Fast Facts: The Essentials of Cytopathology

By P. Dalquen

Cytology is a universally applicable technique that can be used by both general practitioners and specialists. It enables diagnosis of lesions on surfaces such as the skin, mucous membranes or cervix by smear, and the determination of findings from internal organs by fine needle aspiration. The method is technically both simple and inexpensive and can therefore be used in all parts of the world, regardless of their level of development. This book aims to summarize the essential basics of cytology. At the same time, it provides an overview of non-neoplastic and neoplastic lesions, covering both common and rare manifestations across various body sites. The related virtual Case Collection of more than 500 cases from almost all organs provides deeper insights into the morphological spectrum of lesions and into the cytological literature. It is free to access at courses.karger.com/bundles/essentials-of-cytopathology. Table of Contents: • Preparatory methods • Cervix uteri, vagina • Ovary • Breast • Male genital tract • Urinary tract • Respiratory tract • Effusions and tumors of the serous cavities • Lymphoreticular system • Thyroid • Salivary glands • Pancreas, liver and biliary system • Gastrointestinal tract • Central and peripheral nervous system • Soft tissue tumors

• Language: English
• Publisher: S. Karger
• Release date: Nov 3, 2022
• ISBN: 9783318071474

Book preview

Introduction

Cytology is a universally applicable technique that can be used by both general practitioners and specialists. It enables diagnosis of lesions on surfaces such as the skin, mucous membranes or cervix by smear, and the determination of findings from internal organs by fine needle aspiration. The method is technically both simple and inexpensive and can therefore be used in all parts of the world, regardless of their level of development.

Figure 0.1 Non-small-cell lung carcinoma cell showing all the key features of malignancy: enlargement, multinucleation, varying size and shape of nuclei, coarse chromatin structure, atypical macro-nucleoli, and multiple protrusions of cytoplasmic receptors.

This book aims to summarize the essential basics of cytology. At the same time, it provides an overview of non-neoplastic and neoplastic lesions (Figure 0.1, opposite), covering both common and rare manifestations across various body sites. The related virtual Case Collection of more than 500 cases from almost all organs provides deeper insights into the morphological spectrum of lesions and into the cytological literature. It is free to access at courses.karger.com/bundles/essentials-of-cytopathology.

The main motivation for writing this book and compiling a globally accessible Case Collection has been the exchange of ideas over many years with colleagues from around the world, including Bangladesh, Afghanistan and other underserved countries.

Acknowledgments. First and foremost, I would like to thank Guido Sauter, Head and Chairman of the Institute for Pathology, UKE Hamburg, Germany, and also my colleagues there, Andrea Hinsch and Andreas Luebke, who, over many years, made it possible to compile the essential parts of the Case Collection. I thank my friend Michael J. Mihatsch, Basel, Switzerland, for the first critical review of the manuscript, and Lukas Bubendorf, Head of Cytopathology at the Institute of Medical Genetics and Pathology, University Hospital Basel, and his colleagues Daniel Baumhoer, Obinna Chijioke, Jürgen Hench, Spasenija Savic Prince, Luigi Terracciano and Tatjana Vlajnic for their expert review of the individual chapters. Finally, I thank Stephen Batsford for proofreading the English text.

1Preparatory methods

Cytology is based on two fundamental discoveries. The first is Virchow’s statement:

The nucleus is that structure which orders the substances within the cell itself and serves as the inner center of attraction, as the actual center of the preserved activity.

The second is the staining method (Pap staining) developed by George N Papanicolaou from hematoxylin-eosin staining, with which it is possible to show structural changes of the cell nuclei extremely precisely. Since tumors are distinguished from normal cells by aberrations of nuclear chromatin, Pap staining is advantageous in diagnostic cytology. In contrast, other stains, especially variants of the Romanowsky stain such as May-Grünwald-Giemsa (MGG), are more suitable for the diagnosis of hematologic diseases in which cytoplasmic criteria are crucial.

The most important methodological requirements for optimal Pap staining are described here.

Cell sampling methods

Tissue smears. Spatulas or brushes (Figure 1.1a) should be used for tissue smears instead of cotton swabs, to prevent artifacts arising from drying (see also pages 17 and 22). For the same reason, swab preparations made by the imprint method from fresh tissue are not recommended. Preparations that will stain particularly well are obtained by scraping cells with a scalpel from the cut surface of fresh tissue immediately after surgical resection. Such rapidly stained smears are an adjunct or alternative to frozen sections during surgery. The scraped cells are immediately smeared onto a slide with the scalpel blade and fixed.

Figure 1.1 Methods of cell sampling: (a) smear with spatula (top), brush (middle) and aspiration of endometrial cells with a balloon pipette (bottom); (b) example of syringe holder for fine needle aspiration; (c) body cavity fluid; (d) bronchial secretion, viscous with brownish streaks (left bowl) and watery saliva with fine whitish squamous cell aggregates from the oral mucosa (right bowl).

Fine needle aspiration. The fine needle aspiration (FNA) technique is illustrated in Figure 1.2. The use of a syringe holder (see Figure 1.1b) simplifies the puncture procedure. The advantage of FNA over core needle biopsy (CNB) is the ability to make a fan-shaped puncture, which allows access to large parts of the lesion.

Figure 1.2 FNA technique. (a) After disinfecting the skin with a cotton swab moistened with 50% ethanol, the needle is inserted into the tumor. (b) The plunger of the syringe is pulled back as far as it will go to create a negative pressure for aspiration. (c) The needle is then moved back and forth three or four times in different directions. To avoid bleeding from tissue injury, it is important to retract the needle under suction to below the skin surface before changing the needle direction. In the case of a small nodule (1 cm in diameter), the needle is carefully moved back and forth in one direction only.

At the end of the puncture, the plunger is released and the syringe is disconnected from the needle. Only then is the needle removed from the tissue and replaced on the syringe, and the aspirate is applied dropwise to slides as shown in Figure 1.3. The needle contents should not be distributed on to more than three or four slides. The technique for removing cells from the needle hub is illustrated in Figure 1.4a.

Figure 1.3 Preparation of cytological smears. (a) When voiding the needle contents, the needle tip must be in contact with the glass slide to avoid spraying the aspirate into small fast-drying droplets. The needle content should be distributed over a maximum of four slides. (b), (c) The droplet is smeared on each slide in succession and must be fixed immediately to avoid drying artifacts. The ‘unsmeared’ drop is stable for 1–2 minutes. Do not spread out all the drops first and then fix them.¹

Figure 1.4 (a) Apply the cells in the needle hub to the slide by tapping the edge of the hand on the table. (b) A conventional smear and different types of liquid-based preparation.

Cell recovery from body cavity fluids. Since serous fluids (see Figure 1.1c) contain sufficient protein and other cell nutrients, cells can be stored at room temperature for several hours and stored in a refrigerator at 4°C for 24–48 hours without any additives. No anticoagulant (heparin) should be added.

Urine and urinary bladder washings. Only the ‘second’ urine in the morning is suitable for cytological analysis, as the cells in the ‘first’ urine are usually damaged by autolysis. Urine (spontaneously voided or obtained by catheterization) and bladder washings must be sent to the laboratory as a whole. If urine cannot be processed within 1–2 hours after collection, a preservative fluid (for example, an equal volume of 50% denatured ethanol) should be added. This is the only way to prevent bacterial growth and preserve the cells for several days in a refrigerated environment at 4°C. Without the addition of a preservative, the cells will rapidly decompose and bacteria can overgrow the liquid within a very short time (1–2 hours). When centrifuging and preparing urine and bladder washings, similar rules must be observed as for the centrifugation of effusion fluids (see page 94).

Secretion smears. Sputum and bronchial secretions are principally processed in the same way: representative sputum consists of a more viscous bronchial secretion, which may contain brownish streaks (left dish in Figure 1.1d), whereas diagnostically unsuitable aqueous saliva contains predominantly squamous epithelial cells from the oral cavity, recognizable as fine, whitish granules (right dish in Figure 1.1d). To obtain a smear representative of the bronchi, a droplet is taken from the viscous and brownish secretion with forceps and applied to a glass slide. If the secretion is very viscous, the droplet can be separated with nail scissors.

Preparation techniques

Smear preparation technique. Cellular material obtained with a spatula or smear brush should be smeared longitudinally onto the slide. A conventional smear of the sediment obtained by centrifugation or the needle content after FNA is shown in Figure 1.4b.

Liquid-based preparation technique. There are several methods for preparing liquid-based samples. In principle, the swabbed cells or cells obtained by FNA are immediately transferred from the collection instrument to a commercial vial filled with preservative fluid and sent to the cytology laboratory for processing. There, the cells are transferred directly to the glass slide using a special centrifuge and fixed in a moist state. The advantages of this method include standardized processing of samples, resulting in uniform, flat cell distribution and avoidance of drying artifacts. The preparations also allow immunochemical molecular biological tests. Such tests are also possible using the cell-free liquid supernatant.²–⁴

Fixation stabilizes the cells and stops autolysis. Proper fixation is essential for standardized imaging of different cell types. Poor fixation is one of the most common causes of non-evaluable samples and diagnostic pitfalls. Basically, there are two fixation methods: moist fixation and dry fixation.

Moist fixation is performed immediately after spreading the cells while they are still moist. This is necessary to achieve good-quality Pap and hematoxylin and eosin (H&E) staining. Suitable fixatives for this purpose are 96% denatured alcohol (Figure 1.5a), 100% acetone, a 1:1 mixture of alcohol and acetone and commercially available spray fixatives (Figure 1.5b).

Figure 1.5 (a) Fixation of a smear by immersion into 96% denatured ethanol. (b) Spray fixation. (c) Correctly fixed lymphocytes obtained by lymph node FNA.

Important: spreading the cells and fixing them must not take more than 5 seconds.

Fixatives containing alcohol must be stored in a tightly closed bottle. This is particularly important in warm, humid, tropical climates because of the hygroscopic property of alcohol – that is, its tendency to absorb water vapor from the ambient air.

Dry fixation. As a rule, smears of blood or cells from bone marrow are air dried for 10 minutes and then immediately stained with