CHEMICAL EXAMINATION Flashcards by Arriane Cyrel (MTI)

determination of the presence and/or levels of the different analytes in urine associated with pathologic conditions

CHEMICAL EXAMINATION OF URINE

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three methods are available:

(1) reagent strips (2) manual colorimetric/enzymatic (3) automated

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Most frequently tested in urine

GLUCOSE

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Intermediate products of fat metabolism

KETONES

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Increased in carbohydrate deprivation and decreased utilization of carbohydrates

KETONES

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Determined by the concentration of H+ ions which are secreted by the kidneys => ammonium ions, hydrogen phosphate and weak organic acids

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Identification of crystals and determination of unsatisfactory specimens

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Most indicative of RENAL DISEASE

PROTEIN

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Produces white foam when shaken

PROTEIN

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GLUCOSE

Renal Threshold:

160-180 mg/d L

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First morning urine: pH

5.0 to 6.0

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Random urine: pH

4.5 to 8.0

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PROTEIN

Normal value:

<10 mg/dL or <100 mg/24 hr

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Normal Albumin Excretion Rate (AER):

0-20 ug/min

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Microalbuminuria:

20-200 ug/min (or 30 to 300 mg of albumin/24 hours)

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Clinical albuminuria:

> 300 ug/min

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Albumin:

10-150mg/L

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Creatinine:

10-300mg/dL., 0.9-26.5 mmol/L

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– presence of any sugar in urine
– presence of any reducing sugar in urine
– presence of glucose in urine

Mellituria
Glycosuria
Glucosuria

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occurs after meals due to withdrawal of H+ ions for the purpose of secretion of HCl

Alkaline tide

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Clinical proteinuria types

Prerenal/overflow 2. Renal 3. Postrenal

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INTACT RED CELLS

Hematuria

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NO RED CELLS

Hemoglobinuria
Myoglobinuria

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CLOUDY RED URINE

Hematuria

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CLEAR RED URINE

Hemoglobinuria Myoglobinuria

characterized by “cola drink” or “black coffee” urine

Myoglobinuria

bleeding is renal or genitourinary in origin

Hematuria

lysis of RBC produced in urinary tract particularly in dilute, alkaline urine

Hemoglobinuria

result to intravascular hemolysis

Hemoglobinuria

 Not indicative of actual renal disease

 Not detected by reagent strip

Pre-renal(“before”) or Overflow Proteinuria

 Characterized by an increase in low molecular weight proteins which are readily filtered out from the circulation by the glomerulus

Pre-renal(“before”) or Overflow Proteinuria

- Intravascular hemolysis:

↑hemoglobin

- Muscle injury:

↑myoglobin

- Severe infection and inflammation:

↑acute phase reactants

- Multiple myeloma:

↑Bence Jones protein

 Abnormal protein excreted by patients with MULTIPLE MYELOMA

BENCE-JONES PROTEIN

 NOT ALL patients with (?) will excrete detectable levels of BJP

Multiple myeloma

BENCE-JONES PROTEIN  Precipitates at  Dissolves at

40-60 ᴼC 100ᴼC

Indicative of True Renal Disease

Renal

 Most common type

GLOMERULAR PROTEINURIA

 Occurs in primary glomerular diseases

GLOMERULAR PROTEINURIA

 Seen in glomerulonephritis, amyloidosis, exposure to toxic substances, SLE, hypertension

GLOMERULAR PROTEINURIA

 Involves tubular reabsorption dysfunction

TUBULAR PROTEINURIA

 Seen in exposure to toxic substance or heavy metals, severe viral infections, Fanconi’s syndrome, pyelonephritis and acute tubular necrosis

TUBULAR PROTEINURIA

 Not detected by the routine reagent strip

MICROALBUMINURIA

 Associated with diabetic nephropathy and increased risk of CVD

MICROALBUMINURIA

 Proteinuria when standing due to increased pressure to renal veins.

POSTURAL, ORTHOSTATIC, OR CADET PROTEINURIA

 Found during the day but not at night

POSTURAL, ORTHOSTATIC, OR CADET PROTEINURIA

 Screening test: comparison of first morning vs second specimen

POSTURAL, ORTHOSTATIC, OR CADET PROTEINURIA

 First morning urine should be negative for protein

POSTURAL, ORTHOSTATIC, OR CADET PROTEINURIA

 Strip employing antibody-enzyme conjugate that binds albumin

MICRAL TEST

 Principle: MICRAL TEST

Enzyme Immunoassay

 Reagents: MICRAL TEST

Gold-labelled antibody, beta-galactosidase, chlorophenol re galactosidase

 Sensitivity: MICRAL TEST

0-10 mg/dl

sensitive albumin tests related to creatinine concentration to correct for patient hydration

IMMUNODIP Clinitest Microalbumin Strips/MultistixPro

 Protein is added to urine as it passes through the lower urinary tract

Post-renal (“after”)

 May also be due to contamination during menstruation or from prostatic or vaginal secretions

Post-renal (“after”)

urine is coagulated by heat

HEAT AND ACETIC ACID

(REFERENCE METHOD)

HEAT AND ACETIC ACID

SULFOSALICYLIC ACID/SSA  Reagent:  MOST proteins are precipitated by:

3% SSA dilute SSA

 Presence of albumin=  Presence of proteins Other than albumin=

+ SSA; + RGT STRIP + SSA; - RGT STRIP

HEAT AND ACETIC ACID diffuse cloudiness granular, cloudy distinct flocculate 4+ large flocculate, dense, something solid

1+ 2+ 3+

SULFOSALICYLIC ACID/SSA No increase in turbidity Less than 6

Negative

SULFOSALICYLIC ACID/SSA Noticeable turbidity 6–30

Trace

SULFOSALICYLIC ACID/SSA Distinct turbidity, no granulation 30–100

SULFOSALICYLIC ACID/SSA Turbidity, granulation, no flocculation 100–200

SULFOSALICYLIC ACID/SSA Turbidity, granulation, flocculation 200–400

SULFOSALICYLIC ACID/SSA Clumps of protein Greater than 400

SULFOSALICYLIC ACID/SSA False positive

radiographic dyes, tolbutamide metabolites, cephalosporins, penicillins and sulfonamides

SULFOSALICYLIC ACID/SSA False negative

highly alkaline urine, very dilute samples

 Highly pigmented yellow degradation product of hemoglobin

BILIRUBIN

Appearance in urine can provide early indication of liver disease

BILIRUBIN

Detected long before the development of jaundice

BILIRUBIN

Tea-colored/amber urine with yellow foam

BILIRUBIN

Normal value: BILIRUBIN

0.02 mg/dL

Normal value: UROBILINOGEN

< 1 Ehrlich unit or 0.5 to 2.5 mg/24 hours

Colorless and labile substance formed via the conversion of bilirubin in the intestines

UROBILINOGEN

Reabsorbed from the intestines into the blood, some are excreted by the kidneys and the remaining recirculates back to the liver

UROBILINOGEN

Rapid, indirect method for the detection of bacteria capable of reducing nitrate to nitrite

NITRITE

Detects the presence of esterase in WBCs that function as the body’s defense against microorganisms

LEUKOCYTE ESTERASE

source of interference due to its strong reducing property = FALSE NEGATIVE RESULT

ASCORBIC ACID

source of interference due to its strong reducing property = FALSE NEGATIVE RESULT

ASCORBIC ACID

GLUCOSE CLINICAL SIGNIFICANCE

Hyperglycemia-associated Renal-associated

KETONES CLINICAL SIGNIFICANCE

1. Diabetes mellitus 2. Loss of carbohydrate from vomiting 3. Inadequate intake of carbohydrate (starvation and malabsorption) 4. Inborn errors of amino acid metabolism

pH CLINICAL SIGNIFICANCE

1. Acid Urine 2. Alkaline Urine

PROTEIN CLINICAL SIGNIFICANCE

1. Albumin 2. Tamm-horsfall protein

BILIRUBIN CLINICAL SIGNIFICANCE

Liver disorders:  Hepatitis, cirrhosis  Biliary obstruction (gallstones, carcinoma)

NITRITE CLINICAL SIGNIFICANCE

1. Urinary tract infections (cystitis, pyelonephritis) 2. Evaluation of antibiotic therapy 3. Monitoring of patients at high risk for urinary tract infection 4. Screening of urine culture specimens

LEUKOCYTE ESTERASE CLINICAL SIGNIFICANCE

1. Bacterial and non-bacterial urinary tract infections 2. Inflammation of the urinary tract 3. Screening of urine culture specimens

↑ Blood glucose = ↑ urine glucose

Hyperglycemia-associated

Normal blood glucose = ↑ urine glucose

Renal-associated

 Diabetes

Hyperglycemia-associated

 Fanconi’s syndrome  Nephrotic syndrome  Osteomalacia  Pregnancy

Renal-associated

 Mellitus

Hyperglycemia-associated

 Cushing’s syndrome

Hyperglycemia-associated

 Pheochromoc ytoma

Hyperglycemia-associated

 Acromegaly

Hyperglycemia-associated

 Hyperthyroidism

Hyperglycemia-associated

Fanconi’s syndrome

Renal-associated

Nephrotic syndrome

Renal-associated

Osteomalacia

Renal-associated

Pregnancy

Renal-associated

 Metabolic and respiratory acidosis

Acid Urine

 Chronic obstructive pulmonary disease

Acid Urine

 Pyrexia

Acid Urine

 Methanol poisoning

Acid Urine

 Increased protein diet

Acid Urine

 Acid-producing bacteria Cranberry juice

Acid Urine

 Respiratory and metabolic alkalosis

Alkaline Urine

 Renal tubular acidosis

Alkaline Urine

 Urease-producing bacteria

Alkaline Urine

 Vegetable and fruit diet

Alkaline Urine

 Alkaline tide

Alkaline Urine

Old specimens

Alkaline Urine

MAJOR serum protein found in urine

Albumin

produced by the renal tubules and forms MATRIX OF ALL YPES OF CASTS

Tamm-horsfall protein

Most common organisms that infect the urinary tract:

 Proteus spp  E.Coli  Klebsiella pneumoniae  Pseudomonas aeroginos

Reagent strip Oxidizing agent interference on reagent strip (False positive) Non-glucose reducing substance Possible interfering substance for reagent strip (ex: Ascorbic acid)

Small amount of glucose 1+ 4+ Nega

Procedure: Clinitest (?) drops of urine + (?) drops dist. Water + (?)

5 10 Clinitest tablet

Pass-through phenomenon  Occurs when (?) sugar is present  (?) >>>>>>>>> Blue or Greenbrown  to prevent pass-through, use (?)

>2g/dl Blue > Green > Yellow > Brick red 2 drops of urine

Results: Clinitest Small amount of glucose Oxidizing agent interference on reagent strip (False positive) Non-glucose reducing substance Possible interfering substance for reagent strip (ex: Ascorbic acid)

Nega Nega Posi

GLUCOSE METHODS

1. Reagent strip 2. Benedict’s test 3. Clinitest

KETONES METHODS

1. Reagent strip 2. Acetest

pH METHODS

1. Reagent strip 2. Litmus paper 3. Nitrazine paper 4. Liquid indicators such as methyl red

PROTEIN METHODS

None

BLOOD METHODS

1. Reagent strip 2. Microscopic examination 4. Blondheim Test

BILIRUBIN METHODS

1. Reagent strip 2. Ictotest

UROBILINOGEN METHODS

1. Reagent strip 2. Ehrich Tube Test 3. Hoesch Test(Inverse Ehrlich reaction) 4. Watson- Schwartz Test

Acetest Reagents:

Na nitroprusside, glycine, disodium phosphate, lactose

Acetest Sensitivity: acetoacetic acid acetone

5 to 10 mg/dL 20 to 25 mg/dL

Acetest Procedure: (?) drop of urine on tablet

Acetest Results: Posi

lavender to deep purple

DIACETIC ACID/ACTOACETIC ACID

 Gerhard’s test  Rothera’s test  Acetest

- Ferric chloride w/ diacetic acid => PORTWINE/ Bordeaux red color

 Gerhard’s test

- can detect about 1-5 mg/dL if diacetic acid and 10-25 mg/dL of Acetone

 Rothera’s test

- provides sodium nitroprusside, glycine, disodium phosphate, and lactose in tablet form

 Acetest

ACETONE

 Rothera’s test  Legal’s Test  Frommer’s Test  Lange’s Test  Acetest

BHYDROXYBUTYRIC ACID

 Hart’s test Orberg

- INDIRECT METHOD for detection of Bhydroxybutyric acid

 Hart’s test Orberg

 Intact RBCs settle at the bottom of tube

Reagent strip Hematuria

 Red color of urine is lost

Reagent strip Hematuria

Presence of intact RBCs

Microscopic examination Hematuria

Salt precipitation test

Blondheim Test Hematuria

Urine remains red

Reagent strip Hemoglobinuria, Myoglobinuria

Absence of intact RBCs

Microscopic examination Hemoglobinuria , Myoglobinuria

Precipitate is clear

Microscopic examination Myoglobinuria Blondheim Test Myoglobinuria

Red supernatant is positive with reagent strip

Microscopic examination Myoglobinuria Blondheim Test Myoglobinuria

Pale yellow

Microscopic examination Myoglobinuria Blondheim Test Myoglobinuria

Testing mat and tablet: Urine absorbs into the mat while bilirubin stays on the surface Tablet contains p-nitrobenzne-diazoniumptoluenesulfonate, SSA, sodium carbonate, boric acid

Ictotest (BILIRUBIN)

Sensitivity: 0.05 to 0.1 mg/dL

Ictotest (BILIRUBIN)

Procedure: 10 drops urine + 1 dropH2O (60sec)

Ictotest (BILIRUBIN)

Results: Performed in case of questionable results blue to purple

Ictotest (BILIRUBIN)

Tests (BILIRUBIN)

ICTOTEST FOAM-SHAKE TEST HARRISON SPOT TEST

 Ferric hcloride in the prsence of TCA will oxidize yellow bilirubin to

HARRISON SPOT TEST

 MORE sensitive  less subject to interference

ICTOTEST

ICTOTEST  POSITIVE:  NEGATIVE:

 POSITIVE: BLUE TO PURPLE COLOR  NEGATIVE: Other color than blue

 Specimen: 2-hr postprandial urine  Reagents: p-dimethylaminobenzaldehyde and sodium acetate  Procedure: 1 part reagent + 10 parts urine  Positive result: cherry-red color

Ehrich Tube Test

Rapid screening test for urine porphobilinogen (≥2 mg/dL)

Hoesch Test(Inverse Ehrlich reaction)

 Reagent: Ehrlich’s reagent in 6M HCl  Procedure: 2 drops urine + 2ml  Positive result: red  Interferences: highly pigmented urine, methyldopa, indicans

Hoesch Test (Inverse Ehrlich reaction)

 Classic - test to differentiate urobilinogen form porphobilinogen based on miscibility characteristics

Watson- Schwartz Test

Watson- Schwartz Test  Urobilinogen is (?) in both butanol and chloroform  Porphobilinogen is (?) in both butanol and chloroform  Other Ehrlich reactive compounds are (?) in butanol but not in chloroform

extractable immiscible miscible

1. (?) are destroyed in the spleen and liver by phagocytic cells of the RES 2. Liberated hemoglobin is broken down into (?). 3. The RES converts (?). 4. Bilirubin is released into the circulation where it binds to albumin and is transported to the liver (?) 5. In the liver, bilirubin is conjugated with glucoronic acid (?) 6. Conjugated bilirubin is passed into the intestines. Intestinal bacteria reduce (?). 7. A portion of urobilinogen is transported to the kidneys and excreted in urine as (?).

Senescent red cells iron, protein and protoporphyrin protoporphyrin to bilirubin unconjugated bilirubin conjugated bilirubin bilirubin to urobilinogen urobilin

PRE-HEPATIC

NEGATIVE +++

HEPATIC

POSITIVE/NEGATIVE ++

POST-HEPATIC URINE BILIRUBIN : URINE UROBILINOGEN :

+++ NORMAL

Provide a simple, rapid means for performing medically significant chemical analysis of urine, including: pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocytes and specific gravity

REAGENT STRIPS

Multistix, Chemstrip

REAGENT STRIPS

Consist of a chemical-impregnated absorbent pads attached to a plastic strip

REAGENT STRIPS

1. (?) in unmixed specimens 2. Allowing the strip to remain in the urine for an (?) 3. Excess urine remaining on the strip after its removal from the specimen can produce a (?) between chemicals on adjacent pads 4. Inadequate (?) 5. The strip must be (?) to the color chart without actually being placed on the chart 6. (?) from different manufacturers are not interchangeable 7. Specimens that have been refrigerated must be allowed to return to room temperature prior to reagent strip testing, as the (?) on the strips are temperature dependent

Undetected formed elements extended periods runover light source held close Reagent strips and color charts enzymatic reactions

Timing for reaction reading Manufacturers recommend that reactions be read between (?), with the leukocyte esterase reaction read at (?)

60 and 120 seconds 120 seconds

Principle Double sequential enzyme reaction

1. GLUCOSE

Principle Na nitroprusside Reaction (Legal’s Test)

2. KETONES

Principle Change in acid dissociation constant

3. SPECIFIC GRAVITY

Principle Double Indicator System

4. PH

Principle (Sorensen’s) error of indicators

5. PROTEINS

Reagents Multistix: Glucose oxidase, Peroxidase, Potassium iodide (blue to green to brown) Chemstrip: Glucose oxidase, Peroxidase, Tetramethylbenzidine (yellow to green)

1. GLUCOSE

Reagents Sodium nitroprusside / Sodium nitroferricyanide, Glycine

2. KETONES

Reagents Bromthymol blue

3. SPECIFIC GRAVITY

Reagents Methyl red, Bromthymol blue

4. pH

Reagents Multistix: Tetrabromphenol blue Chemstrip: Tetrachlorophenol tertrabromsulfonphthalein

5. PROTEINS

Reaction KETONES

Reaction SPECIFIC GRAVITY

Reaction pH

Reaction PROTEINS

Reaction BLOOD

Reaction BILIRUBIN

Reaction LEUKOCYTE ESTERASE

Reaction GLUCOSE

Reaction UROBILINOGEN

Reaction NITRITE

Principle

Principle Pseudoperoxidase activity of hemoglobin

6. BLOOD

Principle Diazo Reaction

7. BILIRUBIN

Principle Ehrlich Reaction

8. UROBILINOGEN

Principle Greiss Reaction

9. NITRITE

Reagents Multistix: Diisopropylbenzene dehydroperoxide tetramethylbenzidine Chemstrip: Dimethyldihydroperoxyhexane tertamethylbenzidine

6. BLOOD

Reagents Multistix: 2,4-dichloroaniline diazonium salt Chemstrip: 2,6-dichlorobenzene diazonium salt

7. BILIRUBIN

Reagents Multistix: pdimethylaminobenzaldehyde (PDAB or Ehrlich reagent) Chemstrip: 4-methyloxybenzenediazoniumtetrafluoroborate

8. UROBILINOGEN

Reagents Multistix: p-arsinilic acid, tetrahydrobenzo(h)-quinolin-3-ol Chemstrip: Sulfanilamide, hydroxytetrahydro benzoquinoline

9. NITRITE

False Positive strong oxidizing agent, formalin, highly pigmented urine, nitrofurantoin

10. LEUKOCYTE ESTERASE

False Positive Oxidizing agents, detergents

1. GLUCOSE

False Positive Phthalein dyes, highly pigmented red urine, levodopa, medications with sulfhydryl groups

2. KETONES

False Positive High concentrations of protein

3. SPECIFIC GRAVITY

False Positive No known interfering substance; runover from adjacent pads, old specimens

4. pH

False Positive Highly buffered alkaline urine, pigmented specimen, Phenazopyidine, Detergents, Antiseptics, prolonged exposure of strip to specimen, high SG

5. PROTEINS

False Positive Strong oxidizing agents, bacterial peroxidases, menstrual contamination

6. BLOOD

False Positive Highly pigmented urines, phenazopyridine, indican, metabolites of Lodine

7. BILIRUBIN

False Positive Ehrlich reactive compounds, highly pigmented urine

8. UROBILINOGEN

False Positive improperly preserved urine, highly pigmented urine

9. NITRITE

False Negative high specific gravity, protein (>500mg/dL), glucose (>3g/dL), oxalic acid, ascorbic acid, gentamicin, cephalin, cephalothin, tetracycline

10. LEUKOCYTE ESTERASE

False Negative non-nitrate reducing bacteria and yeast, improperly collected specimen, lack of dietary NO3, antibiotics, ascorbic acid, high specific gravity, medications

1. GLUCOSE

False Negative old specimens, formalin and increased nitrite

2. UROBILINOGEN

False Negative improperly stored urine, increase in nitrites and ascorbic acid

3. BILIRUBIN

False Negative No known interfering substance; runover from adjacent pads, old specimens

4. pH

False Negative Proteins other than albumin

5. PROTEINS

False Negative 6. BLOOD Uniform green / blue color = Speckled/Spotted =

High SG, crenated cells, formalin, captopril, high concentrations of nitrite, ascorbic acid (>25mg/dl), unmixed specimen Uniform green / blue color = Hemoglobin/Myoglobin Speckled/Spotted = Hematuria (Intact RBCs)

False Negative improperly stored urine, increase in nitrites and ascorbic acid

7. BILIRUBIN

False Negative old specimens, formalin and increased nitrite

8. UROBILINOGEN

False Negative non-nitrate reducing bacteria and yeast, improperly collected specimen, lack of dietary NO3, antibiotics, ascorbic acid, high specific gravity, medications

9. NITRITE

Sensitivity/ Calibration

Sensitivity/ Calibration Clinistix: 100 mg/dL Multistix: 75 to 125 mg/dL Chemstrip: 40 mg/dL

1. GLUCOSE

Sensitivity/ Calibration Add 0.005 to reading when phH>6.5 due to interference of bromthymol blue indicator

3. SPECIFIC GRAVITY

Sensitivity/ Calibration Indicator is sensitive to albumin

5. PROTEINS

Correlations Glucose

Ketones, Proteins

Correlations

Correlations pH

Nitrite, Leukocyte, Microscopic examination

Correlations PROTEINS

Blood, Nitrite, Leukocytes, Microscopic examination

Correlations BLOOD

Protein, microscopic examination

Correlations BILIRUBIN

Urobilinogen

Correlations UROBILINOGEN

Bilirubin

Correlations NITRITE

Protein, Leukocytes, Microscopic examination

ASCORBIC ACID – 11th reagent pad Causes false negative reactions on:

 Blood  Bilirubin  leukocyte esterase  Nitrite  Glucose

Purple

KETONES

Red to yellow Yellow to Blue

Blue-green

PROTEINS

Yellow Green to Blue

BLOOD

Azodye

BILIRUBIN

Red

UROBILINOGEN

Red azodye

UROBILINOGEN

Diazonium salt Uniform pink

NITRITE

Purple azodye

LEUKOCYTE ESTERASE

blue to green to brown yellow to green

GLUCOSE