The determination of CD4 cells is of crucial clinical importance for patients with AIDS. However, the high costs involved represent limitations for CD4 cell counting in developing countries. In order to provide an affordable technique, we introduced a simplified volumetric counting (SVC) technique without sample manipulations and investigated it in a multicentre study. Blood samples from 434 healthy donors and immunodeficient patients were tested in eight hospital laboratories in Europe, Africa and Asia. CD4 cell counts were compared using in-house flow cytometric methods and the SVC technique. The SVC method was performed on a low-cost flow cytometer (CyFlow SL, Partec, Münster, Germany) after 15 min antibody incubation without pre-analytic manipulations, such as washing or erythrocyte lysing procedures. Linear regression analysis demonstrated a correlation of r=0.942 (Europe), r=0.952 (Africa) and r=0.989 (Asia) between the SVC technique and the in-house methods. Bland Altman plot analysis of all patient data showed a mean bias between the two methods of +26 CD4 cells in favour of the SVC technique (measured range: 6-1905 cells/microl; median CD4 cell count: 388/microl). Three centres used the FACS-count technique (Becton-Dickinson, San José, Calif., USA) as an in-house method dispensing with pre-analytic manipulations. The comparison of SVC and FACS-count method revealed a mean bias of +32 CD4 cells/microl (median CD4 cell count: 349/microl). The accuracy of the SVC was tested on standards with known CD4 cell counts (n=6) and was shown to be 95.2%. The low-cost device and the simplified no-lyse, no-wash test procedure reduces the costs per determination and facilitates the use of flow cytometry in developing countries.

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.

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 no-lyse, 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.-.

Background: It is known from the literature that the use of erythrocyte lysing reagents in flow-cytometric analysis results in a significant loss of immunolabelled cells, e.g. T helper and T supressor lymphocytes (CD4+/CD8+ T cells) or stem cells (CD34+ cells). Although WBC subset-specific variations in the sensitivity to lysing reagents are already described, the same variations are possible for each individual. In the present study we investigate the individual and patient-dependent loss of CD4+ T cells in blood samples by the use of a true volumetric flow cytometer (TVC) and a new no-lyse no-wash procedure. Material and Methods: Blood of 50 individuals was used for flow-cytometric counting of CD4+ T cells, and cells were counted by the use of a lyse no-wash and a no-lyse no-wash protocol (600 single measurements). The reproducibility of the loss of CD4+ T cells was measured by 10 individually prepared samples out of a single blood sample; each sample was measured 3 times (n = 30). Results: The mean lysing-dependent CD4+ T-cell loss out of the sample from a single individual was 15% (range 12-18%). The reproducibility of the determination from the same individual was high (coefficient of variation (CV) = 12%). Measuring the concentration of CD4+ T cells in blood samples of different individuals, the mean loss of CD4+ T cells was 9.8% (range 0-23%), interestingly with a high variability and a CV of 65% In = 600). Therefore, most current flow-cytometric protocols with erythrocyte lysing procedure do not allow reproducible and accurate determinations of CD4+ T cells because of individual leukocyte losses. Conclusion: Lysing-dependent cell loss does not represent a constant or calculable parameter in leukocyte subclass enumeration. Beside the leukocyte subset-specific differences in sensitivity to erythrocyte lysing reagents, patient disease and drug treatment may influence the stability of leukocytes under investigation. Accordingly, the present study shows large lysing-dependent interindividual differences in counting results of CD4+ T cells.

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. (C) 2003 Editions scientifiques et medicales Elsevier SAS. Tous droits reserves.

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.Conclusion : 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.

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 μ m 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, 0.5mm 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 A L 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 γ-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.

Background: Competitive PCR of reverse transcribed mRNA sequences is used to quantify transcripts, but the usual approaches are labor-intensive and time-consuming. We describe the non-gel-based quantification of competitive reverse transcription (RT)-PCR products with use of microparticles and flow cytometry. Methods: PCR products of a target sequence and an internal control sequence (competitor) were labeled during PCR using digoxigenin (DIG)- and dinitrophenol (DNP)-labeled primer, respectively, allowing specific binding to microparticles coated with the corresponding antibody. Both amplification products were biotinylated to enable fluorescence labeling with streptavidin-R-phycoerythrin. The mean fluorescence intensity of each microparticle population, corresponding to the amount of bound PCR product, was measured in a flow cytometer. We constructed microparticles coated with antibodies against DIG and DNP to specifically capture PCR products derived from target and competitor sequences, respectively. Results: As required for a reliable competitive PCR assay, nearly identical kinetics were found for the amplification of target and competitor sequences when using only one competitive primer. The method was applied to examine interleukin-8 expression in human lymphocytes after x-irradiation. One hour after irradiation, the concentration of transcripts decreased by half. Conclusions: The flow cytometric assay for the quantification of competitive RT-PCR products avoids additional hybridization steps and antibody labeling. The use of paramagnetic microparticles would also enable the complete automation of this method.

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.

BACKGROUND Epidemiologic data revealed increased brain tumor incidence in workers exposed to magnetic fields (MFs), raising concerns about the possible link between MF exposure and cancer. However, MFs seem to be neither mutagenic nor tumorigenic. The mechanism of their tumorigenic effect has not been elucidated. METHODS To evaluate the interference of MFs with physical (heat shock, HS) and chemical (etoposide, VP16) induced apoptoses, respectively, we exposed a human glioblastoma primary culture to 6 mT static MF. We investigated cytosolic Ca(2+) ([Ca(2+)](i)) fluxes and extent of apoptosis as key endpoints. The effect of MFs on HS- and VP16-induced apoptoses in primary glioblastoma cultures from four patients was also tested. RESULTS Static MFs increased the [Ca(2+)](i) from a basal value of 124 +/- 4 nM to 233 +/- 43 nM (P < 0.05). MF exposure dramatically reduced the extent of HS- and VP16-induced apoptoses in all four glioblastoma primary cultures analyzed by 56% (range, 28-87%) and 44% (range, 38-48%), respectively. However, MF alone did not exert any apoptogenic activity. Differences were observed across the four cultures with regard to apoptotic induction by HS and VP16 and to MF apoptotic reduction, with an individual variability with regard to apoptotic sensitivity. CONCLUSION The ability of static MFs to reduce the extent of damage-induced apoptosis in glioblastoma cells might allow the survival of damaged and possibly mutated cells.

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-mul samples (prepared from a 20-mul undiluted sample).Results Dilution experiments over a range of 0.5-50 WBC/mul showed a linearity of r = 0.998. The detection limit of this method was 0.83 WBC/mul of red blood cell concentrate (RCC) and 0.67 WBC/mul of platelet concentrate (PC), with an accuracy of 95.5%.Conclusion Residual WBC (< 1 WBC/mu l) can be accurately counted using the CCA within 2 min and at a total cost of less than cent 1 per sample.

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.

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 AL cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The AL 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 AL cells to 100 kV X-rays at 0.1 to 5 Gy. The proportions of X-irradiation-induced CD59− mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5 Gy 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 5 Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6 Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (≤1 Gy). We conclude that the modified AL assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.

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.

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.