Inside new edition of gyn cytopathology guide

November 2024—New this month from CAP Publications is the CAP Practical Guide to Gynecologic Cytopathology—Morphology, Management, and Molecular Methods, second edition. Its 15 chapters cover squamous and glandular epithelial abnormalities, anal cytology, benign changes and mimics of premalignant and malignant epithelial lesions, and much more. In this issue we provide part of the chapter on look-alikes and morphologic spectrums of change (below).

The book’s editors are Christine Noga Booth, MD, of Cleveland Clinic; Michael R. Henry, MD, of Mayo Clinic; and David C. Wilbur, MD, of Harvard Medical School. All three weighed in when CAP TODAY asked a few questions about the new edition. Their answers follow.

You write in your comments about the new edition that practice methods, technologies, and management guidelines have changed substantially since the first edition was published in 2008. Can you describe some of what was done to bring this book up to date?

While cytologic appearances of cells remain as they always have been, as years pass better and more representative presentations of cytologic entities and their mimics are found. This atlas includes examples of these constantly evolving pictures, making for better educational experiences for readers. In that regard, many new images were added to the morphology chapters and several of these chapters were significantly updated to reflect current knowledge. In addition, new information that cytologists must now use every day in their practices is included. This information includes HPV testing, which has undergone significant change since the first edition was published. Methods newly approved are detailed with discussion of their strengths and weaknesses. New methods of artificial-intelligence-driven automated screening have been added to the now routine automated screening methods covered previously. Overall, the second edition now becomes the standard by which practicing cytologists can get the most up-to-date information for their education and use in daily routine.

What sets this gynecologic cytopathology book apart from other textbooks, atlases, and monographs on the topic?
This gynecologic cytopathology atlas is comprehensive in the overall approach to the Pap test, including preanalytic factors, processing, screening, interpretation, and laboratory management. An entire chapter is devoted to human papillomavirus basic information as well as testing methodologies. The atlas goes into great depth in the differential diagnosis of difficult Pap test interpretations, including look-alikes and morphologic spectrums of change. These differential interpretations are illustrated in figures adjacent to one another so that subtle cytomorphologic features can be compared. This side-by-side look-alike approach is unique among all other cytology textbooks in current use today and stems directly from the wealth of slides that are submitted for review by the CAP and from the laboratories of the Cytopathology Committee members.

There are 31 contributors to the guide in all. What can you tell us about your coauthors?
The coauthors of the atlas are current or former members of the CAP Cytopathology Committee as well as several CAP staff members. This group of authors represent pathologists and cytotechnologists from all over the U.S. and Canada who are actively involved in patient care in a variety of practice settings, including service in private practice, academic practice, and military hospitals/Veterans Affairs medical centers. These authors regularly contribute by reviewing patient specimens for gynecologic cytopathology education and proficiency testing as well as publishing findings from the review of data from the programs in Archives of Pathology & Laboratory Medicine.

Is this book a complement to the CAP’s gynecologic cytology glass slide education and proficiency testing programs? If so, how?
The final chapter in the atlas is devoted to the data that the committee has published on slide performance in the CAP education and proficiency testing programs in gynecologic cytopathology. During the past 30 years, the CAP has provided thousands of Pap test glass slide challenges (conventional or liquid-based) through the interlaboratory comparison and proficiency testing programs to thousands of practicing cytotechnologists and pathologists. The process for slide acceptance into the education program as well as slide validation for acceptance into the proficiency testing program is described in detail. The goal of each of the programs is to provide slide challenges that can be reproducibly and correctly interpreted by cytology professionals. As mentioned, the wealth of submitted slides and slides derived from committee members’ laboratories forms the basis for the atlas illustrations.

Here is part of chapter seven in the new book on gynecologic cytopathology. This chapter contains 28 figures; what follows here are figures 1–5 in the section titled “Look-Alikes.” The other figures in the “Look-Alikes” section not published here consist of squamous and glandular lesions. The spectrums pictured in the chapter consist of morphologic squamous spectrums and morphologic glandular spectrums. (Images in the book are larger than those in CAP TODAY.)

The book is $144 for CAP members and $180 for nonmembers (ebook: $129). To order, call 800-323-4040 option 1 and request PUB134 or order online at www.cap.org (Shop tab); for the ebook: ebooks.cap.org.

Look-Alikes and Morphologic Spectrums of Change

Daniel F. I. Kurtycz, MD
Donna K. Russell, MEd, CT(ASCP)HT
Amy C. Clayton, MD; Camilla J. Cobb, MD
Kaitlin E. Sundling, MD, PhD
Michael R. Henry, MD

Introduction

Modern medicine looks back to Morgagni and his 1761 “Seats and Causes of Diseases” for his scientific effort to relate structural changes to disease; less than 100 years later Virchow and his colleagues extended that relation to microscopic anatomy. Countless publications expand on those earlier works. In spite of all the effort that anatomists and pathologists utilize to be as specific as possible in the definition of disease, sometimes there are similarities in gross and microscopic patterns that lead to difficulties in clearly separating disorders. Our morphologic concepts of disease do not allow for perfect resolution. Occasionally either or both gross and microscopic morphology overlap, and the observer needs to dig deeper to understand the processes involved.

Because the affliction of cervical cancer has been widespread and caused the loss of so many lives, disorders of the uterine cervix have been especially well studied. Consistent with medical history, the greater the intensity of inquiry into any area of the body, the more anatomic variation is uncovered in both benign and diseased states. Other chapters in this monograph have shown the numerous morphologic alterations in both benign and neoplastic entities, and in that range of variation there are changes that resemble one another. Our classification schemes have altered based on our growing understanding of the biology of tissue repair and neoplasia. The discovery of the role of high-risk human papillomavirus (hrHPV) and associated morphologic change was a major leap forward, as was the development of the Bethesda System for Reporting Cervical Cytopathology, a diagnostic structure based on hard-won knowledge.

Based on experience and evidence, most of the common diagnostic pitfalls in daily practice can be avoided, and “look-alikes” among benign, dysplastic, and neoplastic processes can be understood. However, some of the processes that the body uses for protection can generate reactive or metaplastic changes that resemble neoplastic events. Repair, metaplasia, and the changes wrought within parakeratotic protective mechanisms can mimic high-grade disease. It is important that individuals performing diagnostic work understand the biology as well as the morphology. It is not enough to circle a cell that “looks odd.” An observer should have an idea of how a cell arrived at that state.

Many of our look-alike pitfalls exist in the problematic spaces in our classifications, where cells and cellular groups fail to sit neatly into the morphologic models that our systems have defined. Degeneration, reactive alterations, microbial-caused cytopathic events, N:C ratio variation, hyperchromatic crowded groups, diathesis, and the varied forms of glandular cells all cause headaches. It is the purpose and intention of this chapter to illustrate and elucidate many of the pitfalls and help the cytologist toward the appropriate interpretation and support their confidence in their analysis.

Look-Alikes: Organisms
Figure 7-1 Trichomonas vaginalis.

Recognizing “Trich” often poses a problem for cytology trainees, but they soon take pride in being able to identify the appropriate backgrounds and small cytoplasmic profiles leading to diagnosis. Experienced cytotechnologists are especially remarkable in their usual prompt recognition of Trichomonas vaginalis (TV) after the examination of just a few microscopic fields. TV is a frequent cause of vaginitis and can be symptomatic with itching and dysuria, but certainly may be found in individuals without symptoms. The most distinctive microscopic feature is the organism’s elliptical nucleus, which stains faintly on Pap stain (A1-4). The nucleus must be identified for definitive diagnosis. Red cytoplasmic granules are also relatively common but are not always present. This protozoal organism is a flagellate and has a back and forth, “falling leaf”-like motility on wet-mount preparations. The flagella are often difficult to identify on Pap-stained preparations, dependent on the state of preservation and background inflammation. Flagella are much more likely to be observed in liquid-based preparations than conventional smears (A3), but organisms may cluster more in the conventional preparations (A1). Look-alikes of TV include cytoplasmic fragments, background detritus, bare nuclei, and degenerated inflammatory or epithelial cells.

Cytoplasmic fragments (B) and degenerate cells (C) are sometimes misidentified as TV. This confusion is most likely to occur in Pap slides showing microbial cytolysis or marked inflammation. Likewise, any small particulate material in the range of 10 µm–20 µm can be mistaken for TV, including: inspissated mucus, lubricant material, or other debris (D). Cytoplasmic fragments, mucus, and lubricant material will typically have variable shapes and sizes and irregular borders, compared to the uniform size and smooth outlines of TV. Trichomonas organisms can vary in size, 7 µm–30 µm, but are usually toward the lower end of the scale; in an individual case, the organisms tend to be uniform in size.

Failure to identify the distinctive elliptical nuclei in suspicious structures may lead to misdiagnosis. One should also be wary of trying to make any diagnosis based on a limited number of examples. Once an observer finds the first “Trich” there will usually be tremendous numbers on the slide. Bare nuclei can also be mistaken for TV and are also typically seen in the background of cytolysis (E). Bare nuclei tend to be rounded or slightly ovoid, instead of the usual appearance of pear-shaped TV, and lack the surrounding cytoplasm with granules. Bare nuclei usually display a small to absent nucleolus, which should not be confused with the elliptical nucleus of TV. TV is not the only protozoa that may be found on cervical samples; in endemic areas one may encounter amoeba, most frequently Entamoeba histolytica (EH). The motile trophozoite form of EH (F) tends to be larger than TV, ranging from 10 µm–60 µm, having a significant amount of cytoplasm and a characteristic Entamoeba nucleus with a central karyosome of heterochromatin and a thickened chromatic rim. Like TV, the EH nucleus will tend to stain faintly on Pap stain. The cyst form of EH may have up to four nuclei and may be more readily found on stool samples.

Figure 7-2 Herpes.

Cytopathic changes due to Herpes simplex are characterized by multinucleation, nuclear molding, and chromatin margination (the “3 Ms”) (A-1,2,3), along with nuclear enlargement and “ground-glass” chromatin. The latter is due to nuclear chromatin replacement by arrays of viral particles. The morphologic changes wrought by herpes can generate patterns of reaction or at times mimic nuclear features of SIL. Multinucleation in squamous and endocervical cells may be seen as a nonspecific reactive change that can mimic herpes, especially when the affected cells degenerate (B). However, compared to herpes, these reactive nuclei tend to be smaller, tend to overlap rather than mold, and do not show chromatin margination. Also, reactive nuclei almost always retain some margin of a granular chromatin pattern and usually possess small nucleoli, instead of having ground-glass-like change or large intranuclear inclusions. In herpes, the prominent inclusions are also termed “Cowdry A” bodies (A-2) and are more condensed arrays of developing virions than are found in the ground-glass nuclei. Degenerating multinucleated cells can pose a morphologic difficulty, because the chromatin may appear washed out and can simulate the homogeneous appearance of herpes-infected nuclei. If classic cytopathic herpetic changes are not identified and herpes can be only suggested, confirmation with other microbial studies should be recommended.

The cells of LSIL are sometimes multinucleated or multilobulated, and if the large dark nuclei are undergoing degeneration, they can be confused with the homogeneous ground-glass appearance of herpes cytopathic changes (C). Again, classic cytopathic change must be present to diagnose herpes. Occasionally, herpetic changes are seen in singly-nucleated cells, and when these cells are of parabasal or squamous metaplastic origin, there can be confusion with cytomegalovirus (CMV) infection or HSIL. CMV usually infects glandular cells or immature squamous cells, and infected cells are much larger than their normal counterparts (D). Singly-nucleated cells with herpetic changes are usually not significantly enlarged, and multinucleated forms are generally present elsewhere on the slide. As mentioned before, it just stands to reason that the more instances of a diagnostic feature on a slide, the more likely is an interpretation to be correct.

Herpes can be associated with marked reactive/reparative epithelial changes that can mimic carcinoma (seen in Fig. 7–10 not published in CAP TODAY). Reactive cells are distinguished by their fine, evenly distributed chromatin, smooth nuclear borders, uniform appearance, and arrangement in orderly sheets (E). When associated herpes cytopathic changes are recognized, caution in interpretation is needed to avoid a false-positive diagnosis of dysplasia or malignancy.
Conventional Smear – A2; B; C; D; E
ThinPrep – A1
SurePath – A3

Figure 7-3 Candida albicans.

The fungus Candida albicans exhibits budding yeasts and pseudohyphae (A1,2) when found on a cervical sample. The pseudohyphae are themselves elongated yeast forms. The structures of Candida can be confused with other organisms, as well as other native and foreign material found in Pap slides. Mucus strands are sometimes confused with pseudohyphae (B). Mucus strands are commonly seen in cervical preparations and usually show variation in size and width, whereas Candida has a more uniform appearance of parallel walls that are typically rounded at the distal end, tapered at the branching end, and are associated with budding yeast forms. Foreign fibers and threads can enter a cervical sample by a number of mechanisms and may even be environmental contaminants that landed on a slide or collection device. Such fibers and threads tend to vary in thickness (generally thicker than Candida), can be long, may curl and knot, and are solid. Candida hyphae are usually not as long as fibrous material, do not curl as tightly or knot, and appear hollow. Trichomes are stellate hairy growths of diverse structure that grow on the surfaces of some higher plants, algae, or lichens and can present in Pap slides as a contaminant (C). The divergent morphology of the trichome, often showing multiple arms and the absence of yeast forms, are helpful discriminators in the differential diagnosis. Other fungal hyphae, such as this example of a branching mold (D), may rarely be seen in Pap slides, most often as a contaminant. These hyphae show true septation (crosswalls) and are not associated with budding yeast. Leptothrix is a thin long bacterium often associated with Trichomonas infection. Leptothrix is much thinner than Candida hyphae and also shows no budding yeast forms (E).
Conventional Smear – C; D
SurePath – A1; E
ThinPrep – A2; B

Reactive Changes
Figure 7-4 Follicular cervicitis.

Follicular cervicitis (FC) typically involves only a portion of the Pap slide and is composed of a polymorphic population of mature lymphocytes and a few tingible body macrophages (i.e. macrophages containing phagocytized debris) (A1, 2). The pattern is actually the result of sampling a follicular nodule of chronic inflammatory cells. FC is readily recognized in well-preserved preparations, but confusion with other small cell lesions is a problem when there is poor preservation and/or degeneration. In cases of lymphoma or leukemia, which rarely present in Pap slides, and is almost always secondary involvement in a patient with a known history, tumor cells usually dominate the specimen and show a more monomorphic population of large lymphoid cells with irregularly distributed granular chromatin and prominent nucleoli (B). Although overall cell appearance may be smaller and rounder in liquid-based preparations, large, atypical lymphocytes with a tendency for similarity in size will still be appreciated in contrast to the spectrum of polymorphous, reactive lymphocytes, including small forms with condensed chromatin in FC. Apoptotic cells are also often seen in cases of lymphoma/leukemia and are typically absent in FC. Likewise, small cell (neuroendocrine) carcinoma would also dominate the Pap slide and show small tumor cells singly and in cell clusters, with molding and chain formation (C). Because of their cohesiveness and epithelial attachments rather than the loose cluster formation seen in follicular cervicitis, hyperchromatic crowded groups of cells such as HSIL (D) or endometrial cells (E) are less often confused with FC.
Conventional Smear – A2; B; C
ThinPrep – A1; D; E

Figure 7-5 Reparative changes.

Reparative change is characterized by cohesive sheets of uniform streaming cells (like a “school of fish”), showing nuclear enlargement, variation in nuclear size, open chromatin, absent to mild hyperchromasia, prominent nucleoli, and rare mitotic figures (A1, 2, 3). Many of these cellular features are also present in malignant cells, such as adenocarcinoma (B) and nonkeratinizing squamous cell carcinoma (C), but the malignant cells also show significant cellular and nuclear pleomorphism, cellular dyscohesion, and loss of polarity within groups of cells. Malignant nuclei also tend to be more hyperchromatic and have irregularly distributed chromatin and irregular nuclear borders. On the other hand, reparative cells have evenly distributed chromatin and smooth nuclear borders. Cells with reparative changes maintain a low N:C ratio, including in radiation change where the overall nuclear and cell size may be markedly enlarged. An increased N:C ratio can be helpful in making a diagnosis of SIL or malignancy, if present. Reparative changes are usually observed in a minority of cells on a slide, except in rare cases such as radiation-induced change, in which many or most of the cells may be affected (A3). In most cases of malignancy, the abnormal cells dominate the smear and are sometimes the only cells present. Reparative/reactive change may be seen in conjunction with SIL, and occasionally dysplastic cells with these additional reactive changes may be seen (D), which is another reason why observers need to pay attention to all the processes occurring on a slide. As mentioned in Fig. 7-2, cells infected with herpes virus may contain large eosinophilic intranuclear inclusions, which may rarely be mistaken for the large nucleoli of repair (E). Overcalling obvious repair can result in return patient visits and additional procedures.
Conventional Smear – A1; E
SurePath – A3; C
ThinPrep – A2; B; D