The microbial threat: Can rare earths help?

Despite an ever increasing demand for reliable and cheap methods in the detection and quantification of microbes, surprisingly few investigations have explored or utilized the luminescence of rare earths in the microbial context, neither in conventional, that is, plating and microscopic imaging techniques, nor in advanced methods like fluorescence flow cytometry. We have thus investigated the potential of some rare earth complexes and hybrid materials for microbiological analysis. We found fairly simple procedures for internal staining (dyes inside the bacterial cell) and external staining (dyes on the cell surface). The present paper is predominantly relying on microscopic imaging and luminescence spectroscopies (excitation, emission, decay times), but also evaluates model rare earth microspheres to estimate an eventual rare earth based stain for a fast and sensitive bacteria enumeration with luminescence flow cytometry.

Luminescence lifetime encoding in time-domain flow cytometry.

Time-resolved flow cytometry represents an alternative to commonly applied spectral or intensity multiplexing in bioanalytics. At present, the vast majority of the reports on this topic focuses on phase-domain techniques and specific applications. In this report, we present a flow cytometry platform with time-resolved detection based on a compact setup and straightforward time-domain measurements utilizing lifetime-encoded beads with lifetimes in the nanosecond range. We provide general assessment of time-domain flow cytometry and discuss the concept of this platform to address achievable resolution limits, data analysis, and requirements on suitable encoding dyes. Experimental data are complemented by numerical calculations on photon count numbers and impact of noise and measurement time on the obtained lifetime values.

A new triplex real time PCR which distinguishes between MRSA, MSSA, and mecA coagulase negative strains by means of melting point analysis using SYTO 9.

BACKGROUND: A new screening method was developed for the detection and identification of methicillin resistant staphylococcus aureus (MRSA) from sterile sites or mixed flora samples (inguinal or nose swabs). METHODS: After rapid treatment of samples, the method consists of two steps, template DNA preparation by a simple and rapid boiling procedure and a multiplex real time PCR. The triplex PCR system simultaneously detects the following targets, (i) the integration site for the open reading frame X (orfX) of the staphylococcal cassette chromosome mec type I - V (SCCmec), (ii) the mecA gene which codes for the penicillin-binding protein PBP-2a, and (iii) an internal control (IC) which can be amplified with the SCCmec primer system. A new buffer system, which contains the fluorescent dye SYTO 9, allows a reproducible real time PCR for the discrimination of the above mentioned PCR products by means of a high resolution melting point analysis (HRM). RESULTS: This new PCR system distinguishes between MRSA, MSSA, and mecA positive but coagulase-negative staphylococci (CoNS) strains. An internal control confirms the integrity of the PCR run. The HRM shows three melting points specific for each amplification product. 78.75 degrees C for the mecA gene, 83.15 degrees C for the SCCmec/orfX fragment and 88.25 degrees C for the internal control. CONCLUSIONS: This new multilocus MRSA PCR system is a fast and inexpensive alternative to commercially available test systems and costs only five to six euros.

An improved PCR method for detection of HIV-1 proviral DNA of a wide range of subtypes and recombinant forms circulating globally.

Proviral DNAs are being measured increasingly as a marker of the efficacy of highly active anti-retroviral therapy (HAART) and is accepted for the early diagnosis of perinatal HIV-1 infections. This requires a standardized test which enables the detection of a wide range of subtypes worldwide including O, N and circulating recombinant forms (CRFs). Based on a previous publication, a PCR - Test for HIV-1 provirus detection in peripheral blood mononuclear cells (PBMCs) was developed. Blood samples from 80 individuals infected with HIV-1 and 20 persons negative for HIV-1&2 from Africa and Germany were tested for the presence of HIV-1 provirus DNA. The primer system used enables the detection of proviral DNA despite the high concentrations of human DNA. The limit of detection was determined to be 5 copies per 10(5) cells. All 20 samples from persons negative for HIV were negative for HIV-1 proviral DNA while provirus DNA was amplified from 76 of the 80 (95%) samples from persons infected with HIV. The amplified products were detected by gel-electrophoresis, flow cytometry and real-time PCR. All three detection systems provided the same results.

A new affordable flow cytometry based method to measure HIV-1 viral load.

Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub-Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV-1-5'LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)-products were captured by anti-DIG and anti-DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin-R-phycoerythrine. The primer system used recognized all HIV-1 subtypes. A coamplified internal standard warranted the functionality of the PCR and allows reproducible viral load measurements. Two drawbacks of current viral load measurements are overcome by the flow cytometry based test described hereof. First, all known worldwide relevant HIV-1 subtypes including subtypes O, N, and new CRFs are quantifiable with high sensitivity (50 to >1 x 10(6) copies per PCR). Second, the cost per test can be reduced to less than 12 US$ instead of the current 50-100 US$. Additionally, the test described in this report offers the possibility to perform complete monitoring program (CD4 T-cell count, CD4% and viral load) for the first time, with the same device for HIV-infected persons.

Evidence for predictive validity of blood assays to evaluate individual radiosensitivity.

PURPOSE: An escalation in standard irradiation dose ensuring improved local tumor control is estimated, but this strategy would require the exclusion of the most sensitive individuals from treatment. Therefore, fast and reliable assays for prediction of the individual radiosensitivity are urgently required. METHODS AND MATERIALS: Seven parameters in lymphocytes of 40 patients with leukemia were analyzed before, during, and after total body irradiation (TBI) and in vitro X-ray irradiation. These were: cell proliferation, nuclear damage, activation of cytokines, and numbers of total leukocytes of CD34+ hematopoietic blood stem cells and of CD4+ and CD8+ lymphocytes. Additionally, antioxidative capacity of blood plasma, uric acid, and hemoglobin levels were measured. Blood samples of 67 healthy donors were used as controls. RESULTS: In vivo and in vitro irradiations showed comparable results. A dose-response relationship was found for most parameters. Three parameters were associated with severe acute oral mucositis (Grade 3 or 4 vs. Grade 0 to 2): leukocytes fewer than 6200/microL after 4 Gy TBI, a rate of >19% lymphocytes with reduced DNA and protein content ("necroses") after 4 Gy in vitro irradiation, and a small antioxidative capacity in blood plasma (<0.68 mMol) after 8 Gy TBI. CONCLUSION: Three simple blood assays were associated with oral mucositis that are posed here hypothetically as an early symptom of enhanced radiosensitivity in leukemic patients: leukocyte count, damaged lymphocyte score, and the antioxidative capacity after exposure.

The impact of erythrocyte lysing procedures on the recovery of hematopoietic progenitor cells in flow cytometric analysis.

Since preanalytic lysing of erythrocytes remains critical in flow cytometry, we investigated the influence of four lysing procedures on the quantification of leukocyte and CD34+ cells in hematopoietic cell transplants (HCTs). Samples were derived from stem cell-enriched mobilized whole blood collected by apheresis (unselected) and immunologically purified stem cell products (selected) and were measured using the dual-platform (2-PF) method with two flow cytometric systems. Additionally, cells were measured by a volume-based technique (single platform [1-PF]). Results were identical in the 2-PF mode (unselected HCTs, r = 0.998; selected HCTs, r = 0.999). In comparison with the 2-PF results, the single-platform (1-PF) measurements revealed a mean decrease of 59.5% for CD34+ cells (50.8% for CD45+ cells) in unselected HCTs and a mean decrease of 52% for CD34+ cells (49.8% for CD45+ cells) in selected HCTs. In order to check the accuracy of cell quantification using the 1-PF method, leukocyte reference values from hematology counter results were compared with flow cytometric (1-PF)-counted nucleated cells. That analysis revealed good congruency, with r = 0.998 for unselected HCTs and r = 0.999 for selected HCTs. In conclusion, all lysing procedures that we used induced substantial loss of leukocytes and CD34+ cells. As demonstrated by the high accuracy of the 1-PF technique, all erythrocyte lysing procedures caused significant cell loss, which led to inconsistent counting of CD34+ cells in nonvolumetric flow cytometric (2-PF) protocols.

Simplified volumetric flow cytometry allows feasible and accurate determination of CD4 T lymphocytes in immunodeficient patients worldwide.

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.

High-grade loss of leukocytes and hematopoietic progenitor cells caused by erythrocyte-lysing procedures for flow cytometric analyses.

All current-flow cytometric techniques use erythrocyte-lysing procedures before leukocyte analysis. We investigated the impact of four lysing procedures with different flow cytometric techniques on the loss of leukocytes and hematopoietic progenitor cells in blood samples. A total of 280 determinations out of 10 samples were measured by two flow cytometers (FCMs), using a FACS-Calibur (Becton Dickinson) and a particle-analyzing system (PAS) with a "true volumetric unit" (Partec). All samples were prepared with four different commercially available erythrocyte-lysing reagents (n = 10, respectively). CD34(+) cells were determined in relation to counted leukocytes with both FCMs (dual platform determinations, 2-PF). In addition, further immunologic and nuclear staining determinations of cells with and without erythrocyte-lysing procedures were performed in the "true volumetric unit" (single platform mode 1-PF) using the PAS system (n = 10, respectively). In the 2-PF mode, both systems showed identical results for CD34(+) cells (r = 0.997). The comparison of 1-PF and 2-PF modes with immunologic stainings revealed a mean decrease of 34.5% for absolute amounts of CD45(+) cells [in detail: Becton-Dickinson (BD) lysis 40%; Ortho Diagnostics (OD) lysis 31%; Uti lyse (UL) 38%; Cylyse (CL) 29%] and of 41.3% for absolute concentration of CD34(+) cells [in detail: BD lysis 45%; OD lysis 40%; UL lysis 45%; CL lysis 34%] by the lysing procedures. In contrast, the nuclear stainings revealed a mean leukocyte loss of only 5% for the nonlysed samples and of 12% for lysed samples. All investigated lysing procedures induced a large loss of leukocytes and progenitor cells, obviously due to cell membrane destruction as demonstrated for identical samples in the 1-PF and 2-PF modes by immunologic and nuclear staining methods.

Static magnetic fields affect calcium fluxes and inhibit stress-induced apoptosis in human glioblastoma cells.

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.

Flow cytometric quantification of competitive reverse transcription-PCR products.

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.