Ruthanne Hyduke, M.A.*
Ruthanne Hyduke, M.A.
1. Describe the urinalysis procedure to CLIA regulations
2. Explain how to collect a urine sample for routine urinalysis.
3. Review the procedures for gross and chemical urinalysis.
4. Develop quality control measures appropriate for the urinalysis in the physician office lab.
5. Interpret the findings in the gross and chemical analysis
6. Define the interfering substances for each chemical analysis.
Changes in the composition of urine occur very early in many disease processes, often before the patient is aware of any symptoms. For this reason and because urine can be obtained relatively easily, the urinalysis was one of the first laboratory tests performed and related to diseases. The urinalysis is a group of qualitative and semiquantitative analyses performed on random or non-timed urine samples. The procedures can be performed manually or by one of several automated methods. Today it is one of the simplest analyses to perform but maintains the sophistication and accuracy of more complicated laboratory tests.
The Clinical Laboratory Improvement Act (CLIA) categorizes the chemical analysis of the urine as a Waived Test only if it is not performed by an instrument. The chemical analysis includes the reagent strip (dipstick) tests for bilirubin, glucose, hemoglobin/blood, ketones, leukocytes, nitrite, pH, protein, specific gravity and urobilinogen. The laboratory must be registered and hold a CLIA certificate of waiver. To perform automated urinalysis procedures, a laboratory must be certified to perform moderate complexity tests. Such a laboratory can also perform waived tests provided they meet those qualifications. Urine sediment examinations are included in the list of Provider Performed Microscopy Prodcedures. This means that urine microscopics can be performed by certain health care providers on his or her own patients.
Ruthanne Hyduke, M.A.
The best sample for a routine urinalysis is a clean-catch or
midstream sample collected after the external
genitalia have been cleansed with an antiseptic solution. In the
clean catch technique, the first portion of voided urine is discarded
and the next portion is collected in a clean (sterile if culturing
for bacteria) container. A random collection is suitable for most
analyses; however, the first morning collection is best for protein
and specific gravity determinations because this urine is the most
concentrated.
The sample should be collected in a clean and clearly labeled container. The urine must be analyzed soon after collection because most urine elements deteriorate at room temperature within an hour. A refrigerated specimen will retain its integrity only up to four hours.
A urine that sits at room temperature for any length of time will change considerably. Bacteria can overgrow the urine, and in the process metabolize any glucose present. The bacteria can also utilize the urine urea, forming ammonia and increasing the pH. Therefore a urine with a strong ammoniacal odor should have an elevated pH (pH 8-9) which can be used as a cross-check between odor and pH. Bilirubin which is light sensitive will decompose and will become unreactive. Casts, red blood cells and white cells also tend to disintegrate after only a short time at room temperature.
Ruthanne Hyduke, M.A.
The preanalytical procedures include the correct collection of the sample in a clean container with the sample being analyzed immediately or stored for no more than 4 hours at refrigerated temperatures. The sample must be clearly labeled with the patient's name on the container and must not be visibly contaminated by fecal material or other debris. Most patients need to be instructed on how to collect the sample so that it won't be contaminated. Because so many personnel are associated with the collection of urine samples, they must be adequately trained on the procedures. Again, written protocol will help in this training process.
The analytical components of quality assurance include equipment maintenance, use of quality reagents, access to procedure manuals and the technical competence of the personnel performing the procedures. Preventive maintenance must be performed on all equipment related to the urinalysis including analyzers, pipettors, refrigerators, microscopes and refractometers. The frequency of this maintenance depends on the equipment used and must meet the minimal standards of the accrediting agency. Inspection checklists from these agencies is an excellent source of information for guiding the development of these procedures.
Procedure manuals must be available in the urinalysis laboratory and include details of all procedures performed, test principles, step-by-step instructions on how to perform every procedure and related calculations, quality assurance guidelines and the reporting of results. These manuals must be continually reviewed and updated and then followed by all personnel. Technical competence of personnel can be assured by proper training, adherence to the procedure manual and quality control checks. Documentation of personnel training and continued competency testing must be kept in the personnel file of the individual.
Quality control materials are used to monitor the accuracy and precision of the procedures. These materials are available commercially and may have recommended values. The control solutions mimic patient samples and monitor the physical, chemical and microscopic analysis of the urine. Tolerance limits for the control results must be defined for each laboratory and, when exceeded, corrective action taken to assure correct results. Results on these solutions must be recorded and filed for easy access.
The post-analytical component of quality assurance includes the efficient reporting of the information and correct interpretation of the data. Standardized report forms including reference ranges make communication effective. Critical or life-threatening values must be established and shared with all personnel interpreting the results. Personnel performing the urinalysis must bring these "panic values" to the attention of the physician treating the patient.
All quality assurance procedures require documentation and must show that they have been reviewed annually. When the tolerance limits are exceeded, corrective action must be taken and documented. Through the use of quality assurance practices, the goal of accurate and reliable urinalysis results can be achieved and, thus, reflect the patient's current condition rather than variances in procedures, equipment, reagents or personnel.
Ruthanne Hyduke, M.A.
Abnormal colors include red, beer-brown, black, orange and blue-green. A red urine can be caused by red blood cells or hemoglobin in the urine or by the red pigments found in beets. Red cells and hemoglobin may be accompanied by seeing red cells in a microscopic examination, by a positive chemical analysis for blood or by both. A beer-brown or yellow-brown urine is most often seen when bilirubin is present; a black urine occurs when melanin is found in the specimen; and orange, blue and green are often associated with the presence of drug, dye or food metabolites.
When the sample is well mixed, note if it is clear or turbid. (Turbidity may be reported as slight, moderate or excessive.) To determine the cause of the turbidity, centrifuge a portion of the urine. If the cloud settles to the bottom leaving a clear supernatant, the turbidity is caused by cells or crystals. If the urine remains cloudy after centrifugation, bacteria are present.
If the odor of the urine is strong, record whether the urine smells urinoid (normal), fruity (like acetone or fingernail polish remover), putrid (fecal smelling) or ammoniacal (like ammonia). Not many people enjoy smelling every urine they analyze, but one should note very strong odors. If the urine smells fruity, the chemical analysis for ketones should be positive. An ammoniacal urine should have a very alkaline pH (8-9).
Additional information about the gross and physical analysis will be found in Part IB: Interpretation of Gross and Chemical Analysis.
Next analyze the specific gravity of the urine. Some reagent strips have a test area for the analysis of specific gravity. It is a good screening method; but when precision and accuracy are desired, a refractometer should be used.
Refractometer
The refractometer measures the refractive index of the total soluble solids. When a beam of light passes through one substance into another, the beam is refracted so that it travels in another direction. The extent to which the beam is refracted depends on the concentration of the total soluble solids. Only a few drops of urine are required; the method is rapid and easy to perform.
An old method for determining the specific gravity used the urinometer. This is a hydrometer which is based on the principle that dissolved substances will cause a body to float.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
1. Store reagent strips in original container at room temperature (less than 30 degrees C). Do NOT store in a refrigerator.
2. Avoid exposing reagent strips to moisture, fluorescent light, sunlight, heat, acids, alkalis or volatile fumes.
3. Do not touch test areas of the reagent strip.
4. Keep reagent test areas away from detergents that may be found in the specimen containers and other contaminating substances found in working areas.
5. Do not remove desiccant from the bottle. Replace cap immediately and tightly after removing reagent strip.
6. Dip test areas in urine completely, but briefly to avoid dissolving reagents off the strip. Keep the strip horizontal after exposing it to the urine.
7. Read the test results carefully at the specified time intervals, against a white background in a good light, and with the test area held near the appropriate color chart on the bottle label. One of the automated instruments that are available for reading these strips may also be used.
Procedure:
1. Mix
Mix the urine to be tested by inverting the sample several times.
2. Insert reagent strip.
Completely immerse all reagent areas of the strip briefly but completely in the urine.
3. Remove excess urine.
To remove excess urine, tap the edge of the strip against the side of the urine container, draw the strip across the top of the container or press the edge of the strip against an absorbent paper.
4. Timing
Time according to manufacturer's directions using a timing device with a second hand.
5. Compare test areas closely with corresponding color charts on the bottle label at the times specified. Hold strip horizontally and close to the color blocks. Read at times listed on the product you are using.
6. Record the results according to the protocol written in your facility.
For good quality control, reagent strips must be tested with known positive and negative solutions (controls) to assure reactivity of all portions of the reagent strip. Controls should be tested daily; but if not they must be analyzed every time a new bottle of strips is opened. Known negative and positive specimens can also be "hidden" in a batch of samples to test the competency of the personnel performing the tests.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
1. Mix the urine to be tested by inverting the sample several times.
Ruthanne Hyduke, M.A.
2. Completely immerse all reagent areas of the strip briefly but completely in the urine.
Ruthanne Hyduke, M.A.
3. Remove excess urine.
3. To remove excess urine, tap the edge of the strip against the side of the urine container, draw the strip across the top of the container or press the edge of the strip against an absorbent paper.
Ruthanne Hyduke, M.A.
4. Time according to manufacturer's directions using a timing device with a second hand.
Ruthanne Hyduke, M.A.
8. Compare test areas closely with corresponding color charts on the bottle label at the times specified. Hold strip horizontally and close to the color blocks. Read at times listed on the product you are using.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
Protein in the urine, proteinuria, is generally confirmed using a precipitation test. Protein is denatured in the presence of many substances and precipitates out of the urine. One of the most common reagents used is sulfosalicylic acid (SSA). In this test equal volumes of centrifuged urine and SSA are added together and the resultant precipitate is read as negative through 4+.
Sulfosalicylic
acid precipitation test.
Detection of low levels of protein in the urine may be accomplished by using one of the sensitive albumin tests that are available. These tests may be used when the reagent strip is negative, but the presence of protein is suspected. Two commercial tests are available at this time: one is a tablet that is based on the same reaction as the reagent strip (double indicator/"protein error of indicators") and the other is a unique reagent strip test based on an immunochemical antibody-enzyme conjugate that reacts with the protein.
Bilirubin in confirmed in the urine most often through the use of tablets such as those produced by the Ames Company (Ictotest Tablets). These tablets are more sensitive to bilirubin and the color change produced is much easier to read visually than the multiparameter strips. With these tablets it is important to read the color change at the indicated time (within 30 seconds) as reading after that may produce an erroneous reading.
Copper sulfate reduction tests are most often used to confirm the presence of glucose or any other reducing sugars such as galactose that might be present. These reactions depend on the ability of the reducing sugar to convert cupric sulfate to cuprous oxide with a resulting color change. Sodium hydroxide is added to the reaction to produce heat which enhances the forward reaction. This test can be performed by using either wet reagents or the commercially available tablets such as Clinitest®. The latter results must be evaluated immediately because any color change after the 15 second incubation lead to interpreting an erroneous result. For example, if analyzing a urine with a very high amount of glucose present, the "pass-through" phenomenon can occur where the cuprous oxide is reoxidized to cupric oxide and a reversal of the reaction occurs. This would give a falsely low result.
Confirmation tests can be performed to confirm the presence of elevated urobilinogen or to differentiate it from porphobilinogens. However, these tests are not usually performed routinely. There are wet chemicals that can be used for this confirmation, called the Watson-Schwartz test. It employs the use of Ehrlich's Reagent, paradimethylaminobenzaldehyde. The mixture of urine and reagent produce a color which, if extracts into chloroform, confirms the presence of urobilinogen.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
Ruthanne Hyduke, M.A.
Specimen Type |
Collection |
Use |
1. Random clean catch or mid-stream collection |
Cleanse external genitalia and discard the first part of the void. |
Routine urinalysis |
2. First morning specimen |
When the patient first arises in the morning. |
Routine urinalysis: it is the most concentrated and best for protein and bilirubin determinations. |
3. Second-voided specimen |
First sample after the first void of the day. |
It may contain cells that are destroyed during stasis in the bladder while the patient sleeps and therefore not seen in the first morning specimen. |
4. Post prandial collection |
At a timed interval (for example, two hours) after the patient has eaten. |
This is the best sample for confirming the presence of elevated urobilinogen. May also be used to detect glucose. |
5. Day specimen |
Usually collected from 9:00 a.m. to 8:00 p.m. |
Used for quantitative determinations of analytes that may undergo a diurnal variation or be diet dependent. |
6. Night specimen |
Usually collected from 8:00 p.m. to 8:00 a.m. |
Used for quantitative determinations of analytes that may undergo a diurnal variation or be diet independent. |
7. Twenty-four hour collection |
Day 1: discard first morning void, mark time, and collect all urine voided until Day 2: void at time marked from Day 1 and include that sample. |
Used when quantitative tests need to be performed. |
8. Catheterized collection |
Spot sample collected via catheter inserted into the bladder. |
Occasionally used for bacteriological study, especially in females. May also be done to obtain urine from infants or small children. |
2. Volume
The volume of urine voided is directly related to the amount of
fluids consumed. Normal adults usually void 1000-1500 mL per 24
hours; the minimum normal excretion vaule is 600 mL. The day volume
is usually 2-4 times that of the night. Children void 3-4 times as
much as adults per kg of body weight.
3. Color
Normally, urine is some shade of yellow. Acid urine is usually darker
thatn alkaline urine. Pathologically, it may be any color. A blue or
green urine is usually the result of ingested dyes (e.g. diagnex
blue) or drugs.
A suggested color scale to use:
4. Transparency:
Normally, freshly voided urine is clear and transparent. It may be
cloudy due to crystals and cells will centrifuge out; bacteria will
not.
Amorphous |
A normal constituent in alkaline or neutral urines; dissolve upon addition of a dilute acid (e.g. acetic). If combined with carbonates, gas will be evolved upon the addition of the acid. |
Amorphous |
A normal constituent in acid urine; often appear as yellow crystals or may be pink ("brick dust" deposit) due to increased uroerythrin. They dissolve upon warming to 40 degrees C. |
Bacteria |
Usually seen as a uniform cloud when an excess of bacteria is present; cannot be removed by ordinary filtration or centrifugation; seen in microscopic examination. |
Blood (red blood cells): |
May give a reddish or brown, smoky appearance to the urine; recognized by seeing red cells upon microscopic examinationor chemical tests for hemoglobin. |
Colloidal Particles |
Cannot be cleared from urine by filtration or centribugation; are not visible in microscopic examinationand not removed by ether. Their cause is unknown. |
Fat Globules |
Will usually give a milky appearance to urine; may be opalescent; are seen in microscopic examiniation and removed by ether. |
Mucus and Epithelial Cells |
Upon cooling and standing, a faint cloud ("nubecula") of mucus, leukocytes and epithelial cells may settle to the bottom. In urine of igh specific gravity (sp gr) it may float near the middle. The nubecula of normal urine is probably due to nucleoprotein (phosphoprotein) and not a mucin or mucoid (glycoprotein). |
Pus (white cells): |
May resemble amorphous phosphates tot he naked eye. Microscopic examination will reveal that the cloud is due to leukocytes. |
5. Urine Odors and Their Causes:
Strong odors of urine need to be identified because many can give clues dto
the origin of diseases.
Ammoniacal |
Occurs especially during decompositoin or urine on standing ("alkaline fermentaion") or retention within urinary bladder; may be related to some bacterial infections. |
Effects of drugs and diet |
Many ingested substances will give the urine a distinct odor as ingested asparagus giving urine its characteristic odor. |
Fecal |
Due to contamination with feces or E. coli; often related to bladder-GI tract fistula. |
Fetid or Putrid |
May be caused by suppurative diseases of the GU tract. Decomposition of urine containing cystine or pus will have the odor of rotten eggs (H2S). |
Fruity or Sweetish |
Usually due to acetone in diabetic acidosis, starvation or dieting. |
Urinoid or Faintly Aromatic; Normal |
Attributed to volatile organic acids most marked in "concentrated" urine specimens. |
Learners may go back to Part IA for the chemical analysis of urine.
Ruthanne Hyduke, M.A.
a.
Blood
Reaction:
The pseudoperoxidase action of erythrocytes and hemoglobin catalyzes
the oxidation of various chromogens to produce the color change.
False negatives:
formalin, excess nitrites, elevated specific gravity and Captopril
may reduce reactivity.
False positives:
oxidizing contaminants (bleach), microbial peroxidase, menstrual
contaminants.
b.
Bilirubin
Reaction:
Bilirubin in the urine couples with a diazonium salt in an acid
medium.
False negatives:
samples exposed to light will show decreased amounts of bilirubin;
excess levels of ascorbic acid.
False positives:
highly colored metabolites of drugs may interfere with reading the
reaction and appear as false positives.
c.
Glucose
Reaction:
The color is produced through a double enzymatic reaction of glucose
oxidase and peroxidase. The latter enzyme reacts with a chromogen to
produce the final color.
False negatives:
elevations of ketones; very elevated specific gravities; excess
levels of ascorbic acid
False positives:
presence of oxidizing agents (bleach)
d.
Ketones
Reaction:
Ketones react with nitroprusside or sodium nitroferricyanide and
glycine to produce a color change.
False positives:
presence of phenylketone or phthalein compounds; highly pigmented
urines; some drug metabolites.
e. Leukocytes
Reaction:
Leukocyte esterase, present in granulocytes, catalyzes the reaction
of the chromogens to produce a color change.
False negatives:
cephalexin and gentamicin concentrations; elevated glucose and
protein concentrations; tetracycline.
False positives:
vaginal contaminants; drugs or foods that color the urine red.
f.
Nitrites
Reaction:
Nitrates in the urine are converted to nitrites by the action of
gram-negative bacteria. These nitrites then react to form a diazonium
salt which in turn reacts with a chromogen to produce the final
color.
False negatives:
excess ascorbic acid
False positives:
presence of red dyes or other chromogens
g.
pH
Reaction:
A double indicator system detects the amount of hydrogen ions in the
urine to produce a color change.
Interferences:
If excess urine is left on the reagent strip, a phenomenon known as
runover may occur. The urine from one reagent area carries reagent
onto the pH test area and changes the result erroneously.
h.
Protein
Reaction:
This reaction is based on the phenomenon known as the "protein error
of indicators" where an indicator that is highly buffered at a pH of
2 will change color in the presence of proteins (anions) as the
indicator releases hydrogen ions to the protein.
False positives:
Strongly basic urine; presence of phenazopyridine,
polyvinylpyrrolidone, chlorhexidine, and bleach.
i.
Specific Gravity
Reaction:
This reaction is based on the change of an indicator color in the
presence of high concentrations of various ions.
False negatives:
Highly alkaline urine
False positives:
Moderate quantities of protein Picture: High Specific Gravity
j.
Urobilinogen
Excretion of urobilinogen is enhanced in alkaline urine; therefore,
the best sample to collect for urobilinogen tests is that voided two
hours after a meal.
Reaction:
Urobilinogen reacts with a chromogen to form an azo dye which appears
as various shades of pink or purple. This reaction occurs best at
room temperature.
False negatives:
Excess nitrites; presence of formalin
False positives:
Presence of phenazopyridine; very warm urine.
Note: You may take Exam I at this point if you wish to check your progress or obtain CE credit.
Ruthanne Hyduke, M.A.
False negatives:
formalin, excess nitrites, elevated specific gravity and Captopril
may reduce reactivity.
False positives:
oxidizing contaminants (bleach), microbial peroxidase, menstrual
contaminants.
Ruthanne Hyduke, M.A.
False negatives:
samples exposed to light will show decreased amounts of bilirubin;
excess levels of ascorbic acid.
False positives:
highly colored metabolites of drugs may interfere with reading the
reaction and appear as false positives.
Ruthanne Hyduke, M.A.
False negatives:
elevations of ketones; very elevated specific gravities; excess
levels of ascorbic acid
False positives:
presence of oxidizing agents (bleach)
Ruthanne Hyduke, M.A.
False positives:
presence of phenylketone or phthalein compounds; highly pigmented
urines; some drug metabolites.
Ruthanne Hyduke, M.A.
False negatives:
cephalexin and gentamicin concentrations; elevated glucose and
protein concentrations; tetracycline.
False positives:
vaginal contaminants; drugs or foods that color the urine red.
Ruthanne Hyduke, M.A.
False negatives:
excess ascorbic acid
False positives:
presence of red dyes or other chromogens
Ruthanne Hyduke, M.A.
Interferences:
If excess urine is left on the reagent strip, a phenomenon known as
runover may occur. The urine from one reagent area carries reagent
onto the pH test area and changes the result erroneously.
Ruthanne Hyduke, M.A.
False positives:
Strongly basic urine; presence of phenazopyridine,
polyvinylpyrrolidone, chlorhexidine, and bleach.
Ruthanne Hyduke, M.A.
False negatives:
Highly alkaline urine
False positives:
Moderate quantities of protein Picture: High Specific Gravity
Ruthanne Hyduke, M.A.
Reaction:
Urobilinogen reacts with a chromogen to form an azo dye which appears
as various shades of pink or purple. This reaction occurs best at
room temperature.
False negatives:
Excess nitrites; presence of formalin
False positives:
Presence of phenazopyridine; very warm urine.
Note: You may take Exam I at this point if you wish to check your progress or obtain CE credit.
Reagent
strip bottles
Reagent
strips
Desiccants
Reagent
strip color chart
Controls
Clinitest© reaction.