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The reagent strip: blood, bile pigments and infection

16 min

Skills you'll build3 skills
  • Compare a positive urine blood pad with the red cells seen on microscopy
  • Check light, delay, and interferents before reporting urine bilirubin or urobilinogen
  • Recognize infections that a negative nitrite or leukocyte esterase pad can miss

At the bench: Read urine chemistry and perform the indicated confirmation · Reconcile urine appearance, chemistry, and microscopy findings

Try first

Try first

A urine strip's blood pad turns positive. What has the pad detected?

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

The blood pad detects heme

The blood pad detects the peroxidase-like activity of heme. Intact red cells lyse on the pad and often leave a speckled color. Free hemoglobin and myoglobin give an even color. The pattern suggests a source and cannot identify it.1

A positive pad calls for microscopy to confirm and count red cells.1,2 A positive pad with few or no red cells suggests hemoglobinuria, myoglobinuria, red cells that lysed in dilute or alkaline urine, or an oxidizing contaminant. Plasma color and hemolysis markers support hemoglobin, and creatine kinase supports myoglobin.1

Bilirubin and urobilinogen

Only conjugated bilirubin enters urine, and the pad detects it with a diazo reaction. Light, delay, ascorbic acid and high nitrite lower its recovery, so test fresh urine kept from light. A negative result from an old or light-exposed specimen can hide bilirubin.1 Intestinal bacteria turn bilirubin into urobilinogen, and a little of it reaches urine. The two results are interpreted together:1,3

BilirubinUrobilinogenPossible cause
PositiveLowBile flow to the intestine reduced, as in biliary obstruction
PositiveIncreasedLiver cells failing to take up and excrete bile pigment
NegativeIncreasedIncreased bilirubin production, as in hemolysis

Nitrite and leukocyte esterase screen for infection

Many gram-negative bacteria reduce dietary nitrate to nitrite, which the pad detects. This requires nitrate-reducing organisms, nitrate in the diet and time in the bladder. Enterococci and many other gram-positive organisms, yeasts and a short bladder time all give a negative nitrite. A negative nitrite cannot exclude a urinary tract infection.1

The leukocyte esterase pad detects an enzyme from granulocytes. Lysed cells still react, so the pad can be positive when fewer intact white cells remain. Its color develops late, so read it at its stated time.1

Follow the bench demonstration

Sources for this step
References
  1. Kouri TT, Hofmann W, Falbo R, et al. The EFLM European urinalysis guideline 2023. Clin Chem Lab Med. 2024;62(9):1653-1786. doi:10.1515/cclm-2024-0070
  2. Barocas DA, Lotan Y, Matulewicz RS, et al. Updates to microhematuria: AUA/SUFU guideline (2025). J Urol. 2025;213(5):547-557. doi:10.1097/JU.0000000000004490
  3. Strasinger SK, Di Lorenzo MS. Urinalysis and Body Fluids. 7th ed. F.A. Davis; 2021.

Watch one

Read each decision and its explanation.

Priya Allcott, 29, gives a midstream clean-catch urine at 13:10. The collection note says she had voided 45 minutes earlier. The urine reaches the bench 20 minutes after collection.

What does the negative nitrite tell you?

TestResultReference intervalFlag
Leukocyte esteraseModerateNegativeNegative
NitriteNegativeNegativeNegative
WBC20–50 /hpf High0–5 /hpf0–5High
RBC0–2 /hpf0–2 /hpf0–2
BacteriaFewNone seenNone seen

Specimen: Not measured on urine. Midstream clean-catch, collected 13:10, received 13:30

  1. Check the specimen: 20 minutes old, collected midstream, acceptable for testing.

    A delayed specimen changes nitrite and cell counts, so the specimen is checked first.

  2. Pair the esterase with the count: a moderate esterase with 20–50 WBC/hpf shows pyuria.

    Two independent findings that agree are stronger evidence than either one.

  3. Check the bladder time: she had voided 45 minutes before this specimen, too short for much nitrite to form.

    Nitrite forms only when nitrate-reducing bacteria sit in the bladder long enough.

  4. Consider the other causes: enterococci, other gram-positive organisms and yeasts also leave the pad negative.

    Some organisms do not produce nitrite.

  5. Report the results as found and follow the laboratory's procedure for culture.

    The screen can miss infection, and culture detects the organism.

The negative nitrite cannot exclude infection. The short bladder time explains it, and the pyuria is supported by the esterase and the white cell count.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

A valid urine blood-pad reaction is positive but microscopy finds few or no intact RBCs. Which interpretation is appropriate?

Correct. The pad detects heme pseudoperoxidase activity, and oxidizing agents or microbial peroxidases can also react. Plasma color, hemolysis markers, and creatine kinase help identify the source.

Incorrect. The pad detects heme activity from free hemoglobin and myoglobin as well as cells. Microscopy establishes intact RBCs.

Incorrect. Repeating the same strip method can reproduce the same reaction. Microscopy on a fresh specimen and hemolysis or muscle-injury studies resolve the discrepancy.

Hint
  1. The pad reacts with heme, and heme has more than one source.
  2. Consider which heme sources leave no cells to count under the microscope.

Review Blood

Problem 2 of 3 · Hints available

A urine specimen has a positive leukocyte esterase reaction, many WBCs, and a negative nitrite. The culture grows Enterococcus faecalis. Which explanation fits the negative nitrite?

Incorrect. Bacteria that keep growing in an unrefrigerated specimen reduce more nitrate, so delay tends to raise nitrite.

Incorrect. Nitrite depends on the organism's ability to reduce nitrate, so a negative result does not show whether the organism is a contaminant.

Correct. The Griess reaction needs bacterial nitrate reduction, and gram-positive organisms such as Enterococcus faecalis usually lack it, so a negative nitrite cannot exclude infection.

Hint
  1. Nitrite comes from bacteria reducing nitrate.
  2. Consider whether this organism reduces nitrate.

Review Nitrite and leukocyte esterase findings

Problem 3 of 3 · On your own

A patient's fresh urine read moderate bilirubin yesterday. Today's urine was collected at 06:00 and waited in a clear cup by a sunny window until testing at 13:00. The bilirubin pad reads negative. What best explains today's result?

The specimen itself explains the change. Seven hours of light and delay destroy bilirubin in the cup.

Reported a light-exposed urine bilirubin as negative

Light, delay, ascorbic acid, and high nitrite lower bilirubin recovery on the diazo pad. A negative result from an old or light-exposed specimen can miss conjugated bilirubin from liver or bile-duct disease.

Light oxidizes bilirubin and delay adds to the loss. A fresh specimen kept from light shows whether bilirubin is still present.

Unconjugated bilirubin is bound to albumin and does not reach urine in appreciable amounts. The handling of this specimen explains the negative pad.

Review Bilirubin and urobilinogen

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Kofi Washburn, 42, is on the medical ward. Today's urine was left in a clear cup on a sunny windowsill.
TestResultPreviousReference intervalFlag
pH8.5 High1 day ago: 6.06.01 day ago4.5–8.04.5–8.0High
BilirubinNegative Delta1 day ago: ModerateModerate1 day agoNegativeNegativeDelta
BloodModerate Delta1 day ago: NegativeNegative1 day agoNegativeNegativeDelta
NitritePositive Delta1 day ago: NegativeNegative1 day agoNegativeNegativeDelta
RBC0–2 /hpf1 day ago: 0–2 /hpf0–2 /hpf1 day ago0–2 /hpf0–2
WBC0–2 /hpf1 day ago: 0–2 /hpf0–2 /hpf1 day ago0–5 /hpf0–5
BacteriaMany1 day ago: None seenNone seen1 day agoNone seenNone seen

Specimen: Not measured on urine. Random urine, collected 06:30, received 12:15 unrefrigerated; previous results from fresh urine

Decision 1 of 4

Why is today's bilirubin negative?

Nearly 6 hours of sunlight and room temperature destroy bilirubin in the cup. The handling of the specimen accounts for the change.

Reported a light-exposed urine bilirubin as negative

Light, delay, ascorbic acid, and high nitrite lower bilirubin recovery on the diazo pad. A negative result from an old or light-exposed specimen can miss conjugated bilirubin from liver or bile-duct disease.

Unconjugated bilirubin does not reach urine, and nothing in this specimen shows a change in his blood. The handling explains the negative pad.

Bilirubin oxidizes in light, and the loss increases with time. A negative pad from this cup cannot show whether bilirubin is still present.

Review Bilirubin and urobilinogen

Decision 2 of 4

The nitrite is positive with many bacteria. What does it show?

Nearly 6 hours unrefrigerated lets bacteria multiply and reduce nitrate in the cup. The alkaline pH fits the same growth. The positive result cannot be attributed to his bladder.

Bacteria growing in the cup give the same nitrite and bacteria. This specimen cannot distinguish the two.

Light destroys bilirubin. The nitrite here comes from bacteria acting on nitrate.

Review Changes during storage

Decision 3 of 4

The blood pad is moderate with 0–2 RBC/hpf. How do you interpret it?

Hematuria means intact red cells, and microscopy finds few. The pad detects heme from any source.

Read every positive blood pad as intact red cells

The blood pad detects the pseudoperoxidase activity of heme in intact red cells, free hemoglobin, and myoglobin, and oxidizing agents can also react. Reporting hematuria from the pad without microscopy can mislabel hemoglobinuria or myoglobinuria as bleeding.

Red cells lyse in alkaline urine over time and leave free hemoglobin that turns the pad positive. A fresh specimen shows whether intact red cells are present.

Free hemoglobin and myoglobin turn the pad positive with no cells to see. The pad result stands, and its source needs a fresh specimen.

Review Blood

Decision 4 of 4

What do you do with this urinalysis?

The comment does not show how much bilirubin was lost or how much of the nitrite and heme came from the cup.

A new strip reads the same altered urine and gives the same results.

The pads read this specimen correctly. The specimen no longer represents the urine Kofi passed.

A fresh specimen shows his actual bilirubin, nitrite and heme. Record the delay and light exposure as the reason.

Review Bilirubin and urobilinogen

Every change traces to the hours on a sunny windowsill. Nearly 6 hours of light and warmth account for the lost bilirubin, the new nitrite and bacteria, the alkaline pH and the heme without red cells, so only a fresh specimen represents Kofi's urine.

For reference

Sources checked