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New Diagnostic Techniques in Anatomic Pathology
Update on Diagnostic Techniques
0025-712:5IHCi $0.00
+ .20
New Diagnostic Techniques in Anatomic Pathology Mark L. Silverman, M.D. *
Describing new developments in anatomic pathology poses a challenge not dissimilar to understanding today's European politics without knowing the events of the First World War. As we approach the 21st century, the discipline in some respects differs little from that of the 18th and 19th centuries in that the vast majority of work is performed using gross observations and techniques of tissue handling and staining developed more than 200 years ago. The pathologist's most trusted tools remain his microscope (a device only slightly more sophisticated, largely in terms of convenience, than that of his predecessors) and his objective application of an understanding of morbid anatomy. Today's pathologist still functions under Rudolf Virchow's 19th-century paradigm, which proposed that human illness could largely be understood through the analysis of an altered morphologic state. Although this premise represented a conceptual advance, it also immediately became an intellectual impediment to further understanding of disease. This article attempts to examine the strengths and weaknesses of Virchow's paradigm, especially its effect on the diagnostic process of today. Morphology Perhaps the most important advance in the application of pathology to clinical practice has been the development of devices to obtain tissue for diagnosis by fiberoptic endoscopy and other closed-biopsy techniques. An overworked joke, well known to all physicians, laments that although the pathologist knows everything and does everything, he is always a day late. Undoubtedly, the root of this cynical view links the study of disease with the lessons of the autopsy table. Today's anatomic pathologist has earned a place beside the clinician by virtue of the ability to diagnose and to objectively monitor the course of disease through the timely interpretation of minute tissue biopsy or cytologic biopsy specimens. For example, the *Chairman, Department of Anatomic Pathology, Lahey Clinic Medical Center, Burlington, Massachusetts
Medical Clinics of North
America~Vol.
70, No. 6, November 1986
1209
1210
MAHK
L.
SILVERMAN
diagnosis of myocarditis (once usually made at autopsy) and the monitoring of its response to therapy through the interpretation of specimens of myocardial biopsy are becoming a standard and straightforward procedure. Probably the majority of endoscopic biopsies are performed by the gastroenterologist, who can not only obtain a diagnosis by this means but can also monitor response to therapy, such as in gluten-induced enteropathy, or can survey for premalignant or frankly malignant change in a host of chronic inflammatory diseases, such as ulcerative colitis. 6 Although the importance of morphologic diagnosis in our understanding cannot be underestimated, other techniques must often be applied even in the face of obvious structural changes that by themselves do not explain the pathophysiology of the disease state observed. Microbiologic techniques, for example, have enabled us to understand that pseudomembranous colitis, a distinct morphologic entity, is often associated with a specific microorganism, Clostridium difficile. 2 In studying diarrheal diseases, the dissociation of the pathologic process from morphologic features becomes noteworthy. Thus, a variety of oligosaccharide deficiencies are manifested in tissue only by biochemical analysis of the enzyme content of the brush border of the villus. Morphologic studies at higher microscopic resolution often enable the pathologist to make more specific diagnoses. The size of objects studied at the practical limit of resolution of the light microscope approximates that of a small bacterium. Thus, the examination of cellular organelles requires a higher useful magnification, which has been brought into reality with the introduction of transmission electron microscopy. A major application of electron microscopy has been in the study of renal disease, in which abnormalities in epithelial, endothelial, and mesangial cells and basement membrane have been correlated with specific disease states. For instance, minimal-change nephropathy, a major cause of nephrotic syndrome in children, has imperceptible morphologic changes when viewed by conventional light microscopy but has a characteristic epithelial lesion when examined ultrastructurally. Electron microscopy is also useful in the study and classification of tumors. The identities of some anaplastic and otherwise unclassified malignant tumors are revealed when studied ultrastructurally. The presence of desmosomes is highly characteristic of squamous cells and may serve as the basis for distinguishing between an epithelial tumor and a sarcoma. 5 Certain characteristics of mesothelial cells help in differentiating between metastatic adenocarcinoma and mesothelioma. 9 Other characteristic organelles are noted in Table 1. The favorable features of high power electron microscopy are offset somewhat by its high cost. The required technical preparation is far more intense than that for normal histologic studies and calls for expensive and sophisticated equipment. The time needed for preparation is also greater. The procurement of fresh tissue is a factor because optimal results often demand special handling by the pathologist. Certain cellular ultrastructural details mav survive formalin fixation and even standard paraffin embedding. 8 Valuabl~ information may therefore be derived from the examination of tissue previously used for routine
1211
DIAC'JOSTIC A]\;ATO:\lIC PATHOLOGY
Table 1. Structures Helpful in Ultrastructural Diagnosis of T11IHOrS TARC;ET STRUCTI'HES
TU\\OR SITEISl
Cellular junctions Pinocytotic vesicles Weibel-Palade bodies Melanosomes Dense-core secretory type granules Myofilaments Mitochondria Microvilli Lysosomes
Epithelial t; nonepilhelial cells Endothelial and smooth muscle cells Endothelial cells Benign and malignant nevocellular lesions Endocrine tissues M uscular tissues Oncocytic tumors Mesothelioma Histiocytic tumors
studies. Such lesions as amelanotic melanomas and carcinoid tumors, for example, may be diagnosed after removal from paraffin. Certainly, electron microscopy may be performed on samples ordinarily in liquid form, such as semen (for certain infertility studies) or blood (buffY coat in the study of leukemias). Techniques adopted for ultrastructural study of fine-needle aspiration biopsy specimens may yield important results, especially in pulmonary disease. 3 Thus, one may be able to more accurately diagnose small-cell tumors and separate oat-cell carcinoma from other poorly differentiated tumors. To date, scanning electron microscopy has enjoyed only limited diagnostic usefulness. Its strength lies in enabling the study of surface features in detail at high resolution. Applications in the field of gastrointestinal disease, for example, include providing insight regarding mucosal changes in inflammatory bowel disease. 4 This technique has also permitted analysis of the effects of surface-active agents, such as laser irradiation in surgery. Greater knowledge of tissue and cellular interactions helps us to better understand certain aspects of physiology, and thus pathophysiology, and will probably lead to increased diagnostic acumen in the future. Cytology Cytology became an important tool when Papanicolau popularized the study of exfoliated cells in the diagnosis of cervical disease. Cytologic preparations at the time of surgery (touch preps) were often used before reliable frozen section became available. Today, there is increasing reliance on a more aggressive approach-that is, fine-needle aspiration biopsy. As a result of improved radiographic imaging techniques, lesions that formerly could have been sampled only at surgery with considerable morbidity and expense are now accessible to aspiration biopsy. This is particularly true in the study of retroperitoneal disease, such as pancreatic cancer. There is also a trend to study more accessible lesions in the outpatient setting, to save time and expense while allowing better planning. l Certainly, aspiration cytology enables rapid analysis of most breast masses and is helpful in studying thyroid disease. Immunology
The recognition that specific molecules could be identified using labeled antibodies is perhaps one of the most important advances since the
1212
~IARK
L.
SIL\'ER\lA:-J
discovery of the microscope. The earliest diagnostic application of this technique was with conjugatcd fluorescent antibodies to specific molecules, such as immunoglobulin G, immunoglobulin M, and fibrin. This technique has become a mainstay in renal pathologic studies and is a mandatory workup study for most, if not all, glomerulopathies. The need to study fresh, unfixed tissue has presented an obvious disadvantage. More recently, however, these techniques have been applied on fixed tissue through the immunoperoxidase method, which takes advantage of the fact (however empirical) that many antigenic proteins retain their antigenicity after fixation. One can study immunoglobulins, peptide hormones, oncofetal antigens, intermediate filaments, and tissue-specific proteins and antigens. Lymphoid lesions, for example, can be studied for evidence of monoclonality, thereby improving evaluation and differentiation of "atypical" lymphoid lesions. Although these mcthods are easily applicable in even fairly rudimentary histology laboratories, the necessity for careful monitoring of technique with appropriate positive and negative control tissue cannot be overemphasized. Attention must be paid to the quality (specificity) of the reagent antibodies in that they represent the "recognition" limb of the method. In addition, care must be taken in the fixation of tissue to preserve critical antigens. Fortunately, high-quality reagents are becoming available commercially. Refinements in monoclonal techniques are in progress that will make recognition more specific than was possible with the early polyclonal antibodies. Antibodies created by monoclonal hybridization techniques are providing powerful tools for the study of specific cellular constituents, such as intermediate filaments. Thus, because of the extreme specificity of the antibodies used, the pathologist can classify tumors with a higher degree of precision than was ever before possible. On the other hand, fresh unfixed tissue is needed, and the developmental costs of reagent antibodies are expensive. In situ hybridization is another technique that will provide identification of specific proteins through mapping of their genetic codes. 7 The study of specific portions of the cell's genetic code and the encoded proteins will be possible, and the study of specific gene function will extend beyond simple identification to the basis for normal and abnormal physiology at the molecular level. The study of "oncogenes" by this technique is beginning to explain the basic alterations associated with neoplastic transformation. The possibilities for diagnosis, and perhaps even treatment, are exciting indeed. .
Special Technologic Advances A variety of technologic advances enables morphologic laboratory studies to be performed in an entirely new fashion. For example, sophisticated fluorometric or photometric techniques with statistical analysis of data, such as those used in flow cytometry, permit large populations of cells to be studied for various parameters. In addition, the DNA content of cells as an index of malignancy can be studied in a screening setting rather than relying solely on subjective assessment by technologists. This
DIAC'JOSTlC ANATO\I\C PATHOLOCY
1213
type of study, which can be automated, could provide a rapid and, most important, a standardized method for screening fluid cytology. By labeling surface structures, such as immunoglobulins or related structural proteins, a population of cells can be sorted and characterized. Flow cytometry is already in use in many large medical centers, and in focusing on various physiologic properties of cells, it may offer rapid and reliable screening of cell populations for specific features. For example, one can find early evidence for circulating leukemic blast cells in numbers that would ordinarily escape detection by routine differential count. A variety of techniques from the physical sciences have found applications in the medical setting. One can, for example, identify metals and minerals within cells by means of electron probe analysis. Other techniques, such as nuclear magnetic resonance imaging, mass spectroscopy, and gas chromatography, may well find routine applications in the future. Conclusion
Today's anatomic pathologist continues the study of disease much like his predecessors, with heavy emphasis on gross and ordinary microscopic analysis. With the increasing availability of specific therapeutic options, however, the need for rapid, precise diagnosis has never been more pressing. The pathologist must enlist a variety of technologic advances to answer specific questions that often extend beyond the simple analysis of deranged structures. He or she is no longer trapped by Virchow's paradigm and may cross a variety of disciplines in the quest for a solid understanding of disease. REFERENCES l. Aretz, H. T., Silverman, M. L., Kolodziejski, J. L., et al.: Fine-needle aspiration: Why it deserves another look. Postgrad. -"fed., 75:49-56, 1984. 2. Bartlett, J. G., Moon, "l., Chang, T. W., et al.: Hole of Clostridium difjicile in antibioticassociated pseudomembranous colitis. Gastroenterology, 75:778-782, 1978 . .3. Di Sant'Agnese, P. A., and De Mesy-Jensen, K. L.: Diagnostic electron microscopy on reembedded ("popped ()ff') areas of large Spurr epoxy sections. U1trastruct. Pathol.,
6:247-253, 1984. 4. Dvorak, A. 1\1., Connell, A. B., and Dickersin, G. H.: Crohn's disease: A scanning electron microscopic study. Hum. Pathol., 10:165-177, 1979 . .5. Erlandson, H. A.: Diagnostic Transmission Electron Microscopy of Human Tumors: The Interpretation of Submicroscopic Structures in Human Neoplastic Cells. New York, Masson Publishing USA., 1981. 6. Hiddell, H. H., Goldman, H., Hansohof[ D. F., et al.: Dysplasia in inflammatory bowel disease: Standardized classification with provisional clinical applications. Hum. Pathol., 14:931-968, 1983. 7. Sklar, .T.: DNA hybridization in diagnostic pathology. Hum. Pathol., 16:654-658, 1985. 8. Van den Bergh Weerman, M. A., and Dingemans, K. P.: Hapid deparaffinization for electron microscopy. Ultrastruct. Pathol., 7:55-.57, 1984. 9. Warhol, M. J., Hickey, W. F., Corson, J. M., et al.: Malignant mesothelioma: Ultrastructural distinction from adenocarcinoma. Am. J. Surg. Pathol., 6:307-314, 1982. Department of Anatomic Pathology Lahev Clinic Medical Center 41 ~iall Hoad Burlington, Massachusetts 01805
0025-712:5IHCi $0.00
+ .20
New Diagnostic Techniques in Anatomic Pathology Mark L. Silverman, M.D. *
Describing new developments in anatomic pathology poses a challenge not dissimilar to understanding today's European politics without knowing the events of the First World War. As we approach the 21st century, the discipline in some respects differs little from that of the 18th and 19th centuries in that the vast majority of work is performed using gross observations and techniques of tissue handling and staining developed more than 200 years ago. The pathologist's most trusted tools remain his microscope (a device only slightly more sophisticated, largely in terms of convenience, than that of his predecessors) and his objective application of an understanding of morbid anatomy. Today's pathologist still functions under Rudolf Virchow's 19th-century paradigm, which proposed that human illness could largely be understood through the analysis of an altered morphologic state. Although this premise represented a conceptual advance, it also immediately became an intellectual impediment to further understanding of disease. This article attempts to examine the strengths and weaknesses of Virchow's paradigm, especially its effect on the diagnostic process of today. Morphology Perhaps the most important advance in the application of pathology to clinical practice has been the development of devices to obtain tissue for diagnosis by fiberoptic endoscopy and other closed-biopsy techniques. An overworked joke, well known to all physicians, laments that although the pathologist knows everything and does everything, he is always a day late. Undoubtedly, the root of this cynical view links the study of disease with the lessons of the autopsy table. Today's anatomic pathologist has earned a place beside the clinician by virtue of the ability to diagnose and to objectively monitor the course of disease through the timely interpretation of minute tissue biopsy or cytologic biopsy specimens. For example, the *Chairman, Department of Anatomic Pathology, Lahey Clinic Medical Center, Burlington, Massachusetts
Medical Clinics of North
America~Vol.
70, No. 6, November 1986
1209
1210
MAHK
L.
SILVERMAN
diagnosis of myocarditis (once usually made at autopsy) and the monitoring of its response to therapy through the interpretation of specimens of myocardial biopsy are becoming a standard and straightforward procedure. Probably the majority of endoscopic biopsies are performed by the gastroenterologist, who can not only obtain a diagnosis by this means but can also monitor response to therapy, such as in gluten-induced enteropathy, or can survey for premalignant or frankly malignant change in a host of chronic inflammatory diseases, such as ulcerative colitis. 6 Although the importance of morphologic diagnosis in our understanding cannot be underestimated, other techniques must often be applied even in the face of obvious structural changes that by themselves do not explain the pathophysiology of the disease state observed. Microbiologic techniques, for example, have enabled us to understand that pseudomembranous colitis, a distinct morphologic entity, is often associated with a specific microorganism, Clostridium difficile. 2 In studying diarrheal diseases, the dissociation of the pathologic process from morphologic features becomes noteworthy. Thus, a variety of oligosaccharide deficiencies are manifested in tissue only by biochemical analysis of the enzyme content of the brush border of the villus. Morphologic studies at higher microscopic resolution often enable the pathologist to make more specific diagnoses. The size of objects studied at the practical limit of resolution of the light microscope approximates that of a small bacterium. Thus, the examination of cellular organelles requires a higher useful magnification, which has been brought into reality with the introduction of transmission electron microscopy. A major application of electron microscopy has been in the study of renal disease, in which abnormalities in epithelial, endothelial, and mesangial cells and basement membrane have been correlated with specific disease states. For instance, minimal-change nephropathy, a major cause of nephrotic syndrome in children, has imperceptible morphologic changes when viewed by conventional light microscopy but has a characteristic epithelial lesion when examined ultrastructurally. Electron microscopy is also useful in the study and classification of tumors. The identities of some anaplastic and otherwise unclassified malignant tumors are revealed when studied ultrastructurally. The presence of desmosomes is highly characteristic of squamous cells and may serve as the basis for distinguishing between an epithelial tumor and a sarcoma. 5 Certain characteristics of mesothelial cells help in differentiating between metastatic adenocarcinoma and mesothelioma. 9 Other characteristic organelles are noted in Table 1. The favorable features of high power electron microscopy are offset somewhat by its high cost. The required technical preparation is far more intense than that for normal histologic studies and calls for expensive and sophisticated equipment. The time needed for preparation is also greater. The procurement of fresh tissue is a factor because optimal results often demand special handling by the pathologist. Certain cellular ultrastructural details mav survive formalin fixation and even standard paraffin embedding. 8 Valuabl~ information may therefore be derived from the examination of tissue previously used for routine
1211
DIAC'JOSTIC A]\;ATO:\lIC PATHOLOGY
Table 1. Structures Helpful in Ultrastructural Diagnosis of T11IHOrS TARC;ET STRUCTI'HES
TU\\OR SITEISl
Cellular junctions Pinocytotic vesicles Weibel-Palade bodies Melanosomes Dense-core secretory type granules Myofilaments Mitochondria Microvilli Lysosomes
Epithelial t; nonepilhelial cells Endothelial and smooth muscle cells Endothelial cells Benign and malignant nevocellular lesions Endocrine tissues M uscular tissues Oncocytic tumors Mesothelioma Histiocytic tumors
studies. Such lesions as amelanotic melanomas and carcinoid tumors, for example, may be diagnosed after removal from paraffin. Certainly, electron microscopy may be performed on samples ordinarily in liquid form, such as semen (for certain infertility studies) or blood (buffY coat in the study of leukemias). Techniques adopted for ultrastructural study of fine-needle aspiration biopsy specimens may yield important results, especially in pulmonary disease. 3 Thus, one may be able to more accurately diagnose small-cell tumors and separate oat-cell carcinoma from other poorly differentiated tumors. To date, scanning electron microscopy has enjoyed only limited diagnostic usefulness. Its strength lies in enabling the study of surface features in detail at high resolution. Applications in the field of gastrointestinal disease, for example, include providing insight regarding mucosal changes in inflammatory bowel disease. 4 This technique has also permitted analysis of the effects of surface-active agents, such as laser irradiation in surgery. Greater knowledge of tissue and cellular interactions helps us to better understand certain aspects of physiology, and thus pathophysiology, and will probably lead to increased diagnostic acumen in the future. Cytology Cytology became an important tool when Papanicolau popularized the study of exfoliated cells in the diagnosis of cervical disease. Cytologic preparations at the time of surgery (touch preps) were often used before reliable frozen section became available. Today, there is increasing reliance on a more aggressive approach-that is, fine-needle aspiration biopsy. As a result of improved radiographic imaging techniques, lesions that formerly could have been sampled only at surgery with considerable morbidity and expense are now accessible to aspiration biopsy. This is particularly true in the study of retroperitoneal disease, such as pancreatic cancer. There is also a trend to study more accessible lesions in the outpatient setting, to save time and expense while allowing better planning. l Certainly, aspiration cytology enables rapid analysis of most breast masses and is helpful in studying thyroid disease. Immunology
The recognition that specific molecules could be identified using labeled antibodies is perhaps one of the most important advances since the
1212
~IARK
L.
SIL\'ER\lA:-J
discovery of the microscope. The earliest diagnostic application of this technique was with conjugatcd fluorescent antibodies to specific molecules, such as immunoglobulin G, immunoglobulin M, and fibrin. This technique has become a mainstay in renal pathologic studies and is a mandatory workup study for most, if not all, glomerulopathies. The need to study fresh, unfixed tissue has presented an obvious disadvantage. More recently, however, these techniques have been applied on fixed tissue through the immunoperoxidase method, which takes advantage of the fact (however empirical) that many antigenic proteins retain their antigenicity after fixation. One can study immunoglobulins, peptide hormones, oncofetal antigens, intermediate filaments, and tissue-specific proteins and antigens. Lymphoid lesions, for example, can be studied for evidence of monoclonality, thereby improving evaluation and differentiation of "atypical" lymphoid lesions. Although these mcthods are easily applicable in even fairly rudimentary histology laboratories, the necessity for careful monitoring of technique with appropriate positive and negative control tissue cannot be overemphasized. Attention must be paid to the quality (specificity) of the reagent antibodies in that they represent the "recognition" limb of the method. In addition, care must be taken in the fixation of tissue to preserve critical antigens. Fortunately, high-quality reagents are becoming available commercially. Refinements in monoclonal techniques are in progress that will make recognition more specific than was possible with the early polyclonal antibodies. Antibodies created by monoclonal hybridization techniques are providing powerful tools for the study of specific cellular constituents, such as intermediate filaments. Thus, because of the extreme specificity of the antibodies used, the pathologist can classify tumors with a higher degree of precision than was ever before possible. On the other hand, fresh unfixed tissue is needed, and the developmental costs of reagent antibodies are expensive. In situ hybridization is another technique that will provide identification of specific proteins through mapping of their genetic codes. 7 The study of specific portions of the cell's genetic code and the encoded proteins will be possible, and the study of specific gene function will extend beyond simple identification to the basis for normal and abnormal physiology at the molecular level. The study of "oncogenes" by this technique is beginning to explain the basic alterations associated with neoplastic transformation. The possibilities for diagnosis, and perhaps even treatment, are exciting indeed. .
Special Technologic Advances A variety of technologic advances enables morphologic laboratory studies to be performed in an entirely new fashion. For example, sophisticated fluorometric or photometric techniques with statistical analysis of data, such as those used in flow cytometry, permit large populations of cells to be studied for various parameters. In addition, the DNA content of cells as an index of malignancy can be studied in a screening setting rather than relying solely on subjective assessment by technologists. This
DIAC'JOSTlC ANATO\I\C PATHOLOCY
1213
type of study, which can be automated, could provide a rapid and, most important, a standardized method for screening fluid cytology. By labeling surface structures, such as immunoglobulins or related structural proteins, a population of cells can be sorted and characterized. Flow cytometry is already in use in many large medical centers, and in focusing on various physiologic properties of cells, it may offer rapid and reliable screening of cell populations for specific features. For example, one can find early evidence for circulating leukemic blast cells in numbers that would ordinarily escape detection by routine differential count. A variety of techniques from the physical sciences have found applications in the medical setting. One can, for example, identify metals and minerals within cells by means of electron probe analysis. Other techniques, such as nuclear magnetic resonance imaging, mass spectroscopy, and gas chromatography, may well find routine applications in the future. Conclusion
Today's anatomic pathologist continues the study of disease much like his predecessors, with heavy emphasis on gross and ordinary microscopic analysis. With the increasing availability of specific therapeutic options, however, the need for rapid, precise diagnosis has never been more pressing. The pathologist must enlist a variety of technologic advances to answer specific questions that often extend beyond the simple analysis of deranged structures. He or she is no longer trapped by Virchow's paradigm and may cross a variety of disciplines in the quest for a solid understanding of disease. REFERENCES l. Aretz, H. T., Silverman, M. L., Kolodziejski, J. L., et al.: Fine-needle aspiration: Why it deserves another look. Postgrad. -"fed., 75:49-56, 1984. 2. Bartlett, J. G., Moon, "l., Chang, T. W., et al.: Hole of Clostridium difjicile in antibioticassociated pseudomembranous colitis. Gastroenterology, 75:778-782, 1978 . .3. Di Sant'Agnese, P. A., and De Mesy-Jensen, K. L.: Diagnostic electron microscopy on reembedded ("popped ()ff') areas of large Spurr epoxy sections. U1trastruct. Pathol.,
6:247-253, 1984. 4. Dvorak, A. 1\1., Connell, A. B., and Dickersin, G. H.: Crohn's disease: A scanning electron microscopic study. Hum. Pathol., 10:165-177, 1979 . .5. Erlandson, H. A.: Diagnostic Transmission Electron Microscopy of Human Tumors: The Interpretation of Submicroscopic Structures in Human Neoplastic Cells. New York, Masson Publishing USA., 1981. 6. Hiddell, H. H., Goldman, H., Hansohof[ D. F., et al.: Dysplasia in inflammatory bowel disease: Standardized classification with provisional clinical applications. Hum. Pathol., 14:931-968, 1983. 7. Sklar, .T.: DNA hybridization in diagnostic pathology. Hum. Pathol., 16:654-658, 1985. 8. Van den Bergh Weerman, M. A., and Dingemans, K. P.: Hapid deparaffinization for electron microscopy. Ultrastruct. Pathol., 7:55-.57, 1984. 9. Warhol, M. J., Hickey, W. F., Corson, J. M., et al.: Malignant mesothelioma: Ultrastructural distinction from adenocarcinoma. Am. J. Surg. Pathol., 6:307-314, 1982. Department of Anatomic Pathology Lahev Clinic Medical Center 41 ~iall Hoad Burlington, Massachusetts 01805