Test 3: Chemical Examination of Urine Flashcards
Reagent strips are a color what
producing chemical reaction takes place when
the absorbent pad containing the reagent(s) contacts
urine
what are the two types of reagent strips
- Single and multitest strips available
Reagent strips provide a
simple, rapid means for
performing routine chemical tests on urine
The brand and number of tests of reagent strips are
matter of
laboratory preference; Specified by urinalysis
Reagent strips
The reactions are interpreted as blank
Several degrees of what are shown
and range of readings
what is the value reordered in
The reactions are interpreted by
comparing the color produced on the pad
within the required time frame with a chart
supplied by the manufacturer
Several degrees of color are shown to
provide semiquantitative readings of neg,
trace, 1+, 2+, 3+, and 4+
Estimates of mg/dL are also provided for
many of the test areas
Reagent strip technique
Dip strip briefly into well-mixed specimen at room temperature
Remove excess urine by touching edge of strip to container as strip is
withdrawn
Blot edge of strip on absorbent pad
Wait specified amount of time
Read using a good light source
Improper techniques errors
Formed elements such as
Allowing the strip to remain blank
Excess urine remaining blank
- Formed elements such as red and white
blood cells sink to the bottom of the
specimen and will be undetected in an
unmixed specimen - Allowing the strip to remain in the urine for
an extended period may cause leaching of
reagents from the pads - Excess urine remaining on the strip after
its removal from the specimen can produce
a runover between chemicals on adjacent
pads, producing distortion of the colors
shake
Improper technique
The timing for reactions to blank
A good what is required
blank charts are different
Specimens must be returned to blank
- The timing for reactions to take place
varies between tests and manufacturers;
the manufacturer’s stated time should be
followed - A good light source is essential for
accurate interpretation of color reactions - Color charts from different
manufacturers are not interchangeable - Specimens that have been refrigerated
must be allowed to return to room
temperature prior to reagent strip testing
Handling and storing reagent strips
Store with desiccant in an opaque, tightly sealed container
Remove strips from container immediately prior to use
Store below 30°C
Do not use past the expiration date
Visually inspect for discoloration/deterioration
Quality control of reagent strips
Run positive and negative controls how often
Run additional controls when
Run positive and negative controls at least once per
24 hours
Run additional controls
When a new lot of strips is opened
When results are questionable
When there are concerns over strip integrity
record control results
Do not use what
Manufactured positive and negative controls are
available
Do not use distilled water as a negative control as
reactions are designed for urine ionic concentration
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Specific gravity
The strip measures the ionic concentration of the sample, which relates to the specific gravity. It is
not perfect, but it is what is used for routine analysis.
The reagent pad contains polyelectrolytes with acid groups that get dissociated to a degree
dependent on the ionic strength (how many ions) of the sample.
The reagent pad contains a pH indicator (bromothymol blue) which then measures the change in
pH. When urine has increased specific gravity, the reagent pad becomes more acidic. The colors
of the reagent pad will range from deep blue-green in urines of low ionic concentration through
green and yellow-green in urines of increasing ionic concentration. The color blocks are in
increments of 0.005 for specific gravity readings between 1.000 and 1.030. Figure 7-8 shows the
correlation between specific gravity ranges and color changes on the reagent strip pad. An
illustration shows the color chart for specific gravity at 45 seconds
Read SG at
45 seconds
Urine pH
what systems are buffers
Renal system, pulmonary system and blood buffers maintain a pH that is
compatible with life
Urine pH
Slightly acidic urine at
5.0-6.0
Postprandial specimens are more
Alkaline
Not physiologically possible pH
pH greater than 8 and less than 4.4 physiologically
impossible
pH reagent strip reactions
indicators
Double-indicator system reaction
Methyl red = 4 to 6 red/orange to yellow
Bromthymol blue = 6 to 9 green to blue
Methyl red + H+ → Bromthymol blue − H+
(Red/Orange → Yellow) (Green → Blue)
what are other things used besides pH reagent strips?
Interferences for pH methods
Can also use a pH meter or pH paper
Interference
– No known substances interfere with urinary pH measurements performed by
reagent strips
pH of 4.5-6.9
Diet and Sleep
Metabolic acidosis (diabetic acidosis)
Respiratory acidosis
Urinary disorders: Renal Failure, Uremia
pH of 7.0-7.9
Diet
Metabolic alkalosis
Respiratory alkalosis
Urinary disorders: UTI, Renal tubular acidosis
Medications
Blood
Hematuria
Hemoglobinuria
Hematuria: intact RBCs
-Cloudy red urine
Hemoglobinuria: product of RBC destruction
-Clear red urine
Blood
Any amount of blood greater then
Chemical test for blank
The microscopic exam can be used to
Any amount of blood greater than five cells per microliter of urine is
considered clinically significant
Chemical tests for hemoglobin provide the most accurate means for
determining the presence of blood
The microscopic examination can be used to differentiate between
hematuria and hemoglobinuria
Hematuria diseases
Glomerular disease
Pyelonephritis or cystitis
Renal calculi
Tumors
Also trauma, hypertension, exercise, smoking, meds and other toxins
Hemoglobinuria
Intravascular hemolysis: transfusion reactions, hemolytic anemia,
paroxysmal nocturnal hemoglobinuria
Infections: malaria
Chemical toxicity: copper, nitrates, nitrites
Exertional hemolysis
Myoglobiuria
Muscles: trauma, ischemia, infections, medications
Seizures
Toxins: spiders and snakes
Hematuria vs hemoglobinuria
Rapid test to distinguish hematuria from hemoglobinuria. The onset of red urine during or
shortly after a blood transfusion may represent hemoglobinuria (indicating an acute
hemolytic reaction) or hematuria (indicating bleeding in the lower urinary tract).
Differentiation of Hemoglobinuria and Myoglobinuria
Both have what positive
If correlation of chemical and microscopic results do not indicate
Difficult to distinguish but
Both heme proteins and toxic to kidneys.
Both will give a positive blood on dipstick
If correlation of chemical and microscopic results do not indicate hematuria, further
studies are needed to distinguish hemoglobinuria from myoglobinuria
Difficult to distinguish but necessary for diagnosis and treatment
Myoglobin in urine produces a
Myoglobin in urine produces a brown urine. Can spin down urine. Then can look at
plasma to diff. myoglobinuria from hemoglobinuria.
Hemoglobin produces a
Hemoglobin produces a reddish coloration in the spun serum, whereas myoglobin does not
discolor the serum (remains clear)
Hematuria produces a
Hemoglobinuria produces a
Myoglobinuria produces a
URINE
Hematuria produces a reddish sediment in spun urine samples.
Hemoglobinuria produces red to brown color in centrifuged specimen.
Myoglobinuria is brown, and often only a few RBCs are present in the urine
Plasma hemoglobin produces a
Hemoglobin produces a reddish or brown coloration in the spun serum, whereas
myoglobin does not discolor the serum
CK levels are markedly what of what
K levels are markedly elevated in myoglobinuria. Results of radioimmunoassays for the
specific measurement of serum or urine myoglobin can be delayed by several days and
are not useful in immediate diagnosis and treatment.
Blood reagent strip reactions of hemoglobin
Principle
Strip pad contains what
gets oxidized by
Principle pseudo peroxidase activity of hemoglobin
Strip pad contains the chromogen tetramethylbenzidine
Gets oxidized by Hgb or Myoglobin and produces a green-blue color.
Pseudoperoxidase activity reaction
H202 + Chromogen with enzymes Hb and MB —–> Oxidized chromogen + H20
- Tetramethylbenzidine. Hb, Hemoglobin, Mb, myoglobin
Blood reagent strip
Two charts corresponding to
Free heme shows
Intact RBCs show
Report and sensitivity
Two charts corresponding to different reactions
Free hemoglobin shows uniform color
Intact RBCs show a speckled pattern on pad
Report: trace, small (1+), moderate (2+), large (3+)
Sensitivity 5 RBCs/μL
False positives of
False positives can occur if menstrual blood present, certain bacteria or high level of Vitamin C
Leukocyte esterase (LE)
A Few of what are blank
Advantages
usually see what
Not considered what
A few WBCs are normal; anything over 20 WBCs/mL indicates pathologic
process: inflammation/response to infection.
Advantage: detects presence of lysed leukocytes.
Usually see bacteria, but could also be in infection with organisms other
than bacteria which may not be readily apparent microscopically
(Chlamydia)
Not considered a quantitative test: do microscopic if positive
LE detects what
LE test detects the presence of leukocyte
esterase in the granulocytes and monocytes
LE is catalyzed by
- LE catalyzes the hydrolysis of acid ester on the pad
to aromatic compound (indoxyl or pyrrole)
and acid; aromatic compound reacts with
diazonium salt on a pad for purple color
LE reaction have to wait
- Note you have to wait 2 minutes.
LE false positive
Presence of vaginal contamination
Highly pigmented urine (nitrofurantoin, beets)
False negatives of LE
High concentrations of protein, glucose, high specific gravity
Presence of the antibiotics; gentamicin, cephalosporins, tetracyclines or other
strong oxidizers
what will not be detected in LE
Detects LE so lymphocytes are not detected because they
don’t produce the enzyme.
Nitrate
Tests ability of bacteria to reduce nitrate
(normal constituent) to nitrite (abnormal)
Correspond with a quantitative bacterial
culture criterion of 100,000 organisms/mL
Does not negate the need for culture.
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Nitrite reagent strip reaction step one
Step one picture
Nitrite reagent strip reaction step two
para-arsanilic acid or sulfanilamide in step one
diazonium salt + tetrahydrobenzoquin—→ pink azodye
Nitrite reaction false positives
Old specimens (bacterial multiplication)
Highly pigmented urine (phenazopyridine or beets)
Nitrite reactions false negatives
Large quantities of bacteria converting nitrite to nitrogen
High concentrations of ascorbic acid
Protein indicates
Most indicative of renal disease
proteinuria seen
Proteinuria seen in early renal disease
Normal protein range
Normal = <20 mg/dL
Low molecular weight proteins are blanked
Low-molecular-weight serum proteins are filtered; many are reabsorbed
what is the primary protein of concern
Albumin is primary protein of concern
Other proteins include
Vaginal, prostatic, and seminal proteins
Tamm-Horsfall (uromodulin), secretory IgA
Proteinuria results from
(1) an increase in the quantity of plasma proteins that are filtered, or
(2) filtering of the normal quantity of proteins but with a reduction in the reabsorptive ability of the renal tub
Pre-renal overflow
Excessive production of low-molecular weight proteins following hemolytic event or sepsis or myoglobins from trauma (rhabdomyolysis). Goes away when condition resolved. Light chain deposition disease/multiple myeloma and amyloidosis produce light chain proteins that pass into urine. The light chains from myeloma are called Bence Jones proteins.
Renal glomerular proteinuria
In disorders that cause glomerular damage. Most common and most serious. This is seen in nephrotic syndrome. Also in chronic glomerulonephritis and sclerotic conditions (focal segmented, diabetic nephropathy, lupus). Very high protein seen in urine.
Renal tubular proteinuria
When tubular function is impaired (Fanconi’s, pyelonephritis, interstitial nephritis and systemic diseases like lupus. Lower protein levels - <2.5 mg/day.
Post-renal proteinuria
Inflammation anywhere in the urinary tract or from injury, tumors or hemorrhage that allows blood proteins into the urinary tract. Usually goes away if condition is resolved.
Postural proteinuria
considered to be a functional proteinuria. Protein in urine only when patient is in an upright (orthostatic) position. considered to be benign, but persistent proteinuria may develop Protein <1.5 g/day. Monitored every 6 months.
Contamination
Dehydration, low blood pressure, inflammation, aspirin, exercise
Vaginal and prostrate secretions
Proteins error of indicators
Certain indicators change color in the presence of protein at a constant pH on the pad is maintained at 3.0 by buffers.
Protein accepts H+ from the indicator, increased sensitivity to albumin due to more amino groups to accept H+ than other proteins
pH of 3.0
Indicator (H+) + Protein (albumin) → H+ ions released from indicator
(Yellow) reactant (Green/Blue)
Protein indicators
Indicator may be sulfanapthalein or tetrabromphenol blue
report proteins as
trace protein values
Report: neg, trace, 1+, 2+, 3+, 4+, or 30, 100, 300, 2000 mg/dL
Trace values are <30 mg/dL
Protein interferes and limitation
Fortunately, the test is designed to be very sensitive to the presence of albumin, but this means globulins, myoglobulin and immunoglobulin light chains (Bence Jones Proteins) are not detected by the strips because they are usually in too low a concentration to be detected.
If urine is very alkaline – can overwhelm the buffering action on the pad and give a false positive
Sulfosalicylic Acid (SSA) Precipitation
Confirmatory test for protein
Proteins are precipitated by 5-sulphosalicylic acid.
Any resulting turbidity will give an estimation of the amount of protein present in the urine which can be subjectively quantitated visually or more precisely quantitated using photometry.
Cells and casts in the urine must be removed by centrifuging before carrying out the test.
The test can detect albumin, hemoglobin, myoglobin, and Bence Jones proteins.
Sulfosalicylic Acid (SSA) Precipitation
results
Negative : No cloudiness
Trace: Faint turbidity.
1+ : definite turbidity
2+ : Heavy turbidity but no flocculation
3+ : Heavy turbidity with light flocculation.
4+ : Heavy turbidity with heavy flocculation.
Microalbuminuria
-Diabetic nephropathy with type 1 and type 2 diabetes mellitus
-Reduced glomerular filtration
-Eventual renal failure
Since the levels are low, labs use specific strips to detect.
Albumin creatine ratio
ACR = Albumin/Creatinine
ACR = 8/0.11 = 72.7 mg/g
The table in the paragraph above shows that a result between 30 and 300 mg/g
means the level of albuminuria is moderately increased,
and the patient places in category A2 of CKD stages.
Urine albumin
Albumin in urine: 8 mg/dL
Creatinine in urine
Creatinine: 110 mg/dL = 0.11 g/dL
further testing of proteins
Once the presence of an increased amount of urine
protein has been established, accurate methods are avail-
able to differentiate and quantify the proteins. Electro-
phoresis, nephelometry, turbidimetry, and radial
immunodiffusion methods are used and are discussed at
length in clinical chemistry textbooks.
Glucose
Clinical significance
Normally, all glucose passes through
Clinical Significance. The presence of glucose in the urine is termed glycosuria (or glycosuria).
Normally, all glucose that passes through the glomerular filtration barrier into the ultrafiltrate is actively reabsorbed by the proximal renal tubules.
However, tubular reabsorption of glucose
is a threshold-limited process with a maximum reabsorptive capacity (Tm) averaging about 350 mg/min
When the level of glucose in the blood exceeds
When the level of glucose in the blood exceeds its renal threshold level of approximately 160 to 180 mg/dL, the ultrafiltrate concentration of glucose exceeds the reabsorptive ability of the tubules, and glucosuria occurs: NORMAL RENAL THRESSHOLD FOR GLUCOSE = 160 to 180 mg/dL
Glucosuria is caused by
(1) a prerenal condition (hyperglycemia), or
(2) a renal condition (defective tubular absorption).
(1) Gestational diabetes is a type of diabetes that happens during pregnancy. Your body
needs a lot of energy as your baby grows, but sometimes it can’t keep up with demand
and doesn’t make enough insulin.
(2) Renal glycosuria is a rare inherited condition where your body eliminates sugar in your
urine even though your blood levels are normal. In this condition, you don’t have too much
glucose but your body gets rid of it anyway.
(3) Fanconi syndrome is a general term for a defect in your kidneys that causes problems
absorbing glucose
Gestational diabetes
is a type of diabetes that happens during pregnancy. Your body
needs a lot of energy as your baby grows, but sometimes it can’t keep up with the demand
and doesn’t make enough insulin
Renal glycosuria
Renal glycosuria is a rare inherited condition where your body eliminates sugar in your
urine even though your blood levels are normal. In this condition, you don’t have too much
glucose but your body gets rid of it anyway
Fanconi syndrome
Fanconi syndrome is a general term for a defect in your kidneys that causes problems
absorbing glucose
Glucose reagent strip reaction
1.) Glucose + O2 with enzyme Glucose oxidase ———> Gluconic acid + H2O2
2.) H202 + chromogen with enzyme peroxidase ——> Oxidized chromogen + H20
Dipstick interferences of glucose
False positive and negatives
False-positive: only if contaminated with peroxide, oxidizing detergents
(unlikely)
False-negative: enzymatic reaction interference
High levels of ascorbic acid
High levels of ketones (unlikely)
High specific gravity and low temperature
Greatest source of error is old specimens
Subjecting the glucose to bacterial degradation
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Copper reduction test Clllinitest
and reaction
The dipstick used in urine testing will only detect glucose. Clinitest
tablets will detect other sugars such as lactose, fructose, galactose,
and pentoses. These other sugars may signify metabolic disorders
when found in urine of small children.
CuSO4 (cupric sulfide) + reducing substance –Heat—>
Cu2O (cuprous oxide) + oxidized substance → color Alkali
(blue/green to orange/red)
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Clllinitest reaction
CuSO4 (cupric sulfide) + reducing substance –Heat—>
Cu2O (cuprous oxide) + oxidized substance → color Alkali
(blue/green to orange/red)
Ketones
Ketones are formed during the catabolism of fatty acids.
Three intermediate products of fat metabolism
Acetone: 2%
Acetoacetic acid: 20%
β-hydroxybutyrate: 78%
Appear in urine when body stores of fat must be metabolized to supply
energy (carbs not available or can’t be used)
May signify diabetic ketoacidosis
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Three intermediate products of fat metabolism
Acetone: 2%
Acetoacetic acid: 20%
β-hydroxybutyrate: 78%
Ketones appear when
Signify what
Appear in urine when body stores of fat must be metabolized to supply
energy (carbs not available or can’t be used)
Ketone test
Primary reagent: sodium nitroprusside
(Nitroferricyanide)
Measure primarily acetoacetic acid
Assumes the presence of β-hydroxybutyrate and acetone
Acetoacetic acid (alkaline) + nitroprusside → purple color
Ketone reaction interferences
Levodopa in large dosage
Medications containing sulfhydryl groups (cancer, hypertension)
May produce atypical color reactions
False-positive results from improperly timed readings
Falsely decreased values in improperly preserved specimens
Breakdown of acetoacetic acid by bacteria
Ketones are volatile and may evaporate if specimen left open.
Acetest for ketones
Not a urine confirmatory test
Tablet = sodium nitroprusside, glycine,
disodium phosphate, lactose (gives
better color)
Can use as a backup or to evaluate
questionable results, but affected by
same interferences seen on dipstick.
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Bilirubin
Urine bilirubin early indicator of liver disease
Normal degradation product of hemoglobin
RBCs destroyed by liver and spleen following 120-day life span
Body recycles iron, protein
Protoporphyrin is broken down into bilirubin
Bilirubin is bound to albumin
Kidneys cannot excrete
Unconjugated bilirubin: water insoluble
Bilirubin is a normal degradation product of what
Normal degradation product of hemoglobin
RBCs destroyed by liver and spleen following 120-day life span
Unconjugated bilirubin is water what
Unconjugated bilirubin: water insoluble
Unconjugated BR and albumins
Can’t get through the glomerulus
Conjugated BR can pass
through glomerulus
Clinical significance of bilirubin
Conjugated bilirubin appears
Conjugated bilirubin appears in urine with bile duct
obstruction, liver disease or damage
Clinical significance of bilirubin
Obstruction
Obstruction: bilirubin backs up into circulation and is excreted
in urine
No urobilinogen is formed
Hepatitis, cirrhosis:
conjugated bilirubin leaks back into
circulation from damaged liver; some bilirubin passes to
intestine
Hemolytic disease
increased unconjugated bilirubin,
increased urobilinogen
Hemolytic disorders (hemolytic
anemia, sickle cell, transfusion
reactions)
Mechinism
Liver function normal. Overproduction of bilirubin from heme. Is
unconjugated and bound to albumin (does not pass through
glomerulus but passes through liver to intestine). In intestine,
some of the excess urobilinogen is reabsorbed and shows up in
urine.
Liver disease
Liver can’t perform normal bilirubin metabolism. Conjugated
bilirubin leaks into circulation and can pass through glomerulus.
Less urobilinogen is removed from circulation by diseased liver,
more goes to kidney
Bile duct obstructions
Liver function normal. Conjugated bilirubin can’t pass through
liver so overflows into circulation. Since no bilirubin is going to
intestines, no urobilinogen is formed. (results in pale stools).
Bilirubin strip reactions
The principle is a diazo reaction
bilirubin glucuronide + *diazonium salt——–> azodye
(tan or pink to violet)
diazonium salt- (2,4-dichloroaniline diazonium salt or 2,6-dichlorobenzene
-diazonium-tetrafluoroborate
Ictotest-diazo tablets
Confirmatory for bilirubin
Tablets containing p-nitrobenzene-diazonium-p-toluenesulfonate, SSA, sodium carbonate, and boric acid
Use the specified mat for the test; mat keeps bilirubin on the surface for reaction, Interfering substances are
washed into the mat, and only bilirubin remains on the surface
The same principle as strip but picks up lower levels
Positive reaction = blue-to-purple color
FALSE positives of bilirubin
Urine pigments
Pyridium (phenazopyridine)
Drugs indican, iodine
False negatives of bilirubin
Old specimens - bilirubin photo oxidized and is otherwise unstable, especially
at room temp
Ascorbic acid >25 mg/dL
Nitrite
Combine with diazonium salt and block bilirubin reaction
Urobilinogen strip reactions
urobilinogen ERC + p-dimethylaminobenzaldehyde (Ehrlich’s reagent) ——→ red color
CHEMSTRIP:
Acid
urobilinogen + diazonium salt —→ red azodye
Urobilinogen reaction interferences
Ehrlich reactive compounds: porphobilinogen, indican, sulfonamides,
methyldopa, procaine, chlorpromazine, p-aminosalicylic acid
Chemstrip: false-negative with high nitrite interferes with diazo reaction.
Colored urine from drugs or beets can mask reaction.
Both tests: urobilinogen is highest after meals (increased bile salts), old
specimens and formalin preservation decrease results
NOTE: These tests cannot reliably indicate a decrease or absence of
urine urobilinogen. Have to interpret using fecal and blood analysis. No
confirmatory test available.
Urobilinogen is very unstable in acid urine (so best to test after meal when
urine is more alkaline) and easily photo oxidizes to urobilin.
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Ehrlich reactive compounds
porphobilinogen, indican, sulfonamides,
methyldopa, procaine, chlorpromazine, p-aminosalicylic acid
Chemstrip for urobilinogen reaction interferences
false-negative with high nitrite interferes with diazo reaction.
Colored urine from drugs or beets can mask reaction.
Urobilinogen is high after what so
Both tests: urobilinogen is highest after meals (increased bile salts), old
specimens and formalin preservation decrease results
Note for urobilinogen test
these tests cannot reliably indicate a decrease or absence of
urine urobilinogen. Have to interpret using fecal and blood analysis. No
confirmatory test available.
Urobilinogen is very unstable in acid urine (so best to test after meal when
urine is more alkaline) and easily photo oxidizes to urobilin.
Reaction interferences
No interference from large molecules, glucose and urea and radiographic
dye and plasma expanders
Reason for difference in refractometer reading
Slight elevation from protein
Decreased readings: urine pH 6.5 or higher
Interferes with indicator; add 0.005 to the reading; readers automatically add
this
Clinical significance
Increased Fat metabolism=
primary causes=
Ketonuria shows=
Increased fat metabolism = inability to metabolize
carbohydrate
Primary causes
- Diabetes mellitus
- Vomiting (loss of carbohydrates)
- Starvation, malabsorption, dieting (↓ intake)
Ketonuria shows insulin deficiency
- Monitor diabetes
Diabetic ketoacidosis=
Diabetic ketoacidosis = increased accumulation of
ketones in the blood
Electrolyte imbalance, dehydration, and diabetic coma
Ketonuria unrelated to diabetes
Inadequate intake/absorption of carbohydrates
Vomiting
Weight loss
Eating disorders
Frequent strenuous exercise
Multisitix Ehlichs aldehyde reaction
Multistix: Ehrlich’s aldehyde reaction
p-dimethylaminobenzaldehyde (Ehrlich reagent); report in Ehrlich units
(EU) 1 EU = 1 mg/dL
Normal readings 0.2 to 1, abnormal 2, 4, 8
Light to dark pink
Chemostrip diazo reaction
Chemstrip: diazo (azo-coupling) reaction
4-Methoxybenzene-diazonium-tetrafluoroborate; more specific than
Ehrlich reaction; report in mg/dL
White to pink
