Numerous stains have been used to enhance the visualization of urine sediment. Each laboratory should have a stain available because stains are inexpensive and can significantly assist in the identification of some urine sediment components. The most commonly used stain is a supravital stain consisting of crystal-violet and safranin, also known as the Sternheimer-Malbin stain (Fig. 7.4). This stain enhances formed element identification by enabling more detailed viewing of internal structures, particularly of WBCs, epithelial cells, and casts. Other formed elements (e.g., RBCs, mucus) stain characteristically, and their descriptions are noted on the package inserts provided with commercially prepared stains. Stabilized modifications of Sternheimer-Malbin stain are available commercially (e.g., Sedi-Stain, Becton, Dickinson and Company, Franklin Lakes, NJ), or it can be prepared by the laboratory if desired.³ One disadvantage of its use is that in strongly alkaline urines, this stain can precipitate, which obstructs the visualization of sediment components.

Another good supravital stain for urine sediment is a 0.5% solution of toluidine blue (Figs. 7.5 and 7.6). The stain is a metachromatic dye that stains various cell components differently; hence, the differentiation between the nucleus and the cytoplasm becomes more apparent. The toluidine blue stain enhances the specific identification of cells and aids in distinguishing cells of similar size, such as leukocytes from renal collecting duct cells.

Sudan III or oil red O is often used to confirm the presence of neutral fat or triglyceride suspected during the microscopic examination (Fig. 7.7). These lipids stain orange or red and may be found (1) free floating as droplets or globules; (2) within renal cells or macrophages, aptly termed oval fat bodies; or (3) within the matrix of casts as droplets or oval fat bodies. An important note is that only neutral fats (e.g., triglycerides) stain. In contrast, cholesterol and cholesterol esters do not stain and must be confirmed by polarizing microscopy. The distinction between triglyceride and cholesterol is primarily academic because the implications for renal disease are the same regardless of the identity of the fat. In other words, changes have occurred in the glomeruli such that triglycerides and cholesterol from the bloodstream are now passing the glomerular filtration barriers with the plasma ultrafiltrate. The urinalysis laboratory can use a fat stain or polarizing microscopy to confirm the presence of fat; the confirmation method selected is usually determined by cost, personnel preference, and convenience.

Although Gram stain is used primarily in the microbiology laboratory, it may at times be used in the urinalysis laboratory. Gram stain provides a means of positively identifying bacteria in the urine and differentiating them as Gram negative or Gram positive (Fig. 7.8). To perform a Gram stain, a dry preparation of the urine sediment is made on a microscope slide by smearing and air drying or by cytocentrifugation. As in the microbiology laboratory, the slide is heat fixed and then stained. Gram-negative bacteria appear pink, whereas Gram-positive bacteria appear dark purple. Because these slides can be viewed using a high-power oil immersion (×100) objective, additional characterization of the bacteria (e.g., cocci, rods) could be made, but this is rarely done by the urinalysis laboratory.

To facilitate the visualization of hemosiderin, free floating or in epithelial cells and casts, the Prussian blue reaction, also known as the Rous test, is used. First described by Rous in 1918 to identify urinary siderosis, the Prussian blue reaction stains the iron of hemosiderin granules a characteristic blue.4 See “Hemosiderin” later in this chapter for more discussion of this reaction and its use.

Hansel stain (methylene blue and eosin-Y in methanol) is used in the urinalysis laboratory specifically to identify eosinophils in the urine (Fig. 7.9). Whereas Wright’s stain or Giemsa stain also distinguishes eosinophils, Hansel stain is preferred.⁵ Urine eosinophils can be present in a variety of renal or urinary tract disorders, such as urinary tract infections (UTIs), acute tubular necrosis, glomerulonephritis, and acute interstitial nephritis (AIN).

Phase contrast microscopy is the preferred technique for microscopic examination of urine sediment because it enables (1) evaluation of RBC morphology and (2) detailed visualization and identification of difficult-to-view (translucent or low-refractile) formed elements such as hyaline casts, RBC ghost cells, and bacteria (Fig. 7.10). An added advantage is that microscopic examinations are generally faster to perform because of the enhanced visualization. See Chapter 18 for a detailed discussion of phase contrast microscopy and how variations in the refractive index of formed elements are converted into variations in contrast, thereby revealing low-refractile components.

In the urinalysis laboratory, polarizing microscopy is often used to confirm the presence of fat, specifically cholesterol. Cholesterol droplets are birefringent (i.e., they refract light in two directions) and, similar to their counterpart triglycerides, they can be found as free-floating droplets or in cells (oval fat bodies) and casts. In droplet form—within cells, free floating, or in casts—cholesterol produces a characteristic Maltese cross pattern with polarized light (Fig. 7.11A). These droplets appear as orbs against a black background divided into four quadrants forming a bright Maltese-style cross. When a first-order red compensator plate is used, the background becomes red to violet and opposing quadrants in the orbs are yellow or blue, depending on their orientation to the light (Fig. 7.11B). Note that starch granules and some drug crystals show a similar pattern, which is called a pseudo-Maltese cross because the four quadrants produced are variable in size (see Chapter 18, Table 18.1). Other neutral fats, such as fatty acids and triglycerides, cannot be identified using polarizing microscopy because they are not optically active—light passes through them unchanged. For triglyceride or neutral fat identification, see the section “Fat or Lipid Stains” earlier in this chapter.

Polarizing microscopy can also assist in differentiating urine sediment components that may look alike (see Table 7.3). RBCs can be distinguished from monohydrate calcium oxalate crystals, casts or mucus from fibers, and amorphous material from coccoid bacteria. See Chapter 18 for additional information, as well as a procedure for converting a brightfield scope for polarizing microscopy (Box 18.3).

Chapter 18 discusses two types of interference microscopy. Differential interference contrast (Nomarski) microscopy and modulation contrast (Hoffman) microscopy provide detailed three-dimensional images of high contrast and resolution (Fig. 7.12). Although their use is suited ideally for microscopic examination of the formed elements found in urine sediment, the increased cost often cannot be justified by the traditional urinalysis laboratory. With experience, however, these microscopic techniques are easy to use and less time-consuming than brightfield microscopy because of the enhanced imaging. In addition, once a brightfield microscope has been modified for modulation contrast microscopy, it can easily be used for brightfield, polarizing, and other techniques by simply removing the specialized slit aperture from the light path.

Because of their small size—approximately 8 μm in diameter and 3 μm in depth—RBCs in urine are viewed and enumerated using high-power magnification. RBCs have no nucleus; they normally appear as smooth biconcave disks, and they are moderately refractile. When suspended in urine sediment, RBCs can be viewed from any angle. When viewed from the side, they have an hourglass shape; when viewed from above, they appear as disks with a central pallor (Fig. 7.13). The size or diameter of RBCs is affected by urine concentration (i.e., osmolality, specific gravity). In hypertonic urine, their diameter can be as small as ~ 3 μm and in hypotonic urine as large as 11.8 μm.⁹

Dysmorphic or distorted forms of RBCs can also be present in urine (Fig. 7.14). At times, these forms are present with normal RBCs in the urine of healthy individuals. Some dysmorphic forms occur because of the urine’s concentration (i.e., osmolality).¹⁰ The most common dysmorphic form is crenated erythrocytes (i.e., echinocytes or burr cells). When RBCs are present in hypertonic urine (osmolality >500 mOsm/L), they become smaller as intracellular water is lost by osmosis, which causes them to become crenated. As they crenate, erythrocytes lose their biconcave disk shape and become spheres covered with evenly spaced spicules or crenations. Because of these reversible membrane changes, the surface of crenated cells appears rough or sometimes grainy, depending on the microscope adjustments, compared with normal erythrocytes. In hypotonic urine (osmolality <180 mOsm/L), erythrocytes swell and will eventually release their hemoglobin to become “ghost” cells, which are cells with intact cell membranes but no hemoglobin. These empty cells, outlined by their membranes, appear as colorless empty circles. Because their hemoglobin has been lost, ghost cells are difficult to see using brightfield microscopy; however, they are readily visible with phase contrast or interference contrast microscopy (Fig. 7.14). Note that alkaline urine promotes RBC lysis and disintegration, which results in ghost cells and erythrocyte remnants.

A variety of dysmorphic erythrocyte forms can be present in a single urine sediment.10 These forms include acanthocytes, schizocytes, stomatocytes, target cells, and teardrop cells (Table 7.5). Some of these forms are reversible and induced by the physical characteristics of the urine as it flows through the nephron (i.e., changes in osmolality, pH and uric acid concentration). However, the presence of acanthocytes (i.e., RBCs in a donut form with one or more protruding cytoplasmic blebs) in urine is particularly noteworthy (Fig. 7.15). The conversion of RBCs into acanthocytes is not induced by changes in osmolality or pH. Rather, the physical forces undergone by RBCs as they pass through the glomerular filtration barrier (i.e., basement membrane) disrupt and permanently alter their cell membranes. Dysmorphic RBCs tend to be smaller, and often RBC fragments and other dysmorphic forms are present with acanthocytes. Studies indicate that when 5% or more of the RBCs in urine sediment are acanthocytes, it is an indicator of hematuria due to a glomerular disorder.^10–12 Rarely observed are sickle cells, which have been seen in the urine sediment of patients with sickle cell disease. Using phase contrast or interference contrast microscopy enhances the ability to evaluate RBC morphology and is recommended.

Table 7.5

Forms of Red Blood Cells in Urine

	Form Name
Category	Common Name	Bessis Nomenclaturea	Phase Microscopy Example	Form Description
Isomorphic	Normal cell	Discocyte		Biconcave disk form of normal size
	Burr cell	Echinocyte or crenated cell		Cell with evenly spaced projections or spicules over cell surface; this “reversible” shape change progresses from a “crenated” disk to a “crenated” sphere.10
	Ghost cell	Ghost cell		Cell with thin membrane and without hemoglobin
Dysmorphic	Acanthocyte or G1 cell	Acanthocyte		Cell in a ring form (donut shape) with one or more cytoplasmic blebs (i.e., vesicle-shaped protrusions or bulges)
	Target cell	Codocyte		Bull’s-eye appearance; can be bell- or cup-shaped
	Schistocyte	Schizocyte		Cell fragment often with two or three pointed ends; size and shape vary
	Stomatocyte	Stomatocyte		Cell with central pallor that appears slit-like; this shape change is “reversible.”10

^aBessis M: Red cell shapes. An illustrated classification and its rationale. Nouvelle Revue Française d’Hématologie 12:721–746, 1972.

Normally, RBCs are found in the urine of healthy individuals and do not exceed 0 to 3 per high-power field or 3 to 12 per microliter of urine sediment.13 Semiquantitation is made by observing 10 representative high-power fields and averaging the number of erythrocytes seen in each. Although RBCs are nonmotile, they are capable of passing through pores only 0.5 mm (500 nm) in diameter.¹⁴ In addition, during inflammation, RBCs can be transported out of capillaries by the same mechanism as inert, insoluble substances.¹² All RBCs in urine originate from the vascular system. The integrity of the normal vascular barrier in the kidneys or the urinary tract can be damaged by injury or disease, causing leakage of RBCs into any part of the urinary tract. Increased numbers of RBCs along with red RBC casts indicate renal bleeding, either glomerular or tubular. These urines also have significant proteinuria. When an increased number of RBCs is present without casts or proteinuria, the bleed is occurring below the kidney or may be caused by contamination (e.g., menstrual, hemorrhoidal).

RBCs observed during microscopic examination should be correlated with physical and chemical examinations (Table 7.6). Macroscopically, the urine sediment may indicate the presence of RBCs when the sediment button is characteristically red in color. Sometimes specimens have a positive chemical test for blood, but the microscopic examination reveals no RBCs. This can be explained by the fact that RBCs readily lyse and disintegrate in hypotonic or alkaline urine; such lysis can also occur within the urinary tract before urine collection. As a result, urine specimens can be encountered that contain only hemoglobin from RBCs that are no longer intact or microscopically visible. However, it is important to note that other substances, such as myoglobin, microbial peroxidases, and strong oxidizing agents, can cause a positive blood chemical test (see Chapter 6). Note that these reactions are considered “false-positive” reactions because RBCs or blood is not present.

Table 7.6

Red Blood Cells: Microscopic Features and Correlations

Microscopic features

• Typical form—smooth, biconcave disks; no nucleus • Size: 6–8 μm in diameter; range possible 3–12 μm9 • Crenated forms—in concentrated urine (high SG) • Ghost cells—in dilute urine (low SG) • Dysmorphic forms and cell fragments (see Table 7.5) Only gold members can continue reading. Log In or Register to continue Share this: Click to share on Twitter (Opens in new window) Click to share on Facebook (Opens in new window) Like this: Like Loading... Related Related posts: Urine Specimen Types, Collection, and Preservation Quality Assessment and Safety Routine Urinalysis—the Physical Examination The Kidney Stay updated, free articles. Join our Telegram channel Join Tags: Fundamentals of Urine and Body Fluid Analysis Oct 18, 2022 | Posted by admin in PATHOLOGY & LABORATORY MEDICINE | Comments Off on Routine Urinalysis–the Microscopic Exam of Urine Sediment Full access? Get Clinical Tree Get Clinical Tree app for offline access Get Clinical Tree app for offline access %d