REAGENT STRIPS
Storage
–
–
–
–
–
Protect from moisture and excessive heat.
Store at room temp. Do not refrigerate.
Keep container tightly capped.
Do not remove desiccant from container.
Do not use beyond expiration date.
Handling
– Review manufacturer’s instructions with each new lot
number
– Remove strips from bottle for immediate use. Recap.
– Check for discoloration.
– Keep away from bleach, acids, fumes, etc.
REAGENT STRIPS (continued)
• Testing
–
–
–
–
–
–
–
–
–
–
–
within 30 minutes to 2 hours
fresh, well-mixed, unspun
at room temperature
Do not touch test pad area
Dip briefly, but completely - app. 1 second
Drain off excess urine, avoid runover
Do not lay strip on bench
Compare test areas to color chart on bottle
Read at specified times
Know sources of error, interfering substances, sensitivity,
and specificity for each strip.
When automated, follow instrument’s operating manual.
Reporting
• Standard terms must be used
– Quantitative
• Concentration mg/dL
• Plus system
– Qualitative
• Small, moderate, large
• Negative/positive/WRR (normal)
pH
• The strip contains the indicators methyl red and
bromthymol blue. The give colors over the pH
range of 5-9. Colors range from orange through
yellow and green to blue.
• Reference value on normal diet - 4.6 to 8.0
CLINICAL SIGNIFICANCE pH
Acid pH <6.0
– Diet (high protein, meat, cranberries)
– Acidifying drugs to prevent alk. stone formation
– Abnormal crystalluria (bilirubin, cystine, tyrosine, leucine,
cholesterol )
– Uric acid stone formers
– Acidosis and uncontrolled Diabetes mellitus
– Hypokalemia
– Starvation
pH (continued)
• Alkaline pH >6.5
– Diet (vegetarian and citrus fruits)
– Alkaline tide produced after a meal
– Metabolic and respiratory alkalosis
– Renal tubular diseases (Fanconi’s syndrome)
– Alkalizing drugs to treat acid calculi formation
– Genitourinary tract infections
• pH >8.0
– Contamination or old urine (not suitable for testing)
Protein
• Reagent strip testing is based on the principle of
protein error of the indicators. Protein in the form
of albumin accepts ions from the indicator,
which is buffered in a very acid medium. Color
changes from shades of green to blue.
• Most significant for early detection of renal
disease.
CLINICAL SIGNIFICANCE PROTEIN
• Reference value
–
–
Random urine negative (not detectable)
<10mg/dL)
24 Hour <150mg or 10 mg/dL
• Composition
–
–
Albumin, 1/3a
Globulins, 2/3
•
Tamm-Horsfall mucoprotein (25%)
• Pathology >30mg/dL or on daily output
–
–
–
Heavy >3g/day
Moderate >1-3 g/day
Minimal <1g/day
CLINICAL SIGNIFICANCE
PROTEIN (CONTINUED)
• Benign
–
Functional
•
–
Orthostatic
•
–
exercise, fever, stress, exposure to cold
positional, renal congestion
Pregnancy
•
transient, investigate cause
• Pathologic
–
Prerenal: overflow of low mole weight proteins
•
•
•
IgG light chains (Bence Jones proteins)
acute phase
hemoglobin, myoglobin
CLINICAL SIGNIFICANCE PROTEIN
(CONTINUED)
• Pathologic
–
Renal Glomerular Pattern
•
•
•
–
Renal Tubular Pattern
•
•
•
–
Group A strep and SLE glomerulonephritis
Hypertensive and diabetic nephropathy
Nephrotic syndrome, tumors, infections, toxic agents
Acute and chronic pyelonephritis
Interstitial nephritis
Renal tubular acidosis, rejection of kidney transplant
Post Renal
•
Inflammation/infection bladder, renal pelvis, ureter,
prostate, external genitalia
SSA Testing for Protein
• When mixed with weak sulfosalicylic acid (SSA),
all urine proteins will denature and precipitate at
room temperature. The degree of which is
graded and reported in semi-quantitative terms.
– Albumin, globulins, glycoproteins, and Bence-Jones
protein are detected.
– False positive caused by x-ray contrast media,
penicillin, sulfonamides, tolbutamides
– False negative caused by highly alkaline urine
Microalbuminuria
• Protein (albumin) that cannot be detected by
routine dipstick for protein
– Sensitive methods needed to detect 10-20mg/L or 12 mg/dL
• Immunochemical
• Dye binding
• Clinical significance
– Early management of kidney disease in diabetes,
hypertension, or peripheral vascular disease
Glucose
• The dipstick determination of glucose is based on a
double sequential enzymatic reaction using the specific
glucose oxidase/peroxidase reaction in the presence of
glucose and a chromogen.
• The Clinitest or Benedict’s Reaction is based on the
ability of reducing substances to reduce copper sulfate
to cuprous oxide in the presence of a chromogen,
which changes color from blue to orange. Test is
performed to screen for non-glucose reducing sugars
in infants and children under 2 years old.
Clinical Significance of Glucose
• Reference value
– No detectable amount present in urine by dipstick
method (<50 mg/dl)
• Positive values found when renal threshold for
glucose is exceeded (160-180 mg/dl)
– Diabetes mellitus (DM)
– Impaired tubular reabsorption
– Pregnancy with latent DM
COMPARISON of REAGENT
STRIP vs. CLINITEST
Strip
Clinitest
Positive
Negative
Negative* Positive
Cause
Sensitivity of methods
Oxidizing contaminants/bleach
Deteriorated Clinitest tablets
Non-glucose reducing substance
Deteriorated reagent strips
Reagent strip interferences
*Ascorbic acid (vitamin C)
Ketones
• Conditions that result in increased and or incomplete
fat metabolism can produce metabolic intermediary fat
products in the urine and blood. The three ketone
bodies present in urine are acetoacetic acid (20%),
acetone (2%), and betahydroxybutyric acid (78%).
• Acetoacetic acid and acetone react with nitroprusside
in an alkaline medium to form a violet dye complex.
Basis of dipstick.
• Betahydroxybutyric acid is not detected with dipstick.
Clinical Significance Acetone
• No detectable ketones present in normal urines.
• Positive values
– Diabetic ketosis (ketonuria)
– Loss of carbohydrates due to fever, vomiting, weight loss,
starvation, diarrhea, stress
– Lactic acidosis caused by liver/renal failure, salicylate
overdose
• Interfering factors
– False positive: pigmented urines
– False negative: delay in testing
Nitrite
• This test depends on the conversion of nitrate to nitrite by the
action on Gram negative bacteria that contain reductase
enzymes in the urine.
• At the acid pH of the reagent area, nitrite in the urine reacts with
an aromatic amine to form a diazonium salt, followed by a
coupling reaction with benzoquinoline to produce a pink color.
• Three factors must be present
– Reductase producing bacteria must be present
– Urine must be retained in bladder long enough to convert nitrates to
nitrites (4 hours)
– Nitrates must be present
CLINICAL SIGNIFICANCE NITRITE
• Reference value
– No nitrites present
– Urine must be FRESH
– Improper storage will result in false positive
• Positive nitrites
– Screen symptomatic and asymptomatic UTI
– Common infecting organisms
•
–
Enterobacter, Citrobacter, Escherichia, Proteus, Klebsiella,
Pseudomonas
Definitive diagnosis made by urine culture
•
Non-reductase producing microorganisms will be negative for nitrites
Leukocyte Esterase
• Granulocytic leukocytes contain esterase activity
that catalyze the hydrolysis of an amino acid
ester to form an aromatic compound which
reacts with a diazonium salt to produce a color
change from beige to purple on the dipstick pad.
• All positive reactions require a microscopic
exam of the sediment.
CLINICAL SIGNIFICANCE LEUKOCYTE
ESTERASE
• Reference value
– 0-5 white cells/hpf
– females 0-8 WBC/hpf or app. 10 WBC/uL (vaginal discharge
can cause false positive)
• Screens for urinary tract inflammation
–
–
–
kidney (pyelonephritis)
bladder (cystitis)
urethra (urethritis)
• Leukocyturia can occur with or without bacteria
LEUKOCYTE-NITRITE
Combination on FRESH urine is
• Cost effective tool to screen for UTI
• Provides 97% predictive value for negative culture
when both tests are negative
• Improved care in asymptomatic patient
Blood
• Dipstick will detect blood by sensing heme that is
present in red cell, hemoglobin, and myoglobin. Based
on the pseudoperoxidase activity of heme in the
presence of an organic peroxide and a benzidine
chromogen.
– Hematuria: in tact red cells present in urine (scattered green
dots)
– Hemoglobinuria: presence of hemoglobin from lysed red cell
in urine (diffuse green color)
– Myoglobinuria: presence of heme protein from muscles in
urine (diffuse green color)
CLINICAL SIGNIFICANCE BLOOD
• Reference value
–
0-5 erythrocytes/mL or 0-2 RBC/hpf
• Hematuria - intact red cells
–
–
renal disease, calculi, tumors, infections
bleeding in kidneys or lower urinary tract
• Hemoglobinuria - free hemoglobin
–
intravascular hemolysis as seen in incompatible blood
transfusions, AIHA, G6PD, etc.
• Myoglobinuria - heme muscle protein
–
–
acute destruction of muscle fibers (rhabdomyolysis)
crush/trauma injuries
• Excessive exercise can cause all above
COMPARISON of URINE FINDINGS
Findings
Hematuria
Hemoglobinuria
Myoglobinuria
Color
pink, red, smoke
clear pink, red, brown
clear red, brown
UA Blood strip
positive dots
positive diffuse
positive diffuse
UA Protein strip
Renal 4+
Nonrenal +/-
pos/neg
pos/neg
UA RBCs
many
occasional
occasional
UA Casts Renal:
RBC
Nonrenal: none
Hemoglobin
Myoglobin
Bilirubin
• The heme released from red cells is converted
to the yellow bile pigment biliribin by a series of
complex reactions in liver. A small amount is
excreted under normal circumstances and is not
detected in the urine with the dipstick.
• When present, the Diazo Reaction is based on
the coupling of bilirubin with a diazonium salt in
an acid medium to form a colored azo-dye
complex.
Ictotest for Bilirubin
• Highly pigmented urines can cause false
positive reactions. Confirmation is required by
testing with the Ictotest tablet test for bilirubin.
• This diazo tablet method is very sensitive to low
levels of bilirubin.
• Pigments will be removed by the absorbent pad
supplied with the test.
CLINICAL SIGNIFICANCE
BILIRUBIN
• Reference value
–
not detected with reagent strips <0.02 mg/dL
• Positive findings
–
obstruction to bile flow from liver
•
–
inflammation and swelling of liver cells
•
•
–
gallstones and neoplasms of pancreas
acute viral hepatitis, drug indued cholestatsis
acute alcoholic hepatits/cirrhosis
congenital hyperbilirubinemias
•
Dublin-Johnson and Rotor
Urobilinogen
• Collectively referred to as the end products of
bilirubin metabolism. Colorless reduction
product of bilirubin which is oxidized by normal
intestinal bacteria to brown pigment that is
excreted in the feces.
• Based on the Ehrlich Reaction in an acid
medium to form a red color.
CLINICAL SIGNIFICANCE UROBILINOGEN
• Reference value
–
–
–
–
up to 1 mg/dL or 1 Ehrlich Unit
greater in PM (alkaline tide after meals)
up to 2 mg/dL transition from normal to abnormal
decrease or absence cannot be determined with strip
• Increased values
–
–
–
–
liver damage: viral hepatitis, cirrhosis, drugs, toxins
infections of biliary tree (cholangitis)
hemolytic anemias and intravascular hemolysis
increased enteric production
• Absent
–
–
obstruction of bile duct
absence of intestinal flora
UA BILIRUBIN & UROBILINOGEN in
UNCOMPLICATED JAUNDICE
Condition
Bilirubin
Urobilinogen
Normal
Hepatic Disease
Obstructive Disease
Hemolytic Disease
Negative
Positive
Positive (+/-)
Negative
up to 2 EU/dL
Increased (+/-)
Absent
Increased
QUALITY ASSURANCE
• Facilities and Resources
–
OSHA compliance
• Proficiency Testing
• Personnel
–
–
Qualifications, education and training,
competency
Review
• Procedure Manual
–
NCCLS GP2-A2
• Controls, Standards, Reagents
• Equipment and Instruments
• Reporting of Results
COMPETENCY ALERTS
REAGENT STRIP TESTING
• Directly Observe
– Followed SOP and manufacturer’s instruction
• labeled date received, opened, expired
• Removed strip immediately before test run
• Replaced cap
–
–
–
–
–
–
Performed test on well mixed, unspun urine
Performed daily maintenance/function checks
Interpretation of color changes for strips or tablets
Performed confirmatory testing as indicated
Performed Clinitest on nursery or pediatric urines
Followed SOP step-by-step
COMPETENCY ALERTS
(continued)
• Monitor and Review
– Compliance with QC as defined in SOP
•
•
•
Results logged on scheduled frequency of use
Parallel testing
PM and service logs signed
–
Critical values reported on interim
worksheets: WHO, WHAT, WHEN.
–
Standard units of measure defined in
SOP are used to report qualitative and
quantitative results.
COMPETENCY ALERTS (continued)
• Assessment of Test Performance
–
–
Proficiency testing
Internal blind samples of known chemical concentration
• Problem Solving
–
–
–
–
Resolve discrepant results
Investigate and resolve delta checked results
Specimen referred for definitive testing (UA culture) based on
reagent strip results. Policy defined in SOP.
Resolution of “out-of-control” results for known reference
controls.
MICROSCOPIC EXAM of URINARY
SEDIMENT
• CLIA’88 Complexity
–
–
Moderate
Provider Performed Microscopy (PPM)
•
•
physician, midlevel practitioner, or dentist
brightfield or phase microscopy
• Specimen of Choice
–
–
–
–
Fresh first morning, midstream, clean catch
Examine within 2 hours
Specific gravity >1.010
pH acid
IDENTIFIABLE SEDIMENT ENTITIES
• Hematopoietic cells
–
Red blood cells (RBC)
•
–
dysmorphic
white blood cells (WBC)
•
•
•
glitter
eosinophils
lymphocytes , histiocytes and macrophages
• Epithelial cells
–
–
–
Transitional (urothelial)
Squamous
Renal tubular
•
oval fat bodies
• Casts
–
–
–
–
–
Matrix : Hyaline and Waxy
Cellular: RBC, WBC, Epithelial, Mixed, Bacteria
Inclusion: Granular, Fatty, Hemosiderin, Crystal
Pigment: Hemoglobin, Myoglobin, Bilirubin
Size: Broad or Wide
• Crystals
• Microorganisms
–
bacteria, yeasts, parasites
• Miscellaneous
–
spermatozoa, mucus, artifacts and contaminants