- Email: [email protected]
Laser Microdissection in Clinical Cardiovascular Research
Laser Microdissection in Clinical Cardiovascular Research* Cristina Chimenti, MD, PhD; Maurizio Pieroni, MD, PhD; Andrea Russo, MD, PhD; Patrizio Sale, MD; Matteo A. Russo, MD; Attilio Maseri, MD; and Andrea Frustaci, MD, FCCP
Laser microdissection (LMD) is an accurate and fast method to procure pure populations of cells from complex, heterogeneous tissues under direct microscopic visualization. It can be applied to a wide range of cell preparation, including paraffin-embedded material. The morphology of the captured cells is retained, and DNA, RNA, and proteins can be extracted for molecular analysis. The potential applications of LMD to human cardiovascular research are multiple, including viral/autoimmune myocarditis and arteritis, atherosclerotic lesions, and myocardial and vascular cell proliferation and death. Molecular and genetic analysis of LMD-procured cells in cardiac and vascular tissues may provide a better understanding of several cardiac diseases. (CHEST 2005; 128:2876 –2881) Key words: cardiovascular diseases; endomyocardial biopsy; inflammatory cardiomyopathy; laser microdissection; myocarditis Abbreviations: EBV ⫽ Epstein-Barr virus; LMD ⫽ laser microdissection; mRNA ⫽ messenger RNA; mtDNA ⫽ mitochondrial DNA; PCR ⫽ polymerase chain reaction
growing number of genomic and proteomic A techniques have become available to assess mes-
senger RNA (mRNA) transcripts and proteins in human cells and tissues. In particular, the differential expression of these molecules between normal and pathologic tissues can provide indications for specific mechanisms of diseases. However, as tissues are usually composed of heterogeneous cell populations, the average information obtained from the molecular analysis of biological samples as a whole may be of limited value. To address this difficulty, several manual or automatic methods have been developed for selecting specific cell populations from a given tissue sample. Among these methods, laser microdissection (LMD) provides considerable advantages and can be successfully applied to human cardiovascular research.
*From the Cardiothoracic and Vascular Department (Drs. Chimenti, Pieroni, and Maseri), Vita e Salute University, Milan; Department of Cardiology, Catholic University (Dr. Frustaci), Rome; Biopathology and Diagnostic Imaging Department (Dr. A Russo), “Regina Elena” Institute, Rome; and Pathology and Experimental Medicine Department (Drs. Sale and M. Russo), “La Sapienza” University, Rome, Italy. Manuscript received February 7, 2005; revision accepted April 11, 2005. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Andrea Frustaci, MD, FCCP, Cardiology Department, Catholic University, Largo A Gemelli 8, 00168, Rome, Italy; e-mail: [email protected] 2876
Laser Microdissection: Methodologic Aspects LMD is a technique that enables the procurement of homogeneous types of cells from both frozen and fixed tissue sections and from cytologic smears, using a laser integrated into a standard microscope. It can be routinely utilized to collect pure populations of targeted cells for subsequent DNA, RNA, and protein analysis. Although the initial cost for the microscope and accompanying computer hardware and software is quite high, LMD has the advantages of being user friendly, precise, rapid, and guarantees the purity of the collected material, which is not feasible using previous manual microdissection techniques. Compared to LMD, in situ techniques such as in situ hybridization and in situ polymerase chain reaction (PCR)1–2 are time consuming and technically complex, while immunohistochemistry lacks comparable sensitivity and specificity and is not suitable for nucleic acid analysis (Table 1). Several systems are available for LMD, and vary in cell-capture method and system configuration (Table 2). The first LMD system, developed in the mid1990s as a research tool at the National Institutes of Health, uses a fusion technique for cell capture. Laser capture microdissection is based on the selective adherence of targeted cells and tissue fragments to a thermoplastic membrane, mounted on a transparent cap and activated by a low-energy infrared Reviews
Table 1—Comparison Between LMD and Other Diagnostic Methods* Method
Duration
Technical Skills
Sensitivity
Specificity
Cell Target Localization
Cost
Source
LMD† In situ hybridization In situ PCR Immunohistochemistry PCR on the whole tissue Manual microdissection
⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹` ⫹⫹ ⫹⫹⫹⫹
⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ no ⫹⫹⫹⫹
⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
Emmert-Buck et al3 Kadkol et al1 Teo and Shaunak2 Leong4 Pan et al5 Eltoum et al6
*⫹ ⫽ low; ⫹⫹ ⫽ intermediate; ⫹⫹⫹ ⫽ high; ⫹⫹⫹⫹ ⫽ very high. †Including the subsequent molecular analysis.
laser pulse.3,7,8 The thermoplastic film lies directly on the surface of the tissue section, mounted on a glass slide, and contains special infrared absorbing dyes that melt and fuse with the selected underlying cell type when the laser is activated. When the film is removed, the selected cells remain bound to the film, leaving behind the remaining tissue. The tissue suffers only brief thermal transients that do not damage DNA, RNA, or proteins. Importantly, this method can be applied also to stained, archival glass slides. The other widely used system is the laser microbeam microdissection system that uses a pulsed ultraviolet laser with a small beam to cut out the cells
of interest by photoablation of the adjacent tissue.9,10 Tissue sections are mounted on a thin polyethylene foil slide, and the cells of interest are microdissected by means of an ultraviolet laser that performs the circumferential dissection of the selected cells following a precisely drawn incision path (Fig 1). By this cold ablation, the material to be extracted is not directly exposed to the laser. The microdissected tissue areas are then collected on the cap of a nanotube for subsequent molecular analysis, using contamination and contact-free methods of cell recovery. When the cells of interest exhibit characteristic disease morphology, LMD can be carried out on
Table 2—Comparison Among Different Commercially Available LMD Systems* Variables
Arcturus Pix Cell IIe
mmi Cellcut
Leica AS LMD
PALM Microbeam
Tissue capture technique
The laser causes the thermoplastic film, attached to the cap, to melt with the target cells Lifting the cap from the tissue section removes the target cells
The laser cuts the specimens, mounted on a supporting membrane, around the target cells
The laser cuts the specimens mounted on a supporting membrane, around the target cells
The force of gravity lands the target cells on the collecting cap
The target cells are catapulted into the microfuge cap
Upright Leica DMLA
Inverted Zeiss Axiovert 200 Nitrogen ultraviolet 337 Membrane-covered glass slides Yes Yes Yes Yes Yes Yes Yes Yes
Microscope laser
Inverted Olympus IX50
Type Wavelength, nm Sections support
Solid-state near infrared 337 Glass slides
The laser cuts the specimens, mounted on a special membranecovered slide, around the target cells Retraction of the adhesive cap, that rests on the membrane, removes the target cells Inverted Nikon TE or others Solid-state ultraviolet 350 Membrane-covered slides
Fluorescence upgrade Applications Frozen sections Fixed sections Cytologic smears Living cells Single cell Micromanipulation Image archiving software
Yes
Yes
Nitrogen ultraviolet 337 Membrane-covered glass slides Yes
Yes Yes Yes Yes Yes No Yes
Yes Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes No Yes
Collection of the selected material
*Arcturus Pix Cell IIe (Arcturus Bioscience; Mountain View, CA); mmi Cellcut (MMI Molecular Machines and Industries; Manchester, NH); Leica AS LMD and Leica DMLA (Leica Microsystems; Bannockburn, IL); PALM Microbeam (P.A.L.M. Microlaser Technologies; Bernried, Germany); Olympus IX50 (Olympus America; Melville, NY); Nikon TE (Nikon Instruments; Melville, NY); Zeiss Axiovert (Carl Zeiss MicroImaging; Thornwood, NY). www.chestjournal.org
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Figure 1. LMD of endomyocardial biopsy tissue using the laser microbeam microdissection system. Top left, A: Giant-cell myocarditis with an extensive inflammatory infiltrate represented by lymphocytes, histiocytes, eosinophils, and multinuclear giant cells associated with cell necrosis. A giant cell is indicated by the arrow (hematoxylin-eosin, original ⫻ 200). Top right, B: The same field in which the path of the giant cell has been traced before the laser activation. Bottom left, C: The same field after microdissection of the selected cell. A hole is visible in the remaining tissue. Bottom right, D: The excised cell on the adhesive cap. All the steps of the procedure are examined with a microscope before proceeding to the next phase.
hematoxylin-eosin–stained tissue sections (Fig 1). However, in order to select cell types that are not readily distinguished by morphologic examination, the expression of specific cell markers by immunohistochemically guided procedures may be employed. In both the laser capture and laser microbeam microdissection systems, the laser is integrated into a standard microscope. The investigator can examine the tissue section microscopically before and after the microdissection procedure and control the homogeneity of the selected material, as the tissue collected on the cap retains its original morphology. These systems also offer the possibility to archive the images of each dissection, allowing the correlation of the histopathologic findings with the subsequent molecular results. Application of LMD to Cardiovascular Research Up to now, the major field of application of this method has been cancer research. Separation of malignant, in situ, and normal cell subpopulations 2878
within a single biopsy specimen allows a direct comparison of tissue DNA, RNA, and protein content and function in both normal and neoplastic tissues.11 This technique has been applied to detect both known and unknown alterations in the genomes of a variety of tumors.12 Moreover, microarray technology has been used to obtain molecular fingerprints of gene expression in microdissected tissue biopsies of human cancer, contributing to identify different disease stages and specific molecular-targeted therapies.13 Recently, this method has been applied to human cardiac and vascular tissue (Table 3). Moniotte et al14 used LMD to selectively capture myocytes and endothelial cells from cardiac frozen sections stained with hematoxylin-eosin. The authors quantitatively assessed the expression of rare mRNAs (ie, 3adrenoceptor mRNAs) in the small number of collected cells using real-time PCR, and demonstrated the efficacy of combining these two methods for gene expression studies in cardiac diseases. Similarly, De Souza et al15 performed LMD on frozen left and right ventricular tissue from explanted hearts to selectively collect blood vessels and myocytes, and analyzed the protein extracts by two-dimensional gel electrophoresis. Their results showed good protein recovery from the microdissected cells and welldistinct protein profiles, demonstrating the feasibility of using LMD to perform proteomic studies of individual cardiac tissue components. LMD has been also employed to analyze the level of deleted mitochondrial DNA (mtDNA) in the conducting myocardium in comparison with working myocardium in a patient with Kearns-Sayre syndrome,16 and showed a greater abundance of mutated mtDNA in the conducting system, consistent with its prevalent clinical involvement. More recently, the LMD technique was applied to endomyocardial biopsy specimens in order to precisely separate and collect the cardiomyocytes and the infiltrating lymphocytes in the cardiac tissue infected by Epstein-Barr virus (EBV).17 Paraffinembedded sections from nine patients with chronic heart failure and a histologic diagnosis of inflammatory cardiomyopathy, with PCR positivity for EBV on whole frozen tissue, were studied. LMD was applied in this series to assess whether the viral genome was localized in the myocytes, the inflammatory cells, or both. The selection of cells was immunohistochemically guided by ␣-sarcomeric actin antibody staining for cardiomyocytes and CD45RO staining for lymphocytes (Fig 2). EBV genome was detected by PCR analysis in cardiomyocytes of all patients and was absent in infiltrating lymphocytes, thus suggesting a cytopathic role of this virus and the opportunity for an antiviral/immunomodulatory therapy. ImporReviews
Table 3—Application of LMD in Human Cardiovascular Diseases Disease
Tissue Samples
Microdissected Cells
Molecular Study
Heart failure
Frozen explanted hearts Myocytes, endothelial cells Real-time PCR for 3adrenoceptor mRNA
Heart failure
Frozen explanted hearts Myocytes, blood vessels
Results
Quantitation of 3Moniotte et al14 adrenoceptor expression in myocytes and endothelial cells Distinct protein profiles De Souza et al15 from myocytes and blood vessels
Analysis of the protein extracts by twodimensional electrophoresis Kearns-Sayre syndrome Fixed autopsy heart Atrioventricular node, Competitive PCR for wild- Higher levels of mutated sinus node, bundle of type and mutated mtDNA in conducting His, and working mtDNA system than in working myocardium myocardium EBV-related myocarditis Fixed endomyocardial Myocytes, lymphocytes PCR for EBV DNA Detection of EBV genome biopsies in myocytes but not in lymphocytes Atherosclerosis Fixed arterial samples Atherosclerotic lesions, Gene expression profiles Identification of normal areas by cDNA array overexpressed genes in macrophage-rich areas Atherosclerosis Fixed carotid Caspase-2 immunoreactive Analysis of the protein Overexpression of caspaseendoarterectomies areas of atherosclerotic extracts by Western 2S with antiapoptotic plaques blotting effect Giant cell arteritis Frozen temporally artery Giant cells and Genomic representational Identification of microbial biopsies inflammatory cells, difference analysis on DNA sequences in the normal areas DNA extracts inflammatory lesions
tantly, this study showed that PCR analysis of nucleic acids extracted from a small number of microdissected cells does not cause a loss of sensitivity compared with PCR performed on the whole frozen tissue, with the advantage to allow the cellular localization of the viral agent. The combination of highly sensitive PCR analysis with the microscopical selection of the targeted cells might be useful in the investigation of the different cellular tropism (ie, cardiomyocytes, endothelial cells, fibroblasts, smooth muscle cells) of the viruses infecting the heart, thus improving our knowledge of viral myocarditis in terms of pathogenesis, clinical manifestations, and possible therapeutic options. Similarly, this method can be applied to human atherosclerotic lesions to investigate the presence of microbial agents in the different cellular components of the plaque (ie, foam cells, smooth-muscle cells, lymphocytes, endothelial cells, fibroblasts). Tuomisto et al18 used LMD to isolate macrophage-rich shoulder areas from human atherosclerotic lesions and compared their gene expression patterns to macroscopically normal diffuse intimal thickening by complementary DNA array. The authors identified several overexpressed genes, such as hydroxymethylglutaryl coenzyme A reductase, colony-stimulating factor receptors, integrin receptors, and nitric oxide synthase, and suggested the usefulness of this approach to identify therapeutic targets for the treatment of atherosclerotic diseases. www.chestjournal.org
Source
Pistilli et al16
Chimenti et al17
Tuomisto et al18
Martinet et al19
Gordon et al21
In another study, Martinet et al19 used LMD to isolate caspase-2—positive macrophage-derived foam cells around the necrotic core of atherosclerotic plaques in human carotid endoarterectomy specimens. The authors analyzed, using Western blotting, the proteins extracted from the targeted cells and identified the overexpression of a 35-kd protein (caspase-2S) with antiapoptotic effect, suggesting that the upregulation of this protein in macrophages can represent a mechanism to survive the increased levels of oxidative DNA damage present in the atherosclerotic plaque. LMD can also be useful to investigate inflammatory diseases of unknown etiology and uncertain treatment, involving the heart and/or the vessels, such as giant-cell myocarditis and arteritis. Giant cell myocarditis is a disease of young, predominantly healthy adults, frequently with a fatal course.20 Its etiology is unknown; it may recur after cardiac transplantation and sometimes responds to immunosuppressive therapy. Histologically, it is characterized by extensive inflammatory infiltrates with the presence of abnormal multinucleated cells, ie, giant cells, of possible myogenic or macrophagic origin. As illustrate in Figure 1, giant cells can be easily dissected from the endomyocardial tissue sample, and molecular analysis of the isolated material can give important information on the knowledge of this heterogeneous disease, providing indications for its treatment that are not established. Similarly, Gordon et al21 used LMD to isolate giant cells from CHEST / 128 / 4 / OCTOBER, 2005
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Figure 2. LMD of endomyocardial biopsy tissue from a patient with EBV myocarditis. LMD was guided by immunohistochemical detection of the targeted cells. Top left, A, and bottom left, D: ␣-Sarcomeric actin-labeled myocytes and CD45RO-labeled infiltrating lymphocytes, respectively (immunoperoxidase, original ⫻ 400). Top center, B, and bottom center, E: The same field after LMD of the selected cells. After the adhesive caps are removed, holes are visible on the remaining tissue. Top right, C, and bottom right, F: The captured cells on the nanotube caps. The homogeneity of the captured material was confirmed by microscopic visualization before DNA extraction.
inflammatory lesions of giant cell arteritis and identified the presence of microbial sequences in the DNA extracted from the collected material, showing that this disease, considered autoimmune in nature, may be associated with a microbial infection. Finally, the fact that the heart is no longer a postmitotic organ and that myocardial regeneration occurs in adult human heart opens a new potential field for application of LMD. Indeed, studies22,23 on the chimerism of the heart after sex-mismatched transplantation have provided consistent results concerning the migration of primitive cells from the host to the graft, showing the presence of Y chromosome in myocytes of female hearts transplanted into male recipients. A population of resident cardiac primitive cells able to differentiate in myocytes, smooth-muscle cells, and endothelial cells has been identified in the adult human heart, and it has been shown that cardiac regeneration is highly activated in acute pathologic conditions, while the regenerative capacity decreases in the chronically decompensated heart 2880
and in aged diseased hearts.24,25 LMD may offer the possibility to selectively study the newly generated myocytes as well as the resident cardiac primitive/ progenitor cells, recognized by specific cell markers. In addition, LMD allows the investigation of the expression of genes that can activate primitive cell growth and differentiation or inhibit cell replication and promote telomere erosion and primitive cell death in the acutely decompensated human heart, as well as in chronic heart failure. In conclusion, LMD is an extremely useful tool that can open new areas in the study of several cardiac diseases, combining advanced molecular biology techniques with the fast and precise selection of targeted cells under direct microscopic visualization of cardiac and vascular tissues. References 1 Kadkol SS, Gage WR, Pasternack GR. In situ hybridization: theory and practice. Mol Diagn 1999; 4:169 –183 Reviews
2 Teo IA, Shaunak S. Polymerase chain reaction in situ: an appraisal of an emerging technique. Histochem J 1995; 27:647– 659 3 Emmert-Buck MR, Bonner RF, Smith PD, et al. Laser capture microdissection. Science 1996; 274:998 –1001 4 Leong AS. Pitfall in diagnostic immunohistology. Adv Anat Pathol 2004; 11:86 –93 5 Pan LX, Diss TC, Isaacson PG. The polymerase chain reaction in histopathology. Histopathology 1995; 26:201–217 6 Eltoum IA, Siegal GP, Frost AR. Microdissection of histologic sections: past, present, and future. Adv Anat Pathol 2002; 9:316 –322 7 Bonner RF, Emmert-Buck MR, Cole K, et al. Laser capture microdissection: molecular analysis of tissue. Science 1997; 278:1481–1483 8 Fend F, Raffeld M. Laser capture microdissection in pathology. J Clin Pathol 2000; 53:666 – 672 9 Gjerdrum LM, Lielpetere I, Rasmussen LM, et al. Laserassisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis: identification of cell populations using immunohistochemistry and in situ hybridization. J Mol Diagn 2001; 3:105–110 10 Zhang L, Yang N, Conejo-Garcia JR, et al. Expression of endocrine gland-derived vascular endothelial growth factor in ovarian carcinoma. Clin Cancer Res 2003; 9:264 –272 11 Michener CM, Ardekani AM, Petricoin EF 3rd, et al. Genomics and proteomics: application of novel technology to early detection and prevention of cancer. Cancer Detect Prev 2002; 26:249 –255 12 Cheng L, MacLennan GT, Zhang S, et al. Laser capture microdissection analysis reveals frequent allelic losses in papillary urothelial neoplasm of low malignant potential of the urinary bladder. Cancer 2004; 101:183–188 13 Ma XJ, Salunga R, Tuggle JT. Gene expression profiles of human breast cancer progression. Proc Natl Acad Sci U S A 2003; 100:5974 –5979 14 Moniotte S, Vaerman JL, Kockx MM, et al. Real-time RT-PCR for the detection of -adrenoceptor messenger RNA’s in small human endomyocardial biopsies. J Mol Cell Cardiol 2001; 33:2121–2133
www.chestjournal.org
15 De Souza AI, McGregor E, Dunn MJ, et al. Preparation of human heart for laser microdissection and proteomics. Proteomics 2004; 4:578 –586 16 Pistilli D, Di Gioia CRT, D’Amati G, et al. Detection of deleted mitochondrial DNA in Kearns-Sayre syndrome using laser capture microdissection. Hum Pathol 2003; 34:1058 – 1061 17 Chimenti C, Russo A, Pieroni M, et al. Intramyocyte detection of Epstein Barr virus genome by laser capture microdissection in patients with inflammatory cardiomyopathy. Circulation 2004; 110:3534 –3539 18 Tuomisto TT, Korkeela A, Rutanen J, et al. Gene expression in macrophage-rich inflammatory cell infiltrates in human atherosclerotic lesions as studied by laser microdissection and DNA array: overexpression of HMG-CoA reductase, colony stimulating factor receptors, CD11A/CD18 integrins, and interleukin receptors. Arterioscler Thromb Vasc Biol 2003; 23:2235–2240 19 Martinet W, Knaapen MW, De Meyer GR, et al. Overexpression of the anti-apoptotic caspase-2 short isoform in macrophage-derived foam cells of human atherosclerotic plaques. Am J Pathol 2003; 162:731–736 20 Cooper LT Jr, Berry GJ, Shabetai R. Idiopathic giant-cell myocarditis: natural history and treatment: Multicenter Giant-Cell Myocarditis Study Group Investigators. N Engl J Med 1997; 336:1860 –1866 21 Gordon LK, Goldman M, Sandusky H, et al. Identification of candidate microbial sequences from inflammatory lesion of giant cell arteritis. Clin Immunol 2004; 111:286 –296 22 Quaini F, Urbanek K, Beltrami AP, et al. Chimerism of the transplanted heart. N Engl J Med 2002; 346:5–15 23 Mu¨ller P, Pfeiffer P, Koglin J, et al. Cardiomyocytes of noncardiac origin in myocardial biopsies of human transplanted hearts. Circulation 2002; 105:31–35 24 Urbanek K, Quaini F, Tasca G, et al. Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy. Proc Natl Acad Sci U S A 2003; 100:10440 –10445 25 Chimenti C, Kajstura J, Torella D, et al. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure. Circ Res 2003; 93:604 – 613
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Laser microdissection (LMD) is an accurate and fast method to procure pure populations of cells from complex, heterogeneous tissues under direct microscopic visualization. It can be applied to a wide range of cell preparation, including paraffin-embedded material. The morphology of the captured cells is retained, and DNA, RNA, and proteins can be extracted for molecular analysis. The potential applications of LMD to human cardiovascular research are multiple, including viral/autoimmune myocarditis and arteritis, atherosclerotic lesions, and myocardial and vascular cell proliferation and death. Molecular and genetic analysis of LMD-procured cells in cardiac and vascular tissues may provide a better understanding of several cardiac diseases. (CHEST 2005; 128:2876 –2881) Key words: cardiovascular diseases; endomyocardial biopsy; inflammatory cardiomyopathy; laser microdissection; myocarditis Abbreviations: EBV ⫽ Epstein-Barr virus; LMD ⫽ laser microdissection; mRNA ⫽ messenger RNA; mtDNA ⫽ mitochondrial DNA; PCR ⫽ polymerase chain reaction
growing number of genomic and proteomic A techniques have become available to assess mes-
senger RNA (mRNA) transcripts and proteins in human cells and tissues. In particular, the differential expression of these molecules between normal and pathologic tissues can provide indications for specific mechanisms of diseases. However, as tissues are usually composed of heterogeneous cell populations, the average information obtained from the molecular analysis of biological samples as a whole may be of limited value. To address this difficulty, several manual or automatic methods have been developed for selecting specific cell populations from a given tissue sample. Among these methods, laser microdissection (LMD) provides considerable advantages and can be successfully applied to human cardiovascular research.
*From the Cardiothoracic and Vascular Department (Drs. Chimenti, Pieroni, and Maseri), Vita e Salute University, Milan; Department of Cardiology, Catholic University (Dr. Frustaci), Rome; Biopathology and Diagnostic Imaging Department (Dr. A Russo), “Regina Elena” Institute, Rome; and Pathology and Experimental Medicine Department (Drs. Sale and M. Russo), “La Sapienza” University, Rome, Italy. Manuscript received February 7, 2005; revision accepted April 11, 2005. Reproduction of this article is prohibited without written permission from the American College of Chest Physicians (www.chestjournal. org/misc/reprints.shtml). Correspondence to: Andrea Frustaci, MD, FCCP, Cardiology Department, Catholic University, Largo A Gemelli 8, 00168, Rome, Italy; e-mail: [email protected] 2876
Laser Microdissection: Methodologic Aspects LMD is a technique that enables the procurement of homogeneous types of cells from both frozen and fixed tissue sections and from cytologic smears, using a laser integrated into a standard microscope. It can be routinely utilized to collect pure populations of targeted cells for subsequent DNA, RNA, and protein analysis. Although the initial cost for the microscope and accompanying computer hardware and software is quite high, LMD has the advantages of being user friendly, precise, rapid, and guarantees the purity of the collected material, which is not feasible using previous manual microdissection techniques. Compared to LMD, in situ techniques such as in situ hybridization and in situ polymerase chain reaction (PCR)1–2 are time consuming and technically complex, while immunohistochemistry lacks comparable sensitivity and specificity and is not suitable for nucleic acid analysis (Table 1). Several systems are available for LMD, and vary in cell-capture method and system configuration (Table 2). The first LMD system, developed in the mid1990s as a research tool at the National Institutes of Health, uses a fusion technique for cell capture. Laser capture microdissection is based on the selective adherence of targeted cells and tissue fragments to a thermoplastic membrane, mounted on a transparent cap and activated by a low-energy infrared Reviews
Table 1—Comparison Between LMD and Other Diagnostic Methods* Method
Duration
Technical Skills
Sensitivity
Specificity
Cell Target Localization
Cost
Source
LMD† In situ hybridization In situ PCR Immunohistochemistry PCR on the whole tissue Manual microdissection
⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹` ⫹⫹ ⫹⫹⫹⫹
⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹ ⫹⫹ ⫹⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹ ⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹⫹⫹ ⫹⫹⫹
⫹⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ no ⫹⫹⫹⫹
⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹
Emmert-Buck et al3 Kadkol et al1 Teo and Shaunak2 Leong4 Pan et al5 Eltoum et al6
*⫹ ⫽ low; ⫹⫹ ⫽ intermediate; ⫹⫹⫹ ⫽ high; ⫹⫹⫹⫹ ⫽ very high. †Including the subsequent molecular analysis.
laser pulse.3,7,8 The thermoplastic film lies directly on the surface of the tissue section, mounted on a glass slide, and contains special infrared absorbing dyes that melt and fuse with the selected underlying cell type when the laser is activated. When the film is removed, the selected cells remain bound to the film, leaving behind the remaining tissue. The tissue suffers only brief thermal transients that do not damage DNA, RNA, or proteins. Importantly, this method can be applied also to stained, archival glass slides. The other widely used system is the laser microbeam microdissection system that uses a pulsed ultraviolet laser with a small beam to cut out the cells
of interest by photoablation of the adjacent tissue.9,10 Tissue sections are mounted on a thin polyethylene foil slide, and the cells of interest are microdissected by means of an ultraviolet laser that performs the circumferential dissection of the selected cells following a precisely drawn incision path (Fig 1). By this cold ablation, the material to be extracted is not directly exposed to the laser. The microdissected tissue areas are then collected on the cap of a nanotube for subsequent molecular analysis, using contamination and contact-free methods of cell recovery. When the cells of interest exhibit characteristic disease morphology, LMD can be carried out on
Table 2—Comparison Among Different Commercially Available LMD Systems* Variables
Arcturus Pix Cell IIe
mmi Cellcut
Leica AS LMD
PALM Microbeam
Tissue capture technique
The laser causes the thermoplastic film, attached to the cap, to melt with the target cells Lifting the cap from the tissue section removes the target cells
The laser cuts the specimens, mounted on a supporting membrane, around the target cells
The laser cuts the specimens mounted on a supporting membrane, around the target cells
The force of gravity lands the target cells on the collecting cap
The target cells are catapulted into the microfuge cap
Upright Leica DMLA
Inverted Zeiss Axiovert 200 Nitrogen ultraviolet 337 Membrane-covered glass slides Yes Yes Yes Yes Yes Yes Yes Yes
Microscope laser
Inverted Olympus IX50
Type Wavelength, nm Sections support
Solid-state near infrared 337 Glass slides
The laser cuts the specimens, mounted on a special membranecovered slide, around the target cells Retraction of the adhesive cap, that rests on the membrane, removes the target cells Inverted Nikon TE or others Solid-state ultraviolet 350 Membrane-covered slides
Fluorescence upgrade Applications Frozen sections Fixed sections Cytologic smears Living cells Single cell Micromanipulation Image archiving software
Yes
Yes
Nitrogen ultraviolet 337 Membrane-covered glass slides Yes
Yes Yes Yes Yes Yes No Yes
Yes Yes Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes No Yes
Collection of the selected material
*Arcturus Pix Cell IIe (Arcturus Bioscience; Mountain View, CA); mmi Cellcut (MMI Molecular Machines and Industries; Manchester, NH); Leica AS LMD and Leica DMLA (Leica Microsystems; Bannockburn, IL); PALM Microbeam (P.A.L.M. Microlaser Technologies; Bernried, Germany); Olympus IX50 (Olympus America; Melville, NY); Nikon TE (Nikon Instruments; Melville, NY); Zeiss Axiovert (Carl Zeiss MicroImaging; Thornwood, NY). www.chestjournal.org
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Figure 1. LMD of endomyocardial biopsy tissue using the laser microbeam microdissection system. Top left, A: Giant-cell myocarditis with an extensive inflammatory infiltrate represented by lymphocytes, histiocytes, eosinophils, and multinuclear giant cells associated with cell necrosis. A giant cell is indicated by the arrow (hematoxylin-eosin, original ⫻ 200). Top right, B: The same field in which the path of the giant cell has been traced before the laser activation. Bottom left, C: The same field after microdissection of the selected cell. A hole is visible in the remaining tissue. Bottom right, D: The excised cell on the adhesive cap. All the steps of the procedure are examined with a microscope before proceeding to the next phase.
hematoxylin-eosin–stained tissue sections (Fig 1). However, in order to select cell types that are not readily distinguished by morphologic examination, the expression of specific cell markers by immunohistochemically guided procedures may be employed. In both the laser capture and laser microbeam microdissection systems, the laser is integrated into a standard microscope. The investigator can examine the tissue section microscopically before and after the microdissection procedure and control the homogeneity of the selected material, as the tissue collected on the cap retains its original morphology. These systems also offer the possibility to archive the images of each dissection, allowing the correlation of the histopathologic findings with the subsequent molecular results. Application of LMD to Cardiovascular Research Up to now, the major field of application of this method has been cancer research. Separation of malignant, in situ, and normal cell subpopulations 2878
within a single biopsy specimen allows a direct comparison of tissue DNA, RNA, and protein content and function in both normal and neoplastic tissues.11 This technique has been applied to detect both known and unknown alterations in the genomes of a variety of tumors.12 Moreover, microarray technology has been used to obtain molecular fingerprints of gene expression in microdissected tissue biopsies of human cancer, contributing to identify different disease stages and specific molecular-targeted therapies.13 Recently, this method has been applied to human cardiac and vascular tissue (Table 3). Moniotte et al14 used LMD to selectively capture myocytes and endothelial cells from cardiac frozen sections stained with hematoxylin-eosin. The authors quantitatively assessed the expression of rare mRNAs (ie, 3adrenoceptor mRNAs) in the small number of collected cells using real-time PCR, and demonstrated the efficacy of combining these two methods for gene expression studies in cardiac diseases. Similarly, De Souza et al15 performed LMD on frozen left and right ventricular tissue from explanted hearts to selectively collect blood vessels and myocytes, and analyzed the protein extracts by two-dimensional gel electrophoresis. Their results showed good protein recovery from the microdissected cells and welldistinct protein profiles, demonstrating the feasibility of using LMD to perform proteomic studies of individual cardiac tissue components. LMD has been also employed to analyze the level of deleted mitochondrial DNA (mtDNA) in the conducting myocardium in comparison with working myocardium in a patient with Kearns-Sayre syndrome,16 and showed a greater abundance of mutated mtDNA in the conducting system, consistent with its prevalent clinical involvement. More recently, the LMD technique was applied to endomyocardial biopsy specimens in order to precisely separate and collect the cardiomyocytes and the infiltrating lymphocytes in the cardiac tissue infected by Epstein-Barr virus (EBV).17 Paraffinembedded sections from nine patients with chronic heart failure and a histologic diagnosis of inflammatory cardiomyopathy, with PCR positivity for EBV on whole frozen tissue, were studied. LMD was applied in this series to assess whether the viral genome was localized in the myocytes, the inflammatory cells, or both. The selection of cells was immunohistochemically guided by ␣-sarcomeric actin antibody staining for cardiomyocytes and CD45RO staining for lymphocytes (Fig 2). EBV genome was detected by PCR analysis in cardiomyocytes of all patients and was absent in infiltrating lymphocytes, thus suggesting a cytopathic role of this virus and the opportunity for an antiviral/immunomodulatory therapy. ImporReviews
Table 3—Application of LMD in Human Cardiovascular Diseases Disease
Tissue Samples
Microdissected Cells
Molecular Study
Heart failure
Frozen explanted hearts Myocytes, endothelial cells Real-time PCR for 3adrenoceptor mRNA
Heart failure
Frozen explanted hearts Myocytes, blood vessels
Results
Quantitation of 3Moniotte et al14 adrenoceptor expression in myocytes and endothelial cells Distinct protein profiles De Souza et al15 from myocytes and blood vessels
Analysis of the protein extracts by twodimensional electrophoresis Kearns-Sayre syndrome Fixed autopsy heart Atrioventricular node, Competitive PCR for wild- Higher levels of mutated sinus node, bundle of type and mutated mtDNA in conducting His, and working mtDNA system than in working myocardium myocardium EBV-related myocarditis Fixed endomyocardial Myocytes, lymphocytes PCR for EBV DNA Detection of EBV genome biopsies in myocytes but not in lymphocytes Atherosclerosis Fixed arterial samples Atherosclerotic lesions, Gene expression profiles Identification of normal areas by cDNA array overexpressed genes in macrophage-rich areas Atherosclerosis Fixed carotid Caspase-2 immunoreactive Analysis of the protein Overexpression of caspaseendoarterectomies areas of atherosclerotic extracts by Western 2S with antiapoptotic plaques blotting effect Giant cell arteritis Frozen temporally artery Giant cells and Genomic representational Identification of microbial biopsies inflammatory cells, difference analysis on DNA sequences in the normal areas DNA extracts inflammatory lesions
tantly, this study showed that PCR analysis of nucleic acids extracted from a small number of microdissected cells does not cause a loss of sensitivity compared with PCR performed on the whole frozen tissue, with the advantage to allow the cellular localization of the viral agent. The combination of highly sensitive PCR analysis with the microscopical selection of the targeted cells might be useful in the investigation of the different cellular tropism (ie, cardiomyocytes, endothelial cells, fibroblasts, smooth muscle cells) of the viruses infecting the heart, thus improving our knowledge of viral myocarditis in terms of pathogenesis, clinical manifestations, and possible therapeutic options. Similarly, this method can be applied to human atherosclerotic lesions to investigate the presence of microbial agents in the different cellular components of the plaque (ie, foam cells, smooth-muscle cells, lymphocytes, endothelial cells, fibroblasts). Tuomisto et al18 used LMD to isolate macrophage-rich shoulder areas from human atherosclerotic lesions and compared their gene expression patterns to macroscopically normal diffuse intimal thickening by complementary DNA array. The authors identified several overexpressed genes, such as hydroxymethylglutaryl coenzyme A reductase, colony-stimulating factor receptors, integrin receptors, and nitric oxide synthase, and suggested the usefulness of this approach to identify therapeutic targets for the treatment of atherosclerotic diseases. www.chestjournal.org
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Pistilli et al16
Chimenti et al17
Tuomisto et al18
Martinet et al19
Gordon et al21
In another study, Martinet et al19 used LMD to isolate caspase-2—positive macrophage-derived foam cells around the necrotic core of atherosclerotic plaques in human carotid endoarterectomy specimens. The authors analyzed, using Western blotting, the proteins extracted from the targeted cells and identified the overexpression of a 35-kd protein (caspase-2S) with antiapoptotic effect, suggesting that the upregulation of this protein in macrophages can represent a mechanism to survive the increased levels of oxidative DNA damage present in the atherosclerotic plaque. LMD can also be useful to investigate inflammatory diseases of unknown etiology and uncertain treatment, involving the heart and/or the vessels, such as giant-cell myocarditis and arteritis. Giant cell myocarditis is a disease of young, predominantly healthy adults, frequently with a fatal course.20 Its etiology is unknown; it may recur after cardiac transplantation and sometimes responds to immunosuppressive therapy. Histologically, it is characterized by extensive inflammatory infiltrates with the presence of abnormal multinucleated cells, ie, giant cells, of possible myogenic or macrophagic origin. As illustrate in Figure 1, giant cells can be easily dissected from the endomyocardial tissue sample, and molecular analysis of the isolated material can give important information on the knowledge of this heterogeneous disease, providing indications for its treatment that are not established. Similarly, Gordon et al21 used LMD to isolate giant cells from CHEST / 128 / 4 / OCTOBER, 2005
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Figure 2. LMD of endomyocardial biopsy tissue from a patient with EBV myocarditis. LMD was guided by immunohistochemical detection of the targeted cells. Top left, A, and bottom left, D: ␣-Sarcomeric actin-labeled myocytes and CD45RO-labeled infiltrating lymphocytes, respectively (immunoperoxidase, original ⫻ 400). Top center, B, and bottom center, E: The same field after LMD of the selected cells. After the adhesive caps are removed, holes are visible on the remaining tissue. Top right, C, and bottom right, F: The captured cells on the nanotube caps. The homogeneity of the captured material was confirmed by microscopic visualization before DNA extraction.
inflammatory lesions of giant cell arteritis and identified the presence of microbial sequences in the DNA extracted from the collected material, showing that this disease, considered autoimmune in nature, may be associated with a microbial infection. Finally, the fact that the heart is no longer a postmitotic organ and that myocardial regeneration occurs in adult human heart opens a new potential field for application of LMD. Indeed, studies22,23 on the chimerism of the heart after sex-mismatched transplantation have provided consistent results concerning the migration of primitive cells from the host to the graft, showing the presence of Y chromosome in myocytes of female hearts transplanted into male recipients. A population of resident cardiac primitive cells able to differentiate in myocytes, smooth-muscle cells, and endothelial cells has been identified in the adult human heart, and it has been shown that cardiac regeneration is highly activated in acute pathologic conditions, while the regenerative capacity decreases in the chronically decompensated heart 2880
and in aged diseased hearts.24,25 LMD may offer the possibility to selectively study the newly generated myocytes as well as the resident cardiac primitive/ progenitor cells, recognized by specific cell markers. In addition, LMD allows the investigation of the expression of genes that can activate primitive cell growth and differentiation or inhibit cell replication and promote telomere erosion and primitive cell death in the acutely decompensated human heart, as well as in chronic heart failure. In conclusion, LMD is an extremely useful tool that can open new areas in the study of several cardiac diseases, combining advanced molecular biology techniques with the fast and precise selection of targeted cells under direct microscopic visualization of cardiac and vascular tissues. References 1 Kadkol SS, Gage WR, Pasternack GR. In situ hybridization: theory and practice. Mol Diagn 1999; 4:169 –183 Reviews
2 Teo IA, Shaunak S. Polymerase chain reaction in situ: an appraisal of an emerging technique. Histochem J 1995; 27:647– 659 3 Emmert-Buck MR, Bonner RF, Smith PD, et al. Laser capture microdissection. Science 1996; 274:998 –1001 4 Leong AS. Pitfall in diagnostic immunohistology. Adv Anat Pathol 2004; 11:86 –93 5 Pan LX, Diss TC, Isaacson PG. The polymerase chain reaction in histopathology. Histopathology 1995; 26:201–217 6 Eltoum IA, Siegal GP, Frost AR. Microdissection of histologic sections: past, present, and future. Adv Anat Pathol 2002; 9:316 –322 7 Bonner RF, Emmert-Buck MR, Cole K, et al. Laser capture microdissection: molecular analysis of tissue. Science 1997; 278:1481–1483 8 Fend F, Raffeld M. Laser capture microdissection in pathology. J Clin Pathol 2000; 53:666 – 672 9 Gjerdrum LM, Lielpetere I, Rasmussen LM, et al. Laserassisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis: identification of cell populations using immunohistochemistry and in situ hybridization. J Mol Diagn 2001; 3:105–110 10 Zhang L, Yang N, Conejo-Garcia JR, et al. Expression of endocrine gland-derived vascular endothelial growth factor in ovarian carcinoma. Clin Cancer Res 2003; 9:264 –272 11 Michener CM, Ardekani AM, Petricoin EF 3rd, et al. Genomics and proteomics: application of novel technology to early detection and prevention of cancer. Cancer Detect Prev 2002; 26:249 –255 12 Cheng L, MacLennan GT, Zhang S, et al. Laser capture microdissection analysis reveals frequent allelic losses in papillary urothelial neoplasm of low malignant potential of the urinary bladder. Cancer 2004; 101:183–188 13 Ma XJ, Salunga R, Tuggle JT. Gene expression profiles of human breast cancer progression. Proc Natl Acad Sci U S A 2003; 100:5974 –5979 14 Moniotte S, Vaerman JL, Kockx MM, et al. Real-time RT-PCR for the detection of -adrenoceptor messenger RNA’s in small human endomyocardial biopsies. J Mol Cell Cardiol 2001; 33:2121–2133
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15 De Souza AI, McGregor E, Dunn MJ, et al. Preparation of human heart for laser microdissection and proteomics. Proteomics 2004; 4:578 –586 16 Pistilli D, Di Gioia CRT, D’Amati G, et al. Detection of deleted mitochondrial DNA in Kearns-Sayre syndrome using laser capture microdissection. Hum Pathol 2003; 34:1058 – 1061 17 Chimenti C, Russo A, Pieroni M, et al. Intramyocyte detection of Epstein Barr virus genome by laser capture microdissection in patients with inflammatory cardiomyopathy. Circulation 2004; 110:3534 –3539 18 Tuomisto TT, Korkeela A, Rutanen J, et al. Gene expression in macrophage-rich inflammatory cell infiltrates in human atherosclerotic lesions as studied by laser microdissection and DNA array: overexpression of HMG-CoA reductase, colony stimulating factor receptors, CD11A/CD18 integrins, and interleukin receptors. Arterioscler Thromb Vasc Biol 2003; 23:2235–2240 19 Martinet W, Knaapen MW, De Meyer GR, et al. Overexpression of the anti-apoptotic caspase-2 short isoform in macrophage-derived foam cells of human atherosclerotic plaques. Am J Pathol 2003; 162:731–736 20 Cooper LT Jr, Berry GJ, Shabetai R. Idiopathic giant-cell myocarditis: natural history and treatment: Multicenter Giant-Cell Myocarditis Study Group Investigators. N Engl J Med 1997; 336:1860 –1866 21 Gordon LK, Goldman M, Sandusky H, et al. Identification of candidate microbial sequences from inflammatory lesion of giant cell arteritis. Clin Immunol 2004; 111:286 –296 22 Quaini F, Urbanek K, Beltrami AP, et al. Chimerism of the transplanted heart. N Engl J Med 2002; 346:5–15 23 Mu¨ller P, Pfeiffer P, Koglin J, et al. Cardiomyocytes of noncardiac origin in myocardial biopsies of human transplanted hearts. Circulation 2002; 105:31–35 24 Urbanek K, Quaini F, Tasca G, et al. Intense myocyte formation from cardiac stem cells in human cardiac hypertrophy. Proc Natl Acad Sci U S A 2003; 100:10440 –10445 25 Chimenti C, Kajstura J, Torella D, et al. Senescence and death of primitive cells and myocytes lead to premature cardiac aging and heart failure. Circ Res 2003; 93:604 – 613
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