1.
pH
One of the
functions of the kidney is to help maintain acid–base balance in the body. To
maintain a constant pH (hydrogen ion concentration) in the blood (about 7.40),
the kidney must vary the pH of the urine to compensate for diet and products of
metabolism. This regulation occurs in the distal portion of the nephron with
the secretion of both hydrogen and ammonia ions into the filtrate, and the
reabsorption of bicarbonate. If sufficient hydrogen ions (H+) are
secreted into the tubule, all of the bicarbonate present will be reabsorbed,
but if fewer H+ are secreted into the tubule, all of bicarbonate
present will be reabsorbed, but if fewer H+ are secreted or if an
excess of bicarbonate is present, some of the bicarbonate will be excreted in
the urine. The continued secretion of H+ after all bicarbonate has
been reabsorbed will drop the pH of the filtrate and result in acidic urine.
The secretion
of H+ in the tubule is regulated by the amount present in the body.
If there is an excess of acid in the body (acidosis), more H+ will
be excreted and the urine will be acidic. When there is an excess base in the
body (alkalosis), less H+ will be excreted and the urine will be
alkaline. The hydrogen ions in the urine are excreted as either free H+,
in association with a buffer such as phosphate, or bound to ammonia as ammonium
ions. The pH of the urine is determined by the concentration of the free H+.
Because pH is
the reciprocal of the hydrogen ion concentration, as the H+
concentration increases, the pH decreases or becomes more acidic. As the H+
concentration decreases, the pH increases or becomes more alkaline. The pH of
the urine may range from 4.6 to 8.0 but averages around 6.0, it is usually
slightly acidic. There is no abnormal range as such, since the urine can
normally vary from acid to alkaline.
Method of determination
All brands of
dipstick uses the same two indicators: methyl red and bromthymol blue, and
measure a range of pH from 5.0 to 8.5.
Most
manufacturers recommend that the pH be read immediately as this will prevent
misreading due to “run–over” effect. This term is used to
describe what happens when excess urine is left on the stick after dipping, and
so the acid buffer from the reagent in the protein area runs onto the pH area.
This type of contamination can cause a false lowering of the pH reading
especially in the case of alkaline or neutral urine.
Recent
advances have been made to prevent “run–over” by integrating a hydrophobic
interpad surface which causes the urine to concentrate. Other brand of dipstick
integrates nylon mesh on the test pad and underlying absorbent papers in place
on the plastic strip. The mesh allows for even diffusion of the urine on the
test pads, and the underlying paper absorbs excess urine to prevent “run over.”
2.
Protein
The presence
of increased amounts of protein in the urine can be an important indicator of
renal disease. It may be the first sign of a serious problem and may appear
long before other clinical symptoms. There are, however, physiologic conditions
such as exercise and fewer that can lead to increased protein excretion in the
urine in the absence of renal disease. There are also some renal disorders in
which proteinuria are absent.
In the normal
kidney, only a small amount of low–molecular weight protein is filtered at the
glomerulus. The structure of the glomerular membrane prevents the passage of
high molecular weight proteins including albumin (MW = 69,000). After
filtration, most of the protein is reabsorbed in the tubules with less than 150
mg/24 hour being excreted. In a child, the normal excretion is less than 100
mg/m2/24 hour. The protein that is normally excreted includes a mucoprotein
called Tamm–Horsfall protein, which is not contained in the
plasma but is secreted by the renal tubules.
Click
here for discussion of Protein in Blood
Method of
determination
a. Screening
Tests
This is based
on the “protein error of indicators” principle or on the ability
of protein to be precipitated by acid or heat. The dipsticks are more sensitive
to albumin than to other proteins.
b. Reagent
Test Strips
This is also
based on the concept of “protein error of indicators,” a
phenomenon which means that the point of color change of some pH indicators is
different in the presence of protein from that observed in the absence of
protein, because protein act as hydrogen ion acceptors at a constant pH. Be
guided also that reagent strips detect albumin primarily and are less sensitive
to globulins.
Since the
dipstick procedure is very sensitive to albumin, the protein that is primarily
excreted as the result of glomerular damage or disease. Other urine proteins
such as gamma globulins, glycoproteins, ribonuclease, lysozyme, hemoglobin,
Tamm–Horsfall mucoprotein, and Bence–Jones protein are much less readily
detected than albumin. Therefore, a negative urinary dipstick result does not
necessarily rule out the presence of these proteins.
Tetrabromphenol
blue, citric acid or sodium citrate is the active ingredient which is usually
present on the dipstick for protein determination.
False positive
result
(1) Highly
buffered alkaline urine. The alkaline pH can overcome the acid buffer in the
reagent and the area may change color in the absence of protein.
(2) If
the dipstick is left in the urine for too long, the buffer will be washed out
of the reagent, the pH will increase, and the strip will turn blue or green even
if protein is not present.
(3) Quaternary
ammonium compounds used in cleaning urine containers will alter the pH and will
result in false positive reaction.
(4) Treatment
with phenazopyridine and after infusion of polyvinylpyrrolidone as a plasma
expander.
(5) Chlorhexidine
gluconate, found in skin cleansers
Common causes
of proteinuria
(1) Transient
proteinuria
is a temporary change in glomerular hemodynamics that causes increase in
protein such in the case of congestive heart failure, dehydration, emotional
stress, exercise or fever. Orthostatic (postural) proteinuria is a benign
condition that can result from prolonged standing; it is confirmed by obtaining
a negative urinalysis result after eight hours of recumbency.
(2) Persistent
proteinuria
is divided into three categories:
(a) In
glomerular proteinuria, albumin is the primary urinary protein as
in the case of Focal segmental glomerulonephritis, IgA nephropathy (Berger’s
disease), IgM nephropathy, Membranoproliferative glomerulonephritis, and
Membranous nepropathy.
(b) Tubular
proteinuria
results when malfunctioning tubule cells no longer metabolize or reabsorb
normally filtered protein. In this condition, low–molecular–weight proteins
predominate over albumin and rarely exceed 2 grams per day as in the case of
Alport’s syndrome, Amyloidosis, Collagen vascular disease, Diabetes Mellitus,
Fabry’s disease, Sarcoidosis, and Sickle Cell Disease.
(c) In
overflow proteinuria, low–molecular–weight proteins overwhelm the
ability of the tubules to reabsorb filtered proteins as in the case of
Hemoglobinuria, Multiple myeloma or myoglobinuria.
3.
Glucose
The presence
of significant amounts of glucose in the urine is called glycosuria
(or glucosuria). The quantity of glucose that appears in the urine is dependent
upon the blood glucose level, the rate of glomerular filtration, and the degree
of tubular reabsorption. Usually, glucose will not be present in the urine
until the blood level exceeds 160 – 180 mg/dL, which is the normal renal
threshold for glucose. When the blood glucose exceeds the renal threshold, the
tubules cannot reabsorb all of the filtered glucose, and so glycosuria occurs.
Normally, this level is not exceeded even after the ingestion of a large
quantity of carbohydrate. A small amount of glucose may be present in normal
urine, but the fasting level in an adult is only about 2 – 20 mg of glucose per
100 mL of urine.
Method of
determination
There are two
basic types of test that used to screen or monitor glycosuria. The procedures
that use the enzyme glucose oxidase are specific for glucose, while the copper
reduction test will detect any reducing substances. The most important correlation
that must be made with urine glucose is the blood glucose level.
Click
here for full discussion on Blood glucose
False negative
result:
a. Reactivity
for glucose can vary with temperature because of the effect of temperature on
enzymatic reactions.
b. An
elevated specific gravity may decrease the sensitivity of glucose oxidase.
c. Alkaline
pH may decrease sensitivity to glucose.
d. The
combination of high specific gravity and alkaline pH may result in false
negatives at low concentrations of glucose.
e. High
urinary concentrations of ascorbate can inhibit the enzymatic reaction which
will result in a reduced or false negative reading. The ascorbic acid will be
oxidized by the hydrogen peroxide in the second part of the enzyme reaction,
and will, therefore, compete with the oxidation of the chromogen, resulting in
the inhibition of the color formation.
Screening for
reducing substances
Reducing
substances are sugars other than glucose that can be found in urine as well
such as galactose, lactose, fructose and maltose. Other reducing substances
include dextrin, homogentisic acid and glucuronates.
A test for
reducing substances is of particular importance for routine urinalysis of all
pediatric patients. This will provide for the early detection of those
metabolic defects which are characterized by the excretion of reducing sugars
such as galactose, which is present in the urine of patients with galactosemia
a. Benedict’s
Test
This test is
based on the fact that in strong alkaline solution and in the presence of heat,
reducing sugars will reduce cupric ions to cuprous oxide. The reaction produces
a color change of blue through green to orange depending upon the amount of
reducing substances present in the urine.
Interfering
factors for this procedure includes the presence of Nalidixic Acid,
cephalosporins and probenecid.
Obsolete
method for test for glucose:
a. Trommer’s
Test – employs 10% NaOH and drops of CuSO4
b. Fehling’s
Test – employs a mixture of Rochelle salt and CuSO4
c. Almen–Nylander
Test – employs a mixture of Rochelle salt dissolved in NaOH and saturated with
bismuth subnitrate. Black precipitate of metallic bismuth is a positive result.
d. Neuman
Test – employs 50% acetic acid saturated with sodium acetate and drops of
phenylhydrazin. This will create crystals of phenylglucosazon and dissolved in
60% alcohol and allowed to recrystallize and melting point for glucose is
determined at 205oC.
4.
Ketones
Ketone or
ketone bodies are formed during catabolism of fatty acids. One of the
intermediate products of fatty acid breakdown is acetyl CoA. Acetyl CoA enters
the citric acid cycle (Krebs cycle) in the body if fat and carbohydrates
degradation is appropriately balanced.
The ketone
bodies are acetoacetic acid (diacetic acid), β– hydroxybutyric acid and
acetone.
The odor of
acetone may be detected in the breath of an individual who has high level of
ketones in the blood because acetone is eliminated via the lungs.
Acetone is
lost in the air if a sample is left standing at room temperature. Therefore,
urines should be tested immediately or refrigerated in a closed container until
testing.
Method of
determination
Most reagent
strips are embedded with sodium nitroprusside that reacts with acetoacetic acid
to form glycine which forms a violet purple color in an alkaline pH.
Obsolete
methods:
a.
Acetone
(1) Legal’s
Test – drops of concentrated sodium nitroprusside with potassium hydroxide is
added to a urine sample.
(2) Gunning’s
Test – Lugol’s iodine is added in urine sediment and observe microscopically
for presence of six sided tablets or stars.
(3) Lieben’s
Test – drops of potassium hydroxide and Lugol’s iodine is added to urine
sediment then warmed then observe for iodoform crystals.
(4) Huppert–Messinger
method
b.
Acetoactic
acid
(1) Gerhardt’s
Test – urine is acidified with H2SO4 with ether and Fe2Cl6
is then added and there is formation of violet red color.
c.
β–hydroxybutyric
acid
(1) Black’s
Qualitative Test – based on the oxidation of β–hydroxybutyric acid in the
presence of ferric chloride.
(2) Bergell
method – urine is made weakly alkaline by addition of sodium carbonate until
condensed. Phosphoric acid and copper sulfate is later added.
(3) Bockelman
and Bouma method – sodium hydroxide and benzoylchloride is added on the urine
then polarized.
5.
Blood
The term
“occult” means “hidden” and the methods used to test for blood in the urine are
capable of detecting even minute amounts not visualized macroscopically.
Another reason for this title is that these procedures actually detect the free
hemoglobin from lysed red blood cells (RBCs).
The chemical
method used in routine urinalysis for detecting blood (hematuria) will also
detect free hemoglobin (hemoglobinuria) and myoglobin (myoglobinuria). The
urine is normally free of all these substances; therefore, a positive test for
occult blood should be followed by determination of the exact cause and origin
of this abnormal finding.
a. Hematuria is the
presence of blood or intact RBCs in the urine. A urine that is highly alkaline
or has a very low specific gravity (<1.007) can cause the red cells to lyse,
thus releasing hemoglobin into the urine. The presence of this type of
hemoglobin is still considered to be hematuria as far as the origin is concerned,
but it is very difficult to distinguish from true hemoglobinuria. When lysing
occurs, the microscopic examination may show the empty red cell membranes which
are often referred to as “ghost” cells. In microhematuria, there is such a
small amount of blood in the urine that the color of the specimen is unaffected
and the hematuria can only be detected chemically or microscopically. On the
other hand, gross hematuria alters the color of the urine and is easily visible
macroscopically.
b. Hemoglobinuria is the
presence of free hemoglobin in the urine as a result of intravascular
hemolysis. The hemolysis that occurs in the urine while in the urinary tract or
after voiding because of a low specific gravity or highly alkaline pH may be
considered to be hemoglobinuria, but it does not bear the same significance as
true hemoglobinuria. Hemoglobinuria without hematuria occurs as a result of
hemoglobinemia and, therefore, it has primarily nothing to do with kidneys even
though it may secondarily result in kidney damage.
Click
here for full discussion on Paroxysmal Cold Hemoglobinuria
c. Myoglobin is the heme
protein of striated muscle. It serves as a reserve supply of oxygen and also
facilitates the movement of oxygen within muscle. Injury to cardiac or skeletal
muscle results in the release of myoglobin into the circulation. Even just subtle
injury to the muscle cells can bring about the release of myoglobin. Myoglobin
has a molecular weight of approximately 17,000 and it is easily filtered
through the glomerulus and excreted in the urine. Because myoglobin is cleared
so rapidly from the circulation, the plasma is left uncolored even though the
urine may be red to brown to black depending on the degree of myoglobinuria.
Method of
determination
a. The
dipstick procedure is based on the peroxidase–like activity of hemoglobin and
myoglobin which catalyzes the oxidation of a chromogen by organic peroxide.
b. The
usual chromogen and oxidant used are: diisoproplbenzene, dihydroperoxide,
tetramethylbenzidine and cumene hydroperoxide.
6.
Bilirubin and
Urobilinogen
Urine normally
does not contain detectable amounts of bilirubin. Unconjugated bilirubin is
water insoluble and cannot pass through the glomerulus; conjugated bilirubin is
water soluble and indicated further evaluation for liver dysfunction and
biliary obstruction when it is detected in the urine.
Normal urine
contains only small amounts of urobilinogen, the end product of unconjugated
bilirubin after it passed through the bile ducts and been metabolized in the
intestine. Urobilinogen is reabsorbed into the portal circulation, and a small
amount eventually is filtered by the glomerulus. Hemolysis and hepatocellular
disease can elevate urobilinogen levels, and antibiotic use and bile duct obstruction
can decrease urobilinogen levels.
Click
here for full discussion on Bilirubin
Method of
determination
a. The
active ingredient in majority of dipstick that is being used in laboratory is
the dichloroanaline diazonium salt for bilirubin and p– diethylaminobenzaldehyde or 4–methloxybenzene–diazonium–
tetrafluoroborate for urobilinogen.
b. Ictotest
is a tablet test that is based on the same diazo reaction as the dipsticks.
However, Ictotest is much more sensitive than the dipsticks, being able to
detect as little as 0.05 mg/dL. Because of this sensitivity, Ictotest is the
recommended procedure when a test for just bilirubin is ordered. It also serves
as a good confirmatory test for a positive dipstick.
The tablet
contains 2,6–dicholorobenzene–diazonium–tetrafluroborate, sulfosalicylic acid
and sodium bicarbonate. The mats that are used in the procedure are made of an
asbestos–cellulose mixture. When the urine is placed on the mat, the absorbent
qualities of the mat cause the bilirubin to remain on the outer surface. The
sulfosalicylic acid provides the acid environment for the reaction. It also
acts with the sodium bicarbonate to provide an effervescence which helps
partially dissolve the tablet. The diazonium salt then couples with the
bilirubin on the mat, giving a blue or purple reaction product.
Urine from
patients receiving large doses of chlorpromazine may give false– positive
reactions. If the urine is suspected of containing a large amount of
chlorpromazine, the washthrough technique can be used where duplicate mats are
prepared with 5 drops of urine each.
c. Foam
Test
If the urine
is a yellowish–brown or greenish–yellow color and bilirubin is suspected, shake
the urine. If yellow or greenish–yellow foam develops, then bilirubin is most
likely present. Bilirubin alters the surface tension of urine and will develop
after shaking. The yellow color is from the bilirubin pigment. A false–positive
foam test occurs when the urine contains phenazopyridine.
7.
Nitrite
Nitrites
normally are not found in urine but result when bacteria reduce urinary
nitrates to nitrites. Many gram negative and some gram positive organisms are
capable of this conversion, and a positive dipstick nitrite test indicates that
these organisms are present in significant numbers (i.e., more than 10,000 per
mL). This test is specific but not highly sensitive. Thus, a positive result is
helpful, but a negative result does not rule out UTI. The nitrite dipstick
reagent is sensitive to air exposure, so containers should be closed
immediately after removing a strip. After one week exposure, one third of
strips give false–positive results. Non– nitrate–reducing organisms also may
cause false–negative results and patients who consume a low–nitrate diet may
have false–negative results.
Method of
determination
Reagent strips
for the detection of nitrite in the urine commonly used p–arsanilic acid a
quinolone compound. Nitrite reacts with p–arsanilic acid to form a diazonium
compound. This compound then couples with the quinoloine compound to produce a
pink color.
8.
Leukocyte
esterase
White blood
cells can be present in any body fluid depending on a cause for their presence.
The most common white blood cell seen in a urine sample is the neutrophil,
which is normally present in low numbers. Increased numbers of neutrophils
usually indicate the presence of a urinary tract infection; and their presence
is indicated by a positive leukocyte esterase test.
Method of
determination
Neutrophils
contain enzymes known as esterases. These esterases can be detected by reagent
strips that contain an appropriate substrate such as indoxycarbonic acid ester.
No comments:
Post a Comment