Biomedical subjects

Dominik Lenz

Publications and source records attributed to Dominik Lenz.

15 recordsLinked to original sources

PubMed2006

Comparison of immunophenotyping by slide-based cytometry and by flow cytometry.

BACKGROUND: Flow cytometry (FCM) is the gold standard for immunophenotyping of peripheral blood leukocytes (PBLs). Slide-based cytometry (SBC) systems, for example the laser scanning cytometer (LSC(R), CompuCyte), can give additional information (repeated staining and scanning, morphology). In order to adequately judge the clinical usefulness of LSC for immunophenotyping it is obligatory to compare it with FCM. AIM: The aim of this study was to systematically compare immunophenotyping by both FCM and LSC methods and to test the correlation of the results. METHODS: PBLs were stained with directly labeled monoclonal antibodies with the whole blood staining method. Aliquots of the same paraformaldehyde fixed specimens were analyzed in parallel by a FACScan (BD-Biosciences) using standard protocols and by LSC with different triggers (forward scatter, CD45 FITC, or 7-AAD). For 7-AAD measurements by LSC, slides were additionally fixed with acetone before 7-AAD staining. RESULTS: Calculating the percentage distribution of PBLs obtained by LSC and by FCM showed very good correlation with regression coefficients close to 1.0 for the major populations and the lymphocyte sub-populations (neutrophils, monocytes, and lymphocytes; T-helper-, T-cytotoxic-, B-, NK-cells). The best trigger for LSC was 7-AAD. CONCLUSION: LSC can be recommended for immunophenotyping of PBLs especially in cases where only limited sample volumes are available or where additional analysis of the cells' morphology is important. The detection of rare leukocytes or weak antigens is limited; in these cases appropriate amplification steps for immunofluorescence should be engaged.

Dactinomycin↗

PubMed2006

Iterative restaining as a pivotal tool for n-color immunophenotyping by slide-based cytometry.

AIM: Slide-based cytometry (SBC) allows to "ask a cell a second time." We used this tool for detailed immunophenotyping of peripheral blood leukocytes (PBLs). METHODS: PBLs primarily stained for CD-markers and DNA were immobilized on a glass slide and analyzed by laser scanning cytometry. Then, iterative restaining was applied for a second and a third analysis. Based on the cells' fixed location, analyses were merged on a single-cell level. RESULTS: We analyzed six virtual immunostainings by "recycling" the PE-channel for four CD-markers. CONCLUSION: Iterative restaining might prove to be a pivotal tool for n-color immunophenotyping exclusive to SBC concepts.

Antibodies↗

PubMed2006

Automated four-color analysis of leukocytes by scanning fluorescence microscopy using quantum dots.

BACKGROUND: Scanning fluorescence microscope (SFM) is a new technique for automated motorized microscopes to measure multiple fluorochrome labeled cells (Bocsi et al., Cytometry A 2004, 61:1-8). AIMS: We developed a four-color staining protocol (DNA, CD3, CD4, and CD8) for the lymphocyte phenotyping by SFM. METHODS: Organic (Alexa488, FITC, PE-Alexa610, CyChrom, APC) and inorganic (quantum dot (QD) 605 or 655) fluorochromes were used and compared in different combinations. Measurements were performed in suspension by flow cytometer (FCM) and on slide by SFM. RESULTS: Both QDs were detectable by the appropriate Axioplan-2 and FCM filters and the AxioCam BW-camera. CD4/CD8 ratios were highly correlated (P = 0.01) between the SFM and FCM. CONCLUSION: Automated SFM is an applicable tool for CD4/CD8 ratio determination in peripheral blood samples with QDs.

Benzimidazoles↗

PubMed2006

Sequential photobleaching of fluorochromes for polychromatic slide-based cytometry.

BACKGROUND: Slide-based cytometry is a key technology for polychromatic cytomic investigations. Here we exploit the relocalization and merge feature of Laser Scanning Cytometry for distinguishing fluorochromes of comparable emission spectra but different photostabilities. METHODS: Blood specimens were stained with the fluorochrome pairs: FITC/ALEXA488, PE/ALEXA532, or APC/ALEXA633. Bleaching was performed by repeated laser excitation. RESULTS: Since ALEXA dyes are photostable as compared to the conventional fluorochromes FITC, PE, and APC, a differentiation within one fluorochrome pair is possible. CONCLUSION: The sequential photobleaching method results in an increased information density on a single cell level and represents an important component to perform polychromatic cytometry.

Antigens, CD19↗

PubMed2006

Laser scanning cytometry in human brain slices.

BACKGROUND: The Laser Scanning Cytometry (LSC) offers quantitative fluorescence analysis of cell suspensions and tissue sections. METHODS: We adapted this technique to immunohistochemical labelled human brain slices. RESULTS: We were able to identify neurons according to their labelling and to display morphological structures such as the lamination of the entorhinal cortex. Further, we were able to distinguish between neurons with and without cyclin B1 expression and we could assign the expression of cyclin B1 to the cell islands of layer II and the pyramidal neurons of layer V of the entorhinal cortex in Alzheimer's disease effected brain. In addition, we developed a method depicting the three-dimensional distribution of the cells in intact tissue sections. CONCLUSIONS: In this pilot experiments we could demonstrate the power of the LSC for the analysis of human brain sections.

Alzheimer Disease↗

PubMed2006

Hyperchromatic cytometry principles for cytomics using slide based cytometry.

BACKGROUND: Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high-content and high-throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non-consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object. METHODS AND RESULTS: We demonstrate various approaches for hyperchromatic analysis on a SBC instrument, the Laser Scanning Cytometer. The different components demonstrated here include (1) polychromatic cytometry (staining of the specimen with eight or more different fluorochromes simultaneously), (2) iterative restaining (using the same fluorochrome for restaining and subsequent reanalysis), (3) differential photobleaching (differentiating fluorochromes by their different photostability), (4) photoactivation (activating fluorescent nanoparticles or photocaged dyes), and (5) photodestruction (destruction of FRET dyes). Based on the ability to relocate cells that are immobilized on a microscope slide with a precision of approximately 1 microm, identical cells can be reanalyzed on the single cell level after manipulation steps. CONCLUSION: With the intelligent combination of several different techniques, the hyperchromatic cytometry approach allows to quantify and analyze all components of relevance on the single cell level. The information gained per specimen is only limited by the number of available antibodies and sterical hindrance.

Animals↗

PubMed2005

Fusion of bone marrow-derived stem cells with cardiomyocytes in a heterologous in vitro model.

OBJECTIVE: Recent studies have demonstrated that transplanted bone marrow-derived stem cells (BMCs) possess a broad differentiation potential and are able to form new cardiomyocytes. However, the identity of BMCs as true cardiomyocytes is still ambiguous. Therefore, we investigated the fate of transplanted fluorescence labeled BMCs and cardiomyocytes in co-culture. METHODS: For cell tracking we used two different fluorescent probes, Vybrant/DiO and Vybrant/DiI. BMCs were taken from human sternal marrow, purified using a Ficoll-gradient-centrifugation, treated with 5-azacytidine and stained with Vybrant/DiO. Furthermore, isolated spontaneous beating cardiomyocytes of neonatal rats (CM) were labeled with Vybrant/DiI. Thereafter, the BMCs were transplanted into CM-cultures and investigated on day 1, 4, 7, 14 and 28 using two-color fluorescence phenotyping by laser-scanning-cytometry (LSC). Two-color positive cells were harvested by patch-clamp technique and beta-MHC mRNA expression was analyzed by single-cell PCR. RESULTS: Two different morphological phenotypes were observed by LSC. First, isolated DiO labeled BMCs without contact or with direct cell contact to DiI labeled CMs. Second, some BMCs and CMs were double positive for DiO/DiI spontaneously forming hybrids. This population increased by 18% from day 1 to 4 and decreased only slightly until day 28. Additionally, few two-color positive cell formations expressed both human and rat specific beta-MHC mRNA as well as only human beta-MHC mRNA indicating that cell-fusion and transdifferentiation has occurred. CONCLUSION: These observations provide in vitro evidence for spontaneous cell fusion and transdifferentiation of BMCs in co-culture, raising the possibility that the observed phenomenons may contribute to development or maintenance of these cell types.

Animals↗

PubMed2005

Effects of exercise and ischemia on mobilization and functional activation of blood-derived progenitor cells in patients with ischemic syndromes: results of 3 randomized studies.

BACKGROUND: Exercise training (ET) has been shown to improve regional perfusion in ischemic syndromes. This might be partially related to a regeneration of diseased endothelium by circulating progenitor cells (CPCs) or CPC-derived vasculogenesis. The aim of the present study was to determine whether ischemic stimuli during ET are required to promote CPC mobilization in patients with cardiovascular diseases. METHODS AND RESULTS: Patients with peripheral arterial occlusive disease (PAOD) were randomized to 4 weeks of daily ischemic ET or control (group A). Successfully revascularized patients with PAOD were randomized to 4 weeks of daily nonischemic ET or control (group B). Patients with stable coronary artery disease were subjected to 4 weeks of subischemic ET or control (group C). At baseline and after 4 weeks, the number of KDR+/CD34+ CPCs was determined by fluorescence-activated cell sorting analysis. Levels of vascular endothelial growth factor (VEGF) were measured by ELISA. A Matrigel assay was used to quantify CPC integration into vascular structures. Expression of the homing factor CXCR4 was determined by reverse transcription-polymerase chain reaction. In group A only, ischemic ET increased VEGF levels by 310% (P<0.05 versus control) associated with an increase in CPCs by 440% (P<0.05 versus control), increased CXCR4 expression, and enhanced integration of CPCs into endothelial networks. In contrast, subischemic ET in groups B and C increased CXCR4 expression and CPC integration. CONCLUSIONS: In training programs, symptomatic tissue ischemia seems to be a prerequisite for CPC mobilization. However, ischemic and subischemic ET programs affect CXCR4 expression of CPCs, which might lead to an improved CPC integration into endothelial networks.

Aged↗

PubMed2005

Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence.

The relationship between the mechanical properties of cells and their molecular architecture has been the focus of extensive research for decades. The cytoskeleton, an internal polymer network, in particular determines a cell's mechanical strength and morphology. This cytoskeleton evolves during the normal differentiation of cells, is involved in many cellular functions, and is characteristically altered in many diseases, including cancer. Here we examine this hypothesized link between function and elasticity, enabling the distinction between different cells, by using a microfluidic optical stretcher, a two-beam laser trap optimized to serially deform single suspended cells by optically induced surface forces. In contrast to previous cell elasticity measurement techniques, statistically relevant numbers of single cells can be measured in rapid succession through microfluidic delivery, without any modification or contact. We find that optical deformability is sensitive enough to monitor the subtle changes during the progression of mouse fibroblasts and human breast epithelial cells from normal to cancerous and even metastatic state. The surprisingly low numbers of cells required for this distinction reflect the tight regulation of the cytoskeleton by the cell. This suggests using optical deformability as an inherent cell marker for basic cell biological investigation and diagnosis of disease.

3T3 Cells↗

PubMed2005

Regenerative and predictive medicine of cardiovascular disease: the 9th Leipziger Workshop and the 2nd International Workshop on slide based cytometry.

Slide-based cytometry (SBC) and related techniques offer unique tools to perform complex immunophenotyping, thereby enabling diagnostic procedures at very early disease stages. Multicolor or polychromatic analysis of cells by SBC is of special importance, not only as a cytomics technology platform but also for patients with low blood volume such as neonates. The exact knowledge of the location of each cell on the slide allows restaining and subsequent reanalysis of the specimen. These separate measurements of the same specimen can be fused to one data file (merging), thus increasing the information obtained per cell. Relocalization and optical evaluation of the cells, a feature typical of SBC, can be of integral importance for cytometric analysis. Due to this feature, artifacts can be excluded and morphology of measured cells can be documented. Predictive medicine aims at the detection of changes in patient's state before the manifestation of the disease or its complications. Such instances concern multiorgan failure in sepsis or noninfectious posttraumatic shock in intensive care patients or the pretherapeutic identification of high-risk patients undergoing cancer cytostatic therapy. Early anti-infectious or antishock therapy and curative chemotherapy in combination with stem cell transplantation may provide better chances of patients' survival at concomitant cost containment. Predictive medicine that guides early individualized decrease or cessation of therapy may lower or abrogate potential therapeutic side effects (individualized medicine). Regenerative medicine concerns patients who have diseased and injured organs and may be treated with transplanted organs. However, there is a severe shortage of donor organs that is worsening yearly given the aging population. Regenerative medicine and tissue engineering apply the principles of cell transplantation, material science, and bioengineering to construct biological substitutes that will restore and maintain normal function in diseased and injured tissues. Neovascularization is promoted by bone marrow-derived endothelial progenitor cells that lead to the formation of entirely new vessels into ischemic tissue. With this knowledge, many therapeutical borders can be skipped. Diseases formerly uncontrolled can be corrected with stem cells to provide causal healing with regeneration processes. The 9th Leipziger Workshop combined with the 2nd International Workshop on SBC aimed to offer new methods in image cytometry and SBC for solutions in clinical research. It moved toward practical applications in clinics and the clinical laboratory. This development will be continued in 2005 at the upcoming Leipziger Workshop and the 3rd International Workshop on SBC.

Cardiovascular Diseases↗

PubMed2005

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

BACKGROUND: Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide-based cytometry, repeated analysis was combined with sequential optical-filter changing. METHODS: Human peripheral blood leukocytes from healthy adult volunteers were labeled with antibodies by direct or indirect staining. Tandem dyes of Cy7 (phycoerythrin [PE]-/allophycocyanin [APC]-Cy7), Cy5.5 (PE-/APC-Cy5.5), and PE-Cy5 and the fluorochromes fluorescein isothiocyanate (FITC), PE, and APC were tested alone and in combinations. Optical filters of the laser scanning cytometer were 555 DRLP/BP 530/30 nm for photomultiplier tube (PMT) 1/FITC, 605 DRLP/BP 580/30 nm for PMT 2/PE, 740 DCXR/BP 670/20 nm for PMT 3/Cy5/APC, and BP 810/90 nm for PMT 4/Cy7. Filter PMT 3 was replaced for detection of PE/Cy5.5 and APC/Cy5.5 by 740 LP/BP 710/20 nm and the sample was remeasured. Both data files were merged into one to combine the different information on a single-cell basis. The combination of eight antibodies against CD3, CD4, CD8, CD14, CD16, CD19, CD45, and CD56 was used to characterize NK and NKT cells and their subsets. RESULTS: In this way Cy5.5 is measurable at 488-nm and 633-nm excitation. Further, with the two different filters it is possible to distinguish Cy5 from Cy5.5 in the same detection channel (PMT 3). With this method we identified NK and NKT cells, subsets of NK (CD3-16+56+, CD3-16+56-, CD3-16-56+) and NKT (CD3+16+56+, CD3+16-56+) and their CD4+8-, CD4-8+, CD4-8- and CD4+8+ subsets. CONCLUSION: With our adaptations it is possible to discriminate tandem conjugates of Cy5, Cy5.5, and Cy7 for eight-color immunophenotyping. Using this method, novel rare subsets of NK and NKT cells that are CD4/CD8 double positive are reported for the first time.

CD3 Complex↗

PubMed2004

Increase of circulating endothelial progenitor cells in patients with coronary artery disease after exercise-induced ischemia.

OBJECTIVE: The concept of neovascularization in response to tissue ischemia has been extended by the finding of postnatal vasculogenesis initiated by endothelial progenitor cells (EPCs). The aim of this study was to analyze whether a maximal stress test in patients with coronary artery disease (CAD) increases the number of circulating EPCs. METHODS AND RESULTS: Blood concentration of EPCs was analyzed by FACS and cell culture assay in CAD patients with (n=16) or without (n=12) exercise-induced myocardial ischemia and in healthy subjects (n=11) for up to 144 hours after maximal stress test. Plasma concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, tumor necrosis factor-alpha, and granulocyte macrophage-colony stimulating factor were determined by ELISA. EPCs increased significantly in ischemic patients, with a maximum after 24 to 48 hours (cell culture: 3.3+/-0.5-fold increase; FACS: 3.1+/-0.6-fold increase) and returned to baseline within 72 hour. In nonischemic patients and healthy subjects, no EPC increase was detectable. VEGF levels in ischemic patients increased significantly after 2 to 6 hours (maximum after 2 hours; 4.0+/-1.1-fold increase) and no change was observed in nonischemic patients and healthy subjects; DeltaVEGF and DeltaEPC correlated significantly (r=0.66). CONCLUSIONS: Patients with symptomatic CAD respond to a single episode of exercise-induced myocardial ischemia with a time-dependent increase in circulating EPCs. This increase may be related to and preceded by an increase in plasma VEGF.

Aged↗

PubMed2003

Protein-losing enteropathy in patients with Fontan circulation: is it triggered by infection?

INTRODUCTION: Protein-losing enteropathy (PLE) is a recognised complication of the Fontan circulation. Its pathogenesis is not fully understood, however, and it is unclear why its onset occurs months or even years after Fontan surgery. PATIENTS: We report a 4.5-year-old girl with Fontan circulation who developed PLE almost 1 year after surgery. At the time of onset the patient had rotavirus enteritis and streptococcal tonsillitis. We have reviewed the records of seven other patients with longstanding PLE. In six of these patients we identified infections at the onset of symptoms. None of our patients had evidence of opportunistic infection. DISCUSSION AND CONCLUSION: The immune system of patients with PLE is compromised, but reports on recurrent opportunistic infections are rare. The present observations suggest that infection and inflammation may be associated with the onset of PLE. The mechanism of how infection may trigger PLE warrants further investigation.

Child↗

PubMed2003

Protein-losing enteropathy after fontan surgery: is assessment of risk patients with immunological data possible?

BACKGROUND: Protein-losing enteropathy (PLE) is a late complication of the Fontan type surgery for univentricular heart characterized by massive enteric protein loss. The pathogenesis of PLE is not fully understood, and it is unclear why the onset of PLE varies widely and occurs months or even years after surgery. Besides characteristic laboratory findings, a typical cellular feature concerns the almost selective loss of CD4(+) lymphocytes at an only slightly changed CD8(+) lymphocyte count. The present pilot study aimed to test whether immunological or laboratory parameters differ in patients at risk for PLE. METHODS: From children (n = 15) with Fontan type circulation, extensive cellular, humoral, and clinical laboratory data were analyzed. Patients without enteric protein loss (group I, n = 8), with transient phases of enteric protein loss in the absence of gastric infections (group II, n = 6), and one PLE patient (group III) were distinguished. The 90 data columns obtained in phases with normal serum protein levels were compared. RESULTS: Clear differences were apparent between patients prior to PLE onset (group III), patients that in at least one occasion exhibited PLE signs (group II), and patients without detectable PLE signs (group I). The most discriminatory parameters between the three patient groups were NK and CD8(+)TCRalphabeta(+), CD8(+)TCRgammadelta(+) cell counts, including sL-selectin, IgE, and Ca(2+) (average recognition index = 91.5%, negative/positive prediction/sensitivity/specificity > 83%). CONCLUSIONS: The results of this study seem to provide access to the early detection of PLE patients.

Adolescent↗

PubMed2002

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry.

BACKGROUND: To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. METHODS: The optical filters of the LSC were replaced-photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. RESULTS: With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. CONCLUSIONS: With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC.

Benzothiazoles↗