Recent advances in pulmonary cytopathology

Recent Advances in Pulmonary C opathology S, DONALD GREENBERG, MD* During the past five years, advances in pulmonary cytopathology have made possible the achievementof several goals, including the diagnosis of pneumonias in immunocompromisedhosts, the detection and localizationof occult lung cancers in high-risk cigarette smokers, the diagnosis of lesions of the lungs and chest wall by fine-needle aspiration, and the detection of asbestos exposure by sputum cytologicstudies. Significantprogress has also been made in the application of cell image analysis to the detection of premalignantbronchial epithelial cells in sputum. For the most part, these advancesare hased on such well-knowncytologic procedures as the preparation of thin and evenly distributed smears, prompt fixation, properly controlled and monitored Papanicolaou staining, and thorough screening of slides. Such diligence in laboratory techniques remains the backbone of excellence in pulmonary cytopathology.Cytologicfindings should be interpreted with the clinical features of each case in mind, for the practice of pulmonary cytopathologyremains an art as well as a science. Hu.~tPATIIOL14:901--912, 1983.

The past five years have been a period of accelerated growth and development in p u h n o n a r y cytopathology. The recent advances, based on investigative as well as applied clinical studies, are many and various. T h e y include new findings concerning the diagnosis and treatment of non-neoplastic and neoplastic lung diseases, the differential diagnostic uses of fine-needle lung aspiration and cytologic examination of pleural fluids, the identification of asbestosrelated diseases, and the cytologic investigation of pre-malignant atypias. Pulmonary cytopathology thus continues to provide the primary laboratory means for diagnosing and treating patients with lung diseases.

ticularly in the distal non-ciliated alveolar ducts and alveoli. In sputum smears, FAMs may contain such varied particulates as hemosiderin, carbon, lipid, and tobacco tars (fig. 2). Additional quantitative studies of FAMs are needed to relate their numbers and cytoplasmic contents to various pulmonary diseases as Friedman-Mot et al. have done in their investigations of FAMs in hemorrhagic shock and heart failure.L2 Sputum smears may also contain artifacts and contaminants. In the spring and fall, spurious pollen grains may give the microscopic a p p e a r a n c e of "prickley pears," and vegetable cells with their hyperchromatic nuclei should not be misinterpreted as cancer cells (fig. 3). 3 At times, sputum specimens may reveal pathogenic fungi, parasites, and viruses (fig. 4). 4-6 This is particularly true in specimens from immunocompromised hosts. DIAGNOSTIC AND THERAPEUTIC CONSIDERATIONS IN NEOPLASTIC DISEASES

Collection

SPUTUM EXAMINATION IN NON-NEOPLASTIC DISEASES Just as urine is examined in the study, of renal diseases, sputum should be examined in the study of pulmonary diseases. Sputum specimens represent a collection o f cells and secretions pooled from not only the tracheobronchial tree but also the upper respiratory tract. T h e adequacy o f a sputum specimen is directly proportional to the number of free alveolar macrophages (FAMs) it contains (fig. 1). A specimen without FAMs is saliva, not sputum, and is unsatisfactory. Free alveolar macrophages are phagocytic cells that are essential in the defense of the lung, par* Professor of Pathology, Baylor College of Medicine, 1200 Moursand, Houston,TX 77030. The cell image and analysisstudy was supported in part by grant CA-27313 from the National Cancer Institute and contract NAS 9-15425 from the National Aeronautics and Space Administration and was performed in collaborationwith the Cell Image Analysis Laboratory,NASA/L.B.J.Space Center, Houston,Texas. Address correspondence and reprint requests to Dr. Greenberg. 901

T h e need for p r o p e r s p u t u m collection continues to provide one o f the foremost challenges in the practice of p u l m o n a r y cytopathology. Despite much study and investigation, controversy as to the best method persists. 7-9 In the author's experience, non-smokers cannot produce satisfactory sputum specimens, because they do not have sufficient bronchial secretions. Chronic cigarette smokers, however, have increased bronchial gland secretions and usually can produce adequate early-morning spontaneous cough specimens. Nevertheless, to obtain consistently comparable sputum specimens, nebulized aerosol induction is recommended. Water, isotonic saline, or 3 or 8 per cent sodium chloride may be used. Aerosolization-is best performed in the laboratory, with the subject sitting next to the nebulizer, breathing the aerosol through a disposable cardboard mouthpiece. Of the several aerosol induction procedures that may be used, the author prefers the subject to breath 8 per cent sodium chloride aerosol for 3 minutes, cough, and expectorate into the fixative and then r-epeat the sequence twice. Sputum induction is relatively harmless, although in atopic persons such as those with asthma, it may occasionally result in transient bronchospasm.

Cytologic Screening Recent reports have reaffirmed the value of repeated sputum cytologic examination in laboratory diagnoses o f m o r e than 80 per cent o f lung cancersJ ~ In the 1970s, the Cooperative Early Lung

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FIGURE 1. [top] Numerous free alveolar maerophages [FAMs] in a sputum smear. These FAMs are the hallmark of an adequate sputum specimen, for they arise within the lungs. [Papanicolaou stain, x 225.] [Reprinted, with permission, from Greenberg SD: Diagnostic Pulmonary Cytopathology [a slide-cassette-study guide program]. New York, Audio Visual Medical Marketing, 1982.] FIGURE 2. [middle]. Left, an FAM with multiple large, coarse hemosiderin granules in the cytoplasm. [Papanicolaou stain, x 600,) Right, M,o FAMs.Notice the fineIy granular tobacco tars in the cytoplasm. {~Papanicolaoustain, x 600.} [Reprinted, with permission, from Greenberg SD: Diagnostic Pulmonary Cytopathology [a slide-cassette-study guide program]. New York. Audio Visual Medical Marketing, 1982,] FIGURE 3. [bo#om]. Left, pollen grains with characteristic peripheral spines. In sputum smears such artifacts are most often present in the spring and fail, when pollen counts are high. They settle on the slides as they are waiting to be stained. (Papanicolaou stain, x 600.] Right, this vegetable fiber contains several irregular, dark-staining nuclei. The specimen was saliva, not sputum. Such vegetable fibers are a potential pitfall in the diagnosis of lung cancer. [Papanicolaou stain, x 600.] [Both parts of this figure are reprinted, with permission, from Greenberg SD: Diagnostic Pulmonary Cytopathology [a slide-cassefte-study guide program], New York, Audio Visual Medical Marketing, 1982.]

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FIGURE 4. Top left, sputum specimens occasionally contain fungi. These fungal hyphae show the branching, root-like pattern of the genus Rhizopus, compatible with Mucor. (Papanicolaou stain, x t25.] Top right, a PAP-stained smear of sputum shows a fungus with septate hyphae, which is most likely AspergiIlus. Aspergillus pneumonia is a common complication in immunosuppressed patients. [Papanicolaou stain, x 225.] Middle left, a specimen of sputum shows the broad-necked, budding yeast of Blastomycosis.A definitive diagnosis of fungi rests on cultural characteristics; however, a working diagnosis can rapidly be established by cytologic examination. [Periodic acid-Schiff stain, x 600.) Middle right, a rounded yeast form of Cryptococcus (arroW. Notice its clear, mucoid capsule; [Papanicolaou stain, x 450.] Bottom le~ parasitic diseases of the lungs are not common. However, nematode larvae migrate through the lungs and on rare occasions may be found in sputum. They can be identified by their characteristic head and tail structures. [Popanicolaou stain. • 450.] Bottom right, in a PAP-stained sputum smear, a multinucleated giant cell contains intranuclear viral inclusions. Such intranuclear inclusions and the prominent nuclear molding ore characteristic of Herpes virus. [Papanicolaou stain, x600.] (All ports of this figure are reprinted, with permission, from Greenberg $D: Diagnostic Pulmonary CytopathoTogy ta slide-cassette-study guide program]. New York, Audio Visual Medical Marketing, 1982.]

HUMANPATHOLOGY Volume14, No. r [October 1983) Cancer Group, funded by the National Cancer Institute and involving T h e Johns Hopkins, Mayo, and Memorial Sloan-Kettering medical institutions, was organized to screen a large c o h o r t of cigarette smoking men, 45 years of/tge and older, who were at high risk for lung cancer. 14-17 This stud), had two well-defined goals. T h e first was to devise techniques to detect, localize, and resect occult lung cancers when sputum specimens were positive for carcinoma but chest radiographs were normal (fig. 5). 14 T h e second was to demonstrate that such treatment would significantly increase five-year survival a m o n g patients with lung cancer. The first goal has been accomplished. Complete data concerning the second goal are not yet available, and the National Cancer Institute has r e c o m m e n d e d that no additional screening programs be begun until this second goal has been proved. Nevertheless, the realization of the first goal is, in the author's opinion, an important accomplishment. When a sputum specimen shows evidence of carcinoma, the patient first undergoes a nose and throat examination, to rule out the possibility of an upper airway lesion, and then a diagnostic fiberoptic bronchoscop),. T h e development of the fiberoptic bronchoscope greatly facilitated the localization of in-situ and stage 1 carcinomas of the lung. 18--2~These occult lung cancers gradually evolve, over 20 to 30 years of a patient's cigarette smoking, from pre-malignant dysplasias to in-situ and invasive carcinomas. 21 Bronchoscopic Localization T h e task of localizing an occult carcinoma of the lung is sometimes difficult, since most in-situ lesions cannot be seen by the naked eye. 16 Such lesions must be localized by selective bronchial brushings and blind spur biopsies. 17 These diagnostic procedures, which involve endobronchial brushings and biopsies of both lungs, usually require 1112to 2 hours and must be p e r f o r m e d u n d e r general anesthesia. 16,17 Recently, however, h e m a t o p o r p h y r i n derivatives injected into veins have been observed to concentrate within the malignant cells of occult lung cancers, and endobronchial fluorescence o f the p o r p h y r i n has been helpful in locating the sites of mucosal lesions. 22 Bronchial Carcinoma In Situ O u r present concepts of the natural history of bronchial carcinoma in situ have been derived largely from the careful morphologic studies of Auerbach et al., 23,24 who prepared multiple tissue sections of bronchial mucosa from lungs obtained at autopsy and then related the various stages of epithelial dysplasias to patients' cigarette smoking histories.23. 24 From these investigations, the concept of a "stairstep" of dysplasias eventually leading to the formation of carcinomas in situ was developed. These observations were subsequently substantiated by the cytologic observations of Saccomanno, 25 who studied the sputum of a cohort o f uranium miners. 25 Saccomanno further determined that the mean duration of bronchial car904

cinomas in situ among his patients was 3.2 years, after which the lesions become invasive. Most recently, he also defined cytologic criteria for the diagnosis of bronchial carcinoma in situ in sputum, and suggested that it should be treated by photoradiation accomplished by endobronchial laser activation of intravenously injected hematoporphyrins, rather than by lobectomy. 25 If proved to be technically feasible, such therapy would appear to have merit, since 80 to 90 per cent of cases of bronchial carcinoma in situ result from the generalized field effect of carcinogens inh a l e d in c i g a r e t t e s m o k e and a s e c o n d (metachronous) primary tumor may eventually occur in 15 to 20 per cent of cases. 17 Survival Following Lobectomy for Stage 1 Lung Cancer Much o f the data on survival following lobectomy for stage 1 lung cancer have been derived from the on-going studies o f the National Cancer Institute Cooperative Early Lung Cancer Group. In patients with stage i occult squamous cell carcinomas, projected five-year survivals have ranged from 70 to 90 per cent.15.z6 Small cell carcinoma is rarely detected as a stage 1 lesion; for lesions of this cell type, multipledrug chemotherapy remains the treatment of choice. Adenocarcinomas and large cell carcinomas are most often peripheral lesions initially detected on chest radiographs. When they are diagnosed as stage I lesions, the treatment of choice is lobectomy, and fiveyear survivals of more than 50 per cent have been reported. 27 Diagnosis of Malignancies Metastatic to the Lung Since the lungs have voluminous arterial and venous blood flow, they are often the site of metastases o f both carcinomas and sarcomas, which may manifest a multiplicity of cell types and masquerade as primary pulmonary neoplasms. Therefore, it is not sufficient simply to diagnose a pulmonary lesion as malignant. One should further attempt to determine whether it is primary or secondary, and the patient's total clinical picture must be considered. In cases of a d e n o c a r c i n o m a s , a general rule is that if after careful clinical examination there is no evidence of an intra-abdominal lesion, the carcinoma may be considered primary in the lung. Cases of squamous cell carcinomas with previous extrapulmonary primary lesions (e.g., o f the larynx or the cervix) similarly must be considered individually, since there are no absolute criteria for diagnosis (fig. 6). From time to time, especially perplexing cases arise---e.g., pulmonary metastases of endometrial stromal sarcoma--for which ultrastructural study may be required to characterize the malignant cell type. 2s FINE-NEEDLE ASPIRATION

Over the past 15 years, fine-needle aspiration cytologic features have assumed an important role in

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O FIGURE 5. Top, a sputum specimen, showing representative cells from an occult squamous cell carcinoma in a 60-year-old man who had smoked 80 pack-years of cigarettes. [Papanicolaou stain, x650.] Bottom, this lesion, seen in a Iobectomy specimen, was~the site of origin of the malignant cells shown above. The growth pattern suggests tllat the carcinoma arose as an in-situ lesion that had extended into the underlying bronchlal glands and lamina propria. [Hematoxylin.--eosin stain, x 20.) [Both parts of this figure are reprinted, with permission, from Greenberg SD: Diagnostic Pulmonary Cytopathology [a slide-cassette-study guide program]. New York, Audio Visual Medical Marketing, 1982.)

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FIGURE 6. Squamous carcinoma ceil in the sputum of a 52oyear-old cigarettesmoking woman. Chest radiographs revealed a right-upper-lobe lesion. The patient had undergone radiation therapy four years previously for stage 1 squamous ceil carcinoma of the cervix, [PapanicoIaou stain, x 450.)

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tion is particularly usefifl in cases of small ("oat") cell carcinomas in which positive cytologic diagnoses preclude thoracotomy. PLEURAL FLUIDS

Non-neoplastic Diseases The examination of pleural fluids continues to be a skilled exercise in differential diangosis, because the list of possible diseases that must be considered is long, including parasitic disease 39 in addition to the more common malignancies.

Neoplastic Diseases

FIGURE 7. Smear of fine needle aspirate from a pleural-based left-upper-lobe mass in a 56-year-old cigarette smoking woman. Notice the cluster of well-defined cells of adenocarcinoma, showing finely reticulated nuclear chromatin and prominant nucleoli. [Papanicolaou stain, x 600.]

the diagnosis of discrete pulmonary lesions. 20-js Fineneedle aspiration is usually performed under local anesthesia by use a long, thin 22- or 23-gauge needle with fluoroscopic guidance. In the author's opinion, the cells obtained should be immediately wet fixed and later stained by the Papanicolaou method. Fineneedle aspiration cytologic examination is most usefifl in diagnoses of peripheral lung masses, particularly those involving the chest wall (fig. 7). Following fineneedle aspiration, there may be minimal transient hemoptysis in 5 to 10 per cent of cases; however, major hemoptysis is extremely rare, occurring in less than 1 per cent. Also, in the first 24 hours following aspiration, a small pneumothorax may be seen on chest radiographs in 20 per cent of cases, but large pneumothoraxes requiring thoracotomy tube insertions are infrequent, occurring in less than 5 per cent. T h e cytologic diagnostic yield from fine-needle aspiration is high among experienced practitioners, who have reported positive results in more than 90 per cent of cases of primary carcinoma of the lung. Since there may be areas of necrosis and/or infection within lung tumors, complete samples are best obtained, once the needle has been introduced into a tumor, by means of multiple thrusts with several aspirations from different angles. Fine needle aspira-

MESOTHELIOMAS. With the increasing incidence o f asbestos-associated diseases, malignant pleural mesotheliomas are no longer rare. The triad o f unilateral chest pain, weight loss, and bloody pleural effusion remains the basis for presumptive clinical diagnoses of malignant mesotheliomas. However, other malignancies metastatic to the pleura may produce similar symptoms, and microscopic examinations of the tumor cells and tissue are essential. Cytologic examinations o f pleural effusions are useful in determining whether it is malignant and possibly of mesothelioma origin40; however, definitive diagnoses o f malignant mesothelioma seem best accomplished by light and electron microscopic examinations of tumor tissues obtained at thoracotomy. If the tumor cells stain with mucicarmine or if they are seen to have short, blunted microvilli on electron microscopic examination, the lesion is a metastatic adenocarcinoma.4M 2 Malignant mesotheliomas are mucicarmine-negative, and have long, slender microvilli and i n t e r m e d i a t e filaments on electron microscopy. 41,42 T o the best o f the author's knowledge, these criteria for the differentiation of malignant mesotheliomas from metastatic adenocarcinomas have not yet been applied to cells of malignant pleural effusions. NON-MESOTHELIOMAS. Pleural fluids examined in the usual manner with Papanicolaou-stained smears, membrane filters, and cell blocks can usually be classified as benign, suspicious for malignancy, or malignant. However, in the absence of a patient's clinical history, the site of origin of malignant cells is often difficult to determine (fig. 8). In such cases, examination o f the cells by transmission electron microscopy is recommended. 43 IDENTIFICATION OF ASBESTOSASSOCIATED DISEASES

Recent advances in the pulmonary cytopathology of asbestos-associated diseases have been based on studies o f asbestosis, p u l m o n a r y carcinomas (predominantly in smokers), and malignant pleural mesotheliomas. Increased industrial use of asbestos from 1940 to 1960 led to the appearance of increasing 906

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FIGURE 8. Top left, cell block of pleural fluid showing two groups of metastatic adenocarcinoma cells. In the group in the upper right corner, there is a darkly stained psammoma body (arrow]. This specimen was from a 62-year-old woman who one year previously had been found to have a papillary serous cystadenocarcinoma of the ovary. (Hematoxylir'~eosin stain, x 400.] Tap right, in this cell bYock of pleural fluid, there is a papillary pattern formed by the malignant epithelial cells. Three years previously, the patient, a 58-year-old woman. had undergone pelvic surgery for papillary cystadenocarcinoma of the ovary. [1-1ematoxylin-eosin stain, x 450.] Bottom left, smear of pleural fluid from a 47-year-old man. showing two malignant ~ells with hyperchromatic irregular nuclei and abundant granular cytoplasm. Two years previously, the patient had undergone a nephrectomy for renal cell carcinoma. [Papanicolaou stain, x 600.] Bottom right, smear of cells from a pleural effusion. Notice the monotonous appearance of the neoplastc oval cells with their rounded nuclei and scant cytoplasm. These are malignant cells of a lymphoblasfic lymphoma that manifested as a mediastinal mass in a 16-year-old boy. (Papanicolaou stain, x600.]

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FIGURE 9. left and' right, asbestos bodies in sputum. Each asbestos body has an asbestos fiber as its central core. The surrounding iron coat is deposited by the free alveolar macrophages [FAMs], which phagocytize the asbestos fibers. Notice that the asbestos bodies are usually accompanied by one or more FAMs. (Both, Papanicolaou stain, x 600.] {The left portion of this figure is reprinted, with permission, from Greenberg SD: Diagnostic Pulmonary Cytopathology [a slide-cassette-study guide program]. New York, Audio Visual Medical Marketing, 1982.]

numbers of asbestos-associated pulmonary diseases, and it has been estimated that more than 5 million construction and insulation workers have been sufficiently exposed to asbestos to warrant surveillance. 44 This discussion will not include the mineralogy of the various serpentine and amphibole asbestos fibers, since their cytologic features are similar. More than 90 per cent of the inhaled asbestos fiber load is removed from the lungs by ciliary clearance and taken into the gastrointestinal tract by swallowing. 45 O f the r e m a i n i n g fibers, a p p r o x i m a t e l y 20 p e r cent are phagocytosed and coated by free alveolar macrophages (FAMs), while some 80 per cent remain uncoated.45. 46 T h e coated fibers (asbestos bodies) are neither fibrogenic nor oncogenic, whereas uncoated fibers more than 10 ~m in length and less than 2 tim in diameter are associated with asbestosis, lung cancer (predominately in smokers), and malignant pleural mesotheliomas. 47 Lung tissue digestion and quantification of the residual asbestos bodies have served as the bases for our understanding of the cause and effect relationships between the pulmonary asbestos load and asbestos-associated diseases. 47-50 In wet lung tissue digests, the finding of more than 100 asbestos bodies p e r gram indicates occupational exposure, whereas non-occupational e x p o s u r e s result in 908

fewer. 47 Histopathologic grades of mild, moderate, and severe asbestosis have been found to correspond to wet lung tissue digests o f approximately 1,000, 5,000 and greater than 10,000 asbestos bodies per gram, respectively, ~7 whereas malignant pleural mesotheliomas usually occur with asbestos lung loads of a p p r o x i m a t e l y 400 asbestos bodies per grain wet weight. 50 The cytologic hallmark of an increased asbestos load in the hmgs is the presence of asbestos bodies in the s p u t u m (fig. 9). 45-48,51 Correlative digestion studies have demonstrated that one asbestos body in the sputum indicates greater than 900 asbestos bodies per gram wet weight of lung. 4s Furthermore, in asbestosis, the quantification of asbestos bodies in_the sputum correlates well with associated chest radiograph findings of interstitial and pleural lung disease as well as with results of pulmonary function studies of restrictive disease. 52.53 In asbestos-associated lung cancers, which occur predominately among asbestos workers who smoke, the pulmonary cytologic findings are no different from those in non-asbestos associated lung cancers. 45 T h e presence of accompanying asbestos bodies in smears should alert the cytopathologist to the presence of a large pulmonary asbestos load.

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FIGURE t0, A Papanicolaou-stained cell of squamous metaplasia (le~ with its computerized analog Crighl). [Left, Papanicolaou stain;

x 900. Right, 1 i~m scanned computer analog.) [Both parts of this figure are reprinted, with permission,from Greenberg SD, HeTgeson NGP: Diagnostic cytopathology, including cytoIogy techniques. In Race GJ [ed]: Laboratory Medicine, tenth edition, revision 9, volume 4. Hagerstown, ME),Harper & Row, 1983, p 34.]

INVESTIGATIONS OF PRE-MALIGNANT ATYPIAS

Cell I m a g e Analysis

The field of automation continues to be one of the frontiers in diagnostic cytopathology. In the 1970s, a major breakthrough was the application of high-resolution cell image analysis techniques in the classification o f leukocytes in peripheral blood smears. 56 Such cell image analysis is an optical scientific method in which the cell is divided into small squares, or pixels, of a resolution of usually 0.5 to 1.0 ~m. Each pixel is digitized to determine its optical density, and these numerical values are recorded and stored in an on-line computer. Subsequently, by use of various algorithms, the stored computer data are analyzed and converted into meaningful diagnostic indexes. Over the past five years, the National Cancer Institute has funded an on-going study to apply such cell image analysis techniques in a search for markers of pre-malignant atypias in sputum (fig. 10). 57-60 These research efforts have recently resulted in tile d e v e l o p m e n t o f several promising a u t o m a t e d methods for the diagnosis of bronchial epithelial dysplasias. T h e research studies have utilized both smokers and non-smokers, matched for age and sex, as well as patients with known lung cancers. Tile investigation was initially directed toward the devel-

Feulgen Staining

Within the past five ),ears, there have been renewed attempts to apply Feulgen-staining DNA analysis to the grading of atypical and malignant bronchial epithelial cells in sputum. 54,55 Such quantitative determinations of nuclear DNA can be performed by destaining diagnostic Papanicolaou-stained smears, restaining them by the Feulgen method, and locating the cells for m e a s u r e m e n t . Good correlations between increasing degrees of aneuploidy, as assessed from DNA values, and increasing cytologic nuclear atypias have been reported. In general, such data have indicated a correlation between progressive atypias and squamous cell carcinomas. Most recently, this m e t h o d of Feulgen-staining quantitative DNA analysis has been adapted to cell image analysis. By means of this latter technique, video signals of optical density points from the Feulgen-stained nuclei are integrated, and the values obtained are related to those derived from known diploid cells. It has been suggested that such cytologic analysis o f Feulgenstained sputum specimens might be used in screening for bronchial epithelial atypias. 909

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FIGURE 11. Representative sputum cells. Top left,, squamous metaplasia, ASI 0.5; top right, mild atypia, ASI 1.6; middle left, moderate atypia, ASI 3.0; middle right, severe atypia, ASI 4.0; bottom, carcinoma, ASI 5.5. [Papanicolaou stain, x 500.] [Reprinted, with permission, from Greenberg el al.6~

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Spivey, T. L. McLemore, and M. L. Mace and cytotechnologists L. C. Mabry and M. L. Farley for their assistance in the cytologic studies of asbestos-associated diseases and Drs. H . J . Spjut, P. R. Swank, G. R. Taylor, and R. G. Estrada, Senior Research Analyst D. G. Winkler, and cytotechnologists N. R. H u n t e r and G.J. Montalvo for their contributions in the application o f cell image analysis to the diagnosis of pre-neoplastic cells in sputum.

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REFERENCES

FREQUENCy 12

1. Friedman-Mor Z, Chalon J, Turndorf H, et al: Tracheobronchial cytologic changes and abnormal serum heme-pigments in hemorrhagic shock. J Trauma 17:829, 1977 2. Friedman-Mot Z, CbalonJ, Turndorf H, et al: Cardiac index and incidence of heart failure cells. Arch Pathol Lab Med 102:418, 1978 3. Weaver KM, Novak PM, Naylor B: Vegetable cell contaminants in cytologic specimens: their resemblance to cells associated with various normal and pathologic states. Acta Cytol 25:210, 1981 4. Humphreys K, Hieger LR: Strongyloides stercorlis in routine Papanicolaou-stained sputum smears. Acta Cytol 23:471, 1979 5. Trumbull ML, Chesney TM: The cytological diagnosis of pulmonary blastomycosis. JAMA 245:836, 1981 6. Johnston WW: Pulmonary cytopathology in the compromised host. Semin Respir Med 4:149, 1982 7. McLarty JW, Greenberg SD, Hurst GA, et al: Statistical comparison of aerosol-induced and spontaneous sputum specimens in the Tyler Asbestos Workers Program. Acta Cytol 24:460, 1980 8. Melamed M, Zaman J, Lazowski W: Obtaining sputum for cytology. N Engl J Med 305:894, 1981 9. Frost JK, Bail WC, Levin ML, et ah Sputum for cytologic diagnosis of lung cancer. N Engl J Med 306:109, 1982 10. Suprun H, Pedio G, RuttnerJR: Tile diagnostic reliability of cytologic typing in primary lung cancer with a review of the literature. Acta Cytol 24:304, 1980 I 1. Chalon J, Tang CK, Klein GS, et ah Routine cytodiagnosis of pulmonary malignancies. Arch Pathol Lab Med 105:11, 1981 12. Johnston WW, Bossen EH: Ten )ears of respiratory cytopathology at Duke University Medical Center. Acta Cyto125:103, 1981 13. Gupta RK: Value of sputum cytology in tile differential diagnosis of alveolar cell carcinoma from bronchogenic adenocarcinoma. Acta Cytol 25:255, 1981 14. Martini N, Melamed MR: Occult carcinoma of the lung. Ann Thorac Surg 30:215, 1980 15. Melamed MR, Flehinger BJ, Zaman MB, et ah Detection of true pathologic stage 1 lung cancer in a screening program, and the effect on survival. Cancer 47:1182, 1981 16. Woolner LB: Earl)' detection and localization of occult lung cancer in symptomless males. In edited by Koss LG, Coleman DV (eds): Advances In Clinical Cytology. Woburn, MA, Butterworth 1981, p 288 17. Woolner LB, Fontana RS, Sanderson DR, et ah Mayo lung project: evaluation of lung cancer screening through December 1979. Mayo Clinic Proc 56:544, 1981 18. Carter D, Marsh BR, Baker RR, et al: Relationships of morphology to clinical presentation in ten cases of early squamous cell carcinoma of the lung. Cancer 37:1389, 1976 19. Jay SJ, Wehr K, Nicholson DP, et al: Diagnostic sensitivity and specificity of pulmonary cytology: comparison of techniques used in conjunction with flexible fiberoptic bronchoscopy. Acta Cytol 24:304, 1980 20. LukemanJM, Hind CRK, Carr DT, et ah New applications of cytologic techniques in cancer diagnosis. In Hickey RC (ed): Current Problems of Cancer. Chicago, Year Book Medical Publishers, 1980, p 22 21. Auerbach O, Saccomanno G, Kuschner M, et ah Histologic findings in the tracheobronclfial tree of uranium miners and non-miners with hmg cancer. Cancer 42:483, 1978

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ATYPIA. SYKTU$ INDEX

FIGURE12. Cell a~ypia profile of a sputum specimen. The closed circles represent orangeophilic squamous cells. The cyanophilic squamous ceils are open circles. More than five of the 100 atypical cells were malignant, and the diagnosis was positive for carcinoma. The distribution of the many pre-malignant atypical cells represents the progressive stages of carcinogenesis, META, squamous metaplasia; MILD,mud a~,pia, MOD, moderate atypia; SEV,severe atypia; MALIG, malignant. [Reprinted with permission, from Kimsey et al.~)

o p m e n t o f a baseline set o f atypical cells to use in t r a i n i n g t h e c o m p u t e r . T o a c c o m p l i s h t h a t goal, t h r e e cytopathologists, working independently, interpreted a p p r o x i m a t e l y 4 , 0 0 0 p r o j e c t e d c o l o r slides o f p h o tomicrographs of various atypical Papanicolaoustained cells in s p u t u m . T h o s e atypical cells with consensus diagnoses were entered into the computer t r a i n i n g set. F o r d e s c r i m i n a n t analysis o f t h e atypical cells, the s t o r e d o n - l i n e c o m p u t e r d a t a w e r e a n a l y z e d f o r 14 f e a t u r e s , i n c l u d i n g n u c l e o c y t o p l a s m i c ratio, irregularity of nuclear membrane, and distribution of nuclear chromatin. From such analyses, an atypia status i n d e x (ASI), was d e r i v e d . 57 T h e A S I is a n u merical m e a s u r e o f cellular a t y p i a a n d r a n g e s f r o m 0.5 ( s q u a m o u s m e t a p l a s i a ) to 4.5 ( c a r c i n o m a ) (fig. 1 1). F o r p u r p o s e s o f p a t i e n t diagnosis a n d f o l l o w - u p care, s p u t u m cell a t y p i a profiles (CAPs), c o n s i s t i n g o f the A S I s o f 100 atypical cells p e r s p e c i m e n , w e r e also d e t e r m i n e d (fig. 12). I n the a u t h o r ' s e x p e r i e n c e , the A S I a n d C A P d e t e r m i n a t i o n s h a v e b e e n f o u n d to be h i g h l y a c c u r a t e a n d m o r e t h a n 90 p e r c e n t r e p r o ducible. T h e y r e q u i r e , h o w e v e r , ]& days p e r s p e c i m e n to process, a n d f o r this r e a s o n t h e y a r e n o t yet applicable in mass s c r e e n i n g o f g r o u p s at h i g h risk f o r l u n g c a n c e r . It is a n t i c i p a t e d t h a t f u t u r e s u b s t i t u t i o n o f a laser s c a n n e r f o r the p r e s e n t television s c a n n e r m i g h t r e d u c e t h e digitizing time p e r s p e c i m e n to less than one hour.

Acknowledgments. T h e a t t t h o r t h a n k s Drs. V . L . Roggli, J. W. McLarty, L.R. Hieger, G.A. Hurst, C.G., 911

HUMAN PATHOLOGY

Volume 14, No. 10 (October '1983}

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