CHEMICAL

EXAMINATION OF
URINE
 CHEMICAL SCREENING
- reagent strips are primarily used
- easy to use and represent multiple complex state-of-the-
art chemical reactions
- can be read manually or using automation (Bayer Atlas) with
a precise amount of urine deposited on the dipstick and read the
reflectance of the color change with excellent reproducibility
(manual method has inconsistencies due to timing and color
discrimination)
- are changed periodically , sensitivities and color reactions
altered with new measurements added
- manufacturers supply tables of interfering substances
 CHEMICAL TESTS: Urine pH
- kidney tubular functions (produce bicarbonate and
ammonium ions; exchange Na+ of ultrafiltrate for H+; metabolic
activities contribute to nonvolatile acids like sulfuric, phosphoric,
and hydrochloric and organic acids like pyruvic, lactic, citric, and
ketone bodies; acids are excreted by glomeruli as salts of Na, K,
Ca and ammonium ions plus ammonia from PCT traps H+ ) and
lungs (excretes CO2) work on pH balance
- average adult with normal diet excretes about 50-100 mEq of H+ per 24
hours to produce a urine pH of 6.0; NV is 4.6 to 8.0
- acid urine may be due high protein diet, cranberries, mild
respiratory acidosis, metabolic acidosis, pharmacologic agents
like NH4Cl and methenamine mandelate used to treat PO4 and
CaCO3 stones, diabetic ketoacidosis, in K depletion due to
hypokalemic alkalosis brought about by vomiting,
hypercortisolism, and prolonged use of diuretics there will be
slightly acidic urine in the presence of metabolic alkalosis
 CHEMICAL TESTS: Urine pH
- alkaline urine is due to fruit (especially citruses) and
vegetable diets producing alkaline tide, treatment of acidic
calculi due to uric acid, cystine and calcium oxalate with alkalis
such as sodium bicarbonate, potassium citrate and
acetazolamide which are further used to activate antibiotics
neomycin, kanamycin and streptomycin in treating UTI, salicylate
poisoning, and sulfonamide therapy, tubular defects leading to
decreased ammonia and impaired capacity to exchange
hydrogen ions, bicarbonate wasting in proximal RTA & Fanconi
syndrome, metabolic alkalosis, and respiratory alkalosis
METHODS
Reagent Strip - indicators methyl red and bromthymol blue
gives orange, green, and blue as the pH increases from 5.0 -
9.0
- requires freshly voided urine, container without dead space
and tightly covered, sample kept cold on ice but not frozen
 CHEMICAL TESTS: Urine pH

On standing, urine pH rises due to loss of CO2 and

production of ammonia from urea due to bacterial action.

pH ELECTRODE - more accurate means of measurement of

pH using the pH meter with glass electrode
- pH usually drifts therefore there is a need to do
standardization using 3 buffers of known pH values
- after standardization, spraying of electrodes with distilled
water and dry with tissue
- immersing the electrode into the urine sample and reporting
the pH at a particular temperature of measurement
 CHEMICAL TESTS: Urine pH

TITRATABLE ACIDITY OF URINE - urine pH is largely

dependent on the amounts of monobasic and dibasic
phosphate present
- measured by titrating an aliquot of a 24-hour urine
(collected on ice) with 0.1N NaOH with pH 7.4 as an endpoint
- normal range is 200-500 mL of 0.1N NaOH (or 6 mL 0.1N
NaOH per kg. of body weight) or 20-40 mEq/24 hours
 CHEMICAL TESTS: Protein
- normally up to 150 mg is excreted in the urine
daily (average range of 2-10 mg/dL depending on urine
volume)
- there are >200 urinary proteins derived from the plasma
and GUT
- about 1/3 is albumin and remaining are small globulins (α-,
β-, and γ-types)
- plasma proteins with MW of 50,000-60,000 pass through the
glomerular basement membrane and reabsorbed in PCT
- albumin has MW of 69,000 are filtered but only in small
amounts
- retinol-binding, β2-microglobulin, Ig light chains and
lysozyme are excreted in small amounts
 CHEMICAL TESTS: Protein
- Tamm-Horsfall protein (uromucoid) secreted by
DCT cells and cells of the ascending loop of Henle is 1/3 or
more of total protein loss
- other sources of proteins are IgA, enzymes, and tubular
epithelial cells' secretions, other desquamated cells, and
leukocytes
- abnormal amount of protein ----> indicator of renal disease
- protein has very low maximal rate of tubular reabsorption
therefore increased amt of protein quickly saturates the
reabsorptive process
- screening methods is to differentiate normal protein
excretion from abnormal & therefore should not detect less
than about 8-10 mg/dL in normal adult with normal urine
flow rate
 CHEMICAL TESTS: Protein
- reagent strip is sensitive to albumin
- Acid precipitation methods detects all urinary
proteins (albumin + globulins)
- very dilute random urine may have falsely low value
- (+) protein in urine is significant thus duplicate testing is
required to confirm
- confirmatory measurements of elevated urine protein
should be accompanied by evaluation of renal function,
urine sediment exam, and urine culture
TYPES OF PROTEINURIA
Functional proteinuria is usually <0.5 g/day due to
dehydration, may accompany CHF, cold exposure and fever
(solved by appropriate treatment and rest for 2-3 days)
- strenuous exercise ---> HMW & LMW types, hyaline and
granular casts present
-
 CHEMICAL TESTS: Protein
Intermittent , Transient Proteinuria can be seen in
normal history, normal PE findings, and normal renal
function, urinalysis is also normal except
occasional proteinuria; need to monitor for hypertension for 6
months & transient proteinuria may also occur in pregnancy
Persistent proteinuria of 1-2 g/day in an asymptomatic or with
hematuria has poorer prognosis than intermittent (transient)
or postural proteinuria
- recent studies focuses on proteinuria in determining risk of
adverse outcomes of CKD classified currently based on GFR
- proteinuria is currently associated with CVD risk
- hereditary proteinuria syndromes are rare and have
heterogeneous forms ranging from congenital nephrotic
syndrome with severe proteinuria to focal segmental GN with
moderate proteinuria
 CHEMICAL TESTS: Protein
- progression to end-stage renal disease is a
common outcome
- specific diagnosis is possible with genetic testing for
mutations in the genes for various structural proteins of the
glomerulus

Postural (Orthostatic) - occurs in 3-5% of healthy young

adults; found in day in recumbent position; associated with
exaggerated lordotic position resulting in renal congestion
and ischemia; and total protein excretion is not >1g/day and
no other evidence of renal disease develops
- evaluation is done by emptying bladder before going to
bed and collect 2 urine samples a) upon waking up and
b)after the pt. has walked and stood for 2 hours; (+) if
sample a is (-) and sample b is (+)
 CHEMICAL TESTS: Protein
TYPES OF PROTEINURIA
Proteinuria in the Elderly - significant proteinuria found
in elderly above 60 years old; generally has 3- to 4-fold
incidence of GN and 1/4 affected responds to steroid
therapy; occult malignancies give rise to membranous GN
with resultant proteinuria
Proteinuria Quantification - 24-hour urine protein
quantification is more useful in diagnosis on renal disease
and monitoring treatment ; adequacy and completeness of
collection is required for accurate results; repeat
measurements are done to decide whether proteinuria is
intermittent or persistent; has 3 types heavy (>4g/day),
moderate (1.0-4.0 g/day) and minimal (<1.0 g/day)
 CHEMICAL TESTS: Protein
Heavy Proteinuria - seen with nephrotic syndrome;
classically, is accompanied by low serum
albumin, generalized edema, and increased lipids
(TAG, cholesterol, and phosphatides); VLDL and LDL are
increased but HDL is found in urine (due to loss of
lipoprotein lipase in urine); susceptibility to infection is due
to γ-globulin loss; when lipid is lost in urine, many granular
casts, fatty casts, fat-filled renal tubular epithelial cells (oval
fat bodies) are found in the sediment; cholesterol ester
droplets may be demonstrated by polarization
Nephrotic syndrome - principally associated with glomerular
dysfunction/damage due to 1)primary renal disease
including idiopathic disease and 2) systemic disease with
renal involvement; transient or mechanical causes include
severe CHF, constrictive pericarditis, and renal vein
thrombosis (due to loss of coagulation factors in urine and
increased fibrinogen)
 CHEMICAL TESTS: Protein
Nephrotic syndrome in children is minimal change disease
(also known as NIL LESION) steroid-responsive glomerular
disorder; other causes of heavy proteinuria are acute,
rapidly progressive and chronic types of glomerulonephritis
accompanied by RBCs and RBC casts; systemic diseases
like diabetes mellitus and lupus erythematosus ( with all cell
types and casts present in urine & hypersensitivity) causing
glomerular injury; malaria; malignant hypertension; toxemia
of pregnancy; heavy metal poisoning (Hg, Au); iatrogenic
(penicillamine); neoplasia; amyloidosis; sickle cell disease;
renal transplant rejection; and rarely primary
antiphospholipid syndrome
Moderate Proteinuria - may be found in a vast majority of
renal diseases in heavy proteinuria; nephrosclerosis,
multiple myeloma; and toxic nephropathies; degenerative,
malignant and inflammatory conditions of LUT including
irritative conditions like presence of calculi
 CHEMICAL TESTS: Protein

Minimal Proteinuria - may be noted in chronic pyelonephritis

which can be intermittent and in relatively inactive phases of
glomerular diseases
- it is found in nephrosclerosis, chronic interstitial nephritis,
congenital diseases like polycystic disease and medullary
cystic disease and renal tubular diseases
- in tubular diseases the urine sediment is normal but
erythrocytes, leukocytes and tubular cells may be seen with
interstitial nephritis with significant sediment findings with
trace protein results
- also present in postural and transient proteinuria
 CHEMICAL TESTS: Protein
Qualitative Categories of Proteinuria
- this is based on detection of protein types in the urine
using electrophoretic separation, and on clinical findings
- proteinuria can be separated into glomerular pattern and
tubular pattern (part of nephron affected) however they tend
to merge as the disease progresses
Glomerular Pattern - caused by glomerular disease with
heavy proteinuria
- albumin permeate into Bowman's space in large amts more
than what the PCT can reabsorb due to loss of reduction in
negative charges of glomerular basement membrane
- when albumin is lost in urine other similarly sized proteins
are also lost (antithrombin, transferrin, prealbumin, α1-acid
glycoprotein, and α1-antitrypsin)
 CHEMICAL TESTS: Protein

Still on Glomerular Pattern

- because tubular function is normal, very small proteins
are still reabsorbed
- large proteins (α2- macroglobulin, β-lipoprotein) are not
seen in urine while the glomerulus is still selective but they
tend to appear when greater damage happens to the
glomerulus and the proteinuria is less selective like with
membranous nephropathy and proliferative GN
- mechanism of proteinuria in diabetic kidney disease is
such that it is a chronic kidney disease (CKD)that is actually
a combination of both glomerular and tubulointerstitial
scarring
 CHEMICAL TESTS: Protein
Tubular Pattern
- associated with loss of a small amount of protein that
would otherwise be reabsorbed
- involves LMW proteins such as α1- microglobulin, β-
globulin, such as β2-microglobulin, light chain
immunoglobulins and lysozyme
- occurs with renal tubular diseases such as Fanconi
syndrome, cystinosis, Wilson's disease, and pyelonephritis
and with renal transplantation rejection
- 1-2g/day protein excreted
- may be missed by reagent strip because of low albumin
but detected by acid precipitation methods
 CHEMICAL TESTS: Protein
Overflow Proteinuria
- due to overflow of excess levels of protein in the circulation
and can be seen with hemoglobin, myoglobin and
immunoglobulins loss into the urine
- the said proteins are linked to glomerular and tubular
diseases but are renal damaging themselves
- myoglobin may cause acute tubular necrosis (ATN) and
hemoglobin in low amounts is not toxic unless hypovolemia
is present
Bence Jones Proteinuria - is associated with multiple
myeloma (50-80% incidence), macroglobulinemia, and
malignant lymphomas; missed by dipstick but detected using
electrophoresis and immunofixation electrophoresis
methods and immunoassay measurement of free lightchains
- high amounts of BJP can cause cast formation and
damage to kidneys leading to nephrotic disease
 CHEMICAL TESTS: Protein

Microalbuminuria
- presence of albumin in urine above the normal level but
below the detectable range of conventional urine dipstick
methods usually ranging from 20-200 mg/L
- indicator of early and possible reversible glomerular
damage
- in diabetic patients, 4-6x increase in cardiovascular
mortality and is an independent risk factor for renal
mortality
-also prevalent in hypertensive subjects
 CHEMICAL TESTS: Protein
METHODS
- urine protein are screened and quantitated
- a (+) screening test may have serious implications thus
there is a need to confirm results with a second different
method
- common screening tests include qualitative,
semiquantitative colorimetric reagent strip, and
precipitation-based methods
- accurate results are obtained with reagent strips only when
albumin is increased; give false positive results with organic
iodides (radiographic contrast medium) and tolbutamide or
other drugs; globulins are screened via acid precipitation
methods
- qualitative screening methods rely on protein precipitation
with heat & HAc, HNO3, TCA, and SSA that precipitates both
albumin and globulins
 CHEMICAL TESTS: Protein
METHODS
- (-) reagent strips with (+) SSA in urine samples is
attributable to radiographic dye, penicillins, and rarely to
isolated increase of globulins
- SSA & TCA are used to precipitate proteins in the cold and
are used as conventional screening method with sensitivity
as low as 0.25 mg/dL depending on the technique used
- to stratify the risk for dev't of diabetic and nondiabetic
nephropathy and other conditions like coronary heart
disease (CHD), recommendations focuses on urine albumin
rather than TP in urine
- urine albumin measurements are much more standardized
and reliable than TP at low conc. for assessment of risk of
progression of chronic renal disease's diagnosis and
planning of treatment
CHEMICAL TESTS: Protein

METHODS
Urine Constituents or Reagent Strip Acid
Condition Precipitation
Screening Test for Detection of Proteinuria
Highly buffered alkaline May cause FP May cause FN
urine
Drug metabolites No effect May cause FP
Radiocontrast media No effect May cause FP
Turbidity No effect May cause FP
Quaternary ammonium May cause FP No effect
groups of chlorhexidine
 CHEMICAL TESTS: Protein
METHODS
REAGENT STRIP
- takes advantage of protein errors of pH indicators
- because proteins carry a charge at physiologic pH thus
their presence will elicit a pH change
- it is impregnated with tetrabromphenol blue buffered to an
acid pH of 3 or tetrachlorophenol-tetrabromosulfonphthalein
that change from yellow (-) to varying shades of green
(trace, +1 to +4) after 30-60 seconds of urine application
- detects 5-20 mg protein/dL
- trace amts may be normal in concentrated urine voided
- high salt levels will lower results
- false (+) result may be seen in excessive wetting of strip
and amidoamines in fabric softeners
 CHEMICAL TESTS: Protein
METHODS
SULFOSALICYLIC ACID (SSA Qualitative)
- precipitation method that detects 5-10 mg/dL
- detects albumin, globulins, BJP and glycoproteins
- high levels of detergents may decrease the result
- SSA precipitate will increase on standing in the presence of
radiographic dye (removed by heat) and typical crystals seen upon
sediment exam
- procedure: centrifuge sample, 3 mL clear supernatant + 3 mL 3% SSA,
invert to mix, let stand for 10 min., invert again 2x, observe degree of
turbidity and grade results as follows:
Negative - no turbidity (5 mg or less); Trace - perciptible turbidity (20 mg/dl);
1+ = distinct turbidity, no granulation (50 mg/dl);
2+ = turbidity with distinct granulation, no flocculation (200 mg/dl);
3+ = turbidity with granulation and flocculation (500 mg/dl);
4+ = clumps of precipitated protein or solid precipitate (1 g/dl or more)
 CHEMICAL TESTS: Protein
QUANTITATIVE PROTEIN DETERMINATIONS &
CONFIRMATORY METHODS
- maybe adaptations of precipitation methods or
colorimetric in nature with SSA and TCA used as precipitants
- resultant turbidity is measured using a photometer or nephelometer
- if a visual interpretation is done, a set of gelled commercial standards
that correspond to 10, 20, 30, 40, 50, 75 and 100 mg/dl are used
- with SSA, the turbidity produced with albumin is 2.4x that produced
with globulin; the polypeptides, glycoprotein and BJP are also ppted.
- of historic note, the Exton's reagent contains SSA, Na2SO4 and an
indicator - bromphenol blue
- TCA method will cause γ- globulins will be ppted with greater turbidity
than albumin with no marked difference
- TCA-Biuret method is used for more precise measurements of small
amounts of protein; TCA ppt is dissolved in NaOH and reacted with
Biuret rgt; tedious; requires a color correction blank
 CHEMICAL TESTS: Protein
QUANTITATIVE PROTEIN DETERMINATIONS &
CONFIRMATORY METHODS
- several dye-binding methods colorimetric methods
are available to quantitate urinary proteins: Coomassie
brilliant blue, Ponceau S, Benzethonium chloride turbidity
methods; Pyrogallol Red-Molybdate reacts with proteins to
produce bluish-purple complex measured at 600nm

Microalbuminuria Determination Methods

- very small amts of albumin & β2- microglobulin are measured using
immunologic (Ig specific to protein), nephelometric and RIA
- Micral II test strip (Boehringer Mannheim) is immunologic, rapid and
semiquantitative for urine albumin (oxytetracycline increases value)
- Clinitek microalbumin (Bayer Dxnostics) is highly sensitive dye-binding
method; with creatinine rgt pad in addition; also reacts with
Tamm-Horsfall mucoprotein
 CHEMICAL TESTS: Protein
QUANTITATIVE PROTEIN DETERMINATIONS &
CONFIRMATORY METHODS

Bence Jones Proteinuria Determination Methods

- measured by protein electrophoresis using Amido Black or Coomassie

brilliant blue stains, immunofixation electrophoresis and immunoassay
for free light chains
- presence of BJG is indicated by a single sharp peak in the globulin
region in protein electrophoresis; represents either κ or λ Ig light chain
- BJP ppts at 40 & 60degC and redissolves near 100degC; other methods
include pptation in the cold with salts, ammonium sulfate and acids (all
will become positive at high levels of BJP); false(+) with heat & HAc but
false (-) with too concentrated BJP with ppt not dissolved @ boiling
point
 CHEMICAL TESTS: Glucose
& other Sugars
urine sugars include glucose (most common), fructose,
■

galactose, lactose, maltose, pentose and sucrose indicating

physiologic and pathologic conditions

GLUCOSE
■presence of detectable amt of glucose is glycosuria

whenever levels in blood surpasses (>180-200 mg/dL) the

renal tubule capacity for reabsorption

glucose appearance is influenced by glomerular blood

■
flow, tubular reabsorption rate, and urine flow
 CHEMICAL TESTS: Glucose
& other Sugars
■ GLUCOSE: Diabetes Mellitus
■glucosuria is not always indicative of DM but necessitates further
workup
■usually accompanied by polyuria and thirst

■inadequate glucose use---> increased ketone from >fat use

■urine test is painless and inexpensive (vs. blood testing)

■most useful for well-controlled diabetics who do not make freq.

adjustments in their insulin/hypoglycemic agents

■there is great variation in renal threshold for glucose among diabetics
thus (-) urine glucose represents a wide range of blood glucose levels
(home blood monitoring more preferred than misleading urine glucose
measurements)
 CHEMICAL TESTS: Glucose
& other Sugars
GLUCOSE: Diabetes Mellitus
■glycosuria monitoring problems: strips are difficult to

interpret at 1-2 g/dl glucose levels - have to look for newer,

more sensitive strips or use >efficacious copper reduction
■Clinitest tablet method. estimation can be up to 10 g/dl
using 1 gtt instead of 2 or 5 gtts og urine
■24-hour urine estimation with glycated Hb is more useful in
some instances for long-term management of DM
■practical and effective screen for 50 or more years old pts
and identifying gravidas at increased risk for gestational
diabetes
 CHEMICAL TESTS: Glucose
& other Sugars
GLUCOSE: Other Causes of Glycosuria with Hyperglycemia
■some endocrine disorders: acromegaly, Cushing's syndrome,
hyperadrenocorticism, functioning α- or β-cell pancreatic
tumors, hyperthyroidism, and pheochromocytoma
■pancreatic disorders like pancreatic CA, pancreatitis and cystic
fibrosis
■CNS disorders: brain tumor or hemorrhage, hypothalamic

disease, and asphyxia

■Disturbances in metabolism: burns, infection, fractures, AMI,

and uremia as well as liver disease, glycogen-storage disease,

obesity and feeding after starvation
■Iatrogenic: thiazides, corticosteroids, ACTH, and birth control

pills
 CHEMICAL TESTS: Glucose
& other Sugars
GLUCOSE: Glycosuria
■in pregnancy, >GFR occurs, and all filtered glucose may not be
reabsorbed ----> glycosuria with low blood glucose
■glucose tolerance may be decreased with aged persons when

pts have poor intake of carbs but not neessarily accompanied

by glycosuria
■glycosuria without hyperglycemia is seen in renal tubular

dysfunction; in renal tubular transport diseases, glycosuria may

be accompanied by impaired reabsorption of water, amino acid,
bicarbonate, phosphate and sodium - a pattern seen in Fanconi
syndrome
■glycosuric renal tubular dysfunction-associated diseases:

galactosemia, cystinosis, lead poisoning, and myeloma

 CHEMICAL TESTS: Glucose
& other Sugars
OTHER SUGARS in URINE
■small amt of disaccharides are excreted in urine - about 50

mg in 24 hrs and level may rise to 250 mg or more in

severe sprue or acute enteritis
■fructose, galactose, lactose, maltose and L-xylulose are
present in urine of pts. with inherited metabolic disorders
identified by TLC
■qualitative confirmatory tests are generally not

satisfactory for sugars

CHEMICAL TESTS: Glucose & other Sugars
OTHER SUGARS in URINE
FRUCTOSE
■appears in urine in association with inherited enzyme deficiences

that cause benign essential fructosuria and serious fructose

intolerance with vomiting, and both liver and kidney diseases
■found in parenteral feeding of fructose

■a marker of sucrose intake in all intervention studies

GALACTOSE
■found in genetic disorders of galactose metabolism associated with

deficiency in galactose-1-phosphate uridyl transferase or

galactokinase where galactose from dietary lactose is not converted
to glucose
■early detection followed by dietary restrictions may control
disease
CHEMICAL TESTS: Glucose & other Sugars
OTHER SUGARS in URINE

LACTOSE
■appears in urine late in normal pregnancy during lactation

■in lactose intolerance, lactose accumulates in the gut and

lactose will be absorbed and excreted unchanged in urine

PENTOSE
■pentosuria may follow ingestion of large amts of fructose
causing the excretion of L-xylulose and L-arabinose
■also seen with certain drug therapies and with benign

essential pentosuria
CHEMICAL TESTS: Glucose & other Sugars
OTHER SUGARS in URINE

SUCROSE
■appears in urine after ingestion of large amts of sucrose

■sucrase deficiency is associated with inherited diseases

such as sprue in the same manner as lactase deficiency
■sucrose intolerance is due to deficiency in sucrase or

α-dextrinase (isomaltase) deficiency

■factitious sucrosuria may cause high spec. grav. urine with

(-) GOD and (-) copper reduction tests

CHEMICAL TESTS: Glucose & other Sugars
METHODS
REAGENT STRIPS
■based on GOD and peroxidase method double sequential

enzyme reaction with various chromogens used

■specific only for glucose without detecting other sugars and drug

metabolites
■can be semiquantitative with results in approx. g/dl

■combination of glucose and ketone strips detect not only ketonuria

but also suppression of the glucose reaction by ketones seen

with other rgt strips
■Glucose + oxygen -----> Gluconic acid + H O
2 2
H2O2 + chromogen -----> oxidized chromogen + HOH
■Clinistix (o-toluidine) color changes from green to purple; detects
100 mg/dl of glucose; sensitive to ascorbic acid interference
CHEMICAL
METHODS
TESTS: Glucose & other Sugars
REAGENT STRIPS
■Multistix (KI chromogen) - color changes from yellow to
brown at 30 seconds
■Chemstrip (aminopropyl-carbazol) - color changes from
yellow to orange-brown at 60 seconds
COPPER REDUCTION TESTS
■used when GOD fails to detect glycosuria when increased
galactose and other sugars in urine esp. for pediatric pts
■with the state-mandated newborn screening, detection of
unstable reducing substance in the urine is rare
■detects sufficient quantities of reducing substances in
urine including most sugars except sucrose
■copper reduction method (+) but GOD (-) rules out
glycosuria that requires drug history and clinical findings
CHEMICAL
METHODS
TESTS: Glucose & other Sugars
COPPER REDUCTION TESTS
■ although detect NGRS, the yield for these sugars is extremely
low
■(+) results are seen in normal neonatal infants during the first

10-14 days of life (because of glucose, galactose, fructose and

lactose) and normal pregnant and postpartum women (because
of lactose)
■Benedict test is more sensitive than Clinitest tablet

■strong reducing substances like ascorbate, gentisic acid or

homogentisic acid inhibits GOD enzyme giving (+) Cu red'n

■very large doses of ascorbate do not affect 2-drop Clinitest but

delays color dev't of GOD test

■cephalosporins and radiographic media give false-(+) with
Clinitest
CHEMICAL
METHODS
TESTS: Glucose & other Sugars
COPPER REDUCTION TESTS
■ copper sulfate, NaOH, sodium carbonate, and citric acid are
incorporated in the Clinitest tablet
++
■reaction includes: Cu ----------> Cu+ in alkaline medium
Cu+ + OH- heated -----------> CuOH
2CuOH further heated -----------> Cu2O + HOH
Clinitest Tablet test
■tablet tests will detect 250mg of reducing substance/dl urine

■both 2-drop and 5-drop methods can be used

■2-drop method was developed in response to a so-called

pass-through phenomenon that may occur if >2g/dl of sugar is

present in urine where tablet goes through the entire range of
colors and back to dark greenish brown (not found in color chart)
and corresponds to a lower result- a false low
CHEMICAL
METHODS
TESTS: Glucose & other Sugars

COPPER REDUCTION TESTS

FIVE-DROP METHOD
■5 gtts urine + 10 gtts water --> add Clinitest tablet not touching
it (contains caustic NaOH)--> watch while boiling
■-->observe 15seconds after boiling stops---> shake the tube

gently and compare color solution to color scale immediately

■results are negative, 0.25g/dl, 0.50g/dl, 0.75g/dl, 1.0g/dl,

2.0g/dl and pass-through

■urine showing pass-through should be retested using the
two-drop method
■for negative and low-level results
CHEMICAL
METHODS
TESTS: Glucose & other Sugars

COPPER REDUCTION TESTS

TWO-DROP METHOD
■2 gtts urine + 10 gtts water----> add Clinitest tablet --> watch
while boiling takes place but do not shake ---> wait 15 seconds
after boiling stops then shake tube gently ---->
■compare color to color chart for 2-drop method

■results are negative, 1 g/dl, 2g/dl, 3 g/dl, 5g/dl and

pass-through
CHEMICAL
METHODS
TESTS: Glucose & other Sugars
COPPER REDUCTION TESTS
Precautions when doing tablet tests
■bottle must be kept tightly closed to prevent moisture

■kept away from sunlight and direct heat in a cool, dry place

■tablets are blue-spotted white that turn to bluish to brown in the

presence of moisture
■individually packaged in aluminum foil to prevent absorption of

moisture
OTHER TESTS FOR SUGARS
■FRUCTOSE is identified by TLC, qualitative resorcinol test and

Benedict's test at low temperatures

■GALACTOSE is identified in urine by TLC, but
galactose-associated diseases are identified by enzyme assay
when suspected
CHEMICAL
METHODS
TESTS: Glucose & other Sugars
OTHER TESTS FOR SUGARS

■LACTOSE is identified by TLC, or a qualitative lactose test

reacting lead acetate, conc. ammonium hydroxide and
urine forming brick-red solution that becomes red ppt with
colorless supernate
■PENTOSES are identified in urine by TLC; L-xylulose will

reduce Benedict's rgt at 50degC within 10 minutes at

concs. 250-300 mg/dl
■SUCROSE will be fermented by yeast and can be separated
by chromatography but needs to be stained with a
substance not dependent on reducing properties
 CHEMICAL TESTS: Ketones in Urine
when defect in carb metabolism or low carb in the
diet (fasting) ---> increased fatty metabolism to compensate
---> urine ketones are produced(products of incomplete fat
metabolism)
■in ketonuria, 3 ketone bodies appear

3-OHbutyrate (78%), acetoacetic acid (20%) & acetone

(2%)
■acetone is nonreversibly formed acetoacetic (diacetic)acid
from removal of CO2
■3-OHbutyrate (β-OHbutyrate) is formed reversibly from
diacetic acid involving removal of 2H
■ total ketone bodies can range as high as 17-42 mg/dl; up to

2 mg diacetic acid/dl is normal

 CHEMICAL TESTS: Ketones in Urine
KETONURIA & KETONEMIA: Diabetic Ketonuria

■ketonuria implies presence of ketoacidosis (ketosis) and

provides a warning of an impending coma
■up to 50 mg of acetoacetic acid/dl may be present but no
evidence of ketosis
■ketonuria is present among diabetic type 1with diabetic
ketoacidosis but not in diabetic type 2 with hyperosmolar
hyperglycemic coma
 CHEMICAL TESTS: Ketones in Urine
NONDIABETIC KETONURIA
■seen in acute febrile states and toxic states accompanied
by vomiting and diarrhea in infants and children
■in severe persistent neonatal ketosis - suspect an inherited
metabolic disease
■also found in hyperemesis of pregnancy, in cachexia and
following anesthesia due to increased fat catabolism in the
face of limited food intake
■normally seen in pregnant woman - low FBS and mild
ketonuria
■also seen in exposure to cold or severe exercise and
low-carb diet for weight reduction
■ total ketone bodies (acetone) can range as high as 17-42

mg/dl; up to 2 mg diacetic acid/dl is normal

 CHEMICAL TESTS: Ketones in Urine
LACTIC ACIDOSIS
■acetoacetic acid and 3-OHbutyrate are elevated in lactic
acidosis (not detected by the usual nitroprusside test)
■lactic acidosis may coexist with shock, DM, renal failure,
liver failure, and infection; in response to drugs like
phenformin and salicylate poisoning
METHODS
■common nitroprusside test and tablet tests

(Rothera-based) detect diacetic acid and acetone

■FeCl (Gerhardt's test) detect acetoacetic acid only
3
■in urine (better for bedside monitoring of diabetic
acidosis) and plasma, reagent strips and tablet test react to
10 mg of acetoacetic acid /dl and are less sensitive to
acetone
 CHEMICAL TESTS: Ketones in Urine
METHODS
■repeated high qualitative determination of acetone and
diacetic acid will not reflect actual changes happening thus
a need to perform semiquantitative tests via reagent strip
and Rothera's tablet test
■false (-) results are usually due to unstable reagents and
labile ketones; in vivo or in vitro bacterial action will cause
of loss of acetoacetic acid
■acetone is lost at RT but not if kept in a closed container in

a refrigerator; reffed samples need to be brought back to

RT before acetone testing
■preservatives do not prevent decay of ketones

■fresh rgts and use of known (+) & (-) controls for best
testing
 CHEMICAL TESTS: Ketones in Urine
REAGENT STRIP METHODS
■ method based on nitroprusside (Na nitroferricyanide)
reaction for ketones (strips without alkali reacts to diacetic
acid but not to acetone; 3+ results need to be diluted and
remeasured reporting “moderate” result and the dilution
factor)
■Chemstrip contains nitroprusside and glycine which reacts
with diacetic acid (about 10 mg/dl) and acetone (about 70
mg/dl) in alkaline medium forming violet color (color changes
from beige to violet) read after 60 seconds
■Chemstrip and Acetest are both sensitive methods

■Multistix contains buffers and sodium nitroferricyanide and

reacts with diacetic acid (5-10 mg/dl) producing
pink-maroon in 15 seconds
 CHEMICAL TESTS: Ketones in Urine
REAGENT STRIP METHODS

■strips correlate only moderately well with quantitative

acetoacetate in plasma and poorly with total blood
ketones
■false (+) occurs after use of phthaleins (PSP or BSP) or in
presence of large amts of phenylketones, preservative
8-OHquinoline or L-dopa metabolites, also
antihypertensives methyldopa and captopril
■acetylcysteine produces red color

■false (-) with strip loss of reactivity

 CHEMICAL TESTS: Ketones in Urine
NITROPRUSSIDE TABLET TEST
■useful when urine has interfering color

■Acetest tablet contains sodium nitoferricyanide, glycine,

and strongly alkaline buffer that is sensitive to moisture and
deteriorate
■used to assay whole blood, plasma, serum or urine

■detects 5-10 mg/dl acetoacetic acid/dl urine and 20-25

mg/dl acetone/dl urine
■gives false (+) with PSP or BSP, phenylketones, and L-dopa

metabolites
■procedure: put tablet on white paper surface, place a drop of

urine/blood/plasma/serum on tablet, compare to color chart at

30 seconds for urine but at 2 minutes for plasma/serum
and at 10 minutes for whole blood with clot removed; (+) is
lavender to deep purple
 CHEMICAL TESTS: Ketones in Urine

NITROPRUSSIDE TABLET TEST

■results are reported as negative, small, moderate, or large

■if large, dilution should be made and report result of

analyses as follows: Undiluted “large”, 1:2 dilution
“large”, 1:4 dilution “moderate”, etc.

OTHER TESTS FOR KETONES

Gerhardt's Ferric Chloride test for acetoacetic acid
■not very specific and sensitivity is low at 25-50 mg/dl

■gives (+) to salicylate and L-dopa

Test Tube Nitroprusside method is sensitive to acetoacetic

acid at about 1-5 mg/dl and to acetone from 10-25 mg/dl
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
■ hematuria is relatively common, hemoglobinuria
uncommon and myoglobinuria rare
HEMATURIA
■asymptomatic microscopic hematuria may be detected by dipstick in
up to 16% of screening populations, many serious diseases of the GUT
release RBCs into urine
■retrospective investigation of microscopic hematuria by renal biopsy
disclosed several histopathologic findings - membranous nephropathy,
non-IgA nephropathy, non-IgA mesangioproliferative GN, focal
glomerulosclerosis, and mild glomerular abnormalities (15% with
normal histology)
■can occur in neoplastic and non-neoplastic dses, trauma incl. calculi
anywhere in GUT, bleeding disorders and anticoagulant use, use of
cyclophosphamide, rare giant cell arteritis, and healthy persons
undertaking excessive exercise (marathon runners with bleeding
originating in bladder mucosa)
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
HEMATURIA
■screening for hemoglobinuria is a useful adjunct to microscopic exam
of sediment for hematuria
■sometimes dipstick for hemoglobinuria is more sensitive test for urine
microscopy for hematuria
■Hb reagent strip is inhibited by ascorbic acid that requires the need for
microscopy
■(+) Hb with normal microscopic sediment exam suggests a fresh
sample be examined for RBCs because alkaline pH and spec. grav. of
<1.010 may lyse erythrocytes
HEMOGLOBINURIA
■caused by hemolysis and significantly intravascularly

■Hemoglobin binds to haptoglobin and free Hb passes thru

the glomerulus as αβ dimers with MW of 32,000, some Hb
reabsorbed and some excreted
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine

HEMOGLOBINURIA
■follows trauma due to exertion with lysis of blood cells

■plasma appears pink at levels of about 50 mg/dl of Hb and

level reaching 1 g/dl shows marked hemolysis
■plasma Hb is marked in acquired than hereditary hemolytic

anemias & moderate levels occur in sickle cell anemia and

homozygous thalassemias ( with unstable Hb causing
brown-pigmented urine thought to be due to a dipyrrole or
bilifuscin with no reaction with strip for heme)

■
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine

HEMOSIDERIN IN URINE
■ filtered free Hb ---> reabsorbed by PCT ----> catabolized

into ferritin and hemosiderin

■hemosiderin appears 2-3 days after an acute hemolytic
episode that caused hemoglobinuria ----> strip often (-) for
Hb but hemosiderin can be found as yellow-brown granules
either as free, or found in epithelial cells and in casts
■also seen in sediments in hemochromatosis, a true siderosis
of kidney parenchyma
■hemosiderinuria indicates a chronic hemolytic state that

requires other tests for correct diagnosis: serum bilirubin,

LDH, and haptoglobin levels
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine

HEMOSIDERIN IN URINE
■ because of the intermittent presence of hemosiderinuria,

urinary Fe may be quantitated to establish chronic

intravascular hemolysis
■increased Fe found in hemosiderosis and RBCs
traumatized by prosthetic valves
■normal urine Fe in pernicious anemia and in hereditary
spherocytosis
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
MYOGLOBINURIA
■ when acute rhabdomyolysis occurs, myoglobin is
released, rapidly cleared from the blood, and excreted in
urine as red-brown pigment
■free myoglobin has MW of 17,000 is quickly removed

■myoglobinuria is seen in strenuous exercise,

dermatomyositis, defect in PFK and ADA enzymes and
mitochondrial trifunctional protein deficiency
■diagnosis (to differentiate from Hburia) includes history and
clinical findings on serum: muscle tenderness and
cramps, voids red-brown urine within 1-2 days after
exertion, strip Hb & protein are markedly (+), clear serum
with high CK, ALS and normal haptoglobin; serum
creatinine may be high, urine clears in 2-3 days
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
METHODS
Reagent Strip for Heme Compounds (Hb & Myoglobin)
■based on liberation of oxygen from peroxide in rgt strip by
peroxidase-like activity of heme in free Hb, lysed RBCs or
myoglobin
■intact RBCs are lysed on the strip for rxn to take place

■well-mixed urine be tested to include intact erythrocytes

■ heme catalyzes the oxidation of buffered

tetramethylbenzidine to produce a green color at 60
seconds following sample application
■Multistix and Chemstrip detect 0.05-0.3 mg Hb/dl urine = 10
lysed RBCs/μl
■normal RBC contains 30 pg of Hb per cell
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
METHODS
Reagent Strip for Heme Compounds (Hb & Myoglobin)
■sensitivity is reduced by high spec. grav. urine,& high
protein levels; false (-) in high ascorbic acid and if formalin
is used as preservative; high nitrite delays rxn; false (+)
results with oxidizing compounds hypochlorite and iodine
as well as presence of UTI due to microbial peroxidase

Other Tests for Hb and Myoglobin

■both can be present in crush injuries, can be protein-bound in

urine, thus difficult to separate using salt precipitation

method and electrophoresis -----> solved by immunoassay
for myoglobin and capillary electrophoresis for urinary Hb
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
METHODS
Other Tests for Hb and Myoglobin
Qualitative test for myoglobin
■use fresh urine, observe color, myoglobin is red if fresh and

turns brown in time, neutralize pH (myoglobin is less stable at

acidic pH)
■1 ml urine + 3 ml 3% SSA to ppt proteins (heat and HAc does
not ppt Hb and myoglobin) and filter, filtrate that is N means
there are no abnormal nonprotein pigment present
■5 ml urine + 2.8g ammonium sulfate, mix to dissolve, urine is

80% saturated with the ammonium salt ---> optimal for

precipitation of Hb, filter and if filtrate is colored red, it is
presumptive for myoglobin
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
METHODS
Hemosiderinuria Test
■the Prussian blue rxn demonstrates presence of iron in
hemosiderin
■Notes: all apparatuses must be Fe-free, deionized water
only, and Prussian blue stain made fresh
■Prussian blue stain: Conc. HCl + 20% potassium
ferrocyanide solution ----> until white ppt forms that
remains stable on shaking; filter thru no. 5 filter paper

Working counterstain: Dilute 1 ml. safranin O stain (0.5 g.

■
in 100 ml dist water) to 50 ml with phosphate buffer (pH 6.4
- 4.7)
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
METHODS
Hemosiderinuria Test
Dry Procedure
■hemosiderin appears as blue granules 1-3 μm occurring

singly or in groups in renal cytoplasm of epithelial cells, as

amorphous sediments, or blue granules in casts
■urine is collected in an iron-free glass container, overlaid with
stain and let stand for 2 hours, decant 3/4 of it, centrifuge
remainder, make a smear of sediment and air dry
■fix smear in meOH for 10 min., rinse with Fe-free water and air

dry, stain with Prussian blue rgt for 30 min., wash gently for 4
min with deionized water and air dry, counterstain with safranin
O for 1-5 min., rinse with Fe-free water and air dry, and mount
coverslip
 CHEMICAL TESTS: Blood, Hemoglobin,
Hemosiderin & Myoglobin in Urine
METHODS
Hemosiderinuria Test
Wet Procedure
■centrifuge a complete morning urine or random urine for 5

minutes and pool sediment; examine several drop of sediment

for coarse yellow-brown granules esp within renal tubular
epithelial cells or casts
■if granules are seen, suspend the rest of the sediment in a

fresh mixture of 5ml 2% potassium ferrocyanide solution and 5

ml of 1% HCl and allow to stand for 10 minutes
■centrifuge and discard supernatant, examine sediment
microscopically ----> coarse granules of hemosiderin
appear blue in cells, casts or amorphous materials (if
granules do not stain reexamine after 30 minutes (delayed rxn)
BILIRUBIN IN URINE
UROBILINOGEN IN URINE
INDIRECT TESTS for UTI
■ NITRITE
■ LEUKOCYTE ESTERASE
MISCELLANEOUS TESTS
■ ASCORBIC ACID
■ 5-HYDROXYINDOLEACETIC ACID
■ MELANIN
■ PORPHYRINS
SPECIAL TESTS
URINARY CALCULI
■ Hypercalciuria & Calcium stones
■ Hyperoxaluria
■ Hyperuricuria
■ Cystine stones
■ Rare calculi
URINE SCREENING OF INHERITED
METABOLIC DISORDERS

■ AMINOACIDURIA
■ PKU
■ ALKAPTONURIA
■ TYROSINURIA
■ MSUD
■ CYSTINURIA
■ HOMOCYSTINURIA
 ADDITIONAL URINE TESTING
MODALITIES
■ Latex agglutination nephelometric assay for urinary
basic fetoprotein (BFP) – nonspecific marker for tumors
in ureter, prostate & bladder; and infection as well
■ Trinder spot test – sensitive screen for salicylates
■ Semiquantitative rapid urine iodide test –survey of
iodine deficiency/thyroid function
■ Monoclonal antibody assay for urine pyridinium
cross-links for bone resorption (detect osteoporosis,
hyperthyroidism, hyperparathyroidism & Paget’s
disease)
■ ELISA & FISH to detect CA in the urinary bladder
(augment cytopathologic urine examination)