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Urinalysis by mean of test strips

Introduction

Introduction
Urine analysis is considered to be the oldest clinical chemistry test. At present, it still remains one of the basic tests regardless of the disease involved. Currently, chemical testing of urine is usually performed by use of a test strip. The routine urine examination includes test strip screening, followed by microscopic examination of urinary sediment if necessary. Other tests are performed where applicable.


Urine sampling
A midstream urine specimen collected after thorough hygiene of external genitals is an optimal sample. Appropriate urine sampling and preparation are of utmost importance for the chemical and microscopic urine tests to produce reliable results. Therefore, the containers used for urine collection must be thoroughly clean and time of analysis strictly defined (within 1-2 hours from urine sampling).

Urine specimen can be obtained by:
1. spontaneous urination,
2. spontaneous urination - midstream urine specimen,
3. catheterization, and
4. percutaneous and suprapubic urinary bladder aspiration.



Types of urine specimen
A healthy adult excretes about 1500 mL of urine daily. The overall amount of urine excreted during 24 hours should preferably be obtained for urinalysis, be it quantitative or qualitative. However, this mode of urine collection is quite questionable, because not all people comply with the instructions and may not present total 24-hour urine for examination. Urine is collected into clean containers in three ways:
1. random specimen (individual specimen),
2. diurnal or nocturnal specimen collected over a certain period of time, and
3. 24-hour urine.
• Random specimen
This type of urine specimen can be obtained at any time of the day, however, the first morning urine is recommended as it is most concentrated and approximates the composition of 24-hour urine. The physicochemical examination of urine is performed on fresh urine without the addition of preservative.
• Diurnal or nocturnal urine specimen or urine specimen collected over a certain period of time
The urine collected in this way is used for urinalysis in some disorders, e.g. alimentary glycosuria, proteinuria, etc. The mode of urine collection, storage and preservation depends on the tests to be performed.
• 24-hour urine
For quantitative assays, 24-hour urine specimens should be carefully collected and preferably refrigerated. Certain preservatives should be added if chemically instable compounds are to be determined. The type of preservative depends on the analyte to be determined. 10% solution of thymol in isopropanol (5 ml for 24-hour urine) is most commonly used as preservative.
For routine urine examination, it is recommended to use the first morning urine obtained at least 8 hours from the last void. For urinalysis by means of test strip, any random urine specimen can be used, however, the time of urine sampling should be considered on result interpretation.


Containers
The containers used for urine collection must be made from a material non-reactive with urine constituents. They must have a wide neck (at least 4 cm in diameter), and be 50 ml in size, preferably disposable. The container used for urine collection must be thoroughly clean. In case of urine specimen transportation, the containers must be provided with appropriate caps and labels containing the following data:
• patient's first and last name,
• date and time of urine sampling, and
• sample storage before transportation to laboratory (in a refrigerator at +2 °C to +8 °C or at room temperature).


Storage of urine specimen
The analysis of a urine specimen should be carried out as soon as possible after urine sampling, due to rapid autolytic changes of the formed elements (cells, cylinders) and propagation of bacteria. Therefore, urine specimens should not be stored at room temperature for more than 2 hours (according to some sources, not more than 30 minutes). It is recommended for urine specimen to be stored at +2 °C to +8 °C, however, the time of storage should be maximally reduced.
If stored in a refrigerator, urinalysis should be carried out within 4 hours from urine sampling.


Consequences of inappropriate urine specimen storage
Until analysis, urine should be protected from direct sunlight, thus avoiding the possibility of erroneous results due to subsequent urine changes. In case of prolonged urine storage beyond the above mentioned time limits, the following changes develop:
• bilirubin - decreased due to sun exposure
• color - changed due to oxidation or metabolite reduction
• bacterial count - increased
• cell and cylinder count - decreased due to disintegration, especially in a diluted or alkaline specimen
• glucose - decreased due to glycolysis and bacterial utilization
• ketones - decreased due to volatilization
• nitrites - increased due to bacterial reduction of nitrates
• pH - increased because of urea decomposition to ammonia due to the bacterial urease activities
• turbidity - increased due to bacterial growth and possible deposition of amorphous salts
• urobilinogen - decreased due to oxidation to urobilin


Procedure of urine sampling - urine collection
1. Urine specimen
Morning urine (midstream)

2. Patient preparation
• The patient is given a urine container, labeled with the patient's name
• The patient is instructed on how to perform urine sampling:
a) hand wash with soap
b) washing external genitals with soap

c) genitals hygiene

Men:
1. Uncircumcised men should be warned to pull the preputium to expose the external ureteral orifice.
2. The glans should be cleaned with a clean (sterile) towel, from the ureteral orifice outward.

Women:
The ureteral orifice and surrounding area should be cleaned with a clean (sterile) towel.

d) the first void should be allowed to pass into the lavatory (1),
e) the midstream urine is voided into the urine container (2),
f) then the remaining urine is voided into the lavatory (3),
g) the container is closed and urine specimen immediately delivered to the laboratory.


Test strip

Test strip - general information
Test strips are used to analyze urine pH and specific gravity, and to demonstrate the presence of glucose, bilirubin, ketones (acetoacetic acid), blood, protein, urobilinogen, nitrites, leukocytes and ascorbic acid in urine (depending on the type of test strip used). Test strips are used for rapid and reliable demonstration and semiquantitative determination of particular analytes in urine by quite a simple procedure.
While working in a medical biochemistry laboratory in Vienna in 1920s, Fritz Feigl began to think about a more rapid testing for certain urine compounds by filter paper impregnation with particular reagents. Some 20 years elapsed from the idea to its implementation. In 1935, a report on rapid urinalysis by test strips appeared in the journal of the Leipzig Academy of Sciences. However, Feigl's invention found routine application years later, when industry manufacture of strips for rapid urine testing started. First strips were intended for a single parameter, e.g. glucose, albumins, ketones, etc. Then, Ames started the manufacture of test strips for simultaneous determination of a number of analytes, e.g. Hema-combistix for protein, glucose, pH and acetone. The shortcoming of these first test strips was their instability due to the reagent susceptibility to the effect of moisture (with filter paper as a medium). Later on, filter paper as a carrier was substituted by inert plastic strips with reagent-impregnated paper slips (6x6 mm slips of filter paper) pasted on.
Currently used test strips are made of plastic foil with reagent paper (impregnated reagents) at particular areas. A special layer to absorb excess fluid is placed underneath the reagent paper. A thin nylon mesh is stretched over the absorbent layer and reagent paper, fixing them to the foil and at the same time protecting them from any undesirable contact and contamination.

The strip allows the reagent paper (6x6 mm) to be uniformly perfused with urine while the reaction is proceeding steadily, together with removal of excess urine. The principles of reagent reactions in dry state for testing particular analytes in urine are identical to those performed in the test tube. A specific chemical reaction for determination of an analyte concentration is combined with an indicator or conjugated reagent to produce a color of varying intensity proportional to the tested analyte concentration.
The first test strips were manufactured in 1950 for diagnostic purpose, i.e. for the detection and follow-up of diabetes mellitus. These test strips contained reagents for demonstration of glucose in urine (Clinistix, Ames; S-Glucotest, Boehringer-Mannheim). Reagents for other tests were then gradually added onto the test strip. At present, 11 different urine parameters can be qualitatively or semiquantitatively determined by manual evaluation or automatically:
1. pH
2. glucose
3. protein
4. nitrites
5. urobilinogen
6. ketones
7. bilirubin
8. blood
9. leukocytes
10. specific gravity
11. ascorbic acid

Test strips have been continuously developed, e.g. new reagents to prevent interferences have been added to the layers with basic reagents (for instance, an iodate-impregnated layer oxidizing ascorbic acid in urine, thus preventing the potentially interfering effect, is being added onto the test strip for determination of blood in urine).
Test strips allow rapid and reliable demonstration and semiquantitative determination of particular analytes in urine. The use of test strips has a number of advantages:
• simple use,
• constant reagent composition,
• specificity for particular parameters, and
• long-term stability.

Disadvantages of the use of test strips include:
• inadequate sensitivity for some analytes (e.g. albumin),
• susceptibility to interferences, and
• semiquantitative results.

The test strip pack and vial are labeled with the type of analyte determined at each individual test strip area. Test strip areas are ready for use immediately upon being taken out from the vial. Test strips can be read manually, without any additional laboratory equipment, or on automated urine test strip readers of a number of manufacturers (e.g. Clinitec, Miditron, Rapimat II, etc.).


Test strip and its use
The manufacturer's instructions provided in each pack should always be strictly followed, because the procedure may vary with different manufacturers.

Urine test strips can be used for:
1. routine examination,
2. monitoring of therapy and possible relapse,
3. self-monitoring, and
4. systematic examinations.

1. Routine examination
Complete chemical urinalysis by means of a test strip covering the following parameters is the most rational procedure for daily routine examination of urine in a general practice or hospital setting:
• nitrites
• pH
• protein
• glucose
• ketones
• urobilinogen
• bilirubin
• blood
• leukocytes
• ascorbic acid
• specific gravity

Early symptoms of the following large groups of diseases can be detected by a single urine test strip:
• carbohydrate metabolism disorders,
• kidney and genitourinary tract diseases (e.g. calculi, tumors, glomerulonephritis, pyelonephritis, etc.), and
• hemolytic and liver diseases.

2. Monitoring of therapy and possible reccurrence
a. Diabetes mellitus

For therapy monitoring, special test strips for this indication are used.
The following tests are performed in diabetes mellitus:

• glucose
• ketones.
These tests will reveal metabolic changes or dietary errors.

b. Kidney and genitourinary tract diseases
The following tests are performed:
• nitrites
• pH
• protein
• blood
• leukocytes
c. Bile pigment demonstration
The following tests are performed:
• urobilinogen
• bilirubin

3. Self-monitoring
Test strips can be given to patients, as recommended by the physician. This especially holds for diabetic self-monitoring (glucose and ketones).

4. Systematic examinations
Urine test strips are used for:
• general screening in systematic examinations,
• general or target screening in epidemiologic studies,
• early detection of pathologic conditions, and
• therapy monitoring.


Test strip sensitivity
Test strip evaluation

Two criteria are essential for the evaluation of test strip usability:
• test strip sensitivity and
• test strip specificity

The level of sensitivity is a very important criterion for test strip evaluation. The level of sensitivity is the concentration of a substance at which at least 90 of 100 different specimens are positive.
The level of sensitivity is the smallest concentration of a substance that can be detected by the test strip.
Detection limit of particular tests is so low that even very small changes in urine composition cause changes in the color of test paper slips, yielding positive reaction.
The limits of detection differ both for particular analytes and for test strips from different manufacturers.


Test strip specificity
Test strip specificity is the characteristic of chemical reaction to react exclusively with the analyte whose concentration is to be determined. If no other substances interfere with the reaction, the chemical reaction is considered highly specific.
• Glucose
A highly specific test, because only glucose reacts with the glucose oxidase enzyme.
• Protein
The reaction is specific for albumins within pH range of 5-9. The test is less sensitive for other proteins (e.g. Bence-Jones protein, mucoproteins such as Tamm-Horsfall protein).
• Ketones
A highly specific reaction for acetoacetic acid and acetone.
• Urobilinogen
A highly specific reaction. No interferences from porphobilinogen, indican, or p-aminosulfosalicylic acid.
• Bilirubin
Specific reaction in a fresh urine specimen.
• Hemoglobin
Specific reaction for hemoglobin and myoglobin. Epithelial cells, leukocytes, and spermatozoids do not interefere with the reaction.
• Nitrites
The reaction depends exclusively on nitrites (pH 5-9).
• Leukocytes
The esterase from granulocytes and histiocytes is specific. Epithelial cells and spermatozoa do not interfere with the reaction.


Test strip storage
Test strips are packed in an aluminum or plastic box. Each box is provided with a moisture absorbing desiccant (inorganic silicate gel or silica gel). With appropriate storage (+4 °C to 30 °C) and usage (the box must be closed immediately upon taking a strip out of it), the test strips remain stable until the expiry date specified on the pack.


Working procedure
Test strips are kept at room temperature in original aluminum boxes. If the box with test strips has been stored in a refrigerator, the strip must be accommodated to room temperature.
The box with the strips must be kept closed all the time.
Urine tests performed with test strips are very simple.
Determination is performed in a freshly obtained, uncentrifuged urine specimen.

• Test strip immersion in urine
The test strip is briefly (maximally 1 second) immersed in the urine so as to wet all test areas.

• Removal of excess urine
Upon taking the test strip out of the urine, excess urine can be removed by wiping the strip edge along the container brim.
However, it is recommended to lean the lower side of the strip against a filter paper or cell-tissue.

• Reading
On visual (manual) reading, time has to be measured because the duration of a particular reaction differs between test strips of different manufacturers. Reading is performed within 30 and 120 seconds (depending on test strip type and manufacturer).
After the time period strictly defined by the manufacturer, the color produced by the reaction is compared with the color of the respective area on the reading scale.
Color changes appearing only along the margins of test areas, or developing after more than 2 minutes have no diagnostic value.
Automated test strip readers read the result within the strictly defined time set according to the manufacturer's instructions.
It is recommended to use automated test strip readers. If it is not possible, a stop-watch should be used on manual reading.

• If some drugs are suspected to influence positive test results, the test should be repeated upon therapy completion.

• Urine assay should be performed within 4 hours.