PH Protein Glucose Ketones Blood Bilirubin Urobilinogen Nitrite Leukocyte Esterase Specific Gravity

Chrissa Mae T.
Catindoy BS Medical Technology 3A

PRELIMS [LECTURE III]: AUBF: Chemical examination of urine
Reagent strip
- Used to perform the routine chemical tests on urine.

- Strips consist of chemical-impregnated absorbent pads on a plastic strip
- Test performed for:
o pH o Bilirubin
o Protein o Urobilinogen
o Glucose o Nitrite
o Ketones o Leukocyte esterase
o Blood o Specific gravity
**NOTE: Wait for 60-120 seconds for the results. Mainly because, all parameter except
leukocyte esterase has to be waited for 120 seconds.
- Single and multi-test strips available.

- 2 major types of reagent strips: Multistix and Chemstrip.
- Some variations occur between the strips with regard to sensitivity and specificity
and interfering substances; users should be familiar with the product literature
- Used with automated instruments readers.
o Principle: “Reflectance photometry”
- Color comparison charts are supplied by the manufacturer.
- Several degrees of color are shown to provide semi-quantitative readings of neg, trace,
1+, 2+, 3+, and 4+.
- Estimates of mg/dL are also provided for many of the test areas.
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• Remove excess urine by touching edge of strip to container as strip is withdrawn.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.
• Compare color reaction to manufacturer’s chart under good lighting.
• Hold the strip horizontally when comparing colors.
Improper technique errors:
• RBCs and WBCs sink to the bottom of an unmixed specimen.

• Enzyme reactions on strip are based on room temperature readings.
• Reagents will leach off a strip remaining in the urine too long - dip briefly into specimen.
• Excess urine on the strip will cause runover of reagents among the pads.
• The amount of time for reactions to occur is specified by the manufacturer; leukocyte
esterase is the longest at 2 minutes.
Handling and storage of the strips:
• Store with desiccant in an opaque, tightly closed container—pads are very hydroscopic.
• Store below 30°C, do not freeze.
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Chrissa Mae T. Catindoy BS Medical Technology 3A
• Do not expose to volatile fumes.
• Do not use past the expiration date.
• Do not use if pads are discolored.
• Remove strips immediately prior to use.
Quality control:
• Run positive and negative controls at least once per 24 hours or on each shift.
• Run controls when a new bottle of strips is opened, results are questionable, or there
is concern over strip integrity.
• Record control results.
• Manufactured positive and negative controls are available
• Do not use distilled water as a negative control as reactions are designed for urine
ionic concentration
• All negative control readings should be negative
• Positive control readings should agree with published control values by +/- one color
block
• Be aware of manufacturer-stated limitations and interfering substances
• Relate chemical readings to each other and physical and microscopic readings
• Liquid and tablet backup tests are available for some reactions
• They must also have positive and negative controls performed whenever these tests are
required:
o Protein: 3% sulfosalycilic acid (SSA) test/acidify specimen
o Galactose: Clinitest
o Ketones: Acetest
o Bilirubin: Ictotest (primary confirmatory test)
Urine pH
- Normal: 4.5–8.0
- Kidneys are major regulator of acid-base balance.
o Acidosis – there is low pH, excreted the acid with the urine to elevate the pH.
o Alkalosis – retain the acid so that the alkaline pH becomes acidic.
- Ideal specimen: First morning specimen normally acidic 5–6.
- Postprandial specimen more alkaline – alkaline tide.
**NOTE: We need to learn alkaline tide mainly because for urobilinogen measurement. 12nn-
4pm best to measure urobilinogen, because it is the highest effect on alkaline tide.
**NOTE: Alkaline tide is encountered not only after meal but also during at night.
- No absolute values are assigned.

- Normal fresh urine cannot reach pH 9.
o If so, it indicates old specimen or bacteria-contaminated urine.
**NOTE: Bacteria will consume the urea present therefore it becomes ammonia.

- Diet and medication regulation:
o Meat = acid pH
o Vegetables = alkaline pH
**NOTE: Exception in vegetables cranberry juice – acidic in nature.
o Medications for urinary tract infection

▪ Maintain an acid pH
**NOTE: Urine test for UTI mainly because the urine is alkaline store in bladder, they will give
their patient any acidic, to kill the bacteria, and maintain the acidity of the urine.
Summary of clinical significance of urine pH:
• Respiratory or metabolic acidosis/ketosis or alkalosis.

• Defects in renal tubular secretion and reabsorption of acids and bases – renal tubular
acidosis.
o There is a consistent alkaline in urine due to impaired renal capacity to excrete
acid in the urine. Acidic blood caused by the tubules in the kidney.
• Renal calculi formation.
**NOTE: Acidic urine characteristic of the stone and exposed to alkaline urine, it will
disintegrate.
• Treatment of urinary tract infections.
**NOTE: Urine pH is mostly alkaline.
• Precipitation/identification of crystals.
**NOTE: Amorphous urates seen in an acidic urine, whilst, amorphous phosphates seen in
an alkaline urine.
• Determination of unsatisfactory specimens.
Reagent strip reactions:
- Principle (pH): “Double indicator reaction”

- Needed to measure between 5 and 9.
o Methyl red: 4-6 pH – red/orange-yellow (positive)
▪ Red or orange – exposed in acidic environment.
▪ Yellow – exposed in alkaline environment.
o Bromthymol blue: 6-9 pH – green-blue (negative)
▪ Yellow – exposed in acidic environment.
▪ Green or Blue – exposed in alkaline environment.
𝑀𝑒𝑡ℎ𝑦𝑙 𝑟𝑒𝑑 + 𝐻 + → 𝐵𝑟𝑜𝑚𝑡ℎ𝑦𝑜𝑙 𝑏𝑙𝑢𝑒 − 𝐻 +
(𝑅𝑒𝑑/𝑂𝑟𝑎𝑛𝑔𝑒 → 𝑌𝑒𝑙𝑙𝑜𝑤) (𝑌𝑒𝑙𝑙𝑜𝑤 → 𝐺𝑟𝑒𝑒𝑛/𝐵𝑙𝑢𝑒)
**NOTE: MacConkey agar (neutral red) if exposed in acidic becomes red or pink, and if expose
to alkaline becomes colorless. If the microorganism is lactose fermenter becomes acid.
- Interference:
o Only runover between acid from protein pad.
Urine Protein
- Normal: <10mg/dL or 100mg/24 hr

- Proteinuria seen in early renal disease.
- Low molecular weight serum proteins are filtered – it will become negative; many are
reabsorbed.
- May cause false-positive result:
o Vaginal proteins o Semen proteins
o Prostatic proteins
- Most sensitive in albumin is primary protein of concern, most sensitive and indicative of
renal disease.
**NOTE: Mainly because, albumin cannot pass through on glomerulus, it can only pass the
glomerulus if it is damaged.
Clinical significance:
- Proteinuria – 30mg/dL or 300mg/24 hr

- Variety of causes:
o Prerenal proteinuria
o Renal proteinuria
o Postrenal proteinuria
Prerenal proteinuria (in blood)
- Conditions affecting the plasma, not the kidney.

- Before the blood goes to the kidney it already has a lot of protein.
- Transient, increase levels of low molecular weight plasma proteins, acute phase
reactants, exceed re-absorptive capacity.
o + APR: elevated proteins when you have an acute inflammation or fever.
o – APR: decreased proteins when you have an acute inflammation or fever.
- Rarely seen on reagent strip (not albumin).
Bence Jones protein (BJP)
- Multiple myeloma
- Overproduction of immunoglobulin light chains of plasma cells.
- Screening test (Presumptive test): coagulates between 40-60°C and dissolves at 100°C.
- Heat and observe turbidity.
- Confirmatory test: serum electrophoresis.
Prerenal tubular disorders:
• Intravascular hemolysis • Acute phase reactants

• Muscle injury • Multiple myeloma
**NOTE: Myoglobin is a protein in muscles.

**NOTE: Hemoglobin is a protein causing prerenal proteinuria and positive in blood in
reagent strip.
Renal proteinuria (in kidney)
- Kidney maybe damaged.
Glomerular proteinuria
- Damage to glomerular membrane.

- Impaired selective filtration causes increased protein filtration.
- Abnormal substances deposit on the membrane.
**NOTE: Diabetes mellitus there is a large deposit of glucose on the glomerular basement
membrane.
o Primarily immune disorders result in immune complex formation.

▪ Systematic lupus erythematosus
▪ Streptococcal glomerulonephritis
- Increased pressure on the filtration mechanism.
o Hypertension
▪ Major regulator is blood pressure.
o Strenuous exercise
▪ Liberation of myoglobin or widening of pores in the glomerulus.
o Dehydration
o Pregnancy (Preeclampsia)
▪ High blood pressure, which pushes the blood proteins in the filter of
glomerulus.
- Benign proteinuria (transient)
o Orthostatic proteinuria
o Exposure to:
▪ Cold ▪ Dehydration
▪ Exercise ▪ High fever
- Orthostatic proteinuria
o Benign (increased pressure on renal vein).
o Occurs in vertical position, disappears in horizontal position.
o Frequently picked up on random specimen.
o Empty bladder before bed.
o Collect specimen immediately on arising.
o Specimen will be negative for protein.
Glomerular disorders:
• Immune complex disorders • Strenuous exercise

• Amyloidosis • Dehydration
• Toxic agents • Hypertension
• Diabetic nephropathy • Preeclampsia

• Orthostatic or postural proteinuria
**NOTE: Serum amyloid A deposited in the glomerular basement membrane.
Tubular proteinuria
- Tubular damage affecting re-absorptive ability.

o Acute tubular necrosis
▪ Toxic substances ▪ Viral infections
▪ Heavy metals ▪ Fanconi syndrome
**NOTE: Major cause of Acute tubular necrosis is – hypoxia. Hypoxia there a depletion or
decreased oxygen supply in our kidneys.
**NOTE: Fanconi syndrome generalized proximal convoluted tubule reabsorption defect.
- Amount of protein.
o Glomerular: up to 4g/day
o Tubular: much lower levels
Postrenal proteinuria (in urinary tract)
- Protein added in the lower urinary and genitourinary tract.

o Ureters o Urethra
o Bladder
- Microbial infections causing inflammations (edema) and release of interstitial fluid
protein.
o Menstrual contamination. o Vaginal secretions.
o Semen/Prostatic fluid. o Traumatic injury.
- Principle (protein): “Protein error of indicators”

- Certain indicators change color in the presence of protein at a constant pH.
- Protein, primarily albumin potently accepts H+ from the indicator.
- Most sensitive to albumin because albumin has more amino groups to accept H+ than
other proteins.
- Present on the reagents:
o Tetra-bromo-phenol blue
o Tetra-chloro-phenol or tetra-brom-sulfon-phthalein
▪ Yellow – in acidic environment.
▪ Blue or green – in alkaline environment.
o Acid buffer
• pH: 3 both indicators are yellow.
• Color: progresses through green-blue.
• Report: neg, trace, 1+, 2+, 3+, 4+, or 30, 100, 300, 2000mg/dL
• Trace values: <30 mg/dL

𝐼𝑛𝑑𝑖𝑐𝑎𝑡𝑜𝑟 (𝐻 + )[𝑦𝑒𝑙𝑙𝑜𝑤] + 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 (𝑝𝐻 3.0) → 𝑃𝑟𝑜𝑡𝑒𝑖𝑛 + 𝐻 +
→ 𝐼𝑛𝑑𝑖𝑐𝑎𝑡𝑜𝑟 (𝑔𝑟𝑒𝑒𝑛/𝑏𝑙𝑢𝑒) − 𝐻 +
Reaction interference:
• Highly buffered alkaline urine overrides acid buffer system.

• Leaving reagent pad in urine too long removes buffer.
• Highly pigmented urine.
• Quaternary ammonium compounds, detergents, antiseptics, chlorhexidine.
• False-negatives: for proteins other than albumin.
• False-positive: trace from high specific gravity.
Sulfosalicylic acid confirmation (SSA)
- Not very common now due to instrumentation.

- SSA precipitates proteins without heat.
- Use centrifuged specimen.
- Compare against standards for 1+, 2+, 3+, 4+.
- Other substances precipitated or interferences:
o Radiographic contrast dye o Tolbutamide
o Antibiotics
Microalbuminurea
- Benign proteinuria.
- Newest testing.
- Diabetic nephropathy.
- Renal failure common in diabetics.
- Control hypertension and glucose levels.
- Early screening for albumin levels lower than seen on routine reagent strip.
- Increased risk of cardiovascular disease.
- Previously required 24-hour collection.
- Reported: mg/24 hr of albumin
Micral test (MT)
- Gold-labeled anti-human antibody-enzyme conjugate.

- Dip strip in urine to marked level for 5 seconds.
- Albumin binds to antibody.
- Bound and unbound conjugates move up strip.
o Unbound removed in captive zone containing albumin.
o Bound continues up strip.
- Reaches enzyme substrate, reacts.
- Colors: white (negative) – red (varying degrees)
- Compare color to chart.
- Results: from 0-10 mg/dL

Immunodip test (IDT)
- Immuno-chromographic technique.
- Specially designed container for strip.
- Place container in controlled amount of specimen for 3 min, urine enters container.
- Albumin binds to blue latex particles coated with anti-human albumin-antibody.
- Bound and unbound migrate up strip.
o Bound continues migrating to an area of immobilized antibody and forms blue
band.
o Unbound encounters area of immobilized albumin on strip and forms blue band.
- Color of band is compared with chart.
o Darker bottom band (unbound ab): <1.2 mg/dL albumin
o Equal band: 1.2–1.8 mg/dL
o Darker top band (bound ab): 2.0–8.0 mg/dL albumin
Microalbumin test (MAT)
- Clinitek microalbumin reagent strips and Multistix Pro reagent strips.

- Simultaneous measurement of albumin and creatinine.
**NOTE: Creatinine measured to see if it is urine and completed the 24-hour collection.
- Provide an estimate of the 24-hour albumin concentrations from random urine.

- Albumin pad uses dye-binding reaction for specific albumin testing.
Albumin Strip Dye
- Bis(3’,3”,diodo-4’,4”-dihydroxy-5’,5”-dinitrophenyl)-3,4,5,6-tetra-bromo-sulphon-
phthalein.
- Specific: albumin
- Sensitivity: 8–20 mg/dL
- Highly buffered alkaline urine interference is controlled by treated paper.
- Polymethyl vinyl glycol carbonate decreases nonspecific binding of poly-amino
acids.
- Results: 10, 30, 80,150 mg/dL
- Abnormal: 30, 80,150 mg/dL
- Visibly bloody urine elevates results
- Abnormally colored urines may interfere with readings.
Creatinine Strip Dye
- Principle: pseudoperoxidase activity of copper-creatinine complexes.

o Copper sulfate (CuSO4,3,3’,5,5’-tetramethylbenzidine) (TMB)
o Diisopropyl benzene dihydroperoxide (DBDH)
- Creatinine in urine combines with copper sulfate to form peroxidase
- Peroxidase reacts with DBDH, releases O2 that oxidizes TMB
- Colors: orange → green → blue

𝐶𝑢 (𝐶𝑅𝐸)𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒 𝐷𝐵𝐷𝐻 + 𝑇𝑀𝐵
𝐶𝑢𝑆𝑂4 + 𝐶𝑅𝐸 → →
(𝑃𝑒𝑟𝑜𝑥𝑖𝑎𝑑𝑠𝑒)
[DBDH + TMB (𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒)]→ 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝑇𝑀𝐵 (𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛) + 𝐻2 𝑂 = 𝑐𝑜𝑙𝑜𝑟 𝑐ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 𝑇𝑀𝐵
- Results: 10, 50, 100, 200, 300 mg/dL or 0.9, 4.4, 8.8, 17.7, 26.5 mmol/L
- No creatinine results are abnormal
- Purpose: determine concentration of random urine
- Correlate creatinine with albumin results to determine the albumin:creatinine ratio
- Elevated: bloody urine, Tagamet (cimetidine), abnormal urine color
Albumin : Creatinine ratio (A:C ratio)
- Automated and manual methods available

- Clinitek microalbumin strips can be read only on Clinitek instruments
o Instrument calculates A:C ratio and prints out albumin, creatinine, and A:C
results
o Results: conventional and SI units
o Abnormal A:C ratio: 30–300 mg/g or 3.4–33.9 mg/mol
- Bayer Multistix Pro 11 strips measure creatinine, protein-high and protein-low
o Protein-high is traditional protein method
o Protein-low is new albumin-dye-binding method
o Urobilinogen is not included on these strips
o Read manually or on instrumentation
o Print-out is protein:creatinine ratio with albumin result included on print-out
- A chart is available for manual calculation
- Specimens with a creatinine reading of 10 are too dilute to interpret; recollect
- Normal: 80 mg albumin/g creatinine or <300 mg/protein/g creatinine
- Result: 15 mg/dL albumin is indicative of clinical albuminuria
Urine glucose
- Clinical significance
o Major screening test for diabetes mellitus
o Renal threshold: 160–180 mg/dL
o Higher blood sugar = glycosuria
- Gestational diabetes
o Placental hormones block action of insulin
▪ High fetal glucose stresses baby’s pancreas
▪ Result: fat baby
▪ Mother prone to type 2 diabetes
- Nondiabetic glycosuria
o Hormonal disorders:
▪ Pancreatitis ▪ Cushing’s syndrome
▪ Pancreatic cancer ▪ Hyperthyroidism
▪ Acromegaly ▪ Pheochromocytoma

o Hormones:
▪ Glucagon ▪ Thyroxine
▪ Epinephrine ▪ Growth hormone
▪ Cortisol oppose glucose
o Insulin: converts glucose to storage glycogen
o Hormones: glycogen back to glucose
o Epinephrine: inhibits insulin; seen with stress, cerebral trauma, and
myocardial infarction
- Renal glycosuria
o Tubular reabsorption disorder
o End-stage renal disease
o Fanconi syndrome
o Temporary lowering of renal threshold in pregnancy
Clinical significance of urine glucose:
• Hyperglycemia-Associated • Central nervous system damage

• Diabetes mellitus • Stress
• Pancreatitis • Gestational diabetes
• Pancreatic cancer • Renal-Associated
• Acromegaly • Fanconi syndrome
• Cushing syndrome • Advanced renal disease
• Hyperthyroidism • Osteomalacia
• Pheochromocytoma • Pregnancy
- Glucose oxidase reaction specific for glucose

- Double sequential enzyme reaction
- On test pad:
o Glucose oxidase o Chromogen
o Peroxide o Buffer
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 𝑜𝑥𝑖𝑑𝑎𝑠𝑒
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂2 → → 𝐺𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝐻2 𝑂2
𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒
𝐻2 𝑂2 + 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 → → 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝑐𝑜𝑙𝑜𝑟𝑒𝑑 𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 + 𝐻2 𝑂
- Neg, trace, 1+, 2+, 3+, 4+

- 100 mg/dL-2 g/dL
- 0.1–2%
- Only false-positive: peroxide, oxidizing detergents

- Negative: enzymatic reaction interference:
o Ascorbic acid and strong reducing agents
o High levels of ketones (unlikely)
o High specific gravity and low temperature
o Biggest error is old specimens due to glycolysis
Copper reduction test (CRT)
- Reduction of copper sulfate to cuprous oxide with alkali and heat

- Clinitest tablets:
o Copper sulfate
o Sodium carbonate
o Sodium citrate
o Sodium hydroxide
𝑆𝑜𝑑𝑖𝑢𝑚 𝑐𝑖𝑡𝑟𝑎𝑡𝑒 + 𝑁𝑎𝑂𝐻 = 𝑯𝒆𝒂𝒕
𝑺𝒐𝒅𝒊𝒖𝒎 𝒄𝒂𝒓𝒃𝒐𝒏𝒂𝒕𝒆 = 𝑪𝑶𝟐 𝒃𝒍𝒐𝒄𝒌𝒔 𝒓𝒐𝒐𝒎 𝒂𝒊𝒓
𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 + 𝐶𝑢𝑆𝑂4 = 𝑪𝒐𝒍𝒐𝒓
𝐴𝑙𝑘𝑎𝑙𝑖
𝐶𝑢𝑆𝑂4 (𝐶𝑢𝑝𝑟𝑖𝑐 𝑠𝑢𝑙𝑓𝑖𝑑𝑒) + 𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 →
𝐻𝑒𝑎𝑡
𝐶𝑢𝑆𝑂4 (𝐶𝑢𝑝𝑟𝑖𝑐 𝑠𝑢𝑙𝑓𝑖𝑑𝑒) + 𝑂𝑥𝑖𝑑𝑖𝑧𝑖𝑛𝑔 𝑠𝑢𝑏𝑠𝑡𝑎𝑛𝑐𝑒 (𝑏𝑙𝑢𝑒/𝑔𝑟𝑒𝑒𝑛 )→ 𝐶𝑜𝑙𝑜𝑟 (𝑜𝑟𝑎𝑛𝑔𝑒/𝑟𝑒𝑑)
Clinitest procedure
- Place a glass test tube in a rack, add 5 drops of urine

- Add 10 drops of distilled water to the urine in the test tube
- Drop 1 Clinitest tablet into the test tube, and observe the reaction until completion
(cessation of boiling)
- CAUTION: The reaction mixture gets very hot; do not touch the bottom area of the test
tube; use glass test tube only
- Wait 15 sec after boiling has stopped, and gently shake the contents of the tube
- Compare the color of the mixture to the Clinitest color chart, and record the result in
mg/dL or %
- Observe for the possibility of the “pass-through” phenomenon
- Repeat, using the two-drop procedure
- Pass through
o High levels of reducing substance
o Color from blue through red back to green-brown. Rapid reaction
o Repeat with two-drop procedure
▪ 10 drops water
▪ 2 drops urine
▪ Values up to 5 g/L vs. 2 g/L
- Sensitivity: 200 mg/dL (lower) than strip
- Hygroscopic tablets: strong blue color and excess fizzing = deterioration
- Not a specific test for glucose

- Reducing substances:
o Galactose o Pentose
o Lactose o Ascorbic acid
o Fructose o Cephalosporin
o Maltose
- Major use is quick screen for galactosemia in newborns
- Newborn screening programs using blood are being incorporated
Summary of Glucose oxidase and Clinitest reactions:
Glucose oxidase Clinitest Interpretation

1+ positive Negative Small amount of glucose present
4+ positive Negative Possible oxidizing agent
Non-glucose reducing, possible interfering
Negative Negative
substance for reagent strip
Urine ketones
- Three intermediate products of fat metabolism

o Acetone
o Acetoacetic acid
o -hydroxybutyric acid
- Appear in urine when fat is broken down to glucose for energy
• Primary causes:
o Diabetes mellitus
o Vomiting (loss of carbohydrates)
o Starvation, malabsorption, dieting (decrease intake)
- Ketonuria shows inadequate insulin
o Monitor diabetes
o Can result in Diabetic ketoacidosis
- Hospitalized patients are often positive
- Illness = decrease intake, poor absorption
- Measure primarily acetoacetic acid

o Acetone in presence of glycine (Chemstrips)
- Primary reagent: sodium nitroprusside
o Nitroferricyanide
𝐴𝑐𝑒𝑡𝑜𝑎𝑐𝑒𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝑁𝑖𝑡𝑟𝑜𝑝𝑟𝑢𝑠𝑠𝑖𝑑𝑒 → 𝑃𝑢𝑟𝑝𝑙𝑒
- Report:
o Neg
o Small (1+) or 5mg/dL, 15mg/dL

o Moderate (2+) or 40mg/dL
o Large (3+) or 80mg/dL or 160mg/dL
Acetest
- Not a confirmatory test

- Can perform serial dilutions and use serum
- Tablet = sodium nitroprusside, glycine, disodium phosphate, lactose (gives better color)
𝐴𝑐𝑒𝑡𝑜𝑎𝑐𝑒𝑡𝑎𝑡𝑖𝑐 𝑎𝑐𝑖𝑑 + 𝑆𝑜𝑑𝑖𝑢𝑚 𝑛𝑖𝑡𝑟𝑜𝑝𝑟𝑢𝑠𝑠𝑖𝑑𝑒 + 𝐺𝑙𝑦𝑐𝑖𝑛𝑒 → 𝑃𝑢𝑟𝑝𝑙𝑒
Acetest procedure:
- Remove the Acetest tablet from the bottle, and place on a clean, dry piece of white
paper
- Place one drop of urine on top of the tablet
- Wait 30 seconds
- Compare the tablet color with the manufacturer-supplied color chart
- Report as negative, small, moderate, or large
- Phthalein dyes in medication, red-colored urine

- Medications with sulfhydryl groups
- False-positives from too long timing
- False-negative from old specimens
o Volatilization and bacterial breakdown of acetoacetic acid
Urine blood
- Reagent strip is more accurate than microscopic for detecting blood

• Hematuria: intact RBCs, cloudy red urine
o Renal calculi o Trauma
o Glomerulonephritis o Exposure to toxic chemicals
o Pyelonephritis o Anticoagulants
o Tumors o Strenuous exercise
• Hemoglobinuria: clear, red urine
o Transfusion reactions o Strenuous exercise
o Hemolytic anemias o RBC trauma
o Severe burns o Brown recluse spider bites
o Infections/malaria
- No RBCs = hemolysis; mixture = lysis
• Hemosiderin: yellow brown granules in sediment
• Myoglobinuria: heme containing protein in muscle tissue; clear, red/brown urine
o Muscular trauma or crush o Drug abuse
syndromes o Extensive exertion
o Prolonged coma o Cholesterol-lowering statin
o Convulsions or trauma medications
o Muscle-wasting diseases o Rhabdomyolysis: muscle
o Alcoholism or overdose destruction
Hemoglobinuria vs. Myoglobinuria
- Both produce clear, red urine

- Both are toxic to renal tubules
- Hemoglobin causes red plasma; whilst myoglobin plasma is clear
- Increased CK enzymes with myoglobin
- Urine separation:
o Myoglobin + NH4SO4 = red supernatant
o Hemoglobin + NH4SO4 = clear supernatant
- Principle: pseudoperoxidase activity of hemoglobin
𝐻𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛
𝐻2 𝑂2 + 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 → → 𝑜𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛 + 𝐻2 𝑂
𝑃𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒
- Intact RBCs show a speckled pattern

- Report:
o Trace
o Small (1+)
o Moderate (2+)
o Large (3+)
- Sensitivity 5 RBCs/μL
Reagent interference:
- False-positive: strong oxidizing agents, bacterial peroxidases, or menstrual

contamination
- False-negative: High specific gravity/crenated cells
- Formalin
- Captopril
- High concentrations of nitrite
- Ascorbic acid >25 mg/dL
- Unmixed specimens

Urine bilirubin
- Urine bilirubin early indicator of liver disease

- RBCs → RE system → protoporphyrin → bilirubin (unconjugated bound to albumin)
circulates to liver → conjugated bilirubin → bile duct → intestine → stercobilinogen
+ urobilinogen
- Stercobilinogen → urobilin → feces
- Urobilinogen → blood → liver (passes through kidney and some is excreted)
- Conjugated bilirubin appears in urine with bile duct obstruction and liver disease
- 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 = jaundice no urine bilirubin,
increased urobilinogen
Reagent strip reaction:
- Principle: A diazo reaction
𝐴𝑐𝑖𝑑
𝐵𝑖𝑙𝑖𝑟𝑢𝑏𝑖𝑛 𝑔𝑙𝑢𝑐𝑢𝑟𝑜𝑛𝑖𝑑𝑒 + 𝑑𝑖𝑎𝑧𝑜𝑛𝑖𝑢𝑚 𝑠𝑎𝑙𝑡 → → 𝐴𝑧𝑜𝑑𝑦𝑒
- Diazonium salt: tan shades, or pink-violet

- Report: neg, small (1+), moderate (2+), large (3+)
- Colors may be difficult to interpret
- Atypical colors can be problem for automated readers

Ictotest
- Confirmatory for bilirubin

- More sensitive: 0.05–0.10 mg/dL
- Reagent strips: 0.40 mg/dL
- May be requested for early disease
- Use specified mat for test; mat keeps bilirubin on surface for reaction
Ictotest procedure:
- Place 10 drops of urine onto one square of the absorbent test mat
- Using forceps, remove one Ictotest reagent tablet, recap the bottle promptly, and place
the tablet in the center of the moistened area
- Place 1 drop of water onto the tablet, and wait 5 sec
- Place a 2nd drop of water onto the tablet so that the water runs off the tablet onto the
mat
- Observe the color of the mat around the tablet at the end of 60 sec. The presence of a
blue-purple color on the mat indicates that bilirubin is present; a slight pink or red
color should be ignored; report as positive or negative
- False-positive: urine pigments, pyridium, indican, Lodine

- False-negative: old specimens (biliverdin does not react), increased ascorbic acid and
nitrite because they combine with diazonium salt and block bilirubin reaction
Urine urobilinogen
- Bilirubin in intestine converted to urobilinogen and stercobilinogen

- Urobilinogen is reabsorbed into circulation and stercobilinogen is not = urobilin
- There is always a small amount of urobilinogen in the urine <1 mg/dL
- Early detection of liver disease

- Liver disorders, hepatitis, cirrhosis, carcinoma
- Hemolytic disorders
o Excess bilirubin being converted to urobilinogen and increased urobilinogen
recirculated to liver
- Negative bilirubin and strong positive urobilinogen are seen in hemolytic disorders
- No urobilinogen is seen in the urine with bile duct obstruction; strip will give a normal
result
- There cannot be a negative urobilinogen reading from a reagent strip
Urine bilirubin and urobilinogen in Jaundice:
Urine bilirubin Urobilinogen

Bile duct obstruction +++ Normal
Liver damage + or - ++

Hemolytic disease Negative +++
- Different principles for Multistix and Chemstrip

- Multistix: p-dimethylaminobenzaldehyde (Ehrlich reagent)
o Report in Ehrlich units (EU) 1 EU = 1 mg/dL
- Normal: 0.2–1
- Abnormal: 2, 4, 8
- Chemstrip: 4-methoxybenzen-diazonium-tetrafluoroborate
o Diazo reaction
o More specific than Ehrlich reaction
o Report in mg/dL
- Ehrlich reactive compounds: porphobilinogen, indican, sulfonamides, methyldopa,

procaine, chlorpromazine, p-aminosalicylic acid
- Both tests: urobilinogen is highest after meals (increased bile salts), old specimens
and formalin preservation decrease results
- Chemstrip: false-negative with high nitrite interferes with diazo reaction
Ehrlich tube test (ETT)
- Differentiation among urobilinogen, porphobilinogen, and Ehrlich reactives

- Addition of Ehrlich reagent to urine produces a cherry red color enhanced by sodium
acetate
- Test is modified to differentiate urobilinogen, porphobilinogen, and Ehrlich reactive
compounds
- Tubes with red color
- Add chloroform; shake
- Add butanol to second tube; shake
- Urobilinogen extracted into both
- Porphobilinogen extracted into none
- Reactive extracted into butanol
Hoesch Screening test (HST)
- For acute porphyria

- Hoesch reagent: Ehrlich reagent in 6 M HCl
- 2 drops urine in 2 mL Hoesch reagent
- Immediate red color on top and throughout when shaken
- High pH inhibits urobilinogen
- False-positive: methyldopa, indican, pigmented urine

Urine nitrite
- Clinical significance:
o Cystitis (bladder)
o Pyelonephritis (tubules)
o Evaluation of antibiotic therapy
o Monitoring of patients at high risk for urinary tract infection
o Screening of urine culture specimens (in combination with LE test)
Reagent strip reaction:
- Tests ability of bacteria to reduce nitrate to nitrite

- Greiss reaction: nitrite reacts with aromatic amine to form a diazonium salt that then
reacts with tetrahydrobenzoquinoline to form a pink azodye
- Sensitive for 100,000 organisms/mL
- Results: negative and positive
𝐴𝑐𝑖𝑑
𝑃𝑎𝑟𝑎 − 𝑎𝑟𝑠𝑎𝑛𝑖𝑙𝑖𝑐 𝑎𝑐𝑖𝑑 𝑜𝑟 𝑠𝑢𝑙𝑓𝑜𝑎𝑛𝑖𝑙𝑎𝑚𝑖𝑑𝑒 + 𝑁𝑂2 → → 𝐷𝑖𝑎𝑧𝑜𝑛𝑖𝑢𝑚 𝑠𝑎𝑙𝑡
𝐴𝑐𝑖𝑑
𝐷𝑎𝑖𝑧𝑜𝑛𝑖𝑢𝑚 𝑠𝑎𝑙𝑡 + 𝑇𝑒𝑡𝑟𝑎ℎ𝑦𝑑𝑟𝑜𝑏𝑒𝑛𝑧𝑜𝑞𝑢𝑖𝑛𝑜𝑙𝑖𝑛𝑒 → → 𝑃𝑖𝑛𝑘 𝑎𝑧𝑜𝑑𝑦𝑒
- False-negative:
o Nonreductase-containing bacteria
o Insufficient contact time between bacteria and urinary nitrate
o Lack of urinary nitrate
o Large quantities of bacteria converting nitrite to nitrogen
o Presence of antibiotics
o High concentrations of ascorbic acid
o High specific gravity
- False-positive:
o Old specimens (bacterial multiplication)
o Highly pigmented urine
o Pink edges or spotting on reagent strip is considered negative
o Check automated readers manually for color interference
Urine leukocyte esterase (LE)
- Purpose is to detect leukocytes so as not to rely on microscopic

- Not considered a quantitative test – do microscopic if positive
- Advantage: detects presence of lysed leukocytes
- Bacterial and nonbacterial urinary tract infection

- Inflammation of the urinary tract

- Screening of urine culture specimens in conjunction with nitrite but a better predictor
than nitrite
- Also seen with Trichomonas, Chlamydia, yeast, interstitial nephritis
- LE catalyzes hydrolysis of acid esterase on pad to aromatic compound and acid.

Aromatic compound reacts with diazonium salt on pad for purple color
𝐿𝑒𝑢𝑘𝑜𝑐𝑦𝑡𝑒
𝐼𝑛𝑑𝑜𝑥𝑦𝑙𝑐𝑎𝑟𝑏𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 𝑒𝑠𝑡𝑒𝑟 → → 𝐼𝑛𝑑𝑜𝑥𝑦𝑙 + 𝐴𝑐𝑖𝑑 𝑖𝑛𝑑𝑜𝑥𝑦𝑙
𝐸𝑠𝑡𝑒𝑟𝑎𝑠𝑒𝑠
𝐴𝑐𝑖𝑑
+ 𝐷𝑖𝑎𝑧𝑜𝑛𝑖𝑢𝑚 𝑠𝑎𝑙𝑡 → → 𝑃𝑢𝑟𝑝𝑙𝑒 𝑎𝑧𝑜𝑑𝑦𝑒
- False-positive:
o Strong oxidizing agents
o Formalin
o Highly pigmented urine, nitrofurantoin
- False-negative:
o High concentrations of protein, glucose, oxalic acid, ascorbic acid,
gentamicin, cephalosporins, tetracyclines
o Crenation from high specific gravity
o Inaccurate timing - must have 2 min
Urine specific gravity
- Based on pKa (dissociation constant) of a polyelectrolyte in alkaline medium

- Polyelectrolyte ionizes releasing H+ in relation to concentration of urine
- Increased concentration = more H+ released
- Indicator bromthymol blue measures pH change
- Blue (alkaline) through green-yellow (acid)

- 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.


PH Protein Glucose Ketones Blood Bilirubin Urobilinogen Nitrite Leukocyte Esterase Specific Gravity

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Chrissa Mae T.

Catindoy BS Medical Technology 3A

- Used to perform the routine chemical tests on urine.

- Single and multi-test strips available.

• Dip strip briefly into well-mixed specimen at room temperature.

Improper technique errors:

• RBCs and WBCs sink to the bottom of an unmixed specimen.

Handling and storage of the strips:

- No absolute values are assigned.

DO NOT COPY WITHOUT PERMISSION

**NOTE: Exception in vegetables cranberry juice – acidic in nature.

o Medications for urinary tract infection

Summary of clinical significance of urine pH:

• Respiratory or metabolic acidosis/ketosis or alkalosis.

• Treatment of urinary tract infections.

**NOTE: Urine pH is mostly alkaline.

• Determination of unsatisfactory specimens.

Reagent strip reactions:

- Principle (pH): “Double indicator reaction”

𝑀𝑒𝑡ℎ𝑦𝑙 𝑟𝑒𝑑 + 𝐻 + → 𝐵𝑟𝑜𝑚𝑡ℎ𝑦𝑜𝑙 𝑏𝑙𝑢𝑒 − 𝐻 +

(𝑅𝑒𝑑/𝑂𝑟𝑎𝑛𝑔𝑒 → 𝑌𝑒𝑙𝑙𝑜𝑤) (𝑌𝑒𝑙𝑙𝑜𝑤 → 𝐺𝑟𝑒𝑒𝑛/𝐵𝑙𝑢𝑒)

- Normal: <10mg/dL or 100mg/24 hr

- Proteinuria – 30mg/dL or 300mg/24 hr

Prerenal proteinuria (in blood)

- Conditions affecting the plasma, not the kidney.

Bence Jones protein (BJP)

Prerenal tubular disorders:

• Intravascular hemolysis • Acute phase reactants

**NOTE: Myoglobin is a protein in muscles.

Renal proteinuria (in kidney)

- Kidney maybe damaged.

- Damage to glomerular membrane.

o Primarily immune disorders result in immune complex formation.

• Immune complex disorders • Strenuous exercise

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- Tubular damage affecting re-absorptive ability.

**NOTE: Fanconi syndrome generalized proximal convoluted tubule reabsorption defect.

Postrenal proteinuria (in urinary tract)

- Protein added in the lower urinary and genitourinary tract.

Reagent strip reactions:

- Principle (protein): “Protein error of indicators”

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• Highly buffered alkaline urine overrides acid buffer system.

Sulfosalicylic acid confirmation (SSA)

- Not very common now due to instrumentation.

Micral test (MT)

- Gold-labeled anti-human antibody-enzyme conjugate.

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Microalbumin test (MAT)

- Clinitek microalbumin reagent strips and Multistix Pro reagent strips.

- Provide an estimate of the 24-hour albumin concentrations from random urine.

Albumin Strip Dye

Creatinine Strip Dye

- Principle: pseudoperoxidase activity of copper-creatinine complexes.

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[DBDH + TMB (𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒)]→ 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝑇𝑀𝐵 (𝑐ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛) + 𝐻2 𝑂 = 𝑐𝑜𝑙𝑜𝑟 𝑐ℎ𝑎𝑛𝑔𝑒 𝑖𝑛 𝑇𝑀𝐵

Albumin : Creatinine ratio (A:C ratio)

- Automated and manual methods available