Summary
Properly collected and stored human specimens offer the unique opportunity to study human diseases at the molecular level in the real in vivo situation. The intention of this chapter is, first, to raise the awareness for important points, which have to be clarified before human tissue samples are collected and stored for molecular studies. Second, detailed protocols are provided for the fixation and processing of bone marrow trephines and the isolation of morphologically and immunohistochemically defined pure cell populations from bone marrow trephine sections using laser-assisted microdissection.
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References
Bauer, K., Taub, S., and Parsi, K. (2004) Ethical issues in tissue banking for research: a brief review of existing organizational policies. Theor Med Bioeth 25, 113–142.
Caulfield, T. (2004) Tissue banking, patient rights, and confidentiality: tensions in law and policy. Med Law 23, 39–49.
Lehmann, U. and Kreipe, H. (2001) Realtime PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies. Methods 25, 409–418.
Fend, F., Bock, O., Kremer, M., Specht, K., and Quintanilla-Martinez, L. (2005) Ancillary techniques in bone marrow pathology: molecular diagnostics on bone marrow trephine biopsies. Virchows Arch 447, 909–919.
Becker, K. F., Schott, C., Hipp, S., Metzger, V., Porschewski, P., Beck, R., Nahrig, J., Becker, I., and Hofler, H. (2007) Quantitative protein analysis from formalin-fixed tissues: implications for translational clinical research and nanoscale molecular diagnosis. J Pathol 211, 370–378.
Fend, F., Emmert-Buck, M. R., Chuaqui, R., Cole, K., Lee, J., Liotta, L. A., and Raf-feld, M. (1999) Immuno-LCM: laser capture microdissection of immunostained frozen sections for mRNA analysis. Am J Pathol 154, 61–66.
Bock, O., Schlué, J., Lehmann, U., von Wasielewski, R., Länger, F., and Kreipe, H. (2002) Megakaryocytes from myeloprolifera-tive disorders show enhanced nuclear bFGF expression. Blood 100, 2274–2275.
Bock, O., Loch, G., Schade, U., von Wasielewski, R., Schlue, J., and Kreipe, H. (2005) Aberrant expression of transforming growth factor beta-1 (TGF beta-1) per se does not discriminate fibrotic from non-fibrotic chronic myeloproliferative disorders. J Pathol 205, 548–557.
Hussein, K., Brakensiek, K., Buesche, G., Buhr, T., Wiese, B., Kreipe, H., and Bock, O. (2007) Different involvement of the meg-akaryocytic lineage by the JAK2 (V617F) mutation in Polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis. Ann Hematol 86, 245–253.
Lehmann, U., Glockner, S., Kleeberger, W., von Wasielewski, H. F., and Kreipe, H. (2000) Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction. Am J Pathol 156, 1855–1864.
Glockner, S., Lehmann, U., Wilke, N., Klee-berger, W., Langer, F., and Kreipe, H. (2001) Amplification of growth regulatory genes in intraductal breast cancer is associated with higher nuclear grade but not with the progression to invasiveness. Lab Invest 81, 565–571.
Lehmann, U., Langer, F., Feist, H., Glock-ner, S., Hasemeier, B., and Kreipe, H. (2002) Quantitative assessment of promoter hyper-methylation during breast cancer development. Am J Pathol 160, 605–612.
Kleeberger, W., Versmold, A., Rothamel, T., Glockner, S., Bredt, M., Haverich, A., Leh-mann, U., and Kreipe, H. (2003) Increased chimerism of bronchial and alveolar epithelium in human lung allografts undergoing chronic injury. Am J Pathol 162, 1487–1494.
Bröcker, V., Langer, F., Fellous, T. G., Mengel, M., Brittan, M., Bredt, M., Milde, S., Welte, T., Eder, M., Haverich, A., Alison, M. R., Kreipe, H., and Lehmann, U. (2006) Fibroblasts of recipient origin contribute to bronchiolitis oblit-erans in human lung transplants. Am J Respir Crit Care Med 173, 1276–1282.
Bock, O., Kreipe, H., and Lehmann, U. (2001) One-step extraction of RNA from archival biopsies. Anal Biochem 295, 116–117.
Tbakhi, A., Totos, G., Pettay, J. D., Myles, J., and Tubbs, R. R. (1999) The effect of fixation on detection of B-cell clonality by polymerase chain reaction. Mod Pathol 12, 272–278.
Wan, X., Cochran, G., and Greiner, T. C. (2003) Removal of mercuric chloride deposits from B5-fixed tissue will affect the performance of immunoperoxidase staining of selected antibodies. Appl Immunohistochem Mol Morphol 11, 92–95.
Suggested Readings
First description of the laser pressure catapulting technology from P.A.L.M.
Schütze, K. and Lahr, G. Identification of expressed genes by laser-mediated manipulation of single cells [see comments]. Nat Biotechnol, 16: 737–742, 1998.
First and comprehensive description of the Actu-rus system, which is based on different principals for dissection and specimen recovery and a very good early review.
Emmert-Buck, M. R., Bonner, R. F., Smith, P. D., Chuaqui, R. F., Zhuang, Z., Goldstein, S. R., Weiss, R. A., and Liotta, L. A. Laser capture microdissection [see comments]. Science, 274: 998–1001, 1996.
Simone, N. L., Bonner, R. F., Gillespie, J. W., Emmert-Buck, M. R., and Liotta, L. A. Laser-capture microdissection: opening the microscopic frontier to molecular analysis. Trends Genet, 14: 272–276, 1998.
A detailed practical guide introducing the Arcturus system can be found in “Current Protocols of Molecular Biology”(eds. Ausubel X et al., Wiley.)
First description of quantitative mRNA analysis in laser-microdissected cells.
Fink, L., Seeger, W., Ermert, L., Hanze, J., Stahl, U., Grimminger, F., Kummer, W., and Bohle, R. M. Real-time quantitative RT-PCR after laser-assisted cell picking. Nat Med, 4: 1329–1333, 1998.
First description of cDNA array based expression profiling of laser-microdissected cells.
Luo, L., Salunga, R. C., Guo, H., Bittner, A., Joy, K. C., Galindo, J. E., Xiao, H., Rogers, K. E., Wan, J. S., Jackson, M. R., and Erlander, M. G. Gene expression profiles of laser-captured adjacent neuronal subtypes [published erratum appears in Nat Med 1999 Mar;5(3):355]. Nat Med, 5: 117–122, 1999.
Two recent reviews about laser-microdissection.
Fink, L., Kwapiszewska, G., Wilhelm, J., and Bohle, R. M. Laser-microdissection for cell type- and compartment-specific analyses on genomic and proteomic level. Exp Toxicol Pathol, 57 Suppl 2 : 25–29, 2006.
Ladanyi, A., Sipos, F., Szoke, D., Galamb, O., Molnar, B., and Tulassay, Z. Laser microdis-section in translational and clinical research. Cytometry A, 69: 947–960, 2006.
Acknowledgments
The authors thank Britta Hasemeier for expert assistance in preparing Figure 1 and Oliver Bock for providing original photomicrographs. The work of the authors is supported by grants from the Deutsche Forschungsgemeinschaft and the Deutsche Kreb-shilfe.
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Lehmann, U., Hans, K. (2009). Tissue Procurement for Molecular Studies Using Laser-Assisted Microdissection. In: Baum, C. (eds) Genetic Modification of Hematopoietic Stem Cells. Methods In Molecular Biology™, vol 506. Humana Press. https://doi.org/10.1007/978-1-59745-409-4_20
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DOI: https://doi.org/10.1007/978-1-59745-409-4_20
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