Dosimetry of soft x-rays in thin liquid layers.

Very thin material layers (<100 microm) partially absorb ionizing radiation of low energy. When irradiating monolayer cell cultures from above, attention must be paid to absorption by the medium. Frequently, the volume of the nutrient medium is variable, and this leads to differences in the radiation doses delivered to the cells. In the present work these conditions were investigated for x-rays of energies between 13 kV and 100 kV in comparison with 60Co gamma rays using chemical dosimetry to measure the absorption by liquid layers between 25 microm and 500 microm thick. When the dose as measured with the ionization chamber was held constant, the dose absorbed in the Fricke solution was shown to increase with decreasing thickness of the layer of liquid because of a dose gradient. The effect of the dose gradient disappeared, however, in thick liquid layers of the Fricke solution by mixing during spectrophotometry. Secondary (photoeffect and Compton) electrons produced in air or filters are responsible for this effect in plastic petri dishes where back scattering at the interface does not occur. This interpretation is suggested by the same results of an analogous experimental setup using gamma rays with a 5-mm-thick Perspex plate. This dose increase in very thin layers, however, could not be verified by irradiating monolayer cells in poured-out plastic petri dishes because the secondary electrons are already absorbed in the remaining liquid film above the cells.

Quantification of PCR products using microparticles and flow cytometry.

The analysis of genetic biomarkers has become an important tool in clinical diagnostics. This includes the identification of disease-related genetic alterations, the detection of pathogenic infective germs on DNA or RNA level and the quantification of the expression of marker genes indicating an altered physiological status. It has been previously described that the combination of polymerase chain reaction (PCR), microparticles and flow cytometry represents a universal platform technology for the routine analysis of such biomarkers. Here we demonstrate the applicability and flexibility of this technology by means of various applications. The quantification of interferon gamma (IFNG) mRNA in irradiated white blood cells is shown as well as the detection of latent infections with cytomegalovirus (CMV). Besides the quantification of single amplification products, the flow cytometric assay is also capable of analysing products of a multiplex PCR. As an example, we describe the identification of spontaneous deletions in the genome of a hybrid cell line using a co-amplified gene (RAB1) essential for the cell survival as an internal control. Furthermore, we show that the use of a green laser (532 nm, 50 mW) substantially increased the sensitivity of the assay compared to conventional flow cytometers using a 488 nm (25 mW) laser. We conclude that the analysis of PCR products using microparticles and flow cytometry fulfils the criteria of clinical routine diagnostics regarding (i) sensitivity, (ii) specificity, (iii) reproducibility and (iv) automatibility.

Mutagenic effect of low energy neutrons on human chromosome 11.

PURPOSE: The shape of the dose-effect curve for neutrons, i.e. the question as to whether the curve is linear or supralinear in the low-dose region, is still not clear. Therefore, the mutagenic effect of very low doses of low-energy neutrons was determined. MATERIALS AND METHODS: Human-hamster hybrid A(L) cells contain human chromosome 11, which expresses the membrane protein CD59. This membrane protein can be detected immunologically and quantified by flow cytometry. The A(L) cells were irradiated with neutrons of 0.565, 2.5 or 14.8 MeV and the results were compared with those after 200 kVp X-rays. Before irradiation, cells spontaneously mutated in the CD59 gene were removed by magnetic cell sorting (MACS). RESULTS: The relative biological effectiveness (RBE) for CD59 mutation induction was 19.8 (+/-2.7) for 0.565 MeV, 10.2 (+/-1.9) for 2.5 MeV, and 10.2 (+/-1.6) for 14.8 MeV neutrons. Linear mutation responses were obtained with all radiations except for 14.8 MeV neutrons where a supralinear curve may be a better fit. The deletion spectrum of mutated cell clones showed 29 Mbp deletions on average after irradiation with 0.069 Gy of 0.565 MeV neutrons. This scale of deletions is similar to that after 3 Gy 100 kV X-rays (=34 Mbp). For 50% cell survival, the RBE of the neutrons was 11 compared with 200 kV X-rays. CONCLUSIONS: Neutrons of low energies (0.565 or 2.5 MeV) produce a linear dose-response for mutation in the tested dose range of 0.015-0.15 Gy. The neutron curve of 14.8 MeV can be approximated by a curvilinear or linear function.

[Microbeads, nanobeads and cytometry: applications to the analysis and purification of cells and biomolecules]. / Microbilles, nanobilles et cytométrie: applications à l'analyse et à la purification cellulaire et moléculaire.

Nano and microspheres are important tools in cytometry. They have been used in first to optimize fluorescent signals detected by flow cytometry and to evaluate phagocytosis. Some antigens were also detected by using nanospheres covalently coupled to antibodies. Specifically dedicated microspheres are now widely used for antigenic quantitation by flow cytometry, and magnetic nano and micropheres are very usefull for cellular and molecular purifications. To date, analytical methods based on the use of microspheres are developed to detect proteins, nucleic acids, and ions. To this end, antibodies, oligonucleotides, or chelating agents are bound to microspheres characterized by different fluorescences. The applications of these multiplexed microspheres assays allow to identify and quantify simultaneously some macromolecules and ions, but they also permit to analyze enzymatic activities and to perform polymorphism analyses. With microspheres used as reactive support, molecular analyses are therefore possible by flow cytometry. Nano and microspheres are also usefull tools for calibration in confocal microscopy as well as for micromanipulations of biomolecules and of living cells. Inovative methods based on the use of nano and microspheres are expected in the fields of biology, medicine, food industry, and environmental sciences.

Mutagenicity of low-filtered 30 kVp X-rays, mammography X-rays and conventional X-rays in cultured mammalian cells.

PURPOSE: To measure the mutagenic effectiveness of low-filtered 30 kVp X-rays, mammography X-rays and conventional (200 kVp) X-rays in mammalian cells. MATERIALS AND METHODS: Two different cell lines and mutation assays were used. Exponentially growing SV40-transformed human fibroblasts were exposed to graded doses of mammography (29 kVp, tungsten anode, 50 microm Rh filter) or conventional X-rays and the frequency of 6-thioguanine-resistent HPRT-deficient mutants was determined. Exponentially growing hamster A(L) cells, which contain a single human chromosome 11 conferring the expression of the human surface protein CD59, were subjected to magnetic cell separation (MACS) in order to remove spontaneous mutants before irradiation with low-filtered 30 kVp (tungsten anode, 0.5 mm Al filter) or conventional X-rays. Fractions of radiation-induced CD59- mutants were quantified by flow-cytometry after immunofluorescence labelling of CD59 proteins. RESULTS: Mammography X-rays were more effective than conventional X-rays at inducing killing of human fibroblasts, whereas 30 kVp X-rays and conventional X-rays were about equally effective at killing Al. cells. Mutant frequencies were linearly related to dose in both mutation assays. An RBE = 2.7 was calculated for the yield of HPRT mutants in human fibroblasts exposed to mammography relative to conventional X-rays and an RBE = 2.4 was obtained for the CD59 mutant frequency in A(L) cells irradiated with low-filtered 30 kVp relative to conventional X-rays. CONCLUSIONS: Both low-filtered 30 kVp and mammography X-rays are mutagenic in mammalian cells in vitro. It is unknown if and how the enhanced mutagenicity of mammography X-rays measured in human cells in vitro translates into breast cancer risk for predisposed women with an enhanced inherited risk for breast cancer. Although the ICRP guidelines attribute the same relative biological effectiveness to all radiations of low LET, including X- and gamma-radiations of all energies for radiobiological protection purposes including the assessment of risks in general terms, they also state that 'for the estimation of the likely consequences of an exposure of a known population, it will sometimes be better to use absorbed dose and specific data relating to the relative biological effectiveness of the radiations concerned and the probability coefficients relating to the exposed population' (ICRP 1991: 32). This latter statement may apply for the population of familial predisposed women. We hope that the presented data on the enhanced mutagenicity of mammography X-rays may stimulate a re-evaluation of the risk assessment of mammography for familial predisposed women. In the meantime, one should be cautious and avoid early and frequent mammography exposure of predisposed women. Alternative examination methods should be applied for these women with an inherited increased risk for breast cancer.

Mutation induction and neoplastic transformation in human and human-hamster hybrid cells: dependence on photon energy and modulation in the low-dose range.

Mutation induction in the HPRT gene of human fibroblasts after irradiation with mammography-like 29 kVp or 200 kVp x-rays shows radiohypersensitivity for doses smaller than approximately 0.5 Gy. Similarly, mutation induction in the CD 59 gene on human chromosome 11 in A(L) cells shows radiohypersensitivity for doses smaller than approximately 0.5 Gy after exposure to 200 kVp x-rays, but not after irradiation with low-filtered 30 kVp x-rays. The RBE values of 29 and 30 kVp x-rays relative to 200 kVp x-rays are strongly dose dependent. For neoplastic transformation of human hybrid (CGL1) cells after irradiation with 29 or 200 kVp x-rays or 60Co gamma rays a linear-quadratic dose relationship was observed with RBE values of approximately four and eight for mammography relative to 200 kVp x-rays and 60Co gamma rays, respectively.

A novel true volumetric method for the determination of residual leucocytes in blood components.

BACKGROUND AND OBJECTIVES: Accurate determination of residual leucocytes [white blood cells (WBC)] in blood components is of high clinical importance. To date, several labour-intensive, time-consuming or expensive techniques have been used for this purpose. MATERIALS AND METHODS: A method for the determination of residual WBC is described using a novel low-cost flow-cytometric cell counter and analyser (CCA). The DNA in WBC was stained using 4'-6-diamidino-2-phenylindole (DAPI) and WBC were automatically analysed by true volumetric counting of 200-microl samples (prepared from a 20-microl undiluted sample). RESULTS: Dilution experiments over a range of 0.5-50 WBC/microl showed a linearity of r = 0.998. The detection limit of this method was 0.83 WBC/microl of red blood cell concentrate (RCC) and 0.67 WBC/microl of platelet concentrate (PC), with an accuracy of 95.5%. CONCLUSION: Residual WBC (< 1 WBC/microl) can be accurately counted using the CCA within 2 min and at a total cost of less than euro 1 per sample.

Identification of a deletion hotspot on distal mouse chromosome 4 by YAC fingerprinting.

Using repetitive elements as probes, genomic DNA fingerprints of four randomly selected yeast artificial chromosome (YAC) clones (two human and two mouse-derived YAC) were analyzed to determine the mutation level following X-ray exposure. Because the repetitive probes were derived from the mammalian host DNA, most of the fingerprint bands originated from the artificial chromosomes and not from the yeast genome. For none of the YAC clones was the mutation frequency elevated following X-ray exposure. However, for one mouse-derived YAC, the mutation level was unusually high (7%; 42 mutants of 607 clones analyzed), whereas for the other three YACs, the mutation level was nearly 0%. Surprisingly, 40 of the 42 mutations were deletions occurring only at three of the 20 mouse specific fingerprint bands. One of the frequently deleted fragments was cloned, sequenced and mapped to distal mouse chromosome 4, which has been repeatedly reported to be the most unstable region of the whole mouse genome, associated with various tumors. Deletion mapping of six YAC mutants revealed this fragment to be completely deleted in four YACs. In the other two mutants, recombination occurred within the fragment, in each case initiated at the same LINE-1 element. In conclusion, the presented YAC fingerprint is a useful tool for detecting and characterizing unstable regions in mammalian genomes.

Frequency of CD59 mutations induced in human-hamster hybrid A(L) cells by low-dose X-irradiation.

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.

The "vanishing counting bead" phenomenon: effect on absolute CD34+ cell counting in phosphate-buffered saline-diluted leukapheresis samples.

BACKGROUND: Using a single-platform protocol to count absolute CD34+ hematopoietic precursor cell (HPC) levels with different reference microbeads, we recorded occasionally artifactually high CD34+ HPC counts in some leukapheresis bags, whereas dual-platform calculations were always consistent. Abnormal countings were observed only when phosphate-buffered saline (PBS)-diluted leukapheresis samples were vortexed before analysis. A large series of blood samples analyzed similarly for CD34+ and CD4+ absolute counts did not show any sample or vortexing effect. With the volumetric absolute counting cytometer Partec-PAS, lower counts were also observed when different reference beads were vortexed before the instrument checking procedures. The counting abnormality was caused by a drop in microbead concentration (the "vanishing bead phenomenon"). This phenomenon reduced the total and relative bead event number in experimental and routine samples and in calibration procedures. This altered the bead denominator used to calculate absolute CD34+ HPC levels and it also reduced the concentration of standard calibration beads. METHODS: Using the Partec-PAS to measure volumetrically the actual bead concentration, we studied the vanishing bead phenomenon. Different types of counting and reference microbeads were resuspended in media with or without proteins or cells. Replicates were submitted either to gentle manual mixing or to vortexing before counting. RESULTS: Vortex agitation almost invariably induced the vanishing bead phenomenon when beads were resuspended in saline media or when an insufficient protein concentration was present, such as in diluted leukapheresis samples. Different bead types showed various degrees of sensitivity to vortexing. The bead disappearance was not caused by bubble formation or disruption. The addition of small amounts of protein completely prevented the vanishing bead phenomenon. The causative effect of the electrostatic charging of tube induced by vortexing is hypothesized. CONCLUSIONS: Sample suspensions containing counting beads for single-platform analysis must be resuspended in media with protein supplements to prevent the vanishing bead phenomenon and to ensure accurate counting.

Effects of single and split doses of cobalt-60 gamma rays and 14 MeV neutrons on mouse stem cell spermatogonia.

The long-term effects of ionizing radiation on male gonads may be the result of damage to spermatogonial stem cells. Doses of 10 cGy to 15 Gy (60)Co gamma rays or 10 cGy to 7 Gy 14 MeV neutrons were given to NMRI mice as single or split doses separated by a 24-h interval. The ratios of haploid spermatids/2c cells and the coefficients of variation of DNA histogram peaks as measures of both the cytocidal and the clastogenic actions of radiation were analyzed by DNA flow cytometry after DAPI staining. The coefficient of variation is not only a statistical examination of the data but is also used here as a measure of residual damage to DNA (i.e. a biological dosimeter). Testicular histology was examined in parallel. At 70 days after irradiation, the relative biological effectiveness for neutrons at 50% survival of spermatogonial stem cells was 3.6 for single doses and 2.8 for split doses. The average coefficient of variation of unirradiated controls of elongated spermatids was doubled when stem cells were irradiated with single doses of approximately 14 Gy (60)Co gamma rays or 3 Gy neutrons and observed 70 days later. Split doses of (60)Co gamma rays were more effective than single doses, doubling DNA dispersion at 7 Gy. No fractionation effect was found with neutrons with coefficients of variation.

Keratinocytes exposed to ultraviolet radiation reveal three down-regulated genes with potential function in differentiation and cell cycle control.

The incidence of skin cancer is increasing in epidemic proportion. Although solar UV radiation is known to be the major risk factor, much information is lacking about the molecular mechanisms leading to skin cancer. To gain a deeper insight into these mechanisms, we have examined cells of a human keratinocyte cell line (HaCat) after exposure to 0.16 minimal erythema doses of UVB radiation. This dose led to an S-phase delay that was reversible 22 h postirradiation. To examine gene expression 10 h after UV irradiation, a nonradioactive differential display was employed. Three genes were identified as being down-regulated significantly. The first encodes for topoisomerase-IIbeta-binding protein 1 (expression level 5% 6 h after irradiation). This protein is associated with human topoisomerase IIbeta and appears to be necessary for DNA replication during the onset of S phase. The second gene product has previously been reported to be involved in differentiation and is therefore known as differentiation-dependent A4 protein (28% 8 h after irradiation). The third gene is XPO1 (also known as CRM1) (5% 8 h after irradiation), whose protein is involved in nuclear export of mRNA molecules. Differential expression of these genes after UV irradiation has not been reported. Because of their potential involvement in cell cycle control and differentiation, these proteins could be important for understanding the reaction of keratinocytes after exposure to UV radiation.

Flow cytometric analysis of reverse transcription-PCR products: quantification of p21(WAF1/CIP1) and proliferating cell nuclear antigen mRNA.

BACKGROUND: Reverse transcription-PCR (RT-PCR) is a powerful tool in clinical diagnostics for analyzing even small amounts of RNA, but sensitive assays for quantifying the amplification products are time-consuming or expensive. Here we describe a novel flow cytometry-based assay for rapid and sensitive determination of relative amounts of RT-PCR products. METHODS: For flow cytometric quantification, PCR products were labeled with both digoxigenin and biotin during amplification. Subsequently, amplicons were simultaneously bound to anti-digoxigenin microparticles and fluorescently labeled with streptavidin-R-phycoerythrin. Fluorescence intensity per bead was determined by flow cytometry. To study this assay, we examined the expression of the p21(WAF1/CIP1) gene and the proliferating cell nuclear antigen (PCNA) gene in ultraviolet irradiation-exposed human keratinocytes lacking functional p53. RESULTS: Fluorescence was linear with 60-10 000 pg of PCR product. As little as 0.4 fmol (40 pg of a 163-bp amplicon) of PCR product could be distinguished from background. The between-run CV of the fluorescent signal for 10 ng of p21 cDNA was 12% (n = 10). The fluorescence-template curve was sigmoidal. p21(WAF1/CIP1) mRNA was decreased after ultraviolet irradiation of keratinocytes, whereas PCNA mRNA was markedly increased. CONCLUSION: The flow cytometric assay permits rapid (25 min) and reproducible identification of changes in mRNA abundance.

The relative biological effectiveness of low doses of 14 MeV neutrons in steady-state murine spermatogenesis as determined by flow cytometry.

The relative biological effectiveness of 14 MeV neutrons in the low-dose range < or =1 Gy has been determined in differentiating and differentiated spermatogonia. Male NMRI mice were exposed to single doses of 2 cGy to 3 Gy of (60)Co gamma rays or neutrons. The ratios of testicular S-phase cells, 4c primary spermatocytes, and elongated spermatids were quantified by DNA flow cytometry 2 to 70 days after irradiation and were found to decrease. Histological samples and testis weight were analyzed in parallel. Doses of 2-5 cGy neutrons and 10-50 cGy gamma rays significantly (P<0.05) decreased the proportions of S-phase cells, spermatocytes and elongated spermatids at 4, 14 and 28 days postirradiation. For S-phase cells, the biphasic shape of the cell survival curves was described with a D(50) of 5 cGy neutrons. The D(50) for (60)Co gamma rays and the relative biological effectiveness could not be determined. The relative biological effectiveness of neutrons at 50% reductions of testis weight, primary spermatocytes, and elongated spermatids were 2.5, 10.0 and 6.1, respectively. This in vivo assay is interesting because of its sensitivity at dose ranges that are relevant for exposures in the environment, the workplace and radiotherapy.

Selection, establishment and characterization of cell lines derived from a chemically-induced rat mammary heterogeneous tumor, by flow cytometry, transmission electron microscopy, and immunohistochemistry.

In order to isolate, characterize, and establish culture cell lines with different diagnostic and prognostic significance, derived from multiclonal neoplasms, a ductal infiltrating mammary tumor was induced in rats by 7,12-dimethylbenz[a]anthracene. Clones with different DNA/protein content, being the DI of 1.16, 1.30, and 1.60, respectively, were observed in the primary tumor. Biparametric flow cytometry suggested that the clone at 1.30 is made up of two subpopulations with different protein and slightly different DNA contents. The culture, after a few passages, exhibited the presence of aneuploid cells and the absence of diploid components, demonstrating that only tumor cells survived. The limiting dilution method gave rise to four lines with DI of 1.16, 1.25, 1.30, and 1.50; a mean chromosome number of 45, 46, 47, and 88, respectively; and different morphological and ultrastructural features. These characteristics were stable during the experimental procedure, that is, for about 20 passages. Conversely, the detection of cytoskeletal proteins indicated that the tumor epithelial cells underwent early dedifferentiation into sarcoma-like cells showing markers of stromal cell type and thus exhibiting phenotypic instability in vitro, a feature reported in many advanced human breast cancers in vivo. In conclusion, this cellular model represents the in vivo situation and appears suitable for in vitro studies of tumor cell characteristics and might be used to predict clinical behavior.

In vitro proliferation and in vivo malignancy of cell lines simultaneously derived from a chemically-induced heterogeneous rat mammary tumor.

Identification of clones in primary tumors responsible for proliferation, invasion, and metastasis was carried out. Four different aneuploid established cell lines derived from a ductal infiltrating mammary rat tumor induced by 7,12-dimethylbenz[a]anthracene were studied for proliferative and growth features in vitro and for tumorigenic and metastatic potential in vivo in nude mice. Clones, named RM1, RM2, RM3, and RM4, were characterized by different proliferative activity. Clone RM1 showed the highest proliferative activity by both tritiated thymidine incorporation and S-phase flow cytometry, followed by clone RM4. Conversely, clones RM2 and RM3 showed a lower proliferation rate. Growth-promoting activity, tested on 3T3 Swiss cells, was high in all clones, although RM1 showed significantly lower growth factors-releasing activity. Nude mice tumorigenesis demonstrated a strong tumor induction of line RM1 (100% of the mice after 47 +/- 7 d) and a slightly lower tumor induction of line RM4 (70% of the mice after 69 +/- 9 d). Line RM3 showed tumor induction in 40% of the mice after 186 +/- 16 d. Lines RM2 showed no tumor induction. Metastasis occurred in mice treated with line RM1 only. Therefore, tumorigenesis and metastasis correlate with proliferation but not with the release of growth factors. In conclusion, flow cytometry monitoring of clones from heterogeneous primary tumors proved to be a suitable model for the study of in vivo malignancy and in vitro proliferation.

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 (R(2) = 0.999). Bland-Altman statistics showed a mean difference of -2 cells/mm(3) [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 flow cytometry of human semen.

The aim of this study was the evaluation of DNA flow cytometry for the analysis of male infertility. 171 ejaculates from 155 patients with fertility problems were analysed by flow cytometry and by conventional microscopical procedures. Using flow cytometry, it was possible to determine the relative proportions of the various cell populations: mature haploid and abnormal diploid mature spermatozoa, cellular fragments, immature germ cells (haploid round spermatids, diploid cells, S phase and 4C cells), and of leukocytes as indicators of infection. A linear association was observed between sperm concentration in semen as quantified by light microscopy and by flow cytometry, even with fewer than 20x10(6) spermatozoa/ml. Eight classes of histograms, each with differing fractions of spermatozoa and other particles, were obtained and correlated with the results of the spermiograms. During the 10 year follow-up, the two patient groups with a low sperm concentration or a high concentration of cellular debris exhibited significantly impaired fertility. The two patient groups with >/=5% diploid spermatozoa and with malcondensed sperm chromatin were also subfertile. No ovulatory disorders were revealed in the 155 female partners. DNA flow cytometry thus provides an additional dimension to semen analysis not easily gained by other methods and has the advantage of being rapidly performed and interpreted. We therefore recommend application of this technique in the diagnosis of male infertility.

DNA/protein flow cytometry as a predictive marker of malignancy in dysplasia-free Barrett's esophagus: thirteen-year follow-up study on a cohort of patients.

Intestinal metaplasia identifies Barrett's esophagus (BE) and is associated with an increased risk for esophageal adenocarcinoma. Dysplasia occurs as an intermediate step. However, progression from metaplasia to neoplasia without the demonstration of dysplasia has been described. The role of dual-parameter flow cytometry (FC) as a predictor of neoplastic risk in dysplasia-free cases was evaluated. DNA/protein FC and histology were performed on 362 samples from 30 dysplasia-free BE patients, followed up since 1985 once every 1-2 years. Nine cases were aneuploid, five of which (group IV) were frankly aneuploid; in the other four cases (group III), aneuploidy was detectable by dual-parameter analysis only. Twenty-one patients were diploid. Twelve (group II) also had an abnormally high G1-phase protein content compared to group I (nine patients), which were diploid with a low-moderate protein content. In three patients of group IV an adenocarcinoma in situ was diagnosed, after 5, 6, and 10 years, respectively. In two patients of group III, a low- and a high-grade dysplasia were observed at 3 and 6 years follow-up, respectively. One patient of group I first acquired a high protein content, then an aneuploid DNA content, and then progressed to adenocarcinoma (12 years). None of the still diploid patients (17 cases) have progressed to dysplasia or cancer compared with 6 of 13 presently aneuploid patients (P < 0.01). In conclusion, DNA/protein FC is a marker of increased malignant potential and thus may be used to detect patients at higher risk in dysplasia-free BE and assist in understanding the various stages of malignant transformation in long-term follow-up studies.

Increased number of cancer cells in bronchial washing fluid detected by combining conventional cytology and high-resolution flow cytometry.

The present study was performed to improve early lung cancer diagnosis in bronchial washing fluid, thereby increasing the diagnostic sensitivity of bronchoscopy by means of high-resolution flow cytometry (FC). We combined dual-parameter DNA/protein FC and conventional cytology in bronchial washing fluid samples from 112 patients with neoplastic and non-neoplastic lung diseases and found 43% of histologically confirmed tumor cases to be cytologically positive; 63% of the tumor samples were aneuploid, 52% of the aneuploid cases were cytologically positive and 48% were negative. In the negative cases, FC was an independent diagnostic factor. In 32% of the cases, FC also failed to detect abnormalities. However, the combination of both techniques increased the sensitivity in detecting neoplastic cells to 73%. Furthermore, simultaneous DNA/protein analysis allowed the recognition of aneuploid cell lines not detectable by single DNA measurement. Identification of aneuploid subpopulations by dual-parameter analysis in cytologically negative one-parameter FC "diploid" samples assumes an important diagnostic value. Dual-parameter DNA/protein FC is a valuable technique that increases the diagnostic yield of bronchoscopy with no risk for the patient and a low additional cost.