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Section 3-1
Preparation and Illumination


3-1. PREPARATION OF URINE SEDIMENT FOR EXAMINATION (UNSTAINED)

A properly performed microscopic examination of urinary sediment can provide valuable information that enables the physician to diagnose renal as well as other abnormalities. The importance of this procedure, which has been compared to a biopsy with respect to its significance, cannot be overemphasized. Thus it is crucial that the many structures occurring in urinary sediment be identified correctly and that the diagnostically significant elements be distinguished from extraneous substances.

3-2. BASIC TECHNIQUE

  1. The urine sample should be examined macroscopically and checked for cloudiness, color, possible blood, and a syrupy consistency that may indicate mucus and casts. Such an examination can provide clues regarding the nature of the sediment.
     
  2. The specimen should be thoroughly mixed to ensure proper dispersal of constituents. Twelve to 15 mL of the specimen is placed in a conical centrifuge tube and centrifuged at 1500 rpm (revolutions per minute) for 5 minutes.
     
  3. The centrifuged tube is then inverted into a clean test tube. The supernatant urine is saved for chemical testing; the sediment that remains in the bottom of the tube is examined microscopically.
     
  4. Next, the sediment is resuspended by "finger-flicking" the tube. Then, one drop of sediment is transferred to a clean, dry glass slide by a pipette or a dropper, and a cover slip is applied.
     
  5. Next, the sediment is scanned by using the low power (10X) objective and is examined in detail by using the high power (43X) objective.

It should be noted that the appearance of the urine does not necessarily correlate with the results of a microscopic analysis. Clear, normal-appearing urine may often reveal abnormal elements of diagnostic importance upon microscopic examination. Urine that gives only slight sediment after centrifugation may contain important structures; on the other hand, cloudy urine that contains heavy sediment may not disclose any clinically significant elements.

3-3. ILLUMINATION OF MICROSCOPIC FIELD

Light Intensity. The technique for use of the microscope must be changed somewhat from ordinary practice due to the transparent nature of urine sediment. Since many of the structures to be examined are hyaline (semi-transparent) in nature, the light should be subdued for ordinary work. Before the structures can be seen and identified, the intensity of light must be reduced to a minimum. The light intensity is reduced by practically closing the iris diaphragm of the microscope.

Scanning and Reporting. The drop should be scanned under the low power objective and with moderate light. Low power is used because the low power objective covers a larger field and thus allows a more rapid scanning of the total preparation. Certain large structures such as parasite ova can be spotted using low power. Due to their large size, casts are also reported as the average number per low power fields. Smaller elements are reported in terms of average number per high field. When switching the objectives from low to high power, the condenser should be raised slightly to bring the light to its former intensity. Red blood cells, white blood cells, and epithelial cells are reported as the average number per high power field, counting 10 fields (for example, 15-19 rbc/HPF). If very few or very many cells are seen, descriptive adjectives are used, for example, "few," "occasional," "too numerous to count." Crystals should be reported when they are observed. However, an actual count is unnecessary; a report of the type of crystal and an indication of relative occurrence, such as "few," "many," "and so forth," is sufficient. If bacteria are seen in a fresh specimen, they are reported as present. Bacteria and epithelial cells are less significant in urine from females than in urine from males, but awareness and reporting of their presence in all specimens is a safe procedure.

3-4. COMMON SOURCES OF ERROR

Delayed Analysis. The urine must be examined while still fresh. As the nature of the sediment changes with the passage of time, the analysis should be performed within 2 hours after voiding. If immediate examination is not possible, the specimen should be refrigerated or preserved with formalin.

Improper Illumination. This is the most common error in the microscopic analysis of urine. As mentioned previously, subdued light is necessary so that hyaline semi-transparent structures are not obscured by intense illumination.

Improper Placement of Sediment on Slide. Placing drops of sediment from too many patients on the same slide is another frequent source of error. As a result, the drops tend to run together. In order to avoid this problem, a slide should not contain sediment from more than two patients.

Dried Slide. Another error is to attempt to identify objects in urine, which has dried on the slide. A valid examination is impossible if this occurs. Not only are the delicate organized structures distorted beyond recognition; however, there is a confusing deposit of urinary salts. After some experience, one can immediately recognize the urine has dried due to the peculiar refraction of the structures.

Confusion Due to Artifacts. Extraneous elements are also a common problem in accurate microscopic analysis. One must become totally familiar with the relevant elements so that extraneous structures are not confused with and reported as significant structures.

David L. Heiserman, Editor

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Revised: June 06, 2015