphological aberrations desintegrated when papain/DTE solutions were used. In this group the abortion rate was high.

Flow cytogenetic studies in chromosomes and whole cells for the detection of clastogenic effects.

Flow cytometric measurements of the chromosomal DNA content have been used to develop a screening method for the detection of chemically- or physically-induced cytogenetic damage. The reproducibility of this flow cytogenetic assay was shown in a series of subcultures of a Chinese hamster cell clone. The accuracy and sensitivity was tested in cultures treated with chemical mutagens and x-rays. The clastogenic effectiveness was quantified and the dose-effect relationship was established by the increase of the coefficient of variation of the peak of the largest chromosome type in the flow histograms. Since structural chromosome aberrations cause an unequal division of the DNA at mitosis, it is expected that clastogenic effects can be detected also in whole cells of growing populations as an increased dispersion of the cellular DNA content. In order to test this feature, high resolution flow cytometric measurements were performed in x-irradiated hamster cells in vitro and mouse bone marrow cells in vivo.

Flow cytogenetics of uncloned and cloned Chinese hamster cells.

Flow cytometry has greatly facilitated the routine use of DNA content as a cellular indicator of the stages of the cell cycle and ploidy. DNA content can also be used to distinguish individual chromosomes. Fluorescent staining of chromosome DNA was done with a combination of ethidium bromide and mithramycin in hypotonic solution. Subsequent detergent treatment of the cells with Triton X-100 facilitated chromosome isolation. DNA flow cytometry of chromosomes of four established uncloned Chinese master cell lines showed 10 to 12 major subpopulations of chromosomes with varying degrees of overlap in the range of low and intermediate DNA content. Cloning of B14F28 cells, the line with the largest heterogeneity in chromosome number and DNA content, considerably reduced the dispersion in chromosome number and improved the resolution of DNA content distributions. Thus, cloned cells with a relatively homogeneous karyotype permit better discrimination of chromosome subpopulations by DNA content than uncloned cells and provide a more sensitive system to study mutagenic effects.

Flow cytometry of human spermatozoa.

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.

[Analysis of spermatozoa with flow cytophotometric determination of DNA]. / Die Analyse von Spermaproben mit Hilfe impulscytophotometrischer DNS-bestimmungen.

Semen samples of fertility patients and fertile men were analysed by an improved technique of flow cytophotometry. The DNA-contents of mature spermatozoa were measured with a high level of accuracy. Besides spermatozoa, peaks of spermiogenetic cells and other seminal cells could be demonstrated. Complementary to the results of semen analyses the complex histograms of pulse cytophotometry investigations may give further informations on the nature of the underlying disturbances of spermatogenesis.