CHAPTER 23 Examination of the Urinary Sediment
Urine must be properly collected to ensure that urinalysis results are reliable. It can be collected in four ways (Table 23-1): catching a sample during urination, expressing the bladder, catheterization, and cystocentesis.1 Cystocentesis and catheterization are the preferred methods because both provide optimal samples for all aspects of urinalysis by avoiding contamination from the lower genital tract and external genitalia. Although it may be easier to express the bladder or catch a sample during urination, urine collected in these ways may be limited for analysis (particularly for a bacterial culture). Urinalysis is usually performed on preprandial, morning samples, because they tend to be the most concentrated, thus increasing the chance of finding abnormalities.
|Voided||Noninvasive, does not require special expertise, can be collected by owner||Contaminated by urethra and prepuce, therefore can have increased RBC and/or WBC due to urethral or prepucial inflammation and is not recommended for culture when cystitis is suspected|
|Expressing the bladder||Noninvasive, requires only minimal expertise, can be performed at time of examination||Contaminated by urethra and prepuce, therefore can have increased RBC and/or WBC due to urethral or prepucial inflammation and is not recommended for culture when cystitis is suspected|
|Catheterization||Semi-invasive, requires less expertise than cystocentesis, can be performed at time of examination||Some contamination, but less than voided or expressed urine, can contribute to development of cystitis after catheterization|
|Cystocentesis||Best for urine culture, less likely to cause cystitis than catheterization, can be performed at time of examination||Invasive but seldom causes cystitis, requires greater expertise than other methods of urine collection|
Courtesy R.D. Tyler and R.L. Cowell.
Fresh urine samples are best for cytologic examination because cell morphology is altered when cells remain in contact with urine for an extended period of time. When applicable, obtaining cells directly from a mass (i.e., traumatic catheterization and ultrasound-assisted fine-needle aspiration biopsy [FNAB]) usually produces a cellular sample with the best morphology for cytologic examination.
Voided samples, which are collected as an animal urinates, are the simplest samples to obtain. A sample collected in this manner usually is not satisfactory for bacteriologic examination because the sample is often contaminated during urination. Occasionally, the voided samples have increased numbers of white blood cells (WBCs) and epithelial cells due to contamination from inflammatory lesions of the external genitalia or reproductive tract, but other evaluations are unaffected. A voided sample is collected in a clean, although not necessarily sterile, container. Ideally, the vulva or prepuce should be washed to decrease sample contamination. A midurination (i.e., midstream) sample is best because there is less chance of contamination.
Urine may be collected from small animals by external, manual compression of the bladder. As with collecting voided samples, the external genitalia should be cleaned before collection. With the animal standing or in lateral recumbency, the bladder is palpated in the caudal abdomen, and urine is expelled by applying gentle, steady pressure. Care must be taken not to exert too much pressure, which could injure or rupture the bladder. This method should never be used on an animal with an obstructed urethra.
Urine may be collected by inserting a catheter of rubber, plastic, or metal into the bladder via the urethra. As in the previous two methods, the external genitalia should be cleaned before the procedure. Sterile catheters are used and sterile gloves worn. Care must be taken to maintain sterility and prevent trauma to the urinary tract. The catheter should pass easily through the urethra. A small amount of sterile, water-soluble lubricating jelly (e.g., K-Y Jelly [Johnson & Johnson Medical Inc, Arlington, Tex.]) should be placed on the tip of the catheter. Because the distal end of many catheters will accept a syringe, urine can be collected with gentle aspiration.
Cystocentesis is often used to collect sterile urine samples from dogs and cats. Sometimes as much urine as possible is removed from the bladder to prevent pressure on the bladder wall and urine leakage though the hole created by the needle. Unless the bladder is markedly extended, however, a volume of urine (5 to 10 ml) sufficient to perform urinalysis and culture can be removed without damaging the bladder or causing urine to leak through the bladder wall.
Microscopic examination of urine sediment is extremely important, especially for recognizing diseases of the urinary tract. In addition, urine sediment examination is occasionally an aid in diagnosing systemic disease. The best urine samples for sediment examination are morning samples or samples obtained after several hours of water deprivation because such samples are more concentrated and the chances of finding formed elements is increased. Urine collected by cystocentesis is the best sample for microscopic examination. The urine sample should be fresh because changes may occur as a sample ages. Samples can be capped and refrigerated for a short time before they are examined, although refrigeration usually increases the numbers of crystals.
Urine obtained from healthy dogs and cats does not contain much sediment. Small numbers of epithelial cells, mucous threads, red blood cells (RBCs), WBCs, hyaline casts, and various types of crystals can be found in the urine of most healthy animals. Bacteria and squamous epithelial cells derived from external genital surfaces may be present in voided and catheterized urine.
In order to semiquantitatively test the formed elements in urine, a standard volume, usually 5 ml, is centrifuged to obtain the sediment on every sample. Then 5 ml of a well-mixed sample is placed in a graduated, conical centrifuge tube and centrifuged for 3 to 5 minutes at approximately 100 G (about 1000 to 2000 rpm depending upon the radius of the centrifuge). The procedure should be standardized for a particular centrifuge to yield uniform results. Some centrifuges such as the Clay-Adams Triac (Becton Dickinson, Parsippany, NJ) are precalibrated to provide the proper force over sufficient time to completely sediment the formed elements in the urine. After centrifugation, the volume of sediment is recorded. The supernatant is gently poured out, leaving about 0.3 ml of urine adhering to the sides of the tube. This urine is allowed to run down the inside of the centrifuge tube, and the sediment is resuspended by gently flicking the bottom of the centrifuge tube.2
The sediment may be examined either stained or unstained. When examining unstained sediment, a small drop of the resuspended sediment is placed on a clean glass slide and covered with a coverslip. Microscopic examination should be conducted immediately. For bright field microscopy, subdued light that partially refracts the elements is used to examine unstained urine sediment. Proper lighting is achieved by partially closing the iris diaphragm and moving the substage condenser downward. Properly adjusted phase-contrast microscopy allows for better distinction of elements than reduced illumination with bright field microscopy. However, phase-contrast microscopes are slightly more expensive than bright field microscopes, and they require more precise adjustment in order to properly illuminate objects in urine sediment.
Stained sediment may also be examined. Satisfactory stains include Sternheimer-Malbin stain (Sedi-Stain, Becton Dickinson, Rutherford, NJ) or 0.5% new methylene blue. A drop of stain is added to and mixed with the suspended sediment before a drop of sediment is placed on a slide. When a stained specimen is examined, illumination is less critical than when an unstained specimen is examined; however, reduced illumination further aids visualization of substances by providing contrast.
The specimen is scanned under low power using the 10× objective in order to find the plane where the material has settled, determine the amount of sediment, and find larger elements such as casts or aggregates of cells. The entire area under the coverslip should be examined because casts tend to float to the coverslip’s edge. Casts and some crystals are usually identified at low power and are usually reported as the average number seen per low-power field (LPF). A higher-power objective (e.g., 40×) is necessary to detect bacteria, identify some crystals, and differentiate cell types. Epithelial cells, RBCs, and WBCs are reported as the average number seen per high-power field (HPF). Bacteria are reported as few, moderate, or many, and their morphology (i.e., cocci, bacilli, filamentous) is noted.
Urine sediment normally contains fewer than 2-3 RBCs/HPF. RBCs are small cells that may have several different appearances, depending on the urine concentration (e.g., specific gravity) and the length of time between collection and examination (Figures 23-4 to 23-9). In fresh samples that have intermediate specific gravities, RBCs usually have smooth edges and are yellow to orange. They can be colorless if their hemoglobin diffused while standing. In concentrated urine, RBCs shrink and crenate (see Figures 23-4 and 23-9). Crenated RBCs have ruffled edges and are slightly darker. Extremely crenated RBCs may even appear granular because of membrane irregularities. In dilute or alkaline urine, RBCs swell and may lyse. Swollen RBCs have smooth edges and are pale yellow or orange. Lysed RBCs may be colorless rings (shadow or ghost cells) that vary in size, but, especially when due to marked alkalinity, they usually dissolve and cannot be identified by microscopic examination.
Figure 23-4 RBCs in urine sediment. All the cells are similar in size, but some have irregular borders because they are crenated because of increased urine osmolarity. (Unstained, original magnification 400×.)
RBCs must be differentiated from WBCs in urine sediments. This is easy on stained sediment smears because RBCs are anucleate, but differences in size and internal structure must be used to differentiate between them on unstained smears. RBCs that maintain their biconcave shape can be easily recognized by their dark central area (see Figure 23-5), but those that shrink or swell in response to urine osmolality may not demonstrate the typical biconcave shape. These RBCs are differentiated from WBCs based on size (approximately half the diameter of a WBC; see Figure 23-6) and lack of internal structure. Although RBCs have no nuclei, they should not be confused with fat globules or yeast. RBCs do not vary much in size, are yellow to orange, and tend to settle onto a slide. Fat globules (Figure 23-10) vary markedly in size and are light green and usually found just under the coverslip. (This last feature causes them to be in a different plane of view from other urine sediment elements.) Yeast organisms are rarely found in fresh urine from dogs and cats, but are typically seen in aged samples with overgrowths of contaminants. When present, yeast organisms are more variable in size than RBCs, and budding can usually be found on some of the organisms.
Figure 23-10 There are many fat droplets present in this sample of urine sediment from a cat. The fat droplets are differentiated from RBCs because of the marked variation in size. The fat droplets also settle on a different plain of focus than the cellular elements. (Unstained, original magnification 50×.)
(Courtesy Oklahoma State University, Clinical Pathology Teaching File.)
RBCs in urine usually indicate bleeding somewhere in the urinary or genital tract. Voided samples from females in proestrus, estrus, or postpartum periods often contain RBCs secondary to contamination with secretions from the genital tract. Similar contamination may be present in urine sediments collected by free catch or manual expression of the bladder from animals with any hemorrhagic condition in the genital system. In such cases, RBCs should not be found in urine collected by cystocentesis. Increased numbers of RBCs along with WBCs are usually found in the urine sediment of animals with inflammatory conditions of the urinary tract. The slight trauma that occurs from catheterization or manual expression, and occasionally with cystocentesis, can increase the number of RBCs in the sediment.
Very few WBCs are present in urine from animals without urinary or genital tract disease. WBCs are about twice the size of RBCs and smaller than epithelial cells (see Figures 23-6 to 23-8). They are round and granular and sometimes brownian movement of the cytoplasmic granules in the neutrophils is seen. The polymorphic neutrophil nuclei can usually be seen in stained sediments and with phase-contrast microscopy and may occasionally be seen with reduced illumination bright field microscopy. WBCs are usually in very low numbers in urine (i.e., 0 to 1/HPF); more than 2 to 3 WBCs/HPF indicates inflammation somewhere in the urinary or genital tract. When increased numbers of neutrophils are found in urine sediment, even if bacteria are not found by microscopic examination, the urine sample should be cultured because microscopic examination is much less sensitive than culture to detecting bacteria in urine.
Squamous epithelial cells, which are derived from the distal urethra, vagina, vulva, or prepuce, are occasionally found in voided samples (Figure 23-11) and are usually not considered significant. They are the largest of the epithelial cells and the largest cells found in urine sediment; they appear flat and often have straight edges and obtuse, angular corners. They usually have a small, round nucleus, although occasionally a nucleus cannot be seen. Squamous epithelial cells are usually not found in samples obtained by cystocentesis. Cells with squamous features can be found in the urine sediment of male dogs with conditions that cause squamous metaplasia of the prostate and occasionally in the sediment of those with urothelial carcinomas (i.e., transitional cell carcinomas) in which there is also squamous metaplasia. In the latter condition, many other features suggestive of epithelial neoplasia, including extreme numbers of epithelial cells with marked pleomorphism, are found.
Urothelial, or transitional, epithelial cells come from the proximal urethra, bladder, ureters, renal pelvis, and renal tubules (see Figures 23-8 and 23-9). They vary in size depending upon their origin. Those originating in the proximal urethra and bladder are the largest, are round to elliptical, and have granular cytoplasm and variably sized nuclei. Epithelial cells of the ureters and renal pelvis are smaller, are round to caudate, and have granular cytoplasm. Renal tubular epithelial cells are small, round cells with a distinct, round nucleus. They have granular cytoplasm that may contain a few small to large fat vacuoles. Fatty renal tubular cells are especially common in cat urine. Renal tubular epithelial cells are only slightly larger than WBCs and are differentiated from WBCs by their round nuclei. A few epithelial cells, especially urothelial cells (i.e., 0 to 1/HPF), are found in sediment from normal animals due to the sloughing of old cells. An increase in epithelial cell numbers may be found in samples obtained by catheterization. Modest to marked increases in epithelial cells are found in inflammatory diseases of the urinary tract because inflammation often causes urothelial hyperplasia. Urine samples from animals with inflammation-induced urothelial hyperplasia also contain increased WBC numbers. Some chemotherapeutic agents (e.g., cyclophosphamide) may induce urothelial hyperplasia with modest pleomorphism. Many epithelial cells are found in sediment from animals with transitional cell carcinomas. Neoplastic urothelial cells usually vary markedly in size. If large numbers of epithelial cells (especially if they vary markedly in size) are found, sediment smears should be made, air-dried, stained with a hematologic stain, and evaluated cytologically.