15.1 Procedural Definition: What Is This Test About?

The urine sediment examination identifies and provides a semiquantitative estimate of the number of erythrocytes, leukocytes, urinary casts, crystals, and bacteria present in a standard volume of urine. Urine sediment examination is necessary to fully interpret the urine reagent strip findings and can assist in identifying underlying causes for abnormalities found in a concurrent CBC and chemistry profile. In most cases, urine contains a relatively low concentration of the formed elements (cells, crystals, and casts). As a result, urine is centrifuged to assist in finding and identifying any formed elements that relate to underlying disease processes. A consistent protocol for urine sediment preparation is necessary to make this in‐house laboratory test semiquantitative rather than simply qualitative.

15.2 Procedural Purpose: Why Should I Perform This Test?

Urinalysis is a component of the “minimum” database often requested when presented with an ill patient. The complete urinalysis includes gross evaluation of the urine color and turbidity, urine specific gravity, reagent strip chemical reactions, and urine sediment examination. The findings in each of these components are essential in interpreting the others. It may be tempting in some cases to just perform the gross examination and reagent strip chemistry test and forego the sediment examination if no abnormalities are seen. However, this can result in missing a significant number of abnormalities on the sediment examination. Approximately 12% of canine urine samples and 6% of feline urine samples with no abnormalities in gross appearance or chemical analysis will have significant abnormalities in the urine sediment, including abnormal numbers of erythrocytes (hematuria), leukocytes (pyuria), and bacteria (bacteriuria) (Barlough et al., 1981). This is especially true in patients with endocrine disease, such as hyperadrenocorticism, where there is increased susceptibility to bacterial cystitis and inhibition of leukocyte movement into tissues.

Urine sediment examination is essential in the interpretation of the gross appearance of the urine and any increased protein concentration noted on the chemical analysis. Red urine can be the result of hematuria, hemoglobinuria, or (less commonly) myoglobinuria. Hematuria is usually associated with a change in clarity from clear to cloudy. Identification of erythrocytes on the urine sediment is necessary to confirm hematuria. Both pyuria and significant crystalluria can result in a grossly cloudy urine sample. Identification of crystalluria or pyuria differentiate between these two causes of change in clarity. Any sample with a significant amount of protein identified on the chemical analysis requires evaluation of the sediment to determine if inflammation of the urinary tract versus glomerular or tubular loss are the cause of the increased proteinuria.

It is unfortunate that many veterinarians choose to send out urinalysis rather perform this valuable test in‐house. By making urinalysis a routine and commonly used diagnostic test, the general practitioner and their well‐trained veterinary technician can develop the necessary skills and the confidence needed to evaluate urine sediment. In addition, there are newer in‐clinic instruments dedicated to urine analysis, such as the IDEXX SediView™, that can assist in performing this valuable procedure. The in‐house instruments still require a good level of skill by the user in identifying formed elements as an internal control for the instrument.

The primary reason for performing urine sediment examination in‐house is the changes that occur when urine is stored prior to analysis. It is optimum to perform a urinalysis within one hour of collection. If a sample cannot be analyzed in this time frame, it can be refrigerated. If left at room temperature for longer than one hour, significant changes can occur in both the chemical and sediment analysis. The primary changes in urine sediment findings seen with prolonged time between collection and analysis include:

• lysis of erythrocytes in dilute urine
  
• degeneration of cellular elements
  
• dissolution and precipitation of crystals
  
• changes between types of crystals (dihydrate calcium oxalate versus monohydrate calcium oxalate)
  
• degeneration of crystals
  
• bacterial growth.

These changes can occur during refrigeration but at a slower rate. If the sample is refrigerated, it should be analyzed within 4–6 hours to prevent significant changes in the sediment. The urine sample should be warmed to room temperature and mixed very well before analysis. Warming to room temperature is essential for the chemical analysis. Some crystal that may have precipitated out during storage may redissolve at room temperature.

The use of a stain with a sediment examination is a controversial issue. Because of the artifacts and alteration in concentration of formed elements that are introduced with the use of a stain such as SediStain™, it is best to refrain from using a stain. If there is concern for whether the operator is seeing bacteria, if the cells present are leukocytes versus erythrocytes, or if there are atypical transitional cells present, a cytological preparation using the “line preparation” method is preferable.

Table 15.1 lists reference intervals for urine sediment formed elements (Rizzi et al., 2017).

15.3 Equipment

Equipment used in this test include:

• centrifuge
  
• conical or round bottom centrifuge tubes
  
• pipettes
  
• glass microscope slides
  
• 25 × 25 mm coverslips
  
• quick Romanowski stain set up
  
• a fresh urine sample collected in a sterile container at room temperature.

15.4 Procedural Steps: How Do I Perform This Test?

• Gather the equipment and supplies for the test (see Figure 15.1).
  
• Gently mix the urine sample before aliquoting a standard volume into a conical or round bottom centrifuge tube. The recommended volume for a urinalysis is 5–10 ml. It is important to use the same sample size in each procedure in order to achieve consistent results (see Figure 15.2).
  
  
  
  • If the standard volume of urine is not available, record the volume used. Semiquantitative interpretation of the formed elements in the sediment examination will be negatively affected and interpretation of the findings should be done with that consideration.
  
  
• Place the centrifuge tube in the centrifuge with an appropriate balance tube containing the same volume of fluid (see Figure 15.3).
  
• Centrifuge for five minutes at 250 or 480 relative centrifugal force (RCF), or 2000 rotations per minute (RPMs) if the centrifuge does not provide for adjustment using RCF (see Figure 15.4a and b).
  
• Once the centrifuge has completely stopped. Decant the supernatant by pouring it off or by removing it using a pipette. Using a pipette allows more control for retaining a standard volume for resuspension of the sediment (see Figures 15.5 and 15.6).
  
  
  
  • Approximately 0.5 mL of fluid should be retained for resuspension of the sediment. Consistency in how much fluid is used for resuspending the sediment is necessary to allow semiquantitative evaluation of the sediment. The sample can be resuspended by gently “flicking” the tube or by using a pipette (see Figure 15.7a and b).
  
  
• Once the sediment is resuspended, place a drop of the sediment on a glass microscope slide and cover it with a 25 × 25 mm coverslip. This size of the coverslip should be used for consistency (see Figure 15.8a and b).

  

  

  
  
• After placing the microscope slide on the stage, to best view the elements in an unstained wet mount, lower the condenser on your microscope, or, if your microscope does not have an adjustable condenser, close the iris diaphragm slightly. This will increase the contrast and make the unstained elements visible (see Figure 15.9).

  

  
  

  The initial evaluation of the sediment is done using the 10x objective to quantify the number of large formed elements, such as epithelial cells, crystals, and casts.

  
  
  
  
  • The average number of each of these elements/10x field is recorded as number/low power field (lpf).
  
  
• Next the sediment is evaluated using the 40x objective to identify the presence and number of RBCs, leukocytes, small epithelial cells, and microorganisms (bacterial and fungal agents).
  
  
  
  • The average number for each of these elements is recorded as number/high power field (hpf).

  

  

  
  
• A line preparation for staining can be prepared if microorganisms and atypical cells are seen in the sediment examination. The line preparation will concentrate formed elements in a single line providing a smaller more concentrated area to review.
  
  
  
  • Place a drop of sediment or, if the urine has high cellularity, nonsedimented urine on a microscope slide.
    
  • Using a spreader slide, draw the spreader slide back to the drop of urine and allow the sample to spread out along the edge of the spreader slide (see Figure 15.10).

    

    
    
  • Push the spreader slide toward the end of the microscope slide (see Figure 15.11).
    
  • About 1/2 to 2/3 of the way to the end, abruptly lift the spreader slide (see Figure 15.12).
    
  • The formed elements of the urine will be carried to a line formed as the spreader slide is lifted (see Figure 15.13).