Electron microscopy for the diagnosis of tumours The history of histopathology is that of ever more precise identification of cells and tkieir constituents by morphologic and cytochemical means. This is of particular importance in tumour pathology. In general, more precise identification leads to more accurate diagnosis and ultimately to more accurate prognosis and more appropriate treatment. The transmission electron microscope is a potent aid in reaching an accurate cellular diagnosis; its use is well established in renal pathology and clinical virology, and is increasingly recognized in modern tumour diagnosis. The value of electron microscopy (EM) in the diagnosis of tumours lies in identifying in poorly differentiated neoplastic tissues structural elements that might be characteristic of the cell line from which the tumour arose. The questions that can be answered with EM are those that require resolution of structural details beyond the reach of the light microscope. The following are among the many clues that may help. 1. Secretory granules and other inclusions, such as melanosomes, APUD (amine precursor uptake and decarboxylation) granules, mucus, lipid and glycogen. 2. Mitochondrial size and shape. Steroid cells have characteristic mitochondria. 3. Lysosomes. Numerous lysosomes of characteristic structure occur in cells of macrophage lineage. 4. Endoplasmic reticulum. Proteinsecreting cells usually have prominent rough endoplasmic reticulum, while steroid-secreting cells have prominent smooth endoplasmic reticulum. 5. Myofibrils. Actin-like microfilaments and characteristic dark bodie*s identify smooth muscle tumours; rhabdomyosarcoma contains both thick and thin filaments. This clue is far more definitive than attempts to identify cross-striations by light microscopy with special stains. 6. Desmosomes. These are often present in epithelial cells; bundles of tonofilaments in squamous carcinoma are particularly prominent. On the other hand, desmosomes are less common in mesenchymal cells. 7. Microvilli. In epithelial cells microvilli are often prominent and relatively straight. Intracellular clusters of microvilli are particularly characteristic of epithelial cells of alimentary tract origin. 8. Normal and abnormal interstitial matrix fibrils. Fibrosarcomas produce
collagen; medullary carcinomas of the thyroid produce amyloid. Both are recognizable ultrastructurally. 9. Basement membrane arrangement. Epithelial cells often cluster in a group in a single basket of basement membrane, whereas mesenchymal cells tend to lie independently, without identifiable basement membrane or with a pericellular arrangement of basementmembrane-like material. 10. Cell relationships in special circumstances. Neoplasms of neurilemmal and meningeal origin have characteristic long, closely interwoven processes. With these clues in mind we have found EM useful in the everyday routine of a surgical pathology service in the differentiation of anaplastic carcinoma and other spindle-cell tumours of doubtful histogenesis; in the identification of amelanotic melanoma, and muscle and other mesenchymal and neural tumours; in the study of endocrine tumours and carcinoids; and in the classification of non-Hodgkin's lymphomas. Such rare granulomatous lesions as malacoplakia and Whipple's disease may be precisely identified by EM, and it is likely that research into the ultrastructure of tumours will lead to further refinement of tissue diagnosis and even to the recognition of new groups of neoplasms. There are difficulties in the use of EM in tumour diagnosis; for example, EM can give grossly misleading information if applied to a heterogeneous structure like a neoplasm without careful localization. A complete picture of the lesion must be obtained by studying the gross specimen, histologic sections and electron micrographs. Because of the small size of the block involved, selection of the appropriate area for EM is vital. This is done either by carefully matching the EM block with the paraffin-embedded material or by selecting the appropriate area from the paraffin block. Finally, EM generally gives little information on proliferative and invasive behaviour; any information on the degree of malignancy is therefore indirect. These difficulties can be overcome by the specialist histopathologist trained in ultrastructure. There are three essentials for the successful use of an electron microscope for tumour diagnosis - speed, reliability and adequate specimens. Material can now be prepared and examined within 24 hours of receipt1 and a report can be provided routinely within 48 hours, and, when necessary, in 24 hours or less. This is at least as fast
as the service provided by conventional histologic techniques. The second essential is reliability; this presupposes adequate maintenance and servicing of an electron microscope and availability of specialist technical staff. The third essential is a good routine flow of material for EM examination, which requires considerable cooperation from pathologists, clinicians, technicians and others. Materials fixed in glutaraldehyde or even formaldehyde can be submitted for light microscopic examination. Ideally a small piece from every biopsy should be fixed in glutaraldehyde or buffered formaldehyde at the time of operation, stored in the buffer and processed further should EM be required. Useful micrographs, however, can often be obtained from material stored routinely in formalin, or even by cutting fragments out of a paraffin block. For best results the EM unit should be dedicated to a service function, unembarrassed by the different priorities of experimental research. The establishment of diagnostic EM units is now taken seriously in the United States.2 Since 1966, the US Veterans Administration has installed 40 high-resolution transmission electron microscopes in its hospitals and 15 more are planned; it is suggested that a better diagnosis is obtained by ultrastructural studies in 4%3 to 8%. of biopsies and we agree. All this is costly. The capital cost is about $60 000 (less than some multichannel analysers); a reasonable cost per biopsy using all possible economy is about $70. This assumes that the unit handles up to 500 cases per annum and services a population of 500 000. US costs4 are $100 per specimen at a rate of 250 cases per annum. This is more than the unit cost for a diagnostic frozen section with paraffin confirmation, about the same as that of barium enema examination and less than that of aortography. Therefore EM cannot be considered an unusually expensive diagnostic aid. These costs could in many places be cut substantially by providing additional technical staff for existing underused capital equipment. For several reasons histopathologists, particularly older histopathologists, do not unanimously accept the diagnostic value of EM in the diagnosis of tumours. There must be few histopathologists over 40 years of age who have received in-depth training in cell ultrastructure; the great explosion of knowledge in this field did not occur until the late 1950s and early 1960s. Electron microscopists have often attempted
CMA JOURNAL/FEBRUARY 19, 1977/VOL. 116 341
to preserve the arcane nature of their discipline rather than to present it as the routine technique it is today. They have often presented their results as beautiful pictures, not integrated with the light microscopic diagnosis and too late to affect the management of the case. And it must be noted that EM is useful in the diagnosis of only a small percentage of tumours. But we would emphasize that, within its limits, we and others4 have found it not only useful but also essential. The indirect results of investment in pathologic ultrastructure are possibly even more important than the direct. Histopathology, the earliest branch of the subject, will not attract talented recruits unless it exploits its own growing points. Pathologic ultrastructure is a major growth point of histopathology; as Virchow saw with the light microscope, so the service pathologist must see now that there is a whole new sphere of pattern recognition and iden-
tification to be explored in the interests of the patient. The advance of ultrastructural pathology will enhance classic histopathologic skills rather than render them outdated, for in the end a diagnosis is reached by looking at the patient, the histologic section and the electron micrograph - in that order. I. CARR, MD, PH D, FRC PATH Department of pathology University of Saskatchewan Saskatoon, Sask. P.G. TONER, MB, D SC, MRC PATH University department of pathology Royal Infirmary Glasgow, Scotland
References 1. ROWDEN G, LEWIS MG: Experience with a three-hour electron microscopy biopsy service. J Clin Pathol 27: 505, 1974 2. BLOODWORTH JM JR, AZAR HA, YO0AIKEN RE (eds): Symposium on electron microscopy in diagnostic pathology. Hum Pathol 6: 403, 1975 3. WILLIAMS MJ: Electron microscopy in the Veterans Administration for service pathology: some administrative aspects of the program. Ibid, p 399 4. GYORKEY
F,
MIN
KW,
KRIsKo
I,
et
al:
The usefulness of electron microscopy in the diagnosis of human tumours. Ibid. p 421
Immunologic mechanisms of hypersensitive lung diseases Hypersensitive lung diseases result from humoral or cellular immune reactions to inhalant antigens in susceptible individuals. Pathophysiologically the disease involves the bronchial airways as in patients with allergic asthma, the terminal lung units as in those with hypersensitivity pneumonitis (also referred to as extrinsic allergic alveolitis) or both anatomic sites as in those with allergic bronchopulmonary aspergillosis. Two key determinants of the development of hypersensitive lung disease are the genetic characteristics of the individual and appropriate environmental exposure. Although atopic diseases mediated by immunoglobulin E (IgE) antibodies often show a strong familial tendency, the exact mode of inheritance is unclear. A number of investigators"2 have reported that IgE antibody production specific for ragweed antigens E and Ra5 is associated with serologically defined loci of the human histocompatibility gene complex (HLA). Others3 have demonstrated the presence of a gene regulating the basal serum concentration of IgE. This gene is not linked to any HLA haplotypes and often masks the effect of HLA-associated immune response genes in controlling the specific IgE-mediated sensitivities to a number of different allergens.3 Direct parent-to-child transmission of IgE-mediated sensitivity to any allergen has not been demonstrated, thus ruling out the possibility
of a single gene model to explain atopic allergies.3 Among patients with hypersensitivity pneumonitis, associations between farmer's lung and HLA-B8 and between pigeon-breeder's lung and HLA-B8, HLA-A2 and HLA-Bwl5 have also been reported.4'5 Even with the necessary genetic factor for hypersensitive responses, clinical disease appears only after appropriate environmental exposure. Apart from the basic immunopathogenetic mechanism, the size of the inhaled organic dust particles also influences the outcome of the allergic lung disease. Spores of thermophilic actinomycetes responsible for farmer's lung are approximately 1 ..t in diameter6 and thus could gain access to the terminal lung units and induce allergic alveolitis. Larger organic dust particles such as grain dust and the spores of most saprophytic fungi would be deposited along the bronchial airways and most likely evoke an asthmatic response in susceptible subjects. The farm environment is a rich source of organic particles of different sizes, which explains the common occurrence of hypersensitive lung diseases among farmers. In this issue (page 391) Warren's practical review of lung diseases in farmers should be of interest, since agriculture and its related industries are economically of great importance in Canada. Allergic asthma is mediated by IgE antibodies (type 1 mechanism accord-
342 CMA JOURNAL/FEBRUARY 19, 1977/VOL. 116
ing to the classification of Gell and Coombs). The inhaled allergens react with specific IgE antibodies on the surface of mast cells in the respiratory mucosa and induce the release of a number of chemical mediators into the surrounding tissue. The initial reaction may occur at the surface of the bronchial mucosa because free mast cells have been demonstrated in the surface mucous blanket.7 Increased tissue permeability induced by mediators released from these superficial mast cells could facilitate transport of the inhaled allergens across the barrier of tight junctions between individual bronchial epithelial cells. Reaction between the allergens and mast cells in deeper mucosal layers results in further release of chemical mediators. A number of primary and secondary mediators of IgE-mediated immunologic reactions have been identified.8 Histamine and bradykinin have profound effects on the microvasculature, causing vasodilatation and increased vascular permeability. In experiments with animals, the administration of histamine and bradykinin causes a sharp increase in respiratory resistance and a decrease in lung compliance, indicating that large and small airways are affected by these mediators.' In allergic dogs undergoing antigenic bronchoprovocation, simultaneous increase of histamine concentration in the plasma and increase in respiratory resistance have been demonstrated.1' However, for a reason that is not clear, antihistamines are not particularly effective in the treatment of allergic asthma. An abrupt increase in the plasma concentration of prostaglandin F2a has also been demonstrated in allergic asthmatics during antigenic bronchoprovocation,'1 and this mediator is capable of causing bronchoconstriction in man and in rodents."12 Slow-reacting substance of anaphylaxis (SRS-A) 'affects mainly the small airways in animals, causing a decrease in lung compliance but no significant change in airway resistance.' The effects of eosinophil chemotactic factor released from mast cells could account for the eosinophilia in the bronchial tissue. Eosinophils could contribute to further inflammatory damage by releasing lysosomal enzymes during phagocytosis of immune complexes and cell debris, but they might also exert beneficial and homeostatic effects because this type of leukocyte contains arylsulfatase B'3 and histaminase,'4 which could inactivate and thus neutralize the effects of SRS-A and histamine. Together these mediators of immediate hypersensitivity could directly cause smooth muscle to contract and could cause mucosal edema and hypersecretion of bronchial mucous
collagen; medullary carcinomas of the thyroid produce amyloid. Both are recognizable ultrastructurally. 9. Basement membrane arrangement. Epithelial cells often cluster in a group in a single basket of basement membrane, whereas mesenchymal cells tend to lie independently, without identifiable basement membrane or with a pericellular arrangement of basementmembrane-like material. 10. Cell relationships in special circumstances. Neoplasms of neurilemmal and meningeal origin have characteristic long, closely interwoven processes. With these clues in mind we have found EM useful in the everyday routine of a surgical pathology service in the differentiation of anaplastic carcinoma and other spindle-cell tumours of doubtful histogenesis; in the identification of amelanotic melanoma, and muscle and other mesenchymal and neural tumours; in the study of endocrine tumours and carcinoids; and in the classification of non-Hodgkin's lymphomas. Such rare granulomatous lesions as malacoplakia and Whipple's disease may be precisely identified by EM, and it is likely that research into the ultrastructure of tumours will lead to further refinement of tissue diagnosis and even to the recognition of new groups of neoplasms. There are difficulties in the use of EM in tumour diagnosis; for example, EM can give grossly misleading information if applied to a heterogeneous structure like a neoplasm without careful localization. A complete picture of the lesion must be obtained by studying the gross specimen, histologic sections and electron micrographs. Because of the small size of the block involved, selection of the appropriate area for EM is vital. This is done either by carefully matching the EM block with the paraffin-embedded material or by selecting the appropriate area from the paraffin block. Finally, EM generally gives little information on proliferative and invasive behaviour; any information on the degree of malignancy is therefore indirect. These difficulties can be overcome by the specialist histopathologist trained in ultrastructure. There are three essentials for the successful use of an electron microscope for tumour diagnosis - speed, reliability and adequate specimens. Material can now be prepared and examined within 24 hours of receipt1 and a report can be provided routinely within 48 hours, and, when necessary, in 24 hours or less. This is at least as fast
as the service provided by conventional histologic techniques. The second essential is reliability; this presupposes adequate maintenance and servicing of an electron microscope and availability of specialist technical staff. The third essential is a good routine flow of material for EM examination, which requires considerable cooperation from pathologists, clinicians, technicians and others. Materials fixed in glutaraldehyde or even formaldehyde can be submitted for light microscopic examination. Ideally a small piece from every biopsy should be fixed in glutaraldehyde or buffered formaldehyde at the time of operation, stored in the buffer and processed further should EM be required. Useful micrographs, however, can often be obtained from material stored routinely in formalin, or even by cutting fragments out of a paraffin block. For best results the EM unit should be dedicated to a service function, unembarrassed by the different priorities of experimental research. The establishment of diagnostic EM units is now taken seriously in the United States.2 Since 1966, the US Veterans Administration has installed 40 high-resolution transmission electron microscopes in its hospitals and 15 more are planned; it is suggested that a better diagnosis is obtained by ultrastructural studies in 4%3 to 8%. of biopsies and we agree. All this is costly. The capital cost is about $60 000 (less than some multichannel analysers); a reasonable cost per biopsy using all possible economy is about $70. This assumes that the unit handles up to 500 cases per annum and services a population of 500 000. US costs4 are $100 per specimen at a rate of 250 cases per annum. This is more than the unit cost for a diagnostic frozen section with paraffin confirmation, about the same as that of barium enema examination and less than that of aortography. Therefore EM cannot be considered an unusually expensive diagnostic aid. These costs could in many places be cut substantially by providing additional technical staff for existing underused capital equipment. For several reasons histopathologists, particularly older histopathologists, do not unanimously accept the diagnostic value of EM in the diagnosis of tumours. There must be few histopathologists over 40 years of age who have received in-depth training in cell ultrastructure; the great explosion of knowledge in this field did not occur until the late 1950s and early 1960s. Electron microscopists have often attempted
CMA JOURNAL/FEBRUARY 19, 1977/VOL. 116 341
to preserve the arcane nature of their discipline rather than to present it as the routine technique it is today. They have often presented their results as beautiful pictures, not integrated with the light microscopic diagnosis and too late to affect the management of the case. And it must be noted that EM is useful in the diagnosis of only a small percentage of tumours. But we would emphasize that, within its limits, we and others4 have found it not only useful but also essential. The indirect results of investment in pathologic ultrastructure are possibly even more important than the direct. Histopathology, the earliest branch of the subject, will not attract talented recruits unless it exploits its own growing points. Pathologic ultrastructure is a major growth point of histopathology; as Virchow saw with the light microscope, so the service pathologist must see now that there is a whole new sphere of pattern recognition and iden-
tification to be explored in the interests of the patient. The advance of ultrastructural pathology will enhance classic histopathologic skills rather than render them outdated, for in the end a diagnosis is reached by looking at the patient, the histologic section and the electron micrograph - in that order. I. CARR, MD, PH D, FRC PATH Department of pathology University of Saskatchewan Saskatoon, Sask. P.G. TONER, MB, D SC, MRC PATH University department of pathology Royal Infirmary Glasgow, Scotland
References 1. ROWDEN G, LEWIS MG: Experience with a three-hour electron microscopy biopsy service. J Clin Pathol 27: 505, 1974 2. BLOODWORTH JM JR, AZAR HA, YO0AIKEN RE (eds): Symposium on electron microscopy in diagnostic pathology. Hum Pathol 6: 403, 1975 3. WILLIAMS MJ: Electron microscopy in the Veterans Administration for service pathology: some administrative aspects of the program. Ibid, p 399 4. GYORKEY
F,
MIN
KW,
KRIsKo
I,
et
al:
The usefulness of electron microscopy in the diagnosis of human tumours. Ibid. p 421
Immunologic mechanisms of hypersensitive lung diseases Hypersensitive lung diseases result from humoral or cellular immune reactions to inhalant antigens in susceptible individuals. Pathophysiologically the disease involves the bronchial airways as in patients with allergic asthma, the terminal lung units as in those with hypersensitivity pneumonitis (also referred to as extrinsic allergic alveolitis) or both anatomic sites as in those with allergic bronchopulmonary aspergillosis. Two key determinants of the development of hypersensitive lung disease are the genetic characteristics of the individual and appropriate environmental exposure. Although atopic diseases mediated by immunoglobulin E (IgE) antibodies often show a strong familial tendency, the exact mode of inheritance is unclear. A number of investigators"2 have reported that IgE antibody production specific for ragweed antigens E and Ra5 is associated with serologically defined loci of the human histocompatibility gene complex (HLA). Others3 have demonstrated the presence of a gene regulating the basal serum concentration of IgE. This gene is not linked to any HLA haplotypes and often masks the effect of HLA-associated immune response genes in controlling the specific IgE-mediated sensitivities to a number of different allergens.3 Direct parent-to-child transmission of IgE-mediated sensitivity to any allergen has not been demonstrated, thus ruling out the possibility
of a single gene model to explain atopic allergies.3 Among patients with hypersensitivity pneumonitis, associations between farmer's lung and HLA-B8 and between pigeon-breeder's lung and HLA-B8, HLA-A2 and HLA-Bwl5 have also been reported.4'5 Even with the necessary genetic factor for hypersensitive responses, clinical disease appears only after appropriate environmental exposure. Apart from the basic immunopathogenetic mechanism, the size of the inhaled organic dust particles also influences the outcome of the allergic lung disease. Spores of thermophilic actinomycetes responsible for farmer's lung are approximately 1 ..t in diameter6 and thus could gain access to the terminal lung units and induce allergic alveolitis. Larger organic dust particles such as grain dust and the spores of most saprophytic fungi would be deposited along the bronchial airways and most likely evoke an asthmatic response in susceptible subjects. The farm environment is a rich source of organic particles of different sizes, which explains the common occurrence of hypersensitive lung diseases among farmers. In this issue (page 391) Warren's practical review of lung diseases in farmers should be of interest, since agriculture and its related industries are economically of great importance in Canada. Allergic asthma is mediated by IgE antibodies (type 1 mechanism accord-
342 CMA JOURNAL/FEBRUARY 19, 1977/VOL. 116
ing to the classification of Gell and Coombs). The inhaled allergens react with specific IgE antibodies on the surface of mast cells in the respiratory mucosa and induce the release of a number of chemical mediators into the surrounding tissue. The initial reaction may occur at the surface of the bronchial mucosa because free mast cells have been demonstrated in the surface mucous blanket.7 Increased tissue permeability induced by mediators released from these superficial mast cells could facilitate transport of the inhaled allergens across the barrier of tight junctions between individual bronchial epithelial cells. Reaction between the allergens and mast cells in deeper mucosal layers results in further release of chemical mediators. A number of primary and secondary mediators of IgE-mediated immunologic reactions have been identified.8 Histamine and bradykinin have profound effects on the microvasculature, causing vasodilatation and increased vascular permeability. In experiments with animals, the administration of histamine and bradykinin causes a sharp increase in respiratory resistance and a decrease in lung compliance, indicating that large and small airways are affected by these mediators.' In allergic dogs undergoing antigenic bronchoprovocation, simultaneous increase of histamine concentration in the plasma and increase in respiratory resistance have been demonstrated.1' However, for a reason that is not clear, antihistamines are not particularly effective in the treatment of allergic asthma. An abrupt increase in the plasma concentration of prostaglandin F2a has also been demonstrated in allergic asthmatics during antigenic bronchoprovocation,'1 and this mediator is capable of causing bronchoconstriction in man and in rodents."12 Slow-reacting substance of anaphylaxis (SRS-A) 'affects mainly the small airways in animals, causing a decrease in lung compliance but no significant change in airway resistance.' The effects of eosinophil chemotactic factor released from mast cells could account for the eosinophilia in the bronchial tissue. Eosinophils could contribute to further inflammatory damage by releasing lysosomal enzymes during phagocytosis of immune complexes and cell debris, but they might also exert beneficial and homeostatic effects because this type of leukocyte contains arylsulfatase B'3 and histaminase,'4 which could inactivate and thus neutralize the effects of SRS-A and histamine. Together these mediators of immediate hypersensitivity could directly cause smooth muscle to contract and could cause mucosal edema and hypersecretion of bronchial mucous
