Reagent Strip

We’ve looked at the urine with our eyes, we’ve measured its concentration, and now it’s time to unleash the workhorse of the urinalysis lab: the reagent strip

Think of the reagent strip as the clinical laboratory’s multi-tool or Swiss Army knife. It’s a plastic strip embedded with chemically-impregnated pads that gives us a rapid, semi-quantitative snapshot of ten different chemical markers. This simple tool is our first line of defense in screening for everything from urinary tract infections to diabetes and kidney disease

But before we can trust our results, we have to perfect our technique. A poorly performed dip can invalidate every result on the strip

Proper Reagent Strip Technique: The Non-Negotiables

1. Check the Expiration Date Never use an expired strip or a container that’s been open too long. The reagents degrade with exposure to light, heat, and humidity
2. Mix the Specimen Gently swirl the urine to ensure a uniform suspension of any formed elements. Don’t centrifuge first!
3. Dip Briefly and Completely Submerge all the pads into the urine, but only for a moment (about 1 second). “Marinating” the strip will cause reagents to leach out of the pads
4. Remove Excess Urine As you withdraw the strip, run the edge against the side of the container. Then, blot the edge of the strip on an absorbent material. This is critical to prevent “run-over,” where chemicals from one pad bleed into the next, causing erroneous results
5. Hold Horizontally While waiting for the reaction times, hold the strip flat to prevent run-over
6. Read at the Correct Time Each reaction has a specific endpoint. Glucose reads at 30 seconds, while leukocytes can take up to 2 minutes. Reading too early or too late will lead to incorrect results. Most automated readers handle this timing perfectly, but it’s essential to know for manual readings
7. Use a Good Light Source Compare the color changes to the chart on the container in well-lit conditions

Now, let’s walk through each pad, one by one

The Reagent Strip Pads: A Detailed Review

pH

• Principle: A double-indicator system (usually methyl red and bromthymol blue) provides a broad range of color changes to measure pH from about 5.0 to 8.5
• Normal Range: A random urine can be slightly acidic, around 6.0, but a range of 4.5-8.0 is considered normal
• Clinical Significance
  • Acidic Urine (pH < 7.0): Seen in metabolic or respiratory acidosis, high protein diets, and with certain medications. An acidic pH is useful for identifying acid-loving crystals (e.g., calcium oxalate, amorphous urates, uric acid)
  • Alkaline Urine (pH > 7.0): Seen in metabolic or respiratory alkalosis, after meals (alkaline tide), with vegetarian diets, and most commonly, in UTIs caused by urea-splitting bacteria (like Proteus spp.). Alkaline pH also indicates an improperly stored specimen, as bacteria will multiply and break down urea into ammonia. This is a good check for specimen integrity!

Protein

• Principle: Based on the “protein error of indicators.” At a fixed, acidic pH (buffered to pH 3.0 on the pad), certain indicators will release H+ ions and change color in the presence of protein (primarily albumin), even though the pH of the solution isn’t actually changing
• Normal Range: Negative to Trace
• Clinical Significance: A positive protein is the single most important indicator of renal disease
  • Proteinuria: The presence of increased protein in the urine
  • Sensitivity: The strip is most sensitive to albumin. It is less sensitive to other proteins like globulins or the Bence Jones protein seen in multiple myeloma
  • Causes: Glomerular damage (glomerulonephritis), nephrotic syndrome, conditions affecting plasma proteins, and some non-pathologic causes like strenuous exercise or postural proteinuria
• Sources of Error
  • False Positive: Highly alkaline urine (pH > 8.0) can override the pad’s acid buffer, causing a color change unrelated to protein
  • Confirmatory Test: A positive protein result is often confirmed with a precipitation test like the Sulfosalicylic Acid (SSA) test, which detects all types of protein

Glucose

• Principle: A double sequential enzyme reaction. Glucose oxidase on the pad reacts with glucose to produce gluconic acid and hydrogen peroxide. Then, peroxidase catalyzes the reaction of that hydrogen peroxide with a chromogen on the pad to produce a color change
• Normal Range: Negative
• Clinical Significance
  • Glycosuria: The presence of glucose in the urine
  • This occurs when the blood glucose level exceeds the renal threshold: for reabsorption (about 160-180 mg/dL)
  • It is a primary screening tool for Diabetes Mellitus
• Sources of Error
  • False Positive: Contamination with strong oxidizing agents, like bleach
  • False Negative: The most common cause is high levels of ascorbic acid (Vitamin C), which is a strong reducing agent that interferes with the peroxidase reaction

Ketones

• Principle: Based on the reaction of acetoacetic acid with sodium nitroprusside in an alkaline medium, which produces a purple color
• Normal Range: Negative
• Clinical Significance
  • Ketonuria: Indicates that the body is metabolizing fat instead of carbohydrates for energy
  • Seen in diabetic ketoacidosis (DKA), a medical emergency. Also found in starvation, low-carbohydrate diets, and with prolonged vomiting
  • Note: The strip primarily detects acetoacetic acid. It is less sensitive to acetone and does not detect the third ketone body, beta-hydroxybutyric acid

Blood

• Principle: Based on the pseudoperoxidase activity of heme. Heme, found in both hemoglobin and myoglobin, acts like the peroxidase enzyme and catalyzes the oxidation of a chromogen, causing a color change
• Normal Range: Negative
• Clinical Significance: Can indicate hematuria, hemoglobinuria, or myoglobinuria
  • Speckled/Spotted Pattern: Suggests intact RBCs have lysed on the pad (hematuria)
  • Uniform Color Change: Suggests the presence of free hemoglobin (hemoglobinuria) or myoglobin (myoglobinuria)
• Sources of Error
  • False Positive: Menstrual contamination, strong oxidizing agents
  • False Negative: High levels of ascorbic acid (Vitamin C), high specific gravity (which can crenate RBCs and prevent lysis)

Bilirubin

• Principle: The diazo reaction. Bilirubin couples with a diazonium salt in an acidic medium to form an azobilirubin complex, which is tan or pink-purple
• Normal Range: Negative
• Clinical Significance
  • Only conjugated (direct) bilirubin: is water-soluble and can appear in the urine
  • Its presence is an early indicator of liver disease: (like hepatitis) or biliary tract obstruction
• Sources of Error & Special Handling
  • False Negative: Bilirubin is very unstable and breaks down upon exposure to light. A specimen waiting for testing must be protected from light
  • Confirmatory Test: The Ictotest is a tablet-based diazo reaction that is more sensitive and specific than the strip test

Urobilinogen

• Principle: Most strips use the classic Ehrlich’s reaction, where urobilinogen reacts with p-dimethylaminobenzaldehyde to form a cherry-red color. Some newer strips use a more specific diazo reaction
• Normal Range: Small amount (0.2 - 1.0 mg/dL or Ehrlich Units). A normal result is a low positive
• Clinical Significance: Correlating bilirubin and urobilinogen is key!
  • Increased Urobilinogen: Seen in hemolytic diseases (increased RBC breakdown leads to more bilirubin, then more urobilinogen) and liver disease (damaged liver can’t re-process the urobilinogen from circulation)
  • Absent Urobilinogen: An absent or negative result (when a bilirubin test is positive) is highly significant and suggests a biliary obstruction. If bilirubin can’t get to the intestine, no urobilinogen can be formed

Nitrite

• Principle: The Griess reaction. Certain bacteria (most commonly gram-negative rods like E. coli) can reduce dietary nitrate to nitrite. The nitrite on the pad reacts with an aromatic amine to form a diazonium salt, which then couples with another chemical to produce a pink color
• Normal Range: Negative
• Clinical Significance: A positive nitrite is a highly specific, though not highly sensitive, indicator of a Urinary Tract Infection (UTI)
• Sources of Error
  • False Negative: A negative result does not rule out a UTI! This can happen if:
    • The bacteria present do not reduce nitrate (e.g., gram-positive cocci)
    • The urine was not held in the bladder long enough (at least 4 hours) for the conversion to occur
    • The patient’s diet is low in nitrates

Leukocyte Esterase (LE)

• Principle: Detects the presence of an enzyme, esterase, which is found in the granules of most white blood cells (neutrophils, eosinophils, basophils, monocytes). The esterase cleaves an ester on the pad, and the resulting product reacts with a diazonium salt to produce a purple color
• Normal Range: Negative
• Clinical Significance: A positive result indicates the presence of WBCs (pyuria), a key sign of inflammation or a UTI. It can detect both intact and lysed WBCs
• Correlation: A positive LE and a positive Nitrite together are a very strong predictor of a UTI

Specific Gravity

• As we discussed previously, this pad measures ionic concentration: based on a pH change in a polyelectrolyte system. It’s a quick estimate but is less accurate than a refractometer, especially in the presence of non-ionic solutes

Putting It All Together: The Power of Correlation

No single test result should be interpreted in a vacuum. The real skill of a Medical Laboratory Scientist is to see the patterns

• Positive Glucose + Positive Ketones: = Probable Diabetic Ketoacidosis (DKA)
• Positive Nitrite + Positive Leukocyte Esterase: = Strong evidence for a UTI
• Positive Protein + Positive Blood: = Possible glomerular bleeding or kidney disease
• Positive Bilirubin + Negative/Normal Urobilinogen: = Suggests biliary obstruction

The chemical reagent strip is your first, best look into the patient’s metabolic and renal health. Master the technique and understand the principles, and you’ll have a powerful diagnostic tool at your fingertips