Analysis of Urine and Body Fluids

Laboratory | Prelims | Week 4

CHEMICAL EXAMINATION OF URINE

This topic focuses on chemical examination of urine GLUCOSE

through the reagent strips. Reagent strips currently • Reagent Strip Reactions
provide a simple, rapid means for performing medically o Glucose oxidase reaction specific for glucose
significant chemical analysis of urine, including pH, o Double sequential enzyme reaction
protein, glucose, ketones, blood, bilirubin, urobilinogen, o Glucose oxidase, peroxide, chromogen, buffer
nitrite, leukocyte and specific gravity. on test pad
o Neg, trace, 1+, 2+, 3+, 4+
SPECIFIC GRAVITY (SG) o Sensitivity:100 mg/dL to 2 g/dL
• Reaction Interference • Reaction Interference
o No interference from large molecules, glucose o False-positive: peroxide, oxidizing detergents
and urea and radiographic dye and plasma o False negative: enzymatic reaction
expanders interference:
o Reason for difference in refractometer reading ▪ Ascorbic acid and strong reducing agents
▪ Slight elevation from protein ▪ High levels of ketones (unlikely)
▪ Decreased readings: urine pH 6.5 or higher ▪ High specific gravity and low temperature
▪ Interferes with indicator; add 0.005 to the ▪ Biggest error is old specimens due to
reading; readers automatically add this glycolysis

URINE PH KETONES
• Reagent Strip Reactions • Reagent Strip Reactions
o Double indicator reaction o Principle: Sodium nitroprusside reaction
o Needed to measure between 5 and 9 o Measure primarily acetoacetic acid
o Methyl red = 4–6 red/orange to yellow o Primary reagent: sodium
o Bromthymol blue = 6–9 green to blue nitroprusside→Acetoacetic acid + nitroprusside
→ purple
Methyl red + H+ → Bromothymol blue – H+ o Report: neg, small (1+), moderate (2+), large
(Red-Orange → Yellow) (Green → Blue) (3+), or small (5,15), moderate (40), large
(80,160) mg/dL
PROTEIN • Reaction Interference
• Reagent Strip Reactions o False Positive
o Protein error of indicators ▪ Phthalein dye, levodopa
o Certain indicators change color in the presence ▪ Highly pigmented red urine
of protein at a constant pH ▪ Medications with sulfhydryl groups
o Protein, primarily albumin, accepts H+ from the o False Negative
indicator ▪ Improperly preserved specimen
o Reagents: Tetrabromophenol blue or ▪ Old specimens
tetrachlorophenol tetrabromsulfonphthalein and
an acid buffer BLOOD
o Report: neg, trace, 1+, 2+, 3+, 4+, or 30, 100, • Reagent Strip Reactions
300, 2000 mg/dL o Principle: pseudoperoxidase activity of
o Trace values are <30 mg/dL hemoglobin
o Normally, <100mg of protein is excreted per o Intact RBCs show a speckled pattern
24hrsClinical proteinuria: > 30mg/dl (300mg/L o Report: trace, small (1+), moderate (2+), large
(3+)  Sensitivity 5 RBCs/μL
• Reaction Interference • Reaction Interference
o Highly buffered alkaline urine overrides acid o False-positive:
buffer system ▪ strong oxidizing agents
o Leaving reagent pad in urine too long removes ▪ bacterial peroxidases
buffer ▪ menstrual contamination
o Highly pigmented urine o False-negative:
o Quarternary ammonium compounds, ▪ High SG/crenated cells
detergents, antiseptics, chlorhexidine ▪ Formalin
o High Specific gravity ▪ Captopril
o Proteins other than albumin ▪ High concentrations of nitrite
o Micoalbuminuria ▪ Ascorbic acid >25 mg/dL
▪ Unmixed specimens
Urine Specific Gravity

BILIRUBIN o False-positive:
• Reagent Strip Reactions ▪ Old specimens (bacterial multiplication)
o Principle is a diazo reaction ▪ Highly pigmented urine
o Reagent: Diazonium salt ▪ Pink edges or spotting on reagent strip is
o Report: neg, small (1+), moderate (2+), large considered negative
(3+) ▪ Check automated readers manually for color
o Colors may be difficult to interpret interference
o Atypical colors can be problem for automated LEUKOCYTE ESTERASE (LE)
readers • Reagent Strip Reactions
• Reaction Interference o LE catalyzes hydrolysis of acid esterase on pad
o False-positive: to aromatic compound and acid. Aromatic
▪ urine pigments compound reacts with diazonium salt on pad for
▪ pyridium purple color
▪ indican • Reaction Interference
▪ Iodine o False-positive:
o False-negative: ▪ Strong oxidizing agents
▪ Old specimens ▪ Formalin
▪ Specimen exposure to light ▪ Highly pigmented urine
▪ Ascorbic acid >25mg/dL ▪ Nitrofurantoin
▪ High concentration of nitrite o False-negative:
▪ ▪ High concentrations of protein
UROBILINOGEN ▪ Glucose
• Reagent Strip Reactions ▪ Oxalic acid
o Different principles for Multistix and Chemstrip ▪ Ascorbic acid
o Multistix: p-dimethylaminobenzaldehyde ▪ gentamicin,
(Ehrlich reagent); report in Ehrlich units (EU) ▪ cephalosporins
▪ 1 EU = 1 mg/dL ▪ tetracyclines
o Normal readings 0.2–1, abnormal 2, 4, 8 ▪ Crenation from high SG
o Chemstrip: ▪ Inaccurate timing-must have 2 min
▪ 4-methoxybenzen-diazonium-
tetrafluoroborate
▪ Diazo reaction
▪ more specific than Ehrlich reaction
▪ report in mg/dL
• Reaction Interference
o Ehrlich reactive compounds: porphobilinogen,
indican, sulfonamides, methyldopa, procaine,
chlorpromazine, p-aminosalicylic acid
o Both tests: urobilinogen is highest after meals
(increased bile salts), old specimens and
formalin preservation decrease results
o Chemstrip: false-negative with high nitrite
interferes with diazo reaction

NITRITE
• Reagent Strip Reaction
o Greiss reaction: nitrite reacts with aromatic
amine to form a diazonium salt that then reacts
with tetrahydrobenzoquinoline to form a pink
azodye
o Sensitive for 100,000 organisms/mL
o Results: negative and positive
• Reaction Interference
o False-negative:
▪ Nonreductase-containingbacteria
▪ Insufficient contact time between bacteria
and urinary nitrate
▪ Lack of urinary nitrate
▪ Large quantities of bacteria converting
nitrite to nitrogen
▪ Presence of antibiotics
▪ High concentrations of ascorbic acid
▪ High specific gravity