Multiplex Capabilities for Serological Immunoassays Using Luminex xMAP® Technology
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F.103 Differences in Telomerase Expression by the CD1a+ Cells in Langerhans Cell Histiocytosis Reflects the Diverse Clinical Presentation of the Disease Cristiana Costa, PhD Student, Leiden University Medical Center, Department of Pediatrics, Leiden, The Netherlands, Maarten Egeler, MD PhD, Leiden University Medical Center, Leiden, The Netherlands, Manja Hoogeboom, Leiden University Medical Center, Leiden, The Netherlands, Ramses Forsyth, N. Goormaghtigh Institute of Pathology, University Hospital Gent, Gent, Karoly Szuhai, PhD, Leiden University Medical Center, Leiden, The Netherlands, Marieke Niesters, Leiden University Medical Center, Department of Pediatrics, Leiden, The Netherlands, Ronald de Krijger, Erasmus Medical Center, Department of Pathology, Rotterdam, Abdellatif Tati, Service de Pneumonologie, Hospital Saint Louis, Paris, Pancras Hogendoorn, Leiden University Medical Center, Department of Pathology, Leiden, The Netherlands, Nicola Annels, PhD, Leiden University Medical Center, Department of Pediatrics, Leiden, The Netherlands Langerhans cell histiocytosis (LCH) is a disease charac- terised by an uncontrolled clonal proliferation of Langerhans cells, whose aetiology is still unclear. The clonal nature of LCH could support the hypothesis that it is a neoplastic disease with unlimited growth potential. One requirement for unlimited proliferation is the maintenance of telomere length. In a group of 70 patients we investigated whether a telomere maintenance mechanism is active by LCH cells. Results showed that LCH cells from all restricted skin LCH lesions (6/6) expressed telomerase as assessed by human telomere reverse transcriptase (hTERT) immunohistochem- istry, whereas LCH cells from the majority of bone lesions analysed did not (26/34). Interestingly, in contrast to solitary bone lesions, LCH cells from lesions of multisystem patients always expressed telomerase (11/11), regardless of the lesional site. In situ telomeric repeat amplification protocol (TRAP) assay performed on different lesional sites showed that telomerase was active. In addition, the telomere length of LCH cells from a hTERT positive skin multisystem lesion was long and homogeneous when compared to the LCH cells from hTERT negative bone single system LCH lesions, which were heterogeneous in length. No evidence for an alter- native lengthening of telomeres mechanism was found in hTERT negative lesions. The difference in telomerase expression and telomere length in the different lesional sites and in biopsies from patients with solitary versus multisystem disease appears to be reflective of the diverse clinical presentation and course of LCH. The results from this study have important implications for understanding the nature of this disease. doi:10.1016/j.clim.2007.03.316 F.104 Multiplex Capabilities for Serological Immunoassays Using Luminex xMAP ® Technology Harold Baker, Senior Scientist, Luminex Corporation, Austin, TX The Luminex xMAP technology incorporates fluorescently encoded microspheres (xMAP microspheres) to perform multiplexed bioassays in conjunction with advanced digital signal processing and proprietary identification techniques. The advantage of the multiplexing technology is the capability to measure several antigens within one sample. The combination of multiple results and reduced sample volume increases the efficiency for testing and sample consumption. Another advantage is that the serological assays can be multiplexed with other immunoassays. Five specific antigens were coupled to xMAP microspheres representing several individual regions. BSA coated micro- spheres were prepared to function as internal assay controls for non-specific reactivity. A total of five regions were used with each antigen to develop a 30-plex assay. The xMAP serological assay format we used was a 60 minute, no wash assay. Diluted patient serum, 10 ƒÝL, was combined with the microsphere mixture, 50 ƒÝL, and incubated 30 min. Diluted PE conjugated antibody, 150 ƒÝL, was added and incubated 30 min before being analyzed with the Luminex system. Equivalent results were obtained with the replicate antigen coated regions. Analytical sensitivity was defined by the maximum dilution before an appreciable decrement in MFI response was observed. This was observed to be at a dilution of 1:1000 representing the use of 0.01 ƒÝL serum for the measurement of antibodies in the serum sample. These results demonstrate that the Luminex xMAP technology can be used to develop multiplex assays such as those required for serological assessment of autoimmune diseases. doi:10.1016/j.clim.2007.03.319 F.105 Immunofluorometrical Exploring of FSH Levels in the Serum of Patients with Histologically Verified Macrocellular Lung Cancer Ivan Milosevic, Primary Care Physician, Dispensary 2, ZZZZR, Kragujevac It is already known in that the cells of macrocellular lung cancer can produce gonadotropins. By following the levels of FSH as one of gonadotropins, in the serum of macrocellular lung cancer patients, we could make certain statistical conclusions how significant these levels would be in eventual future early diagnostic procedures, besides already existing tumor markers and other methods. This work would connect the Oncology, Endocrinology and Immunology fields, contain- ing very interesting immunofluorometrical procedures, hormonal theories and statistical estimates. As an immuno- logical part in this theoretical model, it is represented through figure, time-resolved fluoroimmunoassay for quan- titative detection of FSH. By following of FSH levels in the serum of microcellular lung cancer patients and making determinate statistical conclusions about its importance, that procedure could be eventually used as one of early or advanced diagnostic methods concerning mentioned disease. What would we get with that? The histological verification is very slow and painful, when the macrocellular lung cancer is in the question. This histological type gives the metastases rapidly and it needs to be diagnosed in the fastest possible way in order to be prevented by adequate reaction and therapy. At the same time the biopsy as a very invasive method would be avoided. 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Transcript of Multiplex Capabilities for Serological Immunoassays Using Luminex xMAP® Technology
F.103 Differences in Telomerase Expression by theCD1a+ Cells in Langerhans Cell Histiocytosis Reflectsthe Diverse Clinical Presentation of the DiseaseCristiana Costa, PhD Student, Leiden University MedicalCenter, Department of Pediatrics, Leiden, The Netherlands,Maarten Egeler, MD PhD, Leiden University Medical Center,Leiden, The Netherlands, Manja Hoogeboom, LeidenUniversity Medical Center, Leiden, The Netherlands, RamsesForsyth, N. Goormaghtigh Institute of Pathology, UniversityHospital Gent, Gent, Karoly Szuhai, PhD, Leiden UniversityMedical Center, Leiden, The Netherlands, Marieke Niesters,Leiden University Medical Center, Department of Pediatrics,Leiden, The Netherlands, Ronald de Krijger, ErasmusMedical Center, Department of Pathology, Rotterdam,Abdellatif Tati, Service de Pneumonologie, Hospital SaintLouis, Paris, Pancras Hogendoorn, Leiden University MedicalCenter, Department of Pathology, Leiden, The Netherlands,Nicola Annels, PhD, Leiden University Medical Center,Department of Pediatrics, Leiden, The Netherlands
Langerhans cell histiocytosis (LCH) is a disease charac-terised by an uncontrolled clonal proliferation of Langerhanscells, whose aetiology is still unclear. The clonal nature ofLCH could support the hypothesis that it is a neoplasticdisease with unlimited growth potential. One requirementfor unlimited proliferation is the maintenance of telomerelength. In a group of 70 patients we investigated whether atelomere maintenance mechanism is active by LCH cells.Results showed that LCH cells from all restricted skin LCHlesions (6/6) expressed telomerase as assessed by humantelomere reverse transcriptase (hTERT) immunohistochem-istry, whereas LCH cells from the majority of bone lesionsanalysed did not (26/34). Interestingly, in contrast to solitarybone lesions, LCH cells from lesions of multisystem patientsalways expressed telomerase (11/11), regardless of thelesional site. In situ telomeric repeat amplification protocol(TRAP) assay performed on different lesional sites showedthat telomerase was active. In addition, the telomere lengthof LCH cells from a hTERT positive skin multisystem lesionwas long and homogeneous when compared to the LCH cellsfrom hTERT negative bone single system LCH lesions, whichwere heterogeneous in length. No evidence for an alter-native lengthening of telomeres mechanism was found inhTERT negative lesions. The difference in telomeraseexpression and telomere length in the different lesionalsites and in biopsies from patients with solitary versusmultisystem disease appears to be reflective of the diverseclinical presentation and course of LCH. The results from thisstudy have important implications for understanding thenature of this disease.
doi:10.1016/j.clim.2007.03.316
F.104 Multiplex Capabilities for SerologicalImmunoassays Using Luminex xMAP® TechnologyHarold Baker, Senior Scientist, Luminex Corporation,Austin, TX
The Luminex xMAP technology incorporates fluorescentlyencoded microspheres (xMAP microspheres) to perform
multiplexed bioassays in conjunction with advanced digitalsignal processing and proprietary identification techniques.The advantage of the multiplexing technology is thecapability to measure several antigens within one sample.The combination of multiple results and reduced samplevolume increases the efficiency for testing and sampleconsumption. Another advantage is that the serologicalassays can be multiplexed with other immunoassays. Fivespecific antigens were coupled to xMAP microspheresrepresenting several individual regions. BSA coated micro-spheres were prepared to function as internal assay controlsfor non-specific reactivity. A total of five regions were usedwith each antigen to develop a 30-plex assay. The xMAPserological assay format we used was a 60 minute, no washassay. Diluted patient serum, 10 ƒÝL, was combined with themicrosphere mixture, 50 ƒÝL, and incubated 30 min. DilutedPE conjugated antibody, 150 ƒÝL, was added and incubated30 min before being analyzed with the Luminex system.Equivalent results were obtained with the replicate antigencoated regions. Analytical sensitivity was defined by themaximum dilution before an appreciable decrement in MFIresponse was observed. This was observed to be at a dilutionof 1:1000 representing the use of 0.01 ƒÝL serum for themeasurement of antibodies in the serum sample. Theseresults demonstrate that the Luminex xMAP technology canbe used to develop multiplex assays such as those requiredfor serological assessment of autoimmune diseases.
doi:10.1016/j.clim.2007.03.319
F.105 Immunofluorometrical Exploring of FSHLevels in the Serum of Patients with HistologicallyVerified Macrocellular Lung CancerIvan Milosevic, Primary Care Physician, Dispensary 2, ZZZZR,Kragujevac
It is already known in that the cells of macrocellular lungcancer can produce gonadotropins. By following the levels ofFSH as one of gonadotropins, in the serum of macrocellularlung cancer patients, we could make certain statisticalconclusions how significant these levels would be in eventualfuture early diagnostic procedures, besides already existingtumor markers and other methods. This work would connectthe Oncology, Endocrinology and Immunology fields, contain-ing very interesting immunofluorometrical procedures,hormonal theories and statistical estimates. As an immuno-logical part in this theoretical model, it is representedthrough figure, time-resolved fluoroimmunoassay for quan-titative detection of FSH. By following of FSH levels in theserum of microcellular lung cancer patients and makingdeterminate statistical conclusions about its importance,that procedure could be eventually used as one of early oradvanced diagnostic methods concerning mentioned disease.What would we get with that? The histological verification isvery slow and painful, when the macrocellular lung cancer isin the question. This histological type gives the metastasesrapidly and it needs to be diagnosed in the fastest possibleway in order to be prevented by adequate reaction andtherapy. At the same time the biopsy as a very invasivemethod would be avoided. When the tumor markers are in
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