CHAPTER 9 Urinalysis and Body Fluids Analysis
V. Microscopic Examination of the Urine
A. Urine sediment preparation
1. To ensure the accuracy and reproducibility of the urine microscopic examination, each laboratory must establish a protocol for the preparation of the urine sediment. Bright- field microscopy is the most commonly used method of sediment examination. To see sediments with a low refractive index, such as hyaline casts, sediment should be ex- amined with decreased light obtained by decreasing the illumination, not by lowering the condenser. Phase-contrast microscopy is used to produce a phase difference of wavelengths of light, which gives better contrast when viewing low refractive index sediments (hyaline and cellular casts, mucous threads, and Trichomonas). Polarizing
microscopy involves the use of polarizing filters for observation of sediments that can rotate and refract light in two dimensions at 90◦to each other (e.g., lipids and crystals).
2. Factors that must be considered in urinalysis to help standardize the microscopic examination include the following:
a. Appropriate specimen collection, preservation, and handling. It is best to examine a specimen within 1 hour of individual voiding because many aspects of the chem- ical and microscopic examination change as the urine stands, especially at room temperature.
b. A standard amount of urine, usually 12 mL, is placed in a conical tube and cen- trifuged for a uniform amount of time and speed, typically 5 minutes at a relative centrifugal force of 400.
c. The spun tube is decanted, which leaves approximately 1 mL of fluid in which to resuspend the urinary sediment. If a stain is used, it should be added at this time.
d. A small drop of well-mixed sediment is placed onto a microscope slide and shielded with a coverslip. Slides with uniform wells and coverslips are commercially avail- able for the microscopic examination of urine. Drop size determines the amount of sediment viewed.
e. A consistent method should be used to examine the urinary sediment. The slide is first viewed on low power (10×) to assess the overall composition of the specimen and to observe and count casts. Ten fields are examined at this magnification. The light must be very low to see hyaline casts. Other elements are counted on high power (40×). An average count of sediments in ten fields is usually reported.
f. Microscopic results must be correlated with the color, appearance, and dipstick reactions for each specimen.
3. Microscopic sediment stains (supravital stains) can be used to aid the identification of formed elements.
a. The most commonly used stain is the Sternheimer-Malbin stain, which consists of crystal violet and safranin O. This stain provides a more detailed visualization of the internal structure of cells and casts.
b. Another supravital urine sediment stain is 0.5% toluidine blue. It differentially stains various cell components (e.g., the nucleus, cytoplasm) to help distinguish cells that may be similar in size, such as leukocytes and renal cells.
c. Sudan III or oil red O stains are used to confirm the presence of neutral lipids.
Lipids or fats within renal cells or histiocytes (oval fat bodies) or free-floating triglycerides stain red or orange with these two stains.
d. The Prussian blue stain is used to confirm the presence of hemosiderin (iron) in epithelial cells and casts, as well as hemosiderin that is free-floating. The iron turns a characteristic blue color.
e. Hansel stain consists of methylene blue and eosin-Y in methanol. It is used to identify eosinophils.
f. One to two drops of 2% acetic acid added to a few drops of urine sediment can be used to differentiate RBCs from yeast cells, small WBCs, or epithelial cells. The RBCs lyse; the yeast cells remain intact. The internal structures of WBCs and the epithelial cells are accentuated.
B. Normal and abnormal cells in urine (Figure 9–1)
1. RBCs are small biconcave disks without a nucleus. They are 7–10μm in diameter.
In a hypertonic urine they become crenated, appearing to have a crinkled border. In a hypotonic, alkaline urine, the RBCs swell and may lyse. These lysed cell membranes are called “ghost” or “shadow cells” and appear as faint, colorless circles. Normally, a urine specimen can contain 0 to 3 RBCs per high-power field; increased numbers may indicate renal bleeding or glomerulonephritis (see Web Color Image 9–2).
2. WBCs, usually neutrophils, are larger than RBCs (10–15μm diameter and contain a distinct nucleus. A normal urine sample contains 0 to 5 WBCs per high-power field. An increase is called pyuria and indicates the presence of an infection or in- flammation in the genitourinary tract. Frequent causes of pyuria include bacterial infections (e.g., cystitis, pyelonephritis, prostatitis, urethritis) or nonbacterial disor-
■ Figure 9–1 Characteristic appearance of cells found in urine sediment.
ders (e.g., glomerulonephritis, lupus erythematosus, tumors). In dilute alkaline urines, WBCs can lyse or swell and become glitter cells, in which Brownian movement of their internal granules produces a sparkling appearance (see Web Color Images 9–3 and 9–4).
a. Eosinophils in a urine specimen are identified with the Hansel stain and indicate acute interstitial nephritis caused by hypersensitivity reactions to medications such as penicillin derivatives.
b. Mononuclear cells (histiocytes, lymphocytes, or plasma cells) indicate an inflam- matory process or possible renal transplant rejection.
3. There are three types of epithelial cells found in urine: squamous, transitional, and renal tubular. They are derived from the linings of the urogenital tract. A few of each type can normally be found in urine because of normal sloughing of old cells.
a. Squamous cells are derived from the lining of the vaginal tract and lower por- tions of the female and male urethras. They are the most frequently seen but least significant epithelial cell. Increased numbers in a female urine specimen in- dicate that the specimen was not collected using the midstream clean-catch technique. Squamous cells are large, 30 to 40μm in diameter, and contain abun- dant cytoplasm with a small (7μm in diameter) centrally located nucleus. Their cytoplasmic borders are irregular, and they are often folded over on themselves in a urine specimen. Squamous epithelial cells covered with bacteria are called “clue cells” and represent a vaginal bacterial infection (see Web Color Image 9–5).
b. Transitional or caudate epithelial cells line the urinary tract from the renal pelvis to the proximal two thirds of the urethra. They measure 12 to 20 μm and are characteristically round or pear-shaped with a centrally located nucleus. Unless present in large numbers (>10 per high-power field) with unusual morphology, transitional cells are seldom pathologic. Catheterization often causes these cells to appear in urine. When unusual in morphology or in large numbers, samples of these cells should be referred for cytologic examination and may indicate renal transplant rejection, acute tubular necrosis, ischemic injury to the kidney, or renal carcinoma (see Web Color Image 9–6).
c. Renal tubular epithelial cells line each portion of the renal tubules and are con- sidered the most clinically important. Cells from the proximal or distal convoluted tubules are relatively large (20–60μm). They are oblong or round to oval and contain an eccentric nucleus. Renal tubular cells from the collecting ducts range from 12 to 20μm and are cuboidal, polygonal, or columnar. They have a single, large, dense nucleus that takes up approximately two thirds of its interior. Increased numbers in urine are the result of acute tubular necrosis from heavy metals or drug
toxicity. Large numbers in urine are also caused by all types of renal diseases and are often accompanied by granular, waxy, or renal tubular cell casts and an increased number of blood cells (see Web Color Image 9–7).
d. Renal tubular cells containing fat are called oval fat bodies. They can be stained with Sudan III or oil red O. When visualized with polarized light, these fat globules display a Maltese cross shape. Oval fat bodies often indicate glomerular dysfunction with renal tubular cell death. When present in a urine specimen, these cells are accompanied by increased amounts of protein and cast formation (see Web Color Image 9–8).
4. Tumor cells, platelets, or epithelial cells with viral inclusions are rarely found in urine sediment. Cytologic techniques are more sensitive than conventional urine microscopy in detecting and classifying these kinds of cells.
C. Urine casts (Figure 9–2)
1. Urinary casts are formed in the distal and collecting tubules. Except for a few hyaline or granular casts, which can accompany strenuous exercise (athletic pseudonephritis) or severe stress, casts are not normally present in the urine. The presence of urinary casts is termed cylindroiduria and their appearance is often accompanied by proteinuria.
Acid pH, urinary stasis, elevated protein, and concentrated solutes in urine all favor the formation of casts. Renal disease or damage along with these factors will produce different types of urinary casts. Casts are better identified with the use of a supravital stain and are typically reported as number of casts per low power field through the microscope
2. Tamm-Horsfall protein, which is a mucoprotein secreted only by renal tubular cells, forms the matrix of casts. As the tubular lumen contents become concentrated (often due to stasis of urine flow), Tamm-Horsfall protein forms fibrils that attach it to ductal cells and hold it temporarily in place. As it is held in the tubule, it enmeshes into its matrix any cellular or chemical substance that is present in the filtrate at the time it is formed. Eventually, the cast detaches from the tubular epithelial cells and is flushed into the urine.
3. Because casts form in the tubules, they are cylindrical with parallel sides and rounded ends. Casts formed in the collecting ducts are broader than those formed in the proximal and distal convoluted tubules.
4. The number and type of casts reflect the extent of renal tubule involvement in dis- ease processes. They are classified by the composition of their matrix and the type of substance enmeshed within them. Hyaline casts consist primarily of a homogeneous Tamm-Horsfall protein matrix with a low refractive index similar to urine. Also, they are the hardest to view because they do not contain any inclusions; they must be viewed with subdued light when using bright-field microscopy (see Web Color Image 9–9).
These casts appear in urine after strenuous exercise or stress, although in small numbers they are considered as normal sediment.
5. Cellular casts consist of a matrix of protein covered with different cell types. Red blood cell casts are reddish in color and signify glomerular disease or physical damage to the glomerulus. The outline of an erythrocyte must be observed in part of the cast to identify these (see Web Color Image 9–10). White blood cell (leukocyte) casts are associated with pyelonephritis and infection. The white blood cells are larger than red cells and have multilobed nuclei and granules in the cytoplasm (see Web Color Image 9–11). Renal tubular epithelial cell casts are noted in tubular diseases such as drug toxicity and tubular necrosis; the cells in these casts have a typical RTE cell appearance (see Web Color Image 9–12). Casts containing a mixture of cells are referred to as
“mixed cell casts.”
6. Granular casts may be degenerated cellular casts or they may represent protein aggre- gation on the Tamm-Horsfall cast matrix. They are classified as either finely granular (see Web Color Image 9–13) or coarsely granular (see Web Color Image 9–14) based on the appearance of the inclusions. Granular casts are always associated with renal disease, either glomerular or tubulointerstitial. Waxy casts are the final degenerative stage of finely granular casts. These casts are smooth with blunt ends and cracks along
■ Figure 9–2 Types of urinary casts: their appearance and clinical significance.
the lateral edges. The appearance of waxy casts in urine is a sign of renal failure or severe nephron damage.
7. Other casts include fatty casts, pigmented casts, bacterial, fibrin, and crystal casts depending on the inclusions within the protein matrix.
D. Crystals are commonly found in urine sediment but are rarely clinically significant. Pre- cipitated crystals appear in various forms or as amorphous material. Crystal identification is based on microscopic appearance and urine pH. Normal crystals can be found in acid,
Table 9–5 Summary of Normal Urinary Crystals
Crystal pH Color Solubility
Uric acid Acid Yellow-brown Alkali or heat
Amorphous urates Acid Brick dust or yellow-brown Alkali and heat
Calcium oxalate Acid, neutral, alkaline Colorless (envelopes) Dilute HCI
Amorphous phosphate Alkaline, neutral White-colorless Dilute acetic acid
Calcium phosphate Alkaline, neutral Colorless Dilute acetic acid
Triple phosphate Alkaline Colorless (coffin lids) Dilute acetic acid
Ammonium biurate Alkaline Yellow-brown (thorny apples) Acetic acid with heat
Calcium carbonate Alkaline Colorless (dumbbells) Gas from acetic acid
alkaline, or neutral urine and are reported as few, moderate, many or too numerous to count (TNTC) under microscopic high power.
1. Normal crystals found in acidic urine are urates (i.e., uric acid, amorphous urates) and calcium oxalate (Table 9–5 and Figure 9–3). Microscopically, all urates appear yellow to reddish-brown.
a. Uric acid crystals are yellow to red to orange in color and appear in many shapes, including four-sided and flat; rhombic plates or prisms; ovals with pointed ends;
rosettes; wedges; and needles. They are best identified with polarized light, under which they are multicolored (see Web Color Images 9–15 and 9–16).
b. Amorphous urates are yellow-brown granules, often found in clumps that may ob- scure other elements present in the urine sediment. When present in large amounts, they make the urine specimen appear pink-orange or reddish-brown and turbid.
They are easily solubilized by heating the urine specimen (see Web Color Image 9–17).
c. Calcium oxalate crystals can be seen in acidic or neutral urine. On rare occasions, they are found in alkaline urine. These crystals usually appear under the microscope as small, colorless octahedrals that resemble envelopes or with a cross on their surface. They can also appear in a dumbbell shape (see Web Color Image 9–18).
2. Normal crystals found in alkaline urine (Table 9–5 and Figure 9–3) are predomi- nantly phosphates, which include triple phosphate, amorphous phosphates, and calcium phosphate. Other crystals found in alkaline urine are ammonium biurate and calcium carbonate.
a. Triple phosphate crystals have a distinct colorless, three- to six-sided prism shape with oblique ends and are often called coffin lids (see Web Color Image 9–19).
b. Amorphous phosphates are granular in appearance. When present in large num- bers, they give the urine specimen a white turbidity. They can mask other elements
■ Figure 9–3 Appearance of normal urinary crystals.
■ Figure 9–4 Appearance of abnormal urinary crystals.
present in the urine sediment. Dilute acetic acid dissolves some of the crystals, but can also lyse any RBCs that may be present (see Web Color Image 9–20).
c. Calcium phosphate (see Web Color Image 9–21) appears as colorless, thin prisms, plates, or needles. These crystals are not frequently seen in urine, but when present can be confused with sulfonamide crystals (see image below), which are abnor- mal. The two are distinguished by adding dilute acetic acid to the urine sediment.
Calcium phosphate is soluble; sulfonamides are insoluble.
d. Ammonium biurate crystals are yellow-brown spheres with irregular projections or “thorns” and are referred to as thorny apples. They are often seen in old specimens (see Web Color Image 9–22).
e. Calcium carbonate crystals are small, colorless dumbbells or spheres. They of- ten appear in clumps and can be confused with amorphous phosphates. They are distinguished by the formation of carbon dioxide gas after the addition of acetic acid (see Web Color Image 9–23).
3. Abnormal crystals are found in acidic or neutral urine and have characteristic shapes (Figure 9–4; Table 9–6). No abnormal crystals are found in alkaline urine. Abnormal crystals found in urine are cystine, cholesterol, leucine, tyrosine, bilirubin, sulfon- amides, ampicillin, and radiographic dyes. It is important to check the drug therapy of individuals when unusual crystals are found in their urine specimens.
a. Cystine crystals are colorless hexagonal plates that precipitate in acidic urine.
They result from an inherited metabolic defect that prevents the reabsorption of cystine by the proximal convoluted tubule (see Web Color Image 9–24).
b. Cholesterol crystals in acidic urine resemble rectangular plates with a notch in one or more corners. These crystals are seen in urine from individuals with nephrotic
Table 9–6 Summary of Abnormal Urinary Crystals
Crystal pH Color Solubility
Cystine Acid Colorless Ammonia, dilute HCl
Cholesterol Acid Colorless (notched rectangles) Chloroform
Leucine Acid, neutral Yellow Alkali and heat, alcohol
Tyrosine Acid, neutral Colorless-yellow Alkali or heat
Bilirubin Acid Yellow Acetic acid, HCl, NaOH,
ether, chloroform, acetone
Sulfonamides Acid, neutral Colorless, yellow-brown Ampicillin Acid, neutral Colorless
Radiographic dye Acid Colorless 10% NaOH
syndrome or if the lymphatic system has been damaged. They are seen when urine specimens have been refrigerated and in urine specimens with elevated protein (see Web Color Image 9–25).
c. Leucine appears as yellow-brown oily looking spheres that contain concentric cir- cles with radial striations (see Web Color Image 9–26). These crystals are observed in urine from individuals with liver failure.
d. Tyrosine crystals look like sheaths of fine needles. They are rare, but can occur in individuals who have severe liver disease. They are found in acidic or neutral urine (see Web Color Image 9–27).
e. Bilirubin can precipitate in acidic urine as yellowish spheres with spicules. They appear in urine from individuals with liver disease and are composed of the conju- gated form of bilirubin (see Web Color Image 9–28).
f. Sulfonamides most often look like colorless or yellow-brown bundles of wheat with central bindings in acidic or neutral urine that has been refrigerated. Depending on the form of the sulfonamide drug the individual is taking, these crystals can appear as rosettes, arrowheads, needles, petals, or round forms with striations.
They usually appear when individuals are not adequately hydrated (see Web Color Image 9–29).
g. Ampicillin crystals appear as long, fine colorless needles or form coarse sheaves after refrigeration.
h. Radiographic dyes (contrast media) may have many colorless forms and can be confused with uric acid crystals or calcium carbonate crystals. When radiographic dyes are present in a urine specimen, the specific gravity is very high (>1.040) (Web Color Image 9–30).
E. Microorganisms, artifacts, and miscellaneous
1. Bacteria may or may not be significant, depending on the method of specimen col- lection and how soon after collection the specimen is examined. If WBCs are also present in the sediment with bacteria, an infection may exist. Bacteria will appear as rods, cocci, cocci in chains, or all of these and are typically reported as few, moderate or many under high power (Web Color Image 9–31).
2. Yeasts are usually found in the urine of individuals who have diabetes, but may also have gained access to the urine from places they usually reside (e.g., skin, vaginal tract) as the urine is voided. Airborne yeasts may also contaminate a urine specimen if it is left uncovered. Unless they are budding, yeasts can be confused with RBCs.
To differentiate RBCs and yeast, it is best to add a drop of dilute acetic acid to the urine sediment and re-examine it. RBCs lyse; yeast cells remain intact. Occasionally, mycelial forms of Candida are seen (see Web Color Image 9–32).
3. The parasite Trichomonas vaginalis (8–20μm) is seen in urine specimens as the result of vaginal contamination. Small species can be confused with WBCs, but the parasite has a characteristic undulating flagella.
4. Ova of the parasite Schistosoma haematobium are shed directly into urine. These are large (30×80μm) ovals with a lateral spine (Chapter 9).
5. Occasionally, amoebae can find their way to the bladder through the lymphatics. Enta- moeba histolytica (cyst 10–20μm) is usually accompanied by erythrocytes and leuko- cytes (Chapter 9).
6. Enterobius vermicularis (pinworm) eggs or ova (30×50μm) can contaminate urine when the female migrates to the perianal fold to lay its eggs. Other intestinal parasites can be seen in urine that has been contaminated with feces (Chapter 9).
7. Artifacts seen in urine include muscle fibers and vegetable cells seen with fecal con- tamination, hair, cotton fibers (see Web Color Image 9–33) from diapers or other cloth materials, starch granules from surgical gloves (see Web Color Image 9–34), and oil droplets from lubricants used as catheter lubricants or vaginal creams. If glass particles are on a slide or coverslip used to examine urine sediment, these may appear as bright, refractile, and irregularly shaped objects (see Web Color Image 9–35).
8. Mucus, a protein material produced by glands and epithelial cells in the urogeni- tal tract, is commonly observed in urine specimens, but has no clinical significance.