A urinary sediment examination

Abstract
A urinary sediment examination is an important type of non-invasive, repeatable morphological examination. It is necessary to accurately classify and measure urine components, such as epithelial cells, non-epithelial cells (blood cells), casts, salts/crystals, and microorganisms. The clinical significance of a urinary sediment examination is twofold. First, this examination is used to screen for the presence of a lesion in the kidney or urinary tract; second, it is used as a means to collect information on therapeutic and adverse effects of drugs administered to treat a confirmed lesion in the kidney or urinary tract. Pathological conditions are deduced not only from the results of a urinary sediment examination but also from a comprehensive evaluation of the results from various qualitative urinary examinations, such as urinary protein and occult blood tests, as well as biochemical (blood chemical) examinations. However, advances in diagnostic imaging and immunological examinations have allowed the current use of these methods for evaluating lesions in the kidney and urinary tract, and in consequence, the value of a urinary examination used as a screening test has increased further. Given these circumstances, we wished to conduct examinations with a clear understanding of their purpose; in other words, we hope to effectively conduct urine screening examinations, consider pathological conditions based on urinary findings, and observe and provide information useful to patients and for screening participants. In this part, we will explain the role of a urinary sediment examination and also the related technical methodology.
I  What is Desirable in a Urinary Sediment Examination?
Urinary sediment examinations have played an important role in the diagnosis and assessment of therapeutic effects primarily in patients with urinary tract infections and other diseases of the kidney and urinary system (e.g., diabetic nephropathy or nephrotic syndrome). However, it is necessary to present value-added information to clinical parties in addition to classifying urinary sediment components and reporting calculations to more effectively use a clinical urinary sediment examination. Here “value-added information” refers to information pertinent to etiology. Pressures from medical cost-containment efforts and societal aging have increased the impractical nature of examinations that place large burdens on patients, such as renal biopsy and cystoscopic examinations; in this context, a painless urinary sediment examination has become essential.
However, morphological analysis (except for atypical cells and some other components) in the context of a routine urinary sediment examination has become an auxiliary diagnostic index for pathological conditions of the kidney and urinary tract since the advent of diagnostic imaging (e.g., ultrasonography and magnetic resonance imaging) and various immunological urine biomarkers. Therefore, we suggest a new developmental path for a urinary sediment examination. This path would fully incorporate the characteristics of urine that permit frequent, non-invasive examinations in an effort to understand, based on the detected components, why the components settled down and appeared in the urine, rather than merely performing a simple morphological component analysis.
II  Basic Instructions
Urinary sediment examination results depend on urine sampling and urinary sediment sample preparation methods. Here we will present some examples from day-to-day operations that would require appropriate actions to be taken or that are encountered relatively frequently.
1.  Types of urine specimens
Urine types are distinguished by the time (e.g., morning urine or spot urine) and method (e.g., natural urine or catheter urine) of collection (see p.9 Table 1.1). As it is important to know the type of urine specimen that will be examined, an explicit description of the urine collection time and method should be encouraged.
2.  Urine collection methods
Urine specimens are usually self-collected. To obtain suitable urine specimens, subjects should understand the requirements for a urinary sediment examination and receive guidance about appropriate urine collection methods. In particular, women must receive guidance regarding urine collection from an anatomical point of view, with an emphasis on wiping procedures to avoid contamination by components from the vulva [e.g., red blood cells (RBCs), white blood cells (WBCs), squamous epithelial cells, or bacteria].
However, contamination by irrelevant components may be unavoidable even after providing better urine collection guidance. Therefore, it is desirable to provide relevant comments when reporting such cases.
Examples of comments regarding suspected contamination by vulva-derived components
• The urine specimen is contaminated.
• Contamination of the urine specimen by components from the vulva is suspected.
• Contamination of the urinary sediment by components from the vulva (e.g., RBCs, WBCs, squamous epithelial cells, or bacteria) is indicated.
* In addition to commenting, it is necessary to discuss the need for the resubmission of a urine specimen with the attending physician.
3.  Urinary sediment sample preparation
The quantity of formed elements (i.e., sediment volume) clearly exceeds 0.2 mL in samples from subjects with conditions such as advanced hematuria and pyuria. It is desirable to provide a comment regarding such findings in the report.
Examples of comments when the sediment volume exceeds 0.2 mL
• The quantity of formed elements (sediment volume) exceeds 0.2 mL because of advanced hematuria.
• The quantity of formed elements (sediment volume) exceeds 0.2 mL because of advanced pyuria.
• A large amount of urinary sediment was obtained.
* Such specimens may require a microscopic examination and the reporting of formed elements (sediment elements) after mixing the sediment to the best possible homogeneity after centrifugation and removal of the supernatant with an appropriate procedure.
4.  Description of urinary sediment examination results
While the Japanese Committee for Clinical Laboratory Standards (JCCLS) uses per field units [/high power field (HPF) or /low power field (LPF)] to describe RBCs and WBCs, respectively, in a urinary sediment examination, guidelines from developed countries in Europe, as well as the United States [e.g., National Committee for Clinical Laboratory Standards (NCCLS)1), The European Confederation of Laboratory Medicine (ECLM)2)], recommend describing results in per μL units. The per μL expression, which is based on a counting chamber or uncentrifuged urine method, is also used in the efficacy evaluation criteria for urinary tract infection (UTI) drugs. Therefore, it is important to determine the volume (μL) corresponding to a single field of view during a sample preparation/microscopic examination according to the JCCLS examination method. However, urinary sediment preparation methods must still address the issues of components remaining in the urine supernatant during the centrifugation step or the adsorption of components to the tube wall. In theory, the following consideration is valid.
Area of field of view
 = π × (number of field of view of the eyepiece lens/magnification factor of the objective lens × 1/2)2
Uncentrifuged urine equivalent volume per field of view (μL)
 = area of field of view × urine concentration ratio × sediment load/area of the coverslip
At 20 fields of view, the areas of a single field of view are 3.14 mm2 for a low power field (LPF; objective lens: 10×) and 0.196 mm2 for a high power field (HPF; objective lens: 40×). Therefore, single fields of view correspond to the following amounts of uncentrifuged urine (μL):
  LPF: 7.27 μL, HPF: 0.45 μL
According to the UTI Study Group, the following per μL expressions are based on the counting chamber method/uncentrifuged urine method:
  0–9/μL  10–29/μL  30–99/μL  ≥ 100/μL
III  Staining Techniques
In principle, the specimens without staining is used for microscopic examinations of urinary sediments. Staining procedures may cause hemolysis and interfere with observations of the numbers and shapes of RBCs in urine. The color characteristics of sediment elements may also be lost. Therefore, it is important to use unstained specimens for observation.
However, the use of various suitable staining methods may be useful when urinary sediment elements must be confirmed and identified or differentiated from analogous components.
Basic staining solutions include the Sternheimer staining (S staining) and Sternheimer–Malbin staining (SM staining). When using these staining methods, an approximate 4:1 ratio of urinary sediment and staining solution is recommended while considering possible dilution errors related to the staining solution.
1.  Sternheimer staining (S staining) (Figure 2.1)
<Reagent>
Solution I: 2% Alcian blue 8GS in water
Solution II: 1.5% Pyronin B in water
Solutions I and II are filtered and mixed in a 2:1 ratio. The staining performance of this mixture remains stable for approximately 3 months if the mixture is stored in a cool and dark place.

<Staining procedure>
At the time of the microscopic examination, add a drop of the staining mixture to the sediment and mix.
<Staining behavior>
Red blood cells: unstained or pink/magenta
White blood cells: nucleus, blue; cytoplasm, pink/magenta
Epithelial cells: nucleus, blue; cytoplasm, pink/magenta (note: the stained cytoplasm of mucus-containing cells such as columnar epithelial cells and adenocarcinoma cells is bluish purple or dark magenta)
Macrophages: nucleus, blue; cytoplasm, bluish purple/dark magenta
Casts: hyaline casts, light blue/blue; granular casts and waxy casts, magenta

Figure 2.1 
Sternheimer staining
2.  Sternheimer–Malbin staining (SM staining)
<Reagent>
Solution I: Crystal violet 3.0 g
95% Ethanol 20.0 mL
Ammonium oxalate 0.8 g
Purified water 80.0 mL
Solution II: Safranin O 0.25 g
95% Ethanol 10.0 mL
Purified water 100.0 mL
Solutions I and II are mixed in a 3:97 ratio and filtered before use. A fresh mixture should be prepared every 3 months.
<Staining procedure>
At the time of the microscopic examination, add a drop of the staining mixture to the sediment and mix.
<Staining behavior>
Red blood cells: unstained or pale reddish purple
White blood cells: ① In dark cells, the nucleus is dark purple and cytoplasm is purple. ② In pale cells, the nucleus and cytoplasm are both unstained/light blue.
Epithelial cells: nucleus, purple/dark purple; cytoplasm, pink/purple
Casts: hyaline casts, pale red; granular casts, pale purple/dark purple granules; casts containing different cell elements exhibit unique staining patterns
3.  Sudan III staining (Figure 2.2)
<Reagent>
Dissolve 1.0–2.0 g of Sudan III in 100 mL of 70% ethanol with shaking, and allow this solution to rest in an airtight container in a 56–60°C incubator for 12 h, followed by storage at room temperature.
<Staining procedure>
Add 2–3 drops of the filtered solution to the sediment, allow the mixture to stand at room temperature (15–30°C) for 15–60 min, and evaluate the sediment via a microscopic examination. Sudan IV staining is also useful.
<Staining behavior>
Fat globules, fatty casts, oval fat bodies: yellowish red

Figure 2.2 
Sudan III staining
4.  Prescott–Brodie staining (PB staining) (Figure 2.3)

Figure 2.3 
Prescott–Brodie staining
<Reagent>
Solution I: 2,7-Diaminofluorene 300 mg
Phloxine B 130 mg
95% Ethanol 70 mL
Solution II: Sodium acetate·3H2O 11 g
0.5% Acetic acid 20 mL
Solution III: 3% Hydrogen peroxide water 1 mL
Solutions I, II, and III are mixed and filtered before use.
<Staining procedure>
At the time of the microscopic examination, add 5–10 drops of the staining solution to the sediment and mix well.
<Staining behavior>
Peroxidase-containing cells such as neutrophils, eosinophils, and monocytes: blue/blackish blue
Lymphocytes and other cells: red
5.  Berlin blue staining (Figure 2.4)

Figure 2.4 
Berlin blue staining
<Reagent>
Solution I: 2% Potassium ferrocyanide in water
Potassium ferrocyanide 2.0 g
Purified water 100 mL
Solution II: 1% Hydrochloric acid
Concentrated hydrochloric acid 1.0 mL
Purified water 100 mL
Store solutions I and II in a cool and dark place, and mix them in a 1:1 ratio immediately before use; the resulting clear, pale yellow solution should be used.
<Staining procedure>
Add 10 mL of the staining solution to 0.2 mL of sediment and mix. Allow the mixture to stand for 10–20 min, centrifuge, remove the supernatant, and subject the sediment elements to a microscopic examination.
<Staining behavior>
Hemosiderin granules: blue/indigo
6.  Hansel staining (Figure 2.5)

Figure 2.5 
Hansel staining
<Reagent>
Hansel staining solution:
Methylene blue 0.6 g
Eosin Y 0.2 g
Methanol 60 mL
Phosphate buffered saline (PBS) containing ethanol
Ethanol is added to PBS to a final concentration of 10%.
<Staining procedure>
Add 2 drops of the staining solution to 0.2 mL of the sediment; mix, and allow the mixture to stand for 5 min. Next, add 10 mL of 10% ethanol–PBS, mix/centrifuge, remove the supernatant, and subject the sediment elements to a microscopic examination.
<Staining behavior>
Eosinophil granule components: red
7.  Lugol’s staining (Figure 2.6)

Figure 2.6 
Lugol’s staining
<Reagent>
Lugol’s solution:
After dissolving 2 g of potassium iodide in approximately 10 mL of purified water, add and dissolve 1 g of iodine in this solution; subsequently, add purified water to a total volume of 300 mL (a freshly prepared reagent is preferable).
<Staining procedure>
Mix the sediment and staining solution in a 1:1 ratio, and subject the mixture to a microscopic examination.
<Staining behavior>
Epithelial cells: The cytoplasm of a glycogen-containing cell will be partly or entirely stained brownish red.

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A urinary sediment examination

A urinary sediment examination

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