1koss - Diagnostic - Cytology Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles
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Editors: Koss, Leopold G.; Melamed, Myron R. Title: Koss' Diagnostic Cytology and Its Histopathologic Bases, 5th Edition Copyright 2006 Lippincott Williams & Wilkins
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1 Diagnostic Cytology: Its Origins and Principles

EARLY EVENTS: THE BIRTH OF MICROSCOPY AND CLINICAL CYTOLOGY
Diagnostic cytology is the culmination of several centuries of observations and research. Although it is beyond the scope of this overview to give a detailed account of the past events, the readers may find a brief summary of these developments of interest. Although some cells can be seen with the naked eye, for example, birds' or reptiles' eggs, it was the invention of the microscope that led to the recognition that all living matter is composed of cells. The term microscope was proposed in 1624 by an Italian group of scientists, united at the Academia dei Licei in Florence. The group, among others, included the great astronomer, Galileo, who apparently was also a user of one of the first instruments of this kind (Purtle, 1974). The first microscopes of practical value were constructed in Italy and in Holland in the 17th century. The best instrument, constructed by the Dutchman, Anthony van Leeuwenhoek (1632-1723) allowed a magnification of 275. Leeuwenhoek reported on the miraculous world of microscopy in a series of letters to the Royal Society in London. His observations ranged from bacteria to spermatozoa. Interested readers will find illustrations of Leeuwenhoek's work and further comments on him and his contemporaries in the excellent book entitled History of Clinical Cytology by Grunze and Spriggs (1983). For nearly 2 centuries thereafter, these instruments were costly, very difficult to use and, therefore, accessible only to a very small, wealthy elite of interested scientists, most of whom were amateurs dabbling with microscopy as a diversion. Many of these microscopes were works of art (Fig. 1-1). Using one of these microscopes with a focusing adjustment, the Secretary of the Royal College in London, Robert Hooke, observed, in 1665, that corks and sponges were composed of little boxes that he called cells (from Latin, cellula = chamber) but the significance of this observation did not become apparent for almost 200 years. The great 17th century Italian anatomist, Malpighi, was also familiar with the microscope and is justly considered the creator of histology. The event that, in my judgment, proved to be decisive in better understanding of P.4 cell and tissue structure in health and disease was the invention of achromatic lenses that allowed an undistorted view of microscopic images. In the 1820s, the construction of compound microscopes provided with such optics occurred nearly simultaneously in London (by Lister, the father of Lord Lister, the proponent of surgical antisepsis) and in Paris (by the family of opticians and microscope makers, named Chevalier). These microscopes, with many subsequent improvements, were easy to use, could be mass-produced at a reasonable price, and thus became available to a great many interested professional investigators, leading to a better understanding of cell structure and, indirectly, to an insight into the mechanisms of cell function and, hence, of life processes. Although, even in the age of molecular biology, much remains to be discovered about the interplay of molecules leading to cell differentiation and function, some progress has been made (see Chaps. 3 and 7) and more can be expected in the years to come.
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Figure 1-1 Two beautiful 17th century microscopes. (Courtesy of the Billing's Collection, Armed Forces Institute of Pathology, Washington, DC.)
Nearly all the microscopic observations during the first half of the 19th century were conducted on cells because the techniques of tissue processing for microscopic examination were very primitive. Early on, the investigators observed that animal cells from different organs varied in size and shape and that some were provided with specialized structures, such as cilia. Perhaps the most remarkable record of these observations was an atlas of microscopic images by a French microscopist, Andr Franois Donn, published in Paris in 1845. The atlas was the first book illustrated with actual photomicrographs of remarkable quality (Fig. 1-2), obtained by the newly described method of Daguerre. The observations by many early observers led to the classification of normal cells and, subsequently, tissues as the backbone of normal cytology and histology. In the middle of the 19th century, the pioneering German pathologist, Rudolf Virchow, postulated that each cell is derived from another cell (omnis cellula a cellula). This assumption, which repeatedly has been proved to be correct, implies that at some time in a very distant past, probably many million years ago, the first cell, the mother of all cells, came to exist. How this happened is not known and is the subject of ongoing investigations. By the middle of the 19th century, several books on the use of the microscope in medicine became available. In the book, The Microscope in its Applications to Practical Medicine, P.5 that appeared in two editions (1854 and 1858), Lionel Beale of London described the cells as follows: A cell consists of a perfectly closed sac containing certain contents. The most important structure within the cell wall, in most instances, is the nucleus, upon which the multiplication of the cell (and other functions) depend. It must be borne in mind, however, that in some cells, such as the human blood corpuscles (erythrocytes, comment by LGK) a nucleus is not to be demonstrated. Within the nucleus there usually exists a clear bright spot. This is the nucleolus. Beale further classified cells into several categories according to their shapes (scaly or squamous cells, tesselated cells [epithelial cells lining serous membranes, LGK], polygonal cells, columnar cells, spherical cells,
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spindle-shaped cells, fusiform cells, etc.), thus describing the entire spectrum of cell configuration. He further described cells derived from various organs (including the central nervous system) and reported that some cells were ciliated, notably those of the trachea, bronchus, fallopian tubes and portions of the endocervical canal. Beale also reported that some cells have a remarkable power of multiplication distinguished for the distinctness and number of its nuclei (cancer cells). Beale described the use of the microscope to identify cancer of various organs that he could distinguish from a benign change of a similar clinical appearance. It is evident, therefore, that by the middle of the 19th century, approximately 150 years ago, there was considerable knowledge of the microscopic configuration of human cells and their role in the diagnosis of human disease.
Figure 1-2 Reproduction of Figure 33 from Donn's Atlas, published in 1845. The daguerreotype represents vaginal secreta and shows squamous cells, leukocytes, identified as purulent globules (b), and Trichomonas vaginalis (c). Note the remarkable pictorial quality of the unstained material.
Perhaps the most important series of observations pertinent to this narrative was the recognition that cells obtained from clinically evident cancerous growths differed from normal cells. The initial observations on cancer cells is attributed to a young German physiologist, Johannes Mller, who, in 1838, published an illustrated monograph entitled On the Nature and Structural Characteristics of Cancer and Those Morbid Growth That Can Be Confounded With It. In this monograph, Mller discussed at some length the differences in configuration of cells and their nuclei in cancer when compared with normal cells. Mller's original observations on the differences between normal and cancerous cells were confirmed by several investigators. For example, in 1860, Beale identified and described cancer cells in sputum. It may come as a surprise to some of the readers that as early as 1845 and 1851, a German
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microscopist, working in Switzerland and writing in French, Hermann Lebert, used cell samples aspirated from patients by means of a cannula for the diagnosis of cancer. In 1847, M. Kn of Strasbourg, about whom little is known, described a needle with a cutting edge useful in securing material from subcutaneous tumors, examined as smears (Grunze and Spriggs, 1983; Webb, 2001). Virchow, often considered the father of contemporary pathology, and who was Mller's pupil, was a superb observer at the autopsy table and a good microscopist. He recognized and described the gross and microscopic features of a large number of entities, such as infarcts, inflammatory lesions, leukemia, and various forms of cancer. However, his views on the origin of human cancer were erroneous because he believed that all cancers were derived from connective tissue and not by transformation of normal tissues (Virchow, 1863). For this reason, he had difficulties in accepting the observations of two of his students and contemporaries, Thiersch in 1865 and Waldayer in 1867, who independently advocated the origin of carcinomas of the skin, breast, and uterus from transformed normal epithelium. Because Virchow wielded a tremendous influence in Germany, not only as a scientist but also as a politician (he was a Professor of Pathology in Berlin as well as a Deputy to the German Parliament, a socialist of sorts, who fought with the famous Chancellor, Bismarck), views that were in conflict with his own were often rejected, thus delaying the development of independent scientific thought. It took about 40 years until the confirmation of Thiersch's and Waldayer's concepts of the origin of carcinomas was documented by Schauenstein for the uterine cervix in 1908 (see Chap. 11). It took many more years until the concept of a preinvasive stage of invasive cancer, originally designated as carcinoma in situ by Schottlander and Kermauner in 1912, was generally accepted and put to a good clinical use in cancer detection and prevention. These are but a few of the early contributions that have bearing on diagnostic cytology as it is known today. In addition to the contributors mentioned by name, there were many other heroes and antiheroes who made remarkable contributions to the science of human cytology during the second half of the 19th century, and this brief narrative doesn't do justice to them. The interested reader should consult a beautifully illustrated book on the history of clinical cytology by Grunze and Spriggs (1983). Still, in spite of these remarkable developments, the widespread application of cytology to the diagnosis of human disease did not take place until the 1950s. Although P.6 sporadic publications during the second half of the 19th century and the first half of the 20th century kept the idea of cytologic diagnosis alive, it was overshadowed by developments in histopathology.
THE ERA OF HISTOPATHOLOGY The Beginning

Although cells teased from tissues were the main target of microscopic investigations during the first half of the 19th century, consistent efforts have been made to develop methods of tissue processing. Thus, in the 1858 edition of Beale's book, several pages are dedicated to the methods of hardening soft tissue samples by boiling and to the methods of preparation of transparent, thin sections suitable for microscopic examination with hand-held cutting instruments. Subsequently, various methods of tissue fixation were tried, such as chromium salts, alcohol, and ultimately, formalin and the manual cutting instruments were replaced by mechanical microtomes around 1880. Simultaneously, many methods of tissue staining were developed. There is excellent evidence that, by 1885, tissue embedding in wax or paraffin, cutting of sections with a microtome, and staining with hematoxylin and eosin were the standard methods in laboratories of pathology, as narrated in the history of surgical pathology at the Memorial Hospital for Cancer, now known as the Memorial Sloan-Kettering Cancer Center (Koss and Lieberman, 1997). Two events enhanced the significance and value of tissue pathology. One was the introduction of the concept of a tissue biopsy, initially proposed for diagnosis of cancer of the uterine cervix and endometrium by Ruge and Veit in 1877, who documented that the microscope is superior to clinical judgment in the diagnosis of these diseases. However, the term biopsy is attributable to a French dermatopathologist, Ernest Besnier, who coined it in 1879 (Nezelof, 2000). The second event was the introduction of frozen sections, popularized by Cullen in 1895, which allowed a rapid processing of tissues and became an essential tool in guiding surgeons during surgery (see also Wright, 1985). With these two tools at hand, the study of cells was practically abandoned for nearly a century. Next to autopsy pathology, the mainstay of classification of disease processes during the 18th and 19th centuries, histopathology became the dominant diagnostic mode of human pathology, a position that it holds until today. Histopathology is based on analysis of tissue patterns, which is a much simpler and easier task than the interpretation of smears that often requires tedious synthesis of the evidence dispersed on a slide. Further, histopathology is superior to cytologic samples in determining the relationship of various tissues to each other, for
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example, in identifying invasion of a cancer into the underlying stroma.
Current Status
The introduction of histopathology on a large scale led to the rapid spread of this knowledge throughout Europe and the Americas. The ever-increasing number of trained people working in leading institutions of medical learning was capable of interpretation of tissue patterns supplementing clinical judgment with a secure microscopic diagnosis. Further, the tissue techniques allowed the preparation of multiple identical samples from the same block of tissue, thus facilitating exchanges between and among pathologists and laying down the foundation of accurate classification of disease processes, staging and grading of cancers and systematic follow-up of patients, with similar disorders, leading to statistical behavioral studies of diseases of a similar type. Such studies became of critical importance in evaluating treatment regimens, initially by surgery or radiotherapy and, even more so, after the introduction of powerful antibiotics and anti-cancer drugs that were active against diseases previously considered hopeless. Nearly all clinical treatment protocols are based on histologic assessment of target lesions. Histologic techniques were also essential in immunopathology that allowed the testing of multiple antibodies on samples of the same tissue. Such studies are difficult to accomplish with smears, which are virtually always unique.
THE RETURN OF CYTOLOGY Papanicolaou and the Cytology of the Female Genital Tract
The beginnings of the cytology of the female genital tract can be traced to the middle of the 19th century. The microscopic appearance of cells from the vagina was illustrated by several early observers, including Donn and Beale, whose work was discussed above (see Fig. 1-2). In 1847, a Frenchman, F.A. Pouchet, published a book dedicated to the microscopic study of vaginal secretions during the menstrual cycle. In the closing years of the 19th century, sporadic descriptions and illustrations of cancer cells derived from cancer of the uterine cervix were published (see Chap. 11). However, there is no doubt whatsoever that the current resurgence of diagnostic cytology is the result of the achievements of Dr. George N. Papanicolaou (1883-1962), an American of Greek descent (Fig. 1-3). Dr. Pap, as he was generally known to his coworkers, friends, and his wife Mary, was an anatomist working at the Cornell University with a primary interest in endocrinology of the reproductive tract. Because of his interest in the menstrual cycle, he developed a small glass pipette that allowed him to obtain cell samples from the vagina of rodents. In smears, he could determine that, during the menstrual cycle, squamous cells derived from the vaginal epithelium of these animals followed a pattern of maturation and atrophy corresponding to maturation of ova. He made major contributions to the understanding of the hormonal mechanisms of ovulation and menstruation and is considered to be one of the pioneering contributors to reproductive endocrinology. P.7
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Figure 1-3 George N. Papanicolaou, 1954, in a photograph inscribed to the author.
However, his fame is based on an incidental observation of cancer cells in vaginal smears of women whose menstrual cycle he was studying. Papanicolaou had no training in pathology and it is, therefore, not likely that he himself identified the cells as cancerous. It is not known who helped Papanicolaou in the identification of cancer cells. It is probable that it was James Ewing who was at that time Chairman of Pathology at Cornell and who was thoroughly familiar with cancer cells as a consequence of his exposure to aspiration biopsies performed by the surgeon, Hayes Martin, at the Memorial Hospital for Cancer (see below). Papanicolaou's initial contribution to the subject of New Cancer Diagnosis, presented during an obscure meeting on the subject of the Betterment of the
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Human Race in Battle Creek, MI, in May, 1928, failed to elicit any response. Only in 1939, prodded by Joseph Hinsey, the new Chairman of the Department of Anatomy at Cornell, had Papanicolaou started a systematic cooperation with a gynecologist, Herbert Traut, the Head of Gynecologic Oncology at Cornell, who provided him with vaginal smears on his patients. It soon became apparent that abnormal cells could be found in several of these otherwise asymptomatic patients who were subsequently shown to harbor histologically confirmed carcinomas of the cervix and the endometrium. Papanicolaou and Traut's article, published in 1941 and a book published in 1943, heralded a new era of application of cytologic techniques to a new target: the discovery of occult cancer of the uterus. Papanicolaou's name became enshrined in medical history by the term Pap smear, now attached to the cytologic procedure for cervical cancer detection. The stain, also invented by Papanicolaou and bearing his name, was nearly universally adopted in processing cervicovaginal smears. Papanicolaou's name was submitted twice to the Nobel Committee in Stockholm as a candidate for the Nobel Award in Medicine. Unfortunately, he was not selected. As a member of the jury told me (LGK) many years later, the negative decision was based on the fact that Papanicolaou had never acknowledged previous contributions of a Romanian pathologist, Aureli Babs (Fig. 1-4), who, working with the gynecologist C. Daniel, reported in January 1927 that cervical smears, obtained by means of a bacteriologic loop, fixed with methanol and stained with Giemsa, were an accurate and reliable method of diagnosing cancer of the uterine cervix. On April 11, 1928, Babs published an extensive, beautifully illustrated article on this subject in the French publication, Presse Mdicale, which apparently had remained unknown to Papanicolaou. One of the highlights P.8 of Babs' article was the observation that a cytologic sample may serve to recognize cancer of the uterine cervix before invasion. Babs' observations were confirmed only once, by an Italian gynecologist, Odorico Viana in 1928, whereas Papanicolaou's work stimulated a large number of publications and received wide publicity. Both Babs' and Viana's articles were translated into English by Larry Douglass (1967 and 1970).
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Figure 1-4 Aureli Babs. (Courtesy of Dr. Bernard Naylor, Ann Arbor, MI.)
The reason for Papanicolaou's success and Babs' failure to attract international attention clearly lies in the differences in geographic location (New York City vs. Bucharest) and in timing. If Papanicolaou's 1928 article were his only publication on the subject of cytologic diagnosis of cancer, he would have probably remained obscure. He had the great fortune to publish again in the 1940s and his ideas were slowly accepted after the end of World War II, with extensive help from Dr. Charles Cameron, the first Medical and Scientific Director of the American Cancer Society, which popularized the Pap test. A summary of these events was presented at a meeting of the American Cancer Society (Koss, 1993).
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The Pap Smear: The Beginning

The value of the vaginal smear as a tool in the recognition of occult cancers of the uterine cervix and the endometrium was rapidly confirmed in a number of articles published in the 1940s (Meigs et al, 1943 and 1945; Ayre, 1944; Jones et al, 1945; Fremont-Smith et al, 1947). It soon became apparent that the vaginal smear was more efficient in the discovery of cervical rather than endometrial cancer and the focus of subsequent investigations shifted to the uterine cervix. In 1948, Lombard et al from Boston introduced the concept of the vaginal smear as a screening test for cancer of the uterine cervix. Because the vaginal smear was very tedious to screen and evaluate, the proposal by a Canadian gynecologist, J. Ernest Ayre, to supplement or replace it with a cell sample obtained directly from the uterine cervix under visual control was rapidly and widely accepted. In 1947, Ayre ingeniously proposed that a common wooden tongue depressor could be cut with scissors to fit the contour of the cervix, thus adding a very inexpensive tool that significantly improved the yield of cells in the cervical sample. Ayre's scraper or spatula, now made of plastic, has remained an important instrument in cervical cancer detection. In 1948, the American Cancer Society organized a national conference in Boston to reach a consensus on screening for cervical cancer. The method was enthusiastically endorsed by the gynecologists but met with skepticism on the part of the participating pathologists. Nonetheless, the first recommendations of the American Cancer Society pertaining to screening for cervical cancer were issued shortly thereafter. In 1950, Nieburgs and Pund published the first results of screening of 10,000 women for occult cancer of the cervix, reporting that unsuspected cancers were detected in a substantial number of screened women. This seminal article, followed by a number of other publications, established the Pap test as a standard health service procedure. Further support for the significance of the test was a series of observations that the smear technique was helpful in discovering precancerous lesions (initially collectively designated as carcinoma in situ), which could be easily treated, thus preventing the development of invasive cancer. Unfortunately, no double-blind studies of the efficacy of the cervicovaginal smear have ever been conducted, and it became the general assumption that the test had a very high specificity and sensitivity. The legal consequences of this omission became apparent 40 years later.
The Pap Smear From the 1950 to the 1980s

Although the American pathologists, with a few notable exceptions (Reagan, 1951), were reluctant to acknowledge the value of the cervicovaginal smear, toward the end of the 1960s, an ever-increasing number of hospital laboratories were forced to process Pap smears at the request of the gynecologists. In those years, the number of pathologists trained in the interpretation of cytologic material was very small, and it remained so for many years. The responsibility for screening and, usually the interpretation of the smears, was assumed by cytotechnologists who, although few in number, were better trained to perform this function than their medical supervisors. With the support of the National Cancer Institute, several schools for training of cytotechnologists were established in the United States in the 1960s. These trained professionals played a key role in the practice of cytopathology. This time period has also seen the opening of several large commercial laboratories dedicated to the processing of cervicovaginal smears. New books, journals, and postgraduate courses offered by a number of professional organizations gave the pathologists an opportunity to improve their skills in this difficult field of diagnosis. Several very successful programs of cervix cancer detection were established in the United States and Canada, and it became quite apparent that the mortality from cancer of the uterine cervix could be lowered in the screened populations. As a consequence, by the end of the 1980s, a 70% reduction in the mortality from this disease was recorded in several geographic areas where mass screening was introduced. However, in none of the populations screened was cancer of the cervix completely irradicated.
The Pap Smear From the 1980s to Today

In the 1970s and early 1980s, several articles commenting on the failure of the cervicovaginal smear in preventing the developments of invasive cancer of the uterine cervix appeared in the American literature and in Sweden (Rylander, 1976; Fetherstone, 1983; Koss, 1989; summary in Koss and Gompel, 1999). The reports did not fully analyze the reasons for failure and were generally ignored. In 1987, however, an article in the Wall Street Journal by an investigative journalist, Walt Bogdanich, on failure of laboratories to identify cancer of the cervix in young women, some who were mothers of small children, elicited a great deal of attention. It prompted the Congress of the United States in 1988 to promulgate a law, known as the Amendment to the Clinical P.9
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Laboratory Improvement Act (CLIA 88), governing the practice of gynecologic cytology in the United States. The implications of the law in reference to practice of cytopathology are discussed elsewhere in this book (see Chap. 44). Suffice it to say, cytopathology, particularly in reference to cervicovaginal smears, has become the object of intense scrutiny and legal proceedings against pathologists and laboratories for alleged failure to interpret the smears correctly, casting a deep shadow on this otherwise very successful laboratory test. As a consequence of these events, several manufacturers have proposed changes in collection and processing of the cervicovaginal smears. The collection methods of cervical material in liquid media, followed by automated processing with resulting monolayer preparations, have been approved by the Food and Drug Administration (USA). Other manufacturers introduced apparatuses for automated screening of conventional smears. New sampling instruments were also developed and widely marketed, notably endocervical brushes. All these initiatives were designed to reduce the risk of errors in the screening and interpretation of cervicovaginal smears. These issues are discussed in Chapters 8, 11, 12, and 44.
DEVELOPMENTS IN NONGYNECOLOGIC CYTOLOGY Historical Overview

At the time of early developments in general cytology in the 19th century, summarized above, numerous articles were published describing the application of cytologic techniques to various secreta and fluids, such as sputum, urine, effusions, and even vomit for diagnostic purposes. These contributions have been described in detail in Grunze and Spriggs' book. The recognition of lung cancer cells in sputum by Beale in 1858 was mentioned above. As lung cancer became a serious public health dilemma in the 1930s and 1940s, in Great Britain, Dudgeon and Wrigley developed, in 1935, a method of wet processing of smears of fresh sputum for the diagnosis of lung cancer. The method was used by Wandall in Denmark in 1944 on a large numbers of patients, with excellent diagnostic results. Woolner and McDonald (1949) at the Mayo Clinic and Herbut and Clerf (1946) in Philadelphia also studied the applications of cytology to lung cancer diagnosis. In the late 1940s and early 1950s, Papanicolaou, with several co-workers, published a number of articles on the application of cytologic techniques to the diagnosis of cancer of various organs, illustrated in his Atlas. In the United Kingdom, urine cytology was applied by Crabbe (1952) to screening of industrial workers for cancer of the bladder and gastric lavage techniques by Schade (1956) to screening for occult gastric cancer, a method extensively used in Japan for population screening. Esophageal balloon technique was applied on a large scale in China for detecting precursor lesions of esophageal carcinoma. Screening for oral cancer has been shown to be successful in discovering occult carcinomas in situ. Thus, conventional cytologic techniques, when judiciously applied, supplement surgical pathology in many situations when a tissue biopsy is either not contemplated, indicated, or not feasible. It needs to be stressed that cytopathology has made major contributions to the recognition of early stages of human cancer in many organs and, thus, contributed in a remarkable way to a better understanding of events in human carcinogenesis and to preventive health care. These, and many other applications of cytologic techniques to the diagnosis of early and advanced cancer and of infectious disorders of various organs, are discussed in this text.
THE ASPIRATION BIOPSY (FNA) The Beginning

Ever since syringes or equivalent instruments were introduced into the medical armamentarium, probably in the 15th century of our era, they were used to aspirate collections of fluids. With the introduction of achromatic microscopes and their industrial production in the 1830s, the instrument became accessible to many observers who used it to examine the aspirated material. It has been mentioned above that a French physician, Kn, and a German-Swiss pathologist, Lebert, described, in 1847 and 1851, the use of a cannula to secure cell samples from palpable tumors and used the microscope to identify cancer. Sporadic use of aspirated samples has been described in the literature of the second half of the 19th century and in the first years of the 20th century. An important contribution was published in 1905 by two British military surgeons, Greig and Gray, working in Uganda who aspirated the swollen lymph nodes, by means of a needle and a syringe, of patients with sleeping sickness to identify the mobile trypanosoma (see Webb, 2001 for an excellent recent account of early investigators). In the 20th century, to my knowledge, the first aspiration biopsy diagnosis of a solid tumor of the skin (apparently a lymphoma) was published by Hirschfeld (1912), who was the first person to use a small-caliber needle. He subsequently extended his experience to other tumors, but was prevented by World War I from publishing his results until 1919. Several other early observers reported on the aspiration of lymph nodes and other accessible sites
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(Webb, 2001). The most notable development in diagnostic aspiration biopsy was a paradoxical event. James Ewing, the Director of the Memorial Hospital for Cancer in New York City and also a Professor of Pathology at Cornell University Medical School, was a dominant figure in American oncologic pathology between 1910 and 1940. Although Ewing has made great contributions to the classification and identification of human cancer, he was adamantly opposed to tissue biopsies because they allegedly contributed to the spread of cancer (Koss and Lieberman, 1997). Because of the ban on tissue biopsies, a young surgeon and radiotherapist at the Memorial Hospital, Hayes Martin, who refused to treat patients P.10 without a preoperative diagnosis, began to aspirate palpable tumors of various organs by means of a large-caliber needle and a Record syringe. The material was prepared in the form of air-dried smears, stained with hematoxylin and eosin by Ewing's technician, Edward Ellis. Tissue fragments (named clots) were embedded in paraffin and processed as cell blocks. Palpable lesions of lymph nodes, breast, and thyroid were the initial targets of aspiration. The material was interpreted by Ewing's associate and subsequent successor (and my Chief-LGK), Dr. Fred W. Stewart. In response to a specific query, the reasons for this development were explained many years later in a letter dated June 30, 1980, written by Dr. Fred W. Stewart to this writer. Martin and Ewing were at sword's point on the need for biopsy proof prior to aggressive surgery or radiation (in neck nodes since Hayes Martin dealt exclusively in head and neck stuff) and the needle was a sort of compromise. Ewing thought biopsy hazardousa method of disease spread. The material was seen mostly by me (FWS). Ewing, at the time, was quite inactive. Eddie Ellis merely fixed and stained the slides. He probably looked at themhe was used to looking at stuff with Ewing and really knew more about diagnoses than a lot of pathologists of the period. The needle really spread from neck nodes to the various other regions, especially to the breast, of course. The method proved to be very successful and accurate with very few errors or clinical complications. Martin and Ellis published their initial results in 1930 and 1934. In 1933, Dr. Fred W. Stewart published a classic article, The Diagnosis of Tumors by Aspiration, in which he discussed, at length, the pros and cons of this method of diagnosis, its achievements, and pitfalls, based on experience with several hundred samples. As Stewart himself stated in a letter (to LGK), he was damned by many for having advocated this insecure and potentially harmful method of diagnosis, without a shred of proof. For a detailed description of these events, see Koss and Lieberman (1997). In fact, the method of aspiration pioneered by Martin has remained a standard diagnostic procedure at Memorial SloanKettering Cancer Center until today (2004), the only institution in the world where the procedure has remained in constant use for more than 75 years. There is no evidence that the Memorial style aspiration smear was practiced on a large scale anywhere else in the world. The method was described and illustrated by John Godwin (1956) and again in the first edition of this book (1961) by John Berg, but has met with total indifference in the United States. In Europe, on the other hand, the interest in the method persisted. Thus, in the 1940s, two internists, Paul LopesCardozo in Holland and Nils Sderstrm in Sweden, experimented on a large scale with this system of diagnosis, using small-caliber needles and hematologic techniques to process the smears. Lopes-Cardozo and Sderstrm subsequently published books on the subject of thin-needle aspiration. Although both books were published in English, they had virtually no impact on the American diagnostic scene, but were widely read in Europe.
Current Status
Working at the Radiumhemmet, the Stockholm Cancer Center, the radiotherapist-oncologist, Sixten Franzn, and his student and colleague, Josef Zajicek, applied the thinneedle technique first to the prostate and, subsequently, to a broad variety of targets, ranging from lesions of salivary glands to the skeleton. Franzn et al (1960) described a syringe (initially developed for the diagnosis of prostatic carcinoma) that allowed performance of the aspiration with one hand, whereas the other hand steadied the target lesion (see Chap. 28). As nonpathologists, these observers used air-dried smears, stained with hematologic stains. In the 1970s, special aspiration biopsy clinics were established in Stockholm and elsewhere in Sweden to which patients with palpable lesions were referred for diagnosis. The technique soon became an acceptable substitute for tissue biopsies. An extensive bibliography, generated by the Swedish group, supported the value and accuracy of the procedure (Zajicek, 1974, 1979; Esposti et al, 1968; Lwhagen and Willems, 1981). It can be debated why the aspiration biopsy flourished in Sweden, whereas initially it was unequivocally rejected in the United States (see Fox, 1979). This writer believes that the Swedish success was caused, in part, by inadequate services in biopsy pathology because, by tradition, in the academic Departments of Pathology (that are the mainstay of Swedish pathology), research took precedence over services to patients, a situation quite different from that in the United States (see exchange of correspondence between Koss, 1980, and Sderstrm, 1980). A further reason for
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the Swedish success was the government-sponsored health system, based on salaries, which offered no monetary rewards to surgeons and other clinicians for the performance of biopsies. Therefore, the creation of aspiration diagnostic centers offering credible and rapid diagnoses was greeted with enthusiasm. This is yet another major point of difference with the situation in the United States, where surgeons (and sometimes other specialists) feel financially threatened if the biopsies are performed by people encroaching on their turf. Although the Swedish authors published in English and also contributed to this book (editions 2, 3, and 4), the impact of thin-needle aspiration techniques on the American scene initially has been trivial and confined to a few institutions and individuals. The radical change in attitude and the acceptance of the cytologic aspirates in the United States may be due to several factors. Broad acceptance of exfoliative cytologic techniques (Pap smears) for detection and diagnosis of cervix cancer, subsequently extended to many other organs, clearly played a major role in these developments. The introduction of new imaging techniques, such as imaging with contrast media, computed tomography, and ultrasound, not only contributed to improved visualization of organs but also to roentgenologists' ability to perform a number of diagnostic procedures by aspiration of visualized lesions, hitherto in the domain of surgeons (Ferucci, 1981; Zornoza, 1981; Kamholz et al, 1982). After timid beginnings in the early 1970s, documenting P.11 that the use of a thin needle was an essentially harmless and diagnostically beneficial procedure, a new era of diagnosis began which initially forced the pathologists to accept the cytologic sample as clinically valid and important. In those days, most pathologists had to struggle to interpret such samples. Thus, once again, the pathologists were forced into an area of morphologic diagnosis for which they were not prepared by training or experience. The current enthusiasm for this method in the United States is surely related to the Swedish experience that insisted that the interpreter of the smears (i.e., the cytopathologist) should also be the person obtaining cell samples of palpable lesions directly from patients. In fact, many of the leaders in this field were trained in Sweden, particularly by the late Dr. Torsten Lwhagen. This was the exact opposite of the situation in the 1960s, when Swedish observers repeatedly visited the Memorial Hospital for Cancer in New York City to learn the secrets of the aspiration biopsy. Nowadays, by performing the procedure and by interpreting its results, the pathologists assume an important role in patient care. Without much doubt, aspiration cytology has become an elixir of youth for American pathology, making those who practice it into clinicians dealing with patients, not unlike the pioneers of pathology in the 19th century. At the time of this writing (2004), biopsy by aspiration, also known as thin- or fine-needle aspiration biopsy (FNA), has become an important diagnostic technique, sometimes replacing but often complementing tissue pathology in many clinical situations. The targets of the aspiration biopsy now encompassed virtually all organs of the human body, as discussed in Chapter 28 and subsequent chapters. Within recent years, numerous books, many lavishly illustrated, have been published on various aspects of aspiration cytology. With a few exceptions, these books do not address the key issue of the aspiration biopsy: it is a form of surgical pathology, practiced on cytologic samples (Koss, 1988). Only those who have expertise in tissue pathology are fully qualified to interpret the aspirated samples without endangering the patient. These aspects of aspiration cytology are discussed in Chapter 28.
Figure 1-5 Exfoliative cytology. A schematic representation of the cross section of the vagina, uterine cervix,
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and the lower segment of the endometrial cavity. Cells desquamating from the epithelial lining of the various organs indicated in the drawing accumulate in the posterior vaginal fornix. Thus, material aspirated from the vaginal fornix will contain cells derived from the vagina, cervix, endometrium, and sometimes fallopian tube, ovary, and peritoneum. Common components of vaginal smears include inflammatory cells, bacteria, fungi, and parasites such as Trichomonas vaginalis (see Fig. 1-2). Red indicates squamous epithelium, blue represents endocervical epithelium, and green is endometrium.
CYTOLOGIC SAMPLING TECHNIQUES

Diagnostic cytology is based on four basic sampling techniques: Collection of exfoliated cells Collection of cells removed by brushing or similar abrasive techniques Aspiration biopsy (FNA) or removal of cells from palpable or deeply seated lesions by means of a needle, with or without a syringe. Aspiration biopsy (FNA) procedures are described in Chapter 19 for lung and pleura and Chapter 28 and subsequent chapters for all other organs. Intraoperative cytology (see below)
Exfoliative Cytology
Exfoliative cytology is based on spontaneous shedding of cells derived from the lining of an organ into a body cavity, whence they can be removed by nonabrasive means. Shedding of cells is a phenomenon based on constant renewal of an organ's epithelial lining. Within the sample, the age of these cells cannot be determined: some cells may have been shed recently, others may have been shed days or even weeks before. A typical example is the vaginal smear prepared from cells removed from the posterior fornix of the vagina. The cells that accumulate in the vaginal fornix are derived from several sources: the squamous epithelium that lines the vagina and the vaginal portio of the uterine cervix, the epithelial lining of the endocervical canal, and other sources such as the endometrium, tube, the peritoneum, and even more distant sites (Fig. 1-5). These cells accumulate in the mucoid material and other secretions from the uterus and the vagina. The vaginal smears often contain leukocytes and macrophages that may accumulate in response to an inflammatory process, and a variety of microorganisms such as bacteria, fungi, viruses, and parasites that may inhabit the lower genital tract. Another example of exfoliative cytology is the sputum. The sputum is a collection of mucoid material that contains cells derived from the buccal cavity, the pharynx, larynx, P.12 and trachea, the bronchial tree and the pulmonary alveoli, as well as inflammatory cells, microorganisms, foreign material, etc. The same principle applies to voided urine and to a variety of body fluids (effusions). The principal targets of exfoliative cytology are listed in Table 1-1. It is evident from these examples that a cytologic sample based on the principle of exfoliated cytology will be characterized by a great variety of cell types, derived from several sources. An important feature of exfoliative cytology is the poor preservation of some types of cells. Depending on type and origin, some cells, such as squamous cells, may remain relatively well preserved and resist deterioration, whereas other cells, such as glandular cells or leukocytes, may deteriorate and their morphologic features may be distorted, unless fixed rapidly. In addition, spontaneous cleansing processes that naturally occur in body cavities may take their toll. Most cleansing functions are vested in families of cells known as macrophages or histiocytes and leukocytes. These cells may either phagocytize the deteriorating cells or destroy them with specific enzymes (see Chap. 5). A summary of principal features of exfoliative cytology is shown in Table 1-2. The exfoliated material is usually examined in smears, filters, and cell blocks or by one of the newer techniques of preservation in liquid media and machine processing (see below).
Abrasive Cytology
In the late 1940s and 1950s, several new methods of securing cytologic material from various body sites were developed. The purpose of these procedures was to enrich the sample with cells obtained directly from the surface
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of the target organ. The cervical scraper or spatula, introduced by Ayre in 1947, allowed a direct sampling of cells from the squamous epithelium of the uterine cervix and the adjacent endocervical canal (Fig. 1-6). A gastric balloon with an abrasive surface, developed by Panico et al (1950), led to the development of devices known as esophageal balloons, extensively used in China for the detection of occult carcinoma of the esophagus in high-risk areas (see Chap. 24). A number of brushing instruments, suitable for sampling P.13 various organs, were also developed (see below). Several such instruments were developed for the sampling of the uterine cervix (see Fig. 8-45).
TABLE 1-1 PRINCIPAL TARGETS OF EXFOLIATIVE CYTOLOGY
Target Organ
Techniques*
Principal Lesions To Be Identified
Incidental Benefits
Female genital tract
Smear of material from the vaginal pool obtained by pipette or a dull instrument. Fixation in alcohol or by spray fixative.
Precancerous lesions and cancer of the vagina, uterine cervix, endometrium, rarely fallopian tubes, ovaries
Identification of infectious agents, such as bacteria, viruses, fungi, or parasites (Chapters 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18)
Respiratory tract
Sputum: either fresh or collected in fixative (smears and cell blocks)
Precancerous states mainly carcinoma in situ and lung cancers
Identification of infectious agents, such as bacteria, viruses, fungi, or parasites (Chapters 19 and 20)
Urinary tract
Voided urine; fresh or collected in fixative (smears and cytocentrifuge preparations)
Precancerous states, mainly flat carcinoma in situ and high grade cancers
Identification of viral infections and effect of drugs (Chapters 22 and 23)
Effusions (pleural, peritoneal, or pericardial)
Collection of fluid: fresh or in fixative (smears and cell blocks)
Metastatic cancer and primary mesotheliomas
(Chapters 25 and 26)
Other fluids (cerebrospinal fluid, synovial fluid, etc.)
Collection in fixative Cytocentrifuge preparations
Differential diagnosis between inflammatory processes and metastatic cancer
Identification of infectious agents (viruses, fungi) (Chapter 27)
For further details of sample collection see this and other appropriate chapters. For further technical details, see Chapter 44.
TABLE 1-2 PRINCIPAL FEATURES OF EXFOLIATIVE CYTOLOGY
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The technique is applicable to organs with easy clinical access whence the samples can be obtained. The samples often contain a great variety of cells of various types from many different sources. The cellular constituents are sometimes poorly preserved. The samples may contain inflammatory cells, macrophages, microorganisms, and material of extraneous origin. The signal advantage of exfoliative cytology is the facility with which multiple samples can be obtained.
Figure 1-6 Method of obtaining an abrasive sample (scraping) from the uterine cervix by means of Ayre's scraper. Red indicates squamous epithelium and blue indicates endocervical mucosa.
Endoscopic Instruments
The developments in optics led to the introduction of rigid endoscopic instruments for the inspection of hollow organs in the 1930s and 1940s. Bronchoscopy, esophagoscopy, and sigmoidoscopy were some of the widely used procedures. In the 1960s, new methods of endoscopy were developed based upon transmission of light along flexible glass fibers. This development led to the construction of flexible, fiberoptic instruments permitting visual inspection of viscera of small caliber or complex configuration, such as the secondary bronchi or the distal parts of the colon, previously not accessible to rigid instruments. The fiberoptic instruments are provided with small brushes, biopsy forceps, or needles that permitted a very precise removal of cytologic samples or small biopsies. The introduction of fiberoptic instruments revolutionized the cytologic sampling of organs of the respiratory and gastrointestinal tracts and, to a lesser extent, the urinary tract. The brushes could be used under direct visual control for sampling of specific lesions or areas that were either suspect or showed only slight abnormalities (Fig 17). The method became of major importance in the search for early cancer of the bronchi (including carcinoma in situ) and of superficial cancer of the esophagus and stomach (see Chaps. 20 and 24). Transbronchial aspiration
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biopsies of submucosal lesions could also be performed. The introduction of fiberoptic sigmoidoscopes and colonoscopes contributed to a better assessment of abnormalities that were either detected by roentgenologic examination or were unsuspected. Colonic brush cytology proved to be useful in searching for recurrences of treated carcinoma or in the search for early carcinoma in patients with ulcerative colitis (see Chap. 24).
Figure 1-7 Bronchial brushing under fiberoptic control. Method of securing a brush sample from bronchus. Blue indicates bronchial epithelium.
The cytologic samples obtained by brushings, with or without fiberoptic guidance, differ markedly from exfoliated samples. The cells are removed directly from the tissue of origin and, thus, do not show the changes caused by degeneration or necrosis. Inflammatory cells, if present, are derived from the lesion itself and are not the result of a secondary inflammatory event. The sample is usually scanty and careful technical preparation is required to preserve the cellular material. The methods of smear preparation are described in Chapters 8 and 44. Since fiberoptic instruments can also be used for tissue biopsies of lesions that can be visualized, one must justifiably ask why cytologic techniques are even used. Experience has shown, however, that brush specimens result in sampling of a wider area than biopsies. This is occasionally of clinical value, particularly in the absence of a specific lesion. Brushing and aspiration techniques also allow the sampling of submucosal lesions. A summary of the principal features of abrasive cytology is shown in Table 1-3.
Washing or Lavage Techniques

Washing techniques were initially developed as a direct offshoot of rigid endoscopic instruments. On the assumption that cells could be removed from their setting and collected in lavage fluid from lesions not accessible or not visible to the endoscopist, small amounts of normal saline or a similar solution were instilled into the target organ under visual control, aspirated, and collected in a small container. A pioneering effort by Herbut and Clerf in Philadelphia (1946) P.14 defined the technique of bronchial washings for the diagnosis of lung cancer. The esophagus, colon, bladder, and occasionally other organs were also sampled in a similar fashion (see corresponding chapters).
TABLE 1-3 PRINCIPAL FEATURES OF ABRASIVE CYTOLOGY
The method allows direct sampling of specific targets, such as the surface of the uterine cervix or a bronchus. With the use of fiberoptic instruments direct samples of accessible internal organs may be secured. The cells obtained by abrasive techniques are derived directly from the tissue and thus are better
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preserved than exfoliated cells and require different criteria for interpretation. Subepithelial lesions may be sampled by brushing or aspiration techniques. Care must be exercised to obtain technically optimal preparations.
With the development of flexible fiberoptic instruments, brushings largely replaced the washing techniques. However, several new lavage techniques were developed. The three principal techniques are the peritoneal lavage (described in Chap. 16), bronchoalveolar lavage (described in Chap. 19), and lavage or barbotage of the urinary bladder (described in Chaps. 22 and 23). Because relatively large amounts of fluid are collected during these procedures, the samples cannot be processed by a direct smear technique. The cells have to be concentrated by centrifugation, filtering, or cell block techniques described in Chapter 44. The principal targets of abrasive cytology, washings, and lavage are shown in Table 1-4.
Body Fluids
The cytologic study of body fluids is one of the oldest applications of cytologic techniques, first investigated in the latter half of the 19th century. The purpose is to determine the cause of fluid accumulation in body cavities, such as the pleura, pericardium (effusions), and the abdominal cavity (ascitic fluid). Primary or metastatic cancer and many infectious processes can be so identified (see Chaps. 25 and 26). Other applications of this technique pertain to cerebrospinal fluid and other miscellaneous fluids, described in Chapter 27. The cell content of the fluid samples must be concentrated by centrifugation, sedimentation, or filtration as described in Chapter 44. The material is processed as smears, filter preparations, or cell block techniques.
Aspiration Cytology (FNA)

The technical principles of aspiration cytology are discussed in Chapter 28. The technique of aspiration of the lung and mediastinum is discussed in Chapters 19 and 20. Organ-specific features are described in appropriate chapters. The principal features of the technique are summarized in Table 1-5.
Intraoperative Cytology
Intraoperative consultations by frozen sections are a very important aspect of practice in surgical pathology that is often guiding the surgeon's hand. Supplementing or replacing frozen sections by cytologic touch, scrape, or crush preparations has been in use in neuropathology for many years (Eisenhardt and Cushing, 1930; McMenemey, 1960; Roessler et al, 2002) (see Chap. 42) and more recently has been receiving increased attention in other areas of pathology as well (summary in Silverberg, 1995).
Methods
The smears are prepared by forcefully pressing a clean glass slide to the cut surface of the tissue. Good smears may also be obtained by scraping the cut surface of the biopsy with a small clean scalpel and preparing a smear(s) from the removed material. Crushing small fragments of tissue between two slides and pulling them apart is particularly useful in assessing lesions of the central nervous system where obtaining large tissue samples for frozen sections may be technically difficult, but may also be applied to other organs.
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As with aspiration biopsy samples, the smears may be air-dried and stained with a rapid hematologic stain or fixed and stained with either Papanicolaou or hematoxylin and eosin, depending on the preference and experience of the pathologist. These techniques are described in greater detail in Chapters 28 and 44.
Applications
Intraoperative cytology is applicable to all organs and tissues. As examples, biopsies of the breast (Esteban et al, 1987), parathyroid (Sasano et al, 1988), uterine cervix (Anaastasiadis et al, 2002), and many other tissue targets (Oneson et al, 1989) may be studied. Recently, several communications evaluated the results of cytologic evaluation of sentinel lymph nodes in breast cancer (Viale et al, 1999; Llatjos et al, 2002; Creager et al, 2002a) and malignant melanoma (Creager et al, 2002b).
Advantages and Disadvantages

When compared with a frozen section, the smears are much easier, faster, and cheaper to prepare. Thus, the principal value of intraoperative cytology is a rapid diagnosis. Intraoperative cytology is of special value if the tissue sample is very small and brittle (as biopsies of the central nervous system) but sometimes of other organs, such as the pancreas, that are not suitable for freezing and cutting (Kontozoglou and Cramer, 1991; Scucchi et al, 1997; Blumenfeld et al, 1998). The interpretation of smears is identical to that of material obtained by aspiration biopsy, discussed in appropriate chapters. As is true with other cytologic preparations, the interpretation of intraoperative smears requires training and experience. However, even in experienced hands, a correct diagnosis may be difficult or impossible if the target tissue contains only very small foci of cancer, which can only be identified by special techniques such as immunocytochemistry, as is the case in some sentinel lymph nodes. P.15
TABLE 1-4 PRINCIPAL TARGETS OF ABRASIVE CYTOLOGY WASHINGS AND LAVAGE TECHNIQUES
Principal Lesions to Be Target Organ Technique* Identified Incidental Benefits
Female Genital Tract
Uterine cervix, vagina, vulva, endometrium
Scrape or brush; smear with immediate fixation in alcohol or spray fixative
Precancerous states and early, cancer and their differential diagnosis
Cancerous processes in other organs or the female genital tract may be identified (ovary, tube); identification of infectious processes (Chapters 12, 14, and 16)
Peritoneal fluid collection and washings
Fluid sample: collect in fixative
Residual or recurrent cancer of ovary, tube, endometrium, or cervix
(Chapter 16)
Respiratory Tract
Bronchial brushing;
Identification of
Recognition of
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bronchial washings and lavage; bronchoalveolar lavage
precancerous states, lung cancer, and infections
infectious agents; chemical and immunologic analysis of fluids in chronic fibrosing lung disease (Chapters 19 and 20)
Buccal Cavity and Adjacent Organs
Direct scrape smear; fixation as above
Identification of precancerous states and cancer
(Chapter 21)
Urinary Tract
Bladder washings or barbotage; processed fresh or fixed
Identification of carcinoma in situ and related lesions
Monitoring of effect of treatment; DNA analysis by flow cytometry or image analysis (Chapter 23)
Gastrointestinal Tract
Esophagus
Brush or balloon smears; fixation as above
Identification of precancerous states (mainly carcinoma in situ and dysplasia), early cancer, or recurrent cancer after treatment
Stomach
Brush, rarely balloon; smears, fixation as above
Colon
Brush; smears, fixation as above
Monitoring of ulcerative colitis
(Chapter 24)
Bile ducts and pancreas
Aspiration of pancreatic juice (essentially obsolete); brushing
Diagnosis of cancer of the biliary tree and pancreas
Techniques of collection of cell samples in liquid media and processing by specially constructed machines or apparatuses are described in Chapter 44.
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P.16
TABLE 1-5 PRINCIPAL FEATURES OF THIN-NEEDLE ASPIRATION BIOPSY
Impeccable aspiration and sample preparation techniques are required for optimal results.* Virtually any organ in the body can be sampled using either palpation or imaging techniques. Thorough knowledge of surgical pathology is required for the interpretation of the sample. The technique is well tolerated, easily adaptable as an outpatient procedure, rapid, and costeffective.
See Ljung et al., 2001, and Chapter 28.
By nearly unanimous consensus of the authors of numerous articles on this topic, false-positive cancer diagnoses are very rare in experienced hands (specificity approaches 100%), but failures to recognize a malignant tumor are not uncommon. The sensitivity and overall accuracy of the method are approximately 80% to 85%. Clearly, in many cases of cancer, the intraoperative cytology will obviate the need for frozen sections and will replace frozen sections in special situations.
Application of Cytologic Techniques at the Autopsy Table

It is gratifying that several observers proposed the use of cytologic techniques at the autopsy table, as first described by Suen et al in 1976. The technique, based on touch preparations or needle aspiration of visible lesions, offers the option of a rapid preliminary diagnosis that may be of value to the clinicians and pathologists. Further, this approach is an excellent teaching tool of value in training house officers in cytology. Ample evidence has been provided that this simple and economical technique should be extensively used (Walker and Going, 1994; Survarna and Start, 1995; Cina and Smialek, 1997; Dada and Ansari, 1997).
TELECYTOLOGY
New developments in microscopy, image analysis, and image transmission by microwaves, telephone, or the Internet have generated the possibility of exchange of microscopic material among laboratories and the option of consultations with a distant colleague. The concept was applied to histopathology (summary in Weinstein et al, 1996, 1997) and expanded to cytology (Raab et al, 1996; Briscoe et al, 2000; Allen et al, 2001; Alli et al, 2001). As a consultation system, the method is particularly appealing for solo practitioners in remote areas who can benefit from another opinion offered by a large medical center in difficult cases. On an experimental basis, the system was applied to cervicovaginal smears (Raab et al, 1996), breast aspirates (Briscoe et al, 2000), and a variety of other types of specimens (Allen et al, 2001). The accuracy of the system in reference to cervicovaginal smears was tested by Alli et al (2001) comparing the diagnoses established by several pathologists on glass slides and digital images. The diagnostic agreement in this study was low to moderate, although the levels of disagreement were relatively slight. Discrepancies were also reported in reference to other types of material (Allen et al, 2001). Although theoretically very appealing and possibly useful in select situations such as the diagnosis of breast cancer in a patient in the Antarctica, cut off from access to medical facilities for 6 months a year, there are significant problems with telecytology. A smear contains thousands of images that should be reviewed before reaching a diagnostic verdict. Transmitting and receiving this large number of images is time consuming at both ends. Finding a suitable consultant who would be willing and able to spend hours reviewing microscopic images on a television screen would not be practical as a daily duty. Reservations about the use of preselected fields of view in diagnostic telecytology were also expressed by Mairinger and Geschwendter (1997).
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On the other hand, telecytology as a teaching tool has already achieved much success and will continue to be a desirable addition to any teaching system.
THE ROLE OF CLINICIANS IN SECURING CYTOLOGIC SAMPLES

The quality of the cytologic diagnosis depends in equal measure on the excellence of the clinical procedure used to secure the sample, the laboratory procedures used to process the sample, and the skills and experience of the interpreter. The clinical procedures used to secure cytologic samples from various body sites and organ systems are discussed in appropriate chapters. The success and failure of the method often calls for close collaboration between the clinician and the cytopathologist. Experience and training cannot be described in these pages except for outlining of a few basic principles: Familiarity with diagnostic options available for the specific organ or organ system; Securing in advance all instruments and materials needed for the procedure; If necessary or in doubt, a discussion between and among colleagues to determine the optimal procedure, which may be of benefit to the patient. The choice of methods depends on the type of information needed. Cancer detection procedures, for example, P.17 those used for detecting precursor lesions of carcinoma of the uterine cervix, have a different goal than diagnostic procedures required to establish the identity of a known lesion. The issue of turf, that is, who is best qualified to perform the procedure, is often dictated by clinical circumstances. A skilled endoscopist or interventional radiologist cannot be replaced and must be thoroughly familiar with the optimal technique of securing diagnostic material. In many ways, the diagnostic cytologic sample is similar to a biopsy where the territories are well defined, that is, the clinician obtaining the sample for the pathologist to interpret. However, in diagnostic cytology, there are gray areas, such as the needle aspiration of palpable lesions (FNA), where special skills must be applied for the optimal benefit to the patient. In such situations, optimal training and experience should prevail (see Chap. 28). In general, material for cytologic examination is obtained either as direct smears, prepared by the examining physician, gynecologist, surgeon, trained cytopathologist, or paramedical personnel from instruments used to secure the samples at the time of the clinical examination, or as fluid specimens, that are forwarded to the laboratory for further processing. Regardless of the method used, it is essential for the clinician to provide accurate clinical and laboratory data that are often extremely important in the interpretation of the material. Of the two procedures, the preparation of smears is by far the more difficult.
Preparation of Smears
Smears can be prepared from material obtained directly from target organs by means of simple instruments (e.g., the uterine cervix) or from brushes used to sample hollow organs (e.g., the bronchi or organs of the gastrointestinal tract). For most diagnostic purposes, well-prepared, well-fixed, and stained smears are easier to interpret than air-dried smears, which have different microscopic characteristics, unless the observer is trained in the interpretation of this type of material. Still, many practitioners of aspiration biopsies (FNAs), particularly those who follow the Swedish school, favor air-dried smears fixed in methanol and stained with hematologic stains (see Chap. 28). In this book, every effort has been made to present the cytologic observations based on the two methods side-by-side. It is important to place as much as possible of the material obtained on the slide and to prepare a thin, uniform smear. Thick smears with overlapping cell layers are difficult or impossible to interpret. Considerable skill and practice are required to prepare excellent smears by a single, swift motion without loss of material or air drying. Preparation of smears from small brushes used by endoscopists to investigate hollow organs may be particularly difficult. A circular motion of the brush on the surface of the slide, while rotating the brush, may result in an adequate smear. Too much pressure on the brush may result in crushing of material. If the person obtaining diagnostic material is not familiar with the technical requirements of smear preparation, competent help must be secured in advance. If none is available, the brushes can be put into liquid fixative and forwarded to the laboratory for smear preparation. Except in situations in which the preparation of air-dried material is desirable (see above and Chap. 28), immediate fixation of material facilitates correct interpretations. Two types of fixatives are commonly used: fluid
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fixatives and spray fixatives. Both are described in detail in appropriate chapters and summarized in Chapter 44. In addition to the customary commonly available fixatives, such as 95% alcohol, new commercial fixatives have become available. One such fixative is CytoRich Red (TriPath Corp., Burlington, NC) that has found many uses in the preparation of various types of smears. This fixative preserves cells of diagnostic value while lysing erythrocytes (see Chaps. 13 and 44 for further discussion of this fixative). In general fixation of smears, 15 minutes is more than adequate to provide optimal results. Errors of patient identification or occurrence of floaters, or free-floating cells, may cause serious diagnostic mishaps. If automated processing of a cytologic sample is desired, the commercial companies provide vials with fixatives accommodating collection devices or cell samples. For further discussion of these options, see Chapters 8 and 44. Spray fixatives provide another option. Their makeup and mode of use are described in detail in Chapter 44. When correctly used, spray fixatives protect the smears from drying by forming an invisible film on the surface of the slides. If spray fixatives are selected (and they usually are easier to handle than liquid fixatives), they should be applied immediately after the process of smear preparation has been completed. The use of spray fixative requires some manual dexterity, described in detail in the appendix to Chapter 8.
Collection of Fluid Specimens

Fluid specimens may be obtained from a variety of body sites, such as the respiratory tract, gastrointestinal tract, urinary tract, or effusions, and the clinical procedures used in their collection are described below. As is discussed in detail in Chapter 44, unless the laboratory has the facilities for immediate processing of fluid specimens, it is advisable either to collect such specimens in bottles with fixative prepared in advance or to add the fixative shortly after collection. The common fixative of nearly universal applicability to fluids is 50% ethanol or a fixative containing 2% carbowax in 50% ethanol (see Chap. 44). It is sometimes advisable to collect bloody fluids with the addition of anticoagulants, such as heparin. Ether-containing fixatives should never be added to fluids. The volume of the fluid rarely need be larger than 100 ml. Screw cap bottles of 250-ml content, containing 50 ml of fixative, are suitable for most specimens. Generally, the volume of the fixative should be the same or slightly in excess of the volume of the fluid to be studied. The fluids P.18 may be processed either as smears or cell blocks. The methods of preparation are described in Chapter 44.
BASIC PRINCIPLES OF THE INTERPRETATION AND REPORTING OF CYTOLOGIC SAMPLES

Diagnostic cytology is the art and science of the interpretation of cells from the human body that either exfoliate (desquamate) freely from the epithelial surfaces or are removed from tissue sources by various procedures, summarized above. The cytologic diagnosis, which is often more difficult than histologic diagnosis, must be based on a synthesis of the entire evidence available, rather than on changes in individual cells. If the cytologic material is adequate and the evidence is complete, a definitive diagnosis should be given. Clinical data are as indispensable in cytologic diagnosis as they are in histologic diagnosis. Definitive cytologic diagnosis must be supported by all the clinical evidence available. Of the greatest possible importance in maintaining satisfactory results in diagnostic cytology is the uniformity of the technical methods employed in each laboratory. The cytologic diagnoses are frequently based on minute alterations of cytoplasmic and nuclear structure. These alterations may not be very significant, per se, unless one can be sure that variations due to the technique employed can be safely eliminated. However, as in any laboratory procedure, situations may arise in which the evidence is too scanty for an opinion, and this fact must be reported appropriately. The imposition of rigid reporting systems, such as the Bethesda system for reporting cervicovaginal material, summarized in Chapter 11, and found to be of value in securing epidemiologic or research data, may sometimes deprive the pathologist of diagnostic flexibility. These issues are discussed at length in reference to all organs and organ systems. Before attempting the cytologic diagnosis of pathologic states, it is very important to acquire a thorough knowledge of normal cells originating from a given source. Normal includes variations in morphology caused by physiologic changes that depend on the organ of origin. Moreover, the cells may show a variety of morphologic changes that, in the absence of cancer, may result in substantial cellular abnormalities. Among these, one should mention primarily inflammatory processes of various types; proliferative, metaplastic, degenerative, and benign neoplastic processes; and, finally, iatrogenic alterations that occasionally may create a truly malicious confederacy of cellular changes set on misleading the examiner. The understanding of the basic principles of cell structure and function, although perhaps not absolutely essential in
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the interpretation of light microscopic images, nevertheless adds a major dimension to the understanding of morphologic cell changes in health and disease. Furthermore, basic sciences have already been of value in the diagnosis of human disease. For these reasons, in the initial chapters of this book, there is a reasonably concise summary of some of the basic knowledge of cells and tissues.
QUALITY CONTROL
Much has been said lately about quality control in cytology. On the assumption that this branch of human pathology is practiced with the skill and technical expertise similar to that observed elsewhere in medicine today, the best quality control is generated by the follow-up of patients. Constant referral to tissue evidence and the clinical course of the disease and, if death intervenes, to the postmortem findings, are the only ways to secure one's knowledge. It is a pity that currently there is a pervasive tendency to regard a postmortem examination as a tedious and generally wasteful exercise. There is abundant evidence that, in spite of enormous technical progress, the autopsy still provides evidence of clinically unsuspected disease in a significant percentage of patients. Diagnostic cytology must be conceived of and practiced as a branch of pathology and of medicine. Any other approach to this discipline is not beneficial to the patients.
BIBLIOGRAPHY
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1 - Diagnostic Cytology- Its Origins and Principles

1 Diagnostic Cytology: Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles

THE ERA OF HISTOPATHOLOGY The Beginning

1 - Diagnostic Cytology- Its Origins and Principles

example, in identifying invasion of a cancer into the underlying stroma.

1 - Diagnostic Cytology- Its Origins and Principles

Figure 1-3 George N. Papanicolaou, 1954, in a photograph inscribed to the author.

1 - Diagnostic Cytology- Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles

The Pap Smear: The Beginning

The Pap Smear From the 1950 to the 1980s

The Pap Smear From the 1980s to Today

1 - Diagnostic Cytology- Its Origins and Principles

DEVELOPMENTS IN NONGYNECOLOGIC CYTOLOGY Historical Overview

THE ASPIRATION BIOPSY (FNA) The Beginning

1 - Diagnostic Cytology- Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles

CYTOLOGIC SAMPLING TECHNIQUES

1 - Diagnostic Cytology- Its Origins and Principles

TABLE 1-1 PRINCIPAL TARGETS OF EXFOLIATIVE CYTOLOGY

Principal Lesions To Be Identified

Female genital tract

Sputum: either fresh or collected in fixative (smears and cell blocks)

Precancerous states mainly carcinoma in situ and lung cancers

Voided urine; fresh or collected in fixative (smears and cytocentrifuge preparations)

Identification of viral infections and effect of drugs (Chapters 22 and 23)

Effusions (pleural, peritoneal, or pericardial)

Collection of fluid: fresh or in fixative (smears and cell blocks)

Metastatic cancer and primary mesotheliomas

(Chapters 25 and 26)

Other fluids (cerebrospinal fluid, synovial fluid, etc.)

Collection in fixative Cytocentrifuge preparations

Differential diagnosis between inflammatory processes and metastatic cancer

Identification of infectious agents (viruses, fungi) (Chapter 27)

TABLE 1-2 PRINCIPAL FEATURES OF EXFOLIATIVE CYTOLOGY

1 - Diagnostic Cytology- Its Origins and Principles

1 - Diagnostic Cytology- Its Origins and Principles

Washing or Lavage Techniques

TABLE 1-3 PRINCIPAL FEATURES OF ABRASIVE CYTOLOGY

1 - Diagnostic Cytology- Its Origins and Principles

Aspiration Cytology (FNA)

1 - Diagnostic Cytology- Its Origins and Principles

Advantages and Disadvantages

Principal Lesions to Be Target Organ Technique* Identified Incidental Benefits

Female Genital Tract

Uterine cervix, vagina, vulva, endometrium

Scrape or brush; smear with immediate fixation in alcohol or spray fixative

Precancerous states and early, cancer and their differential diagnosis

Peritoneal fluid collection and washings

Fluid sample: collect in fixative

Residual or recurrent cancer of ovary, tube, endometrium, or cervix

1 - Diagnostic Cytology- Its Origins and Principles

bronchial washings and lavage; bronchoalveolar lavage

precancerous states, lung cancer, and infections