CHAPTER 2. Clinical Pathology: Cytology
Craig A. Thompson
I. Comparison and contrast of cytology and biopsy with histologic examination
A. Benefits of cytology over histology
1. Speed. A cytologic specimen can be obtained, stained, and evaluated in minutes rather than the many hours to days that are required for fixation, processing, staining, and evaluation for traditional histology (excluding frozen sections, which are uncommon in veterinary medicine)
2. Affordability
a. A needle, collection tube, slide, and stain generally cost fractions of a dollar for cytology
b. A biopsy instrument, formalin, tissue processing, and staining cost considerably more for histology
3. Invasiveness
a. Obtaining cells from an animal, whether by fine needle aspiration or impression of a draining tract, is nearly always less traumatic and induces less morbidity
b. Obtaining a tissue specimen by incisional or excisional biopsy is more likely to require sedation or general anesthesia; induces more trauma; and carries a higher risk of hemorrhage, morbidity, and mortality
4. Detail. Oil immersion lenses are readily available and can be used in the evaluation of cytologic specimens, which allow for detection of fine details such as chromatin patterns, nucleolar features, and minute organisms
5. Fluids. Histology is not generally applicable to fluid evaluation, such as serous cavity effusions, cerebrospinal fluid, and synovial fluid
B. Benefits of histology over cytology
1. Architecture. Cytologic examination provides an excellent evaluation of the cell populations, their morphology, and the background in which they lie. Histology affords a glimpse of the tissue as it was in situ, which includes important features such as invasion (vascular and/or basement membranes) and spatial arrangement of cells in relation to other features, such as inflammation
2. Volume. For the most part, the volume of the sample obtained from comparable locations for histologic evaluation is appreciably greater compared with cytology
3. Durability. For samples obtained from similar sites, fixed tissues will last longer and can be manipulated more readliy than cytologic specimens
a. Paraffin-embedded tissues are essentially inert except in temperature extremes
b. Cytologic specimens will degrade over time unless coverslipped and kept in a dark, dry place
c. If additional stains are needed later, more sections can be cut from the paraffin blocks, whereas cytologic specimens have to be de-stained and re-stained
II. Stains commonly used for cytology
A. Gram stain
1. Used to classify bacteria
2. Gram-positive (G+) bacteria have teichoic acid in their cell walls and stain deep blue
3. Gram-negative (G−) bacteria have lipopolysaccharides in their cell wall and are either unstained or take on the counterstain (safranin or fuchsin), which is generally red
B. Romanowsky type
1. For use on air-dried specimens
2. Aqueous-based stains
3. Includes a number of stains: Wright’s, Giemsa, Wright’s Giemsa and May-Grünwald-Giemsa, Leishman’s, Diff-Quik (Harleco, Gibbstown, NJ) and DipStat (Medichem, Inc., Santa Monica, CA)
4. Frequently used as a mainstay in cytopathology because they impart different colors to cytoplasm and the nucleus (i.e., polychromatic stains), allowing good visualization of each
C. New methylene blue
1. An aqueous stain; therefore, can be used to visualize some lipids
2. Poor cytoplasmic detail with excellent nuclear and nucleolar visualization
3. Good for detection of nucleated cells, all bacteria, fungi, and yeast
4. Because erythrocytes stain poorly, it can be used to evaluate nucleated cell populations when heavy blood contamination is present
5. Also frequently used as a stain for urine sediment (Sedi-Stain)
6. Used in hematologic specimens to enumerate reticulocytes and identify some organisms
D. Papanicolaou stain
1. Similar to hematoxylin and eosin (H&E), bichrome, trichrome, and Sano’s modified stain
2. Provide excellent nuclear details and delicate cytoplasmic details
3. Allow visualization of cells in clumps and layers
4. Laborious methods and availability limit its usefulness in veterinary medicine
E. Prussian blue
1. Stains ferric (Fe 3+) iron
2. Detects loosely bound iron such as that in hemosiderin
3. Will not identify iron tightly bound, such as with hemoglobin
4. Does not stain hematoidin
F. Fat stains
1. Oil-Red-O (ORO) can be used on fresh, non–alcohol-fixed smears to identify fat
2. Sudan Black B (SBB) stains fat a blue-black color
G. Immunocytochemical stains
1. Use antibodies against epitopes, such as clusters of differentiation (CD antigens) to identify the cell line of origin of unknown cells
2. Can be very specific and powerful
3. Limited only by the antibodies available, many of which are conserved across species, therefore allowing the use of human antibodies
4. Similar technology is used for flow cytometry, but using a fluid medium (e.g., effusions)
INFLAMMATION
The first thing any diagnostic sample should be evaluated for is inflammation. Without the benefit of architecture, the relationship of the inflammatory cells with the non-inflammatory cells is unknown. Inflammation can induce a variety of hyperplastic, dysplastic, and metaplastic changes in the noninflammatory components of a smear. These changes could be misinterpreted as neoplastic changes (see criteria of malignancy). The type of inflammation present can help shape a differential diagnosis list, especially when combined with other clinical data. By ruling out inflammation, the background, other populations present, and their morphologies can then be evaluated with confidence that the changes seen are a spontaneous change and not induced by the inflammation.
I. Types of inflammatory cells and their morphology
A. Neutrophils
1. 13 to 16 μm in diameter
2. Three to five nuclear lobes connected by thin intranuclear bridges
3. Female animals occasionally have a Barr body, often described as a small drumstick on the end of one nuclear lobe
4. The cytoplasm is clear (dogs, cats, horses) to pink (ruminants)
B. Macrophages
1. Medium- to large-sized cells, often greater than 25 μm
2. Nuclei can range in shape from round to markedly pleomorphic
3. Can take on different morphologies
a. Mononuclear, variably vacuolated cell
b. Epithelioid macrophages: Smooth blue cytoplasm with eccentrically placed nucleus
c. Multinucleated giant cell
C. Eosinophils
1. 13 to 16 μm in diameter
2. Three to five nuclear lobes connected by thin intranuclear bridges
3. Variable numbers of granules. Vivid pink, discrete and round to rod-shaped, pink granules (Feline)
D. Lymphocytes
1. 7 to 13 μm in diameter
2. Single round nucleus
3. Uniformly high nucleus-to-cytoplasm (N:C) ratio
E. Plasma cells
1. 9-15 μm long
2. Ovoid shaped
3. Eccentrically placed nucleus
4. Dark blue cytoplasm due to the abundant amount of rough endoplasmic reticulum (RER)
5. Variably sized perinuclear pale to clear zone
6. Mott cells are plasma cells that are congested with immunoglobulin, contained within pale blue Russell bodies
II. Suppurative-neutrophilic-purulent inflammation
A. Denotes a population that is more than 85% neutrophils
1. Suggests an active, likely (but not necessarily) acute process; however, can be a chronic process
2. Often seen with infectious etiologies (bacteria mainly; fungal and viral also)
3. Also seen with traumatic, toxic insults, immune-mediated diseases, and neoplastic processes, among others
4. Neutrophils are not normal tissue residents; they have been called there for some reason
B. Degenerate changes
1. Represented by a set of morphologic changes that are primarily nuclear
3. Karyolysis
a. Indicates rapid cell killing and is an unmistakable sign of degeneration
b. Often seen with bacterial infections, especially gram-negative (G−), when endotoxin is present
c. Swelling of the nuclear lobules and intranuclear bridges
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