Microscopic Examination of Urinary Sediment, Graff's Textbook of Urinalysis and Body Fluids, cells in urine
The microscopic examination is a vital part of the rou- tine urinalysis. It is a valuable diagnostic tool for the detection and evaluation of renal and urinary tract disorders as well as other systemic diseases. The value of the microscopic examination is dependent on two main factors: the examination of a suitable specimen and the knowledge of the person performing the examination.
The best specimen for the routine urinalysis is the first morning specimen. Casts and red blood cells (RBCs) tend to dissolve or lyse in specimens with a low specific gravity or alkaline pH. The first morning specimen usually provides the concentrated and acidic environment needed to maintain these structures. The sediment should be examined as soon as possible after collection, but it may be refrigerated for a few hours if the examination cannot be performed immediately.
There have been some advances made in an effort to aid the technologist with the microscopic examination. These include the use of stains and the development of the phase and interference contrast microscopy techniques and automated computerized imaging. The most common stain for urinary sediments is the Stemheimer–Malbin supravital stain.1–3 Stemheimer–Malbin contains crystal violet and safranin stains and can be used as a general stain for most urinary structures. Some of the other staining techniques that can be used to differentiate certain urinary components include Sudan III, Sudan IV, and Oil Red O, which are used to stain fat a pink to red color; eosin, which stains RBCs and helps distinguish them from yeast cells which will not pick up the stain; and iodine, which can be used to stain starch granules and vegetable fibers a dark brown.
MICROSCOPY
The intent of this book is to familiarize the reader with the appearance of unstained urinary sediment structures viewed with the bright field microscope. The use of phase contrast microscope, polarized light, filtered light, and the interference contrast microscope aids in viewing unstained sediment material.
Phase microscopy and interference contrast microscopy make transparent objects visible by changing the amplitude of light waves as they pass through the objects. Phase microscopy artificially retards diffracted light by one fourth of a wavelength, and this produces a halo where the surfaces of slightly differing refractile indices meet one another. The interference contrast microscope produces its image by the splitting of light into two distinct beams. One beam passes through the object while the other serves as a reference. The beams are then recombined before being received, and this gives the object a relief or “three-dimensional” appearance. Phase microscopy should be used for the routine microscopic examination of urine. Interference contrast microscopy is useful in teaching morphologic identification of structures in the urinary sediment.
Polarized light is used for the identification of fat, crystals, and other anisotropic substances. This can be done by the use of two polarizing filters, one is placed in the condenser and the other is placed on the ocular. The field is then darkened by rotating one of the filters, crossing the polarizing filters at 90 . Colored filters can be placed below the condenser to help bring out the details of some structures. Filters can be very helpful when trying to photograph objects such as hyaline casts that tend to blend in with the background.
The photomicrographs in this book include not only the abnormal structures found in the urine but also those elements that have no pathological significance. Mastering the identification of normal urinary sediment allows the technologist to know when abnormal sediment is present. The magnification given for photomicrographs is approximately the magnification of the print itself. The value of the photomicrograph is limited in that only one focal plane can be seen, whereas in practice, individuals are able to see what is on all planes by constantly focusing up and down.
SEDIMENT PREPARATION AND USE OF THE MICROSCOPE
The microscopic examination should be performed on a centrifuged sample. If the volume of the specimen is too small to be centrifuged, then examine the sample directly, but note in the report that the results are from an uncentrifuged urine. Mix the specimen and then place approximately 10–15 mL of urine into a centrifuge tube and centrifuge at 2000 rpm for about 5 minutes. In an attempt to standardize the microscopic examination, the laboratory should adopt a regulated speed, time, and amount for the centrifugation of the urine specimens. Pour off the supernatant fluid (this can be used for confirmatory protein testing) and resuspend the sediment in the urine that drains back down from the sides of the tube. Some laboratories leave exactly 1 mL of sediment and supernatant in the tube. Flick the bottom of the tube to mix the sediment and place a drop of sediment on a clean slide or in a
counting chamber. Cover with a coverslip and examine immediately.
The first rule for examining unstained urinary sediment with the bright field microscope is that subdued light must be used to provide adequate contrast. This is obtained by partially closing the iris diaphragm and then adjusting the condenser downward until optimum contrast is achieved. If there is too much light, some of the structures will be missed. For example, hyaline casts, which are gelled protein, have a very low refractive index and will be overlooked if the light is too bright or if there is not enough contrast.
Figure 5-1. Amorphous phosphates and hyaline cylindroid.
The cylindroid is not visible in A but appears in B when the focus is adjusted (200x).
The second important rule is that the fine adjustment should be continuously adjusted up and down to enable the viewer to see the depth of the object as well as other structures that may be on a different focal plane. Figure 5-1A is an example of why the focus should be constantly adjusted. The field seems to contain only amorphous phosphates (pH is 7.5); but when the fine adjustment knob is moved slightly, a hyaline cylindroid appears (Fig. 5-1B).
Sediment should be viewed first under low power magnification (100). Scan the slide and observe for casts, crystals, and elements that are present in only a few fields. Enumerate the number of casts. Switch to high dry power (400) when necessary to delineate the structures that are seen. Casts have a tendency to move toward the edge of the coverslip, so the entire periphery of the coverslip should be scanned. Casts are reported as the average number that is present in 10–15 fields under low power magnification (100). For example, if the number of hyaline casts in 10 different fields is 1, 3, 2, 1, 1, 2, 2, 3, 1, and 3, then the report would be 1–3 hyaline casts/low power field (LPF). Some laboratories use ranges for reporting casts: 0–2, 2–5, 5–10. Other laboratories may report casts as rare, few, moderate, or many. Cells are enumerated using high dry power (400) and are reported in ranges (0–2, 2–5, 5–10, 10–20, 20–50, 50) or as rare, few, moderate, many, and packed.
Crystals, bacteria, parasites, and other rare sediments may be reported as being present, or may be reported as rare, occasional, moderate, and many.
CELLS
Cells that can be present in the urine include erythrocytes (RBCs), leukocytes (white blood cells or WBCs), and epithelial cells from anywhere in the urinary tract from the tubules to the urethra or as contaminants from the vagina or vulva. Microscopic evaluation of urine is important for detection of these cells not only for confirmation of chemical findings but also for detection of RBCs and WBCs in specimens that may contain interfering substances for these cells. In addition, no chemical test detects the presence of renal epithelial cells.
ERYTHROCYTES
Red blood cells in the urine may have originated in any part of the urinary tract from the glomerulus to the urethral meatus, and in the female they may be the result of menstrual contamination. They can appear in a variety of forms depending upon the environment of the urine (Fig. 5-2). When the urine specimen is fresh, the red cells have a normal, pale, or yellowish appearance and are smooth, biconcave disks approximately 7 microns in diameter and 2 microns thick. They contain no nuclei and, when viewed from the side, they have an hourglass appearance. In dilute or hypotonic urine, the red cells swell up and can lyse, thus releasing their hemoglobin into the urine. Lysed cells,
Figure 5-2. Red blood cells. The field also contains a white cell and several “ghost” cells (400x)
which are referred to as “ghost” or “shadow” cells, are faint, colorless circles and are actually the empty red cell membranes. Lysing will also occur in alkaline urine. Red blood cells will crenate in hypertonic urine and sometimes the crenations may resemble granules.
There are some structures that can be confused with RBCs in the microscopic examination. Swollen or crenated RBCs can sometimes be mistaken for WBCs, even though they are larger and contain nuclei. This is especially true if there is only one type of cell present in the sediment not allowing for comparisons to be made among cells. The presence of a positive test for occult blood is often helpful inmaking a decision.
Simple adjustments of the microscope can aid in the differentiation of cells. In Figure 5-3A, which shows a field with both red cells and white cells, there should not be any problem differentiating the two types of cells. The red cells in the figure resemble those that are seen in a blood smear. Now, by
turning the fine adjustment up and down, the result is that the red cells “pop out” at the viewer as black circles, and this
Figure 5-3. RBCs and WBCs (A). Changing the focus causes
the red cells to appear as black circles (B) (400x).
is seen in Figure 5-3B. This occurs because RBCs are very refractile and are thicker on the edges than in the center. This phenomenon will not occur if the red cells are grossly distorted by a hypotonic or hypertonic urine environment.
The best way to differentiate red cells is by the addition of a few drops of 2% acetic acid. The red cells will lyse in dilute acetic acid, but white cells will not. The addition of the acid will also emphasize the nuclei of the WBCs. Because the acid will lyse the red cells, it is important to count the cells that are present before adding the acid. Scan the entire slide before the acid is added, otherwise, structures such as red cell casts will also dissolve, or new crystals will precipitate out.
Yeast cells can be mistaken for RBCs. Yeast cells are ovoid, rather than round, and they frequently contain buds which are smaller than themselves in size. The doubly refractile border of the yeast cell tends to resemble the doughnut appearance of the red cell. Yeast cells will not dissolve in 2% acetic acid, nor will they stain with eosin.
Normally, RBCs do not appear in the urine, although the presence of 1–2 RBC/HPF is usually not considered abnormal.8–11 The mechanism whereby red cells enter the urine is not entirely clear.12 Unlike white cells, red cells do not possess ameboid characteristics and, therefore, they must stay within the blood vessels. Injury or rupture of the blood vessels of the kidney or urinary tract releases RBCs into the urine, but this does not account for the acceptance of the normal presence of a few RBCs in the urine.
Hematuria is the presence of an increased number of RBCs in the urine and the blood reagent pad will reflect the presence of RBCs or free hemoglobin (see Chapter 4). In addition, the protein test will be positive if large amounts of blood are present. As always, a correlation should be made between the chemical tests and the results of microscopic examination.
LEUKOCYTES
White blood cells can enter the urinary tract anywhere from the glomerulus to the urethra. On average, normal urine can contain up to 2 WBCs/HPF.13–15 White blood cells are approximately 10–12L in diameter16 and are larger than RBCs but smaller than renal epithelial cells.
White blood cells are usually spherical and can appear dull gray or greenish-yellow in color (Fig. 5-4). WBCs may appear singly or in clumps (Fig. 5-5). The WBCs that are seen in urine are mostly neutrophils, which can be identified by their characteristic granules and nuclear lobulations. Figure 5-6 shows a field of packed WBCs. The addition of 2% acetic acid was used to accentuate the nuclei.
Leukocytes shrink in hypertonic urine and swell or are lysed in hypotonic or alkaline urine. The number of WBCs in an alkaline and hypotonic urine decreases by 50% within 1 hour after collection if the specimen is kept at room temperature.
When WBCs expand in a dilute or hypotonic urine, their granules may demonstrate Brownian movement. Cells that develop this characteristic are called “glitter cells.” Glitter cells were previously considered to be specific for pyelonephritis, but they can occur in a variety of conditions if the cells are exposed to a hypotonic environment. An increase of WBCs in urine is associated with an inflammatory process in or adjacent to the urinary tract. Leukocytes are attracted to any area of inflammation and, because of their ameboid properties, can penetrate the areas adjacent to the inflammatory site. Sometimes pyuria (pus in the urine) is seen in conditions such as appendicitis and pancreatitis. Pyuria is also found in noninfectious conditions such as acute glomerulonephritis, lupus nephritis, renal tubular acidosis, dehydration, stress,
Figure 5-4. White blood cells in a
hypotonic urine. The nuclei and granules
are easily recognized (800x)
Figure 5-5. White cell clumps (200x).
fever, and in noninfectious irritation to the ureter, bladder, or urethra. The presence of many white cells in the urine, especially when they are in clumps, is strongly suggestive of acute infection such as pyelonephritis, cystitis, or urethritis.
White blood cell casts are evidence that the WBCs originated in the kidney. White blood cell clumps are also strongly suggestive of renal origin, but they are not conclusive evidence.20 Because of the importance of WBC clumps, their presence should be reported.
A few leukocytes can normally be found in secretions from the male and female genital tracts, so the possibility of a contaminated urine should be considered.
EPITHELIAL CELLS
The epithelial cells in the urine may originate from any site in the genitourinary tract from the proximal convoluted tubule to the urethra, or from the vagina. Normally, a few cells from these sites can be found in the urine as a result of the normal sloughing off of old epithelial cells. A marked increase indicates inflammation of that portion of the urinary tract from which the cells are derived.
Making a distinction between the epithelial cells that arise in the various portions of the urinary tract is difficult.12 For this reason, many a laboratory reports the presence of epithelial cells without attempting to differentiate
Figure 5-6. Numerous white cells.
Acetic acid (2%) was added to accentuate the nuclei (400)
Figure 5-7. Transitional cell (A), Renal
epithelial cells (B) and WBCs (C) (800x)
them. When distinction is possible, three main types of epithelial cells may be recognized: renal tubular, transitional, and squamous.
Renal Tubular Epithelial Cells
Renal tubular epithelial cells are slightly larger than leukocytes and contain a large round nucleus. They may be flat, cuboidal, or columnar. Figure 5-7 shows a field containing WBCs, renal tubular epithelial cells, and a transitional cell. Note the variation in the sizes of these cells as well as the relative size of their nuclei. Increased numbers of tubular epithelial cells suggest tubular damage. This damage can occur in pyelonephritis, acute tubular necrosis, salicylateintoxication, and kidney transplant rejection.
Transitional Epithelial Cells
Transitional epithelial cells are two to four times as large as white cells. They may be round, pear-shaped, or may have taillike projections. Occasionally, these cells may contain two nuclei. Transitional cells line the urinary tract from the pelvis of the kidney to the upper portion of the urethra. Figure 5-8 shows pear-shaped transitional cells, and Figure 5-9 demonstrates the size of a transitional cell in proportion to the size of WBCs.
Figure 5-8. Transitional epithelial cells (500x).
Figure 5-9. Transitional epithelial cell (large arrow),
several squamous epithe- lial cells, and white cells (200x)
Squamous Epithelial Cells
Squamous epithelial cells are easily recognized as large, flat, irregularly shaped cells. They contain small central nuclei and abundant cytoplasm (Fig. 5-10). The cell edge is often folded over and the cell may be rolled up into a cylinder. Squamous epithelial cells occur principally in the urethra and vagina. Many of the squamous cells present in the female urine are the result of contamination from the vagina or vulva, and as such, they have little diagnostic significance.
Figure 5-10. Squamous epithelial cells (160x).
REFERENCES
Lillian A. Mundt and Kristy Shanahan, Graff's Textbook of Urinalysis and Body Fluids, Second Edition 2011
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