Portal de Periódicos da CAPES

Tissue microarray construction from bone marrow biopsies

2005; Future Science Ltd; Volume: 39; Issue: 6 Linguagem: Inglês

10.2144/000112073

1940-9818

Ellen C. Obermann, Joerg Marienhagen, Robert Stoehr, Peter H. Wuensch, Ferdinand Hofstaedter,

Monoclonal and Polyclonal Antibodies Research

BioTechniquesVol. 39, No. 6 BenchmarksOpen AccessTissue microarray construction from bone marrow biopsiesEllen C. Obermann, Joerg Marienhagen, Robert Stoehr, Peter H. Wuensch & Ferdinand HofstaedterEllen C. Obermann*Address correspondence to: Ellen C. Obermann, Institute of Pathology, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany. e-mail: E-mail Address: ellen.obermann@klinik.uni-regensburg.deUniversity of Regensburg, Regensburg, Joerg MarienhagenUniversity of Regensburg, Regensburg, Robert StoehrUniversity of Regensburg, Regensburg, Peter H. WuenschClinical Center Nuremberg, Nuremberg, Germany & Ferdinand HofstaedterUniversity of Regensburg, RegensburgPublished Online:30 May 2018https://doi.org/10.2144/000112073AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInReddit Tissue microarray (TMA) methodology has accelerated tissue analysis by in situ technologies. This technology allows rapid evaluation of large numbers of tissue samples (1–4). In this technique, small tissue samples are taken from tissue blocks containing routinely processed specimens and inserted into a recipient block that may contain up to 1000 specimens (5). Any antibody staining, in situ hybridization, and other molecular detection technology, which is applicable to whole tissue sections, can also be adapted to TMA slides (6). However, TMAs have, so far, mostly been made from paraffin-embedded tissue samples in which the original block contained relatively large specimens. It has been said that "it is not a major problem for a skilled technician to produce tissue microarrays from decalcified bone marrow biopsies" (7), but as yet, no study has been published proving the applicability of this method.Availability of TMAs from bone marrow biopsies (BMBs) would be especially useful in hematopathologic research, because a large number of neoplasms of the lymphatic and hematopoietic system involve the bone marrow. In some entities, such as leukemia, BMBs can be the only type of tissue containing neoplastic cells that is available for the histopathologist. The high-throughput, time- and cost-efficient TMA technology is therefore highly desirable to facilitate research in this field.There are several draw backs when dealing with BMBs. The major challenge is that the average BMB is not more than 2.0 mm wide and 2.5 cm long; however, a thickness of the donor tissue of 3–4 mm is usually required for TMA construction (6).Therefore, we modified the conventional technique of constructing TMAs and adjusted it to the special requirements of BMBs. Basically, we followed the protocol for manufacturing TMAs as described previously (8) and modified it according to the needs of the material used. Briefly, routinely processed BMBs were retrieved from the archives of the Institutes of Pathology at the University of Regensburg and the Clinical Center of Nuremberg. Areas of interest were marked on the original hematoxylin and eosin (H&E)-stained slide. Biopsies were taken from the preselected areas in the original blocks and inserted into premade holes in the acceptor block at defined array coordinates using a specially manufactured stainless steel tube with a wall thickness of 0.1 mm and a diameter of 1.6 mm (Sueddeutsche Feinmechanik GmbH, Waechtersbach, Germany). This size had been found to give the best results regarding preservation of the punch biopsy. Most importantly, instead of inserting the tissue samples next to each other in different holes in the donor block, as it is usually done, we stacked up to three punch biopsies obtained from one donor block on top of each other into one single hole in the acceptor block (Figure 1). Using this "stack method" a final thickness of tissue in the donor block of up to 4 mm can be achieved. Equal thickness of almost all the samples in a TMA block is especially desirable, since it avoids the well-known problem of cores being not yet or not any more cut from a TMA (9). However, a minor "loss of tissue" might be observed in sections between individual stacks.Figure 1. Construction of a tissue microarray(TMA) using the stack method.Core biopsies are punched from the original paraffin block (left) using a stainless steel needle and put on top of each other into prenade holed in the TMA block (right).Despite the small size of the samples, it was still possible in most cases to construct three cores from the same case in the final TMA; this is desirable because additional cores of identical cases increase the agreement in results between conventional sections and TMA (10). So far, we have successfully constructed four TMAs from BMBs, altogether containing 75 cases.In order to prove the validity of the stack method, we assessed the expression of several cell cycle markers by immunohistochemistry—both in whole tissue samples and in the core biopsies in one of our newly constructed TMAs containing 33 core biopsies from 11 cases of chronic lymphocytic leukemia (CLL) (Figure 2).Figure 2. Tissue microarrays (TMAs) constructed from bone marrow biopsies (BMBs) utilizing the stack method.This TMA contains 33 cores of 11 BMBs infiltratedby chronic lymphocytic leukemia (CLL). (A) This overview of a hematoxylin and eosin (H&E)-stained slide shows four core biopsies. The architecture of both marrow and bone structures are well preserved. Cellularity and the disiribution of cells can easily be appreciated. A nodular infiltrate by CLL is clearly recognizable (arrow). (B) This close-up view of a core biopsy stained for the proliferation marker Ki-67 shows disiribution of fat cells and hematopoietic/lymphatic cells. There is excellent preservation of the bone trabecula (arrow).Whole sections from the donor blocks before construction of the microarray and sections from the final microarrays were stained for Ki-67 (mouse monoclonal, clone MIBI; DAKO, Hamburg, Germany; final dilution 1:50), BM28 for detection of Mcm2 (mouse monoclonal, clone 46; BD Biosciences, San Jose, CA, USA; final dilution 1:3000), and Cyclin E (mouse monoclonal, clone 13A3; NovoCastra, Newcastle, UK; final dilution 1:5) using standard procedures with heat-induced antigen retrieval (microwave for 30 min at 320 W) and visualization of staining by the avidinbiotin peroxidase method with diaminobenzidine chromatogen.Ki-67 was chosen because it is the standard proliferation marker in histopathological research (11), while Mcm2 denotes the proliferative potential of cells and has been exploited as a novel marker of tumor growth (12). Cyclin E overexpression has been reported in several neoplasms including hematological neoplasias (13). Therefore, these markers are likely to be used in further studies utilizing TMAs of BMBs.One hundred cells were assessed in both whole sections and tumor cores, and the labeling index (LI) for each protein (percentage of cells with a distinct nuclear staining) was calculated. If more than one core biopsy was the available from the same case, the results were averaged. Statistical analyses were performed using SPSS version 10.0 (SPSS, Chicago, IL, USA). Evaluation of immunohistochemical staining was possible in 96 of 99 (97%) core biopsies. Noninformative cases were attributable to the array technology (i.e., loss of tissue) (9). However, more than one core biopsy of the same BMB was available in these cases. The comparison of LIs for both whole section and core biopsies showed correlations for all markers (intraclass correlation coefficient: 0.881 for Ki-67, 0.619 for Mcm2, and 0.858 for Cyclin E), which were all significant (P values: <0.001 for Ki-67, 0.013 for Mcm2, and <0.001 for Cyclin E).In summary, we show that construction of a TMA from BMBs is possible using the stack method and prove the validity of our approach by comparing immunohistochemical results obtained from whole tissue sections and core biopsies. This method can easily be performed by anyone experienced in the construction of conventional TMAs. Furthermore, the stack method may be transferred to other fields of research where only small tissue samples, such as renal or liver biopsies, are available.AcknowledgmentsThe authors thank A. Vielberth and R. Jung for excellent technical assistance. This study was supported by a grant of the University of Regensburg to E.C.O. (Regensburger Forschungsförderung in der Medizin: ReForMA).Competing Interests StatementThe authors declare no competing interests.References1. Bubendorf, L., J. Kononen, P. Koivisto, P. Schraml, H. Moch, T.C. Gasser, N. Willi, M.J. Mihatsch, et al.. 1999. Survey of gene amplifications during prostate cancer progression by high-throughout fluorescence in situ hybridization on tissue microarrays. Cancer Res. 59:803–806.Medline, CAS, Google Scholar2. Kononen, J., L. Bubendorf, A. Kallioniemi, M. Barlund, P. Schraml, S. Leighton, J. Torhorst, M.J. Mihatsch, et al.. 1998. Tissue microarrays for high-throughput molecular profiling of tumor specimens. Nat. Med. 4:844–847.Crossref, Medline, CAS, Google Scholar3. Barlund, M., F. Forozan, J. Kononen, L. Bubendorf, Y. Chen, M.L. Bittner, J. Torhorst, P. Haas, et al.. 2000. Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis. J. Natl. Cancer Inst. 92:1252–1259.Crossref, Medline, CAS, Google Scholar4. Bubendorf, L., M. Kolmer, J. Kononen, P. Koivisto, S. Mousses, Y. Chen, E. Mahlamaki, P. Schraml, et al.. 1999. Hormone therapy failure in human prostate cancer: analysis by complementary DNA and tissue microarrays. J. Natl. Cancer Inst. 91:1758–1764.Crossref, Medline, CAS, Google Scholar5. Bubendorf, L., A. Nocito, H. Moch, and G. Sauter. 2001. Tissue microarray (TMA) technology: miniaturized pathology archives for high-throughput in situ studies. J. Pathol. 195:72–79.Crossref, Medline, CAS, Google Scholar6. Kallioniemi, O.P., U. Wagner, J. Kononen, and G. Sauter. 2001. Tissue microarray technology for high-throughput molecular profiling of cancer. Hum. Mol. Genet. 10:657–662.Crossref, Medline, CAS, Google Scholar7. Packeisen, J., E. Korsching, H. Herbst, W. Boecker, and H. Buerger. 2003. Demysiified…tissue microarray technology. Mol. Pathol. 56:198–204.Crossref, Medline, CAS, Google Scholar8. Tzankov, A., A. Zimpfer, A.C. Pehrs, A. Lugli, P. Went, R. Maurer, S. Pileri, and S. Dirnhofer. 2003. Expression of B-cell makers in classical hodgkin lymphoma: a tissue microarray analysis of 330 cases. Mod. Pathol. 16:1141–1147.Crossref, Medline, Google Scholar9. Mengel, M., H. Kreipe, and R. Von Wasielewski. 2003. Rapid and large-scale transition of new tumor biomarkers to clinical biopsy material by innovative tissue microarray systems. Appl. Immunohistochem. Mol. Morphol. 11:261–268.Crossref, Medline, CAS, Google Scholar10. Camp, R.L., L.A. Charette, and D.L. Rimm. 2000. Validation of tissue microarray technology in breast carcinoma. Lab. Invest. 80:1943–1949.Crossref, Medline, CAS, Google Scholar11. Endl, E., C. Hollmann, and J. Gerdes. 2001. Antibodies against the Ki-67 protein: assessment of the growth fraction and tools for cell cycle analysis. Methods Cell Biol. 63:399–418.Crossref, Medline, CAS, Google Scholar12. Stoeber, K., T.D. Tlsty, L. Happerfield, G.A. Thomas, S. Romanov, L. Bobrow, E.D. Williams, and G.H. Williams. 2001. DNA replication licesencing and human cell proliferation. J. Cell Sci. 114:2027–2041.Crossref, Medline, CAS, Google Scholar13. Schraml, P., C. Bucher, H. Bissig, A. Nocito, P. Haas, K. Wilber, S. Seelig, J. Kononen, et al.. 2003. Cyclin E overexpression and amplification in human tumours. J. Pathol. 200:375–382.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByRecommendations for Tissue Microarray Construction and Quality AssuranceApplied Immunohistochemistry & Molecular Morphology, Vol. 28, No. 4Tissue microarrays of bone marrow aspirate clot allow assessment of multiple samplesPathology - Research and Practice, Vol. 216, No. 1Tissue microarray technique is applicable to bone marrow biopsies of myeloproliferative neoplasms20 August 2016 | Histochemistry and Cell Biology, Vol. 147, No. 1Genomic Copy Number Variations Characterize the Prognosis of Both P16-Positive and P16-Negative Oropharyngeal Squamous Cell Carcinoma After Curative ResectionMedicine, Vol. 94, No. 50Human papillomavirus in oropharyngeal squamous cell carcinomas in korea: Use of G1 cycle markers as new prognosticators2 November 2011 | Head & Neck, Vol. 34, No. 10Applications of Tissue Microarray Technology21 July 2010Tissue Microarrays from Biopsy Specimens21 July 2010In situ analysis of the antigen-processing machinery in acute myeloid leukaemic blasts by tissue microarray15 January 2009 | Leukemia, Vol. 23, No. 5Modern techniques for the diagnostic evaluation of the trephine bone marrow biopsy: Methodological aspects and applicationsProgress in Histochemistry and Cytochemistry, Vol. 42, No. 4Feasibility of constructing tissue microarrays from diagnostic prostate biopsies1 January 2007 | The Prostate, Vol. 67, No. 10 Vol. 39, No. 6 Follow us on social media for the latest updates Metrics History Received 15 September 2005 Accepted 17 October 2005 Published online 30 May 2018 Published in print December 2005 Information© 2005 Author(s)AcknowledgmentsThe authors thank A. Vielberth and R. Jung for excellent technical assistance. This study was supported by a grant of the University of Regensburg to E.C.O. (Regensburger Forschungsförderung in der Medizin: ReForMA).Competing Interests StatementThe authors declare no competing interests.PDF download