AUBF311: ANALYSIS OF URINE & BODY FLUIDS

Lecture Module First Semester

Week 04 S.Y. 2023-2024

CHEMICAL EXAMINATION OF URINE

TOPIC OUTLINE ERRORS CAUSED BY IMPROPER TECHNIQUE
1 Reagent Strips unmixed specimens where formed elements are
2 pH 1 at the bottom of the specimen which cannot be
3 Protein detected by the reagent strip
4 Glucose 2 extended period in urine that cause leaching
5 Ketones excess urine in the strip after its removal from
3
6 Blood the specimen produces run-over
7 Bilirubin improper timing (don’t know the timeframe per
4
8 Urobilinogen parameter)
9 Nitrite 5 inadequate light source (uses color chart)
10 Leukocyte Esterase reagent strip not touching the color chart to
6
11 Specific Gravity prevent contamination and carry-over of urine
reagent strips & color charts from different
7
manufacturers are not interchangeable
REAGENT STRIPS specimens that have been refrigerated should
8
return first to room temperature before testing
 provide a simple, rapid means for performing
medically significant chemical analysis of urine HANDLING & STORING REAGENT STRIPS
including pH, protein, glucose, ketones, blood,
bilirubin, urobilinogen, nitrite, leukocytes & SG  must be protected from deterioration caused by
 consist of chemical impregnated absorbent pads moisture, volatile chemicals, heat & light
attached to a plastic strip  packaged in opaque containers w/ desiccant to
 color producing chemical reaction takes place protect them from light & moisture
when the absorbent pad comes in contact w/ urine  strips are removed prior to testing and bottle is
sealed immediately
1. MULTISTIX  stored at room temp below 30C (not refrigerated)
o Siemens Healthcare Diagnostics  do not use past the expiration date
Deerfield, IN
TYPES  regular inspection of the strip
2. CHEMSTRIP
o Roche Diagnostics, Indianapolis, QUALITY CONTROL & CONFIRMATORY TESTING
IN  strips must be checked w/ both positive &
negative controls once every 24 hrs (preferably
REAGENT STRIP TECHNIQUE before shift)
 confirmatory testing uses different reagents /
dip the reagent strip briefly into a well-mixed methodologies to detect same substances
 uncentrifuged urine specimen at room
temperature
remove excess urine by touching the edge of the

strip to the container as the strip is withdrawn
blot the edge of the strip on a disposable

absorbent pad
wait the specified amount of time for the reaction

to occur
compare the color reaction of the strip pads to

the manufacturer’s color chart in good lighting
 semiquantitative value of trace, 1+, 2+, 3+, or 4+
 estimate of the mg/dL

College Of Medical Laboratory Science │Ranz Barron Bamba

 AUBF311: ANALYSIS OF URINE & BODY FLUIDS
Lecture Module First Semester
Week 04 S.Y. 2023-2024

pH  prolonged exposure of the strip to the

specimen reagent
 important in crystal identification & determination  high specific gravity
of unsatisfactory specimen  False Negative:
 proteins other than albumin
First Morning pH 5.0-6.0  microalbuminuria
Random pH 4.5-8.0
CLINICAL SIGNIFICANCE
CAUSES OF ACID & ALKALINE URINE
 intravascular hemolysis
ACID URINE ALKALINE URINE  muscle injury
 Emphysema  Hyperventilation Prerenal
 acute-phase reactants
 Diabetes mellitus  Vomiting  multiple myeloma
 Starvation  Renal Tubular  glomerular disorder
 Dehydration Acidosis  immune complex
 Diarrhea  Presence of Bacteria  amyloidosis
 Presence of Bacteria  Vegetarian Diet  toxic agent
 High Protein Diet  Old Specimens  diabetic nephropathy
 Cranberry Juice Renal
 strenuous exercise
 Medications  dehydration
 hypertension
PRINCIPLE double indicator system  preeclampsia
REAGENTS methyl red & bromothymol blue  orthostatic/postural proteinuria
COLOR & TIME red[4-6] yellowblue [6-9] (60s)  lower UTI/inflammation
 injury/trauma disorders
INTERFERENCE Postrenal  menstrual contamination
 no known interfering substances  prostatic fluid/spermatozoa
 runover from adjacent pads  vaginal secretions
 old specimens  Fanconi syndrome
Tubular
 bacterial growth  toxic agents/heavy metals
Disorders
 severe viral infectios

CLINICAL PROTENURIA
PROTEIN  indicated at ≥ 30 md/dL or 300 mg/L
not indicative or actual renal
 most indicative of renal disease is the protein disease; increased levels of low-
determination molecular-weight plasma proteins
and acute-phase reactants
Protenuria presence of protein in urine
BENCE JONES PROTEIN
PRE-RENAL
people with multiple myeloma
protein (Sorensen’s) error of PROTENURIA
PRINCIPLE elevated levels of monoclonal
indicator (sensitive only to albumin) immunoglobulin light chains
Multistix: tetrabromophenol blue GLOMERULAR PROTEINURIA
Chemstrip: 3’,3”,5’,5”- present with amyloid material, toxic
REAGENTS
tetrachlorophenol / 3,4,5,6- substances, and immune complexes
tetrabromosulfophthalein MICROALBUMINURIA
COLOR & TIME blue-green (60s) development of diabetic
nephropathy; associated with
INTERFERENCE increased risk of cardiovascular
 False Positive: RENAL disease
 highly buffered interference alkaline urine PROTENURIA ORTHOSTATIC PROTEINURIA
 pigmented specimens, phenazopyridine persistent benign proteinuria
 quaternary ammonium compounds/detergent TUBULAR PROTEINURIA
 antiseptics, chlorhexidine (loss of buffer form) affecting tubular reabsorption;
caused by exposure to toxic

College Of Medical Laboratory Science │Ranz Barron Bamba

 AUBF311: ANALYSIS OF URINE & BODY FLUIDS
Lecture Module First Semester
Week 04 S.Y. 2023-2024

substances and heavy metals, IMMUNODIP

severe viral infections, and Fanconi sensitive albumin tests related
syndrome PRINCIPLE to creatinine concentration to
POSTRENAL passes through the structure of the correct for patient hydration
PROTENURIA lower urinary tract dye albumin, CuSO4, 3,3’,5,5’-
REAGENTS
TMB & DBDH creatinine
SULFOSALICYLIC ACID (SSA) TEST visibly bloody/abnormal
INTERFERENCE
 cold precipitation test that reacts equally with all colored urine (false positive)
forms of proteins
 all types of protein can be detected including
albumin (only albumin will be positive in strip)
GLUCOSE

 most frequently performed chemical analysis of

urine
Renal Threshold 160-180 mg/dL

glucose oxidase reaction / double

PRINCIPLE
sequential enzyme reaction
Multistix: glucose oxidase,
peroxidase, potassium iodide
REAGENTS
Chemstrip: glucose oxidase,
peroxidase, tetramethylbenzidine
Multistix: green-brown (30s)
COLOR & TIME
Chemstrip: yellow-green (30s)

INTERFERENCE
 False Positive:
 contamination by oxidizing agents and
INTERFERENCE detergents
 False Positive:  False Negative:
 radiographic contrast dye (x-ray film)  high levels of ascorbic acid
 drugs (penicillin, sulfonamide, cephalosporin)  high levels of ketones
 False Negative:  high specific gravity
 very alkaline urine  low temperatures
 increase pH  improperly preserved specimens

TESTING FOR MICROALBUMINURIA CLINICAL SIGNIFICANCE

 semi-quantitative reagent strip method  diabetes mellitus
 random or first morning specimen  pancreatitis
MICRAL-TEST  pancreatic cancer
PRINCIPLE enzyme immunoassay  acromegaly
gold-labeled antibody, beta- Hyperglycemia-  cushing syndrome
REAGENTS galactosidase, chlorophenol associated  hyperthyroidism
red galactoside  pheochromocytoma
INTERFERENCE dilute urine (false negative)  CNS damage
 Stress
IMMUNODIP  gestational diabetes
PRINCIPLE immunochromographics  Fancony syndrome
REAGENTS antibody-coated blue latex Renal-  advanced renal disease
INTERFERENCE dilute urine (false negative) associated  osteomalacia
 pregnancy

College Of Medical Laboratory Science │Ranz Barron Bamba

 AUBF311: ANALYSIS OF URINE & BODY FLUIDS
Lecture Module First Semester
Week 04 S.Y. 2023-2024

COPPER REDUCTION TEST (CLINITEST) CLINICAL SIGNIFICANCE

 relies on the ability of glucose and other  diabetic acidosis
substances to reduce copper sulfate to cuprous  insulin dosage monitoring
oxide in presence of alkali and heat  starvation
 color change from negative blue (CuSO4) through  malabsorption/pancreatic disorders
green, yellow, and orange/red (Cu2O)  cold exposure
main reagent: CuSO4  strenuous exercise
REAGENTS NaOH, sodium citrate (heat)  vomiting
NaCO3 (eliminate O2)  inborn errors of amino acid metabolism
 alcoholism
INTERFERENCE  febrile state in children

 False Positive: ACETEST TABLETS

 other reducing sugars  confirmatory test
 ascorbic acid  addition of lactose gives better color differentiation
 drug metabolites
 cephalosporin
 False Negative: BLOOD
 oxidizing agents (detergents)
 pass-through phenomenon  normal count is 0-2
PASS-THROUGH PHENOMENON
o color passes through orange/red stage and pseudoperoxidase activity of
returns to green-brown color PRINCIPLE
hemoglobin
o occurs when >2g/dL sugar is present Multistix: diisopropylbenzene
o prevention: use 2 drops of urine dihydroperoxide & 3,3’,5,5’-
REAGENTS tetramethylbenzidine
Chemstrip:dimethyldihydroperoxy-
KETONE hexane & tetramethylbenzidine
(-) yellow ; (+) blue-green (60s(
 represents 3 intermediate products of fat COLOR & TIME uniformed green/blue = Hgb/Mgb
metabolism: speckled / spotted = hematuria
 2% acetone
 20% acetoacetic acid INTERFERENCE
 78% beta-hydroxybutyrate  False Positive:
Renal Threshold 70 mg/dL  strong oxidizing agents
 bacterial peroxidase
PRINCIPLE sodium nitroprusside reaction  menstrual contamination
Multistix: sodium nitroprusside  False Negative:
REAGENTS Chemstrip: sodium nitroprusside  high specific gravity / crenated cells
and glycine (detect acetone)  formalin
COLOR & TIME purple (40s)  captopril
 high concentrations of nitrite
INTERFERENCE  ascorbic acid >25 mg/dL
 False Positive:  unmixed specimens
 phthalein dyes
 highly pigmented red urine CLINICAL SIGNIFICANCE
 levodopa  renal calculi
 medications containing free sulfhydryl groups  glomerulonephritis
 False Negative:  pyelonephritis
 Improperly preserved specimen Hematuria  tumors
 trauma
 exposure to toxic chemicals
 anticoagulants

College Of Medical Laboratory Science │Ranz Barron Bamba

 AUBF311: ANALYSIS OF URINE & BODY FLUIDS
Lecture Module First Semester
Week 04 S.Y. 2023-2024

 strenuous exercise RHABDOMYOLYSIS

 transfusion reactions  trauma
 hemolytic anemias  crush syndromes
 severe burns  prolonged coma
Hemoglobinuria  infections/malaria  convulsions
 strenuous exercise/RBC  muscle-wasting disease
trauma  alcoholism
 brown recluse spider bites  heroin abuse
 muscular trauma  extensive exertion
 crush injuries
 prolonged coma HEMOGLOBINURIA VS. MYOGLOBINURIA
 convulsions larger hemoglobin molecules are
 muscle-wasting diseases precipitated by ammonium sulfate
Myoglobinuria PRINCIPLE
 alcoholism/overdose and myoglobin remain in the
 drug-abuse supernatant
 extensive exertion
 cholesterol-lowering statin
medications BILIRUBIN

HEMATURIA  early indication of liver disease

 intact RBCs in urine  often detected long before patient exhibits hepatic
jaundice
HEMOGLOBINURIA NORMAL VALUE 0.02 mg/dL
 lysed RBCs in urine
 result from intravascular hemolysis and PRINCIPLE diazo reaction
subsequent filtering of hemoglobin Multistix: 2,4-dichloroaniline
 caused when free hemoglobin present exceeds diazonium salt
REAGENTS
the haptoglobin content Chemstrip: 2,6-dichlorobenzene
 occurs in: diazoniumtetrafluoroborate
 hemolytic anemias COLOR & TIME tan, pink, or violet (30s)
 severe burns
 bites from brown recluse spider INTERFERENCE
 infections  False Positive:
 strenuous exercise  highly pigmented urines, phenozopyridine
 indicant (intestinal disorder)
HEMOGLOBIN-HAPTOGLOBIN COMPLEXES  metabolites of iodine
 prevents glomerular filtration of hemoglobin  False Negative:
 specimen exposure to light
 ascorbic acid >25 mg/dL
HEMOSIDERIN
 high concentrations of nitrite
 large yellow-brown granules of denatured ferritin
appearing as a result of reabsorption of filtered
CLINICAL SIGNIFICANCE
hemoglobin
 hepatitis
 cirrhosis
MYOGLOBINURIA  other liver disorders
 caused by muscle lysis  biliary obstruction (gallstones, carcinoma)
 myoglobin, heme-containing protein produces a
clear red-brown urine ICTOTES TABLETS
 suspected in patients with conditions associated  confirmatory test
with muscle destruction (rhabdomyolysis)  blue to purple color indicates presence of bilirubin
 myoglobin is toxic to renal tubules  detect 0.05 - 0.1mg/dL bilirubin
 components:
 p-nitrobenzene-diazonium-p-toluenesulfonate

College Of Medical Laboratory Science │Ranz Barron Bamba

 AUBF311: ANALYSIS OF URINE & BODY FLUIDS
Lecture Module First Semester
Week 04 S.Y. 2023-2024

 sulfosalicylic acid NITRITE

 sodium carbonate
 boric acid  rapid screening test for presence of UTI
 most commonly caused by gram-negative
bacteria
UROBILINOGEN SPECIMEN 1st morning or 4-hour urine
NORMAL VALUE <1 mg/dL or 1 Ehrlich unit
PRINCIPLE Greiss reaction
afternoon urine (2-4pm due to Multistix: p-arsanilic acid &
SPECIMEN
alkaline tide) tetrahydrobenzo(h)-quinolin-3-ol
REAGENTS
Chemstrip: sulfanilamide,
PRINCIPLE Ehrlich reaction hydroxytetra-hydrobenzoquinoline
Multistix:p- dimethylaminobenzaldehyde (+) uniform pink [60s] = 100,000
REAGENTS Chemstrip: 4-methoxybenzene- COLOR & TIME
org/mL (-) not pink spots / edge
diazonium-tetrafluoroborate
COLOR & TIME red (60s) INTERFERENCE
 False Positive:
INTERFERENCE (MULTISTIX)  nonreductase-containing bacteria
 False Positive:  insufficient contact time between bacteria and
 porphobilinogen urinary nitrate
 indicant  lack of urinary nitrate
 p-aminosalicylic acid  large quantities of bacteria converting nitrite to
 sulfonamides nitrogen
 methyldopa  presence of antibiotics
 procaine  high concentrations of ascorbic acid
 chlorpromazine  high specific gravity
 highly pigmented urine  False Negative:
 False Negative:  improperly preserved specimens
 old specimens  highly pigmented urine
 preservation in formalin
CLINICAL SIGNIFICANCE
INTERFERENCE (CHEMSTRIP)  cystitis
 False Positive:  pyelonephritis
 highly pigmented urine  evaluation of antibiotic therapy
 False Negative:  monitoring of patients at high risk of UTI
 old specimens  screening of urine culture specimens
 preservation in formalin
 high concentration of nitrite
LEUKOCYTE ESTERASE
CLINICAL SIGNIFICANCE  Trichomonas, Clamydia, yeast, and inflammation
 early detection of liver disease of renal tissues (interstitial nephritis) produce
 liver disorders, hepatitis, cirrhosis, carcinoma leukocyturia without bacteria
 hemolytic disorders
NORMAL VALUE 0-2 to 0-5/hpf

WATSON-SCWARTZ DIFFERENTIATION TEST PRINCIPLE granulocytic esterase reaction

 used to differentiate urobilinogen to Multistix: derivatized pyrrole
porphobilinogen amino acid ester & diazonium salt
REAGENTS
Chemstrip: indoxylcarbonic acid
ester & diazonium salt
COLOR & TIME purple (120s)

College Of Medical Laboratory Science │Ranz Barron Bamba

 AUBF311: ANALYSIS OF URINE & BODY FLUIDS
Lecture Module First Semester
Week 04 S.Y. 2023-2024

INTERFERENCE
 False Positive:
 strong oxidizing agents
 formalin
 highly pigmented urine, nitrofurantoin
 False Negative:
 high concentration of protein, glucose, oxalic
acid, ascorbic acid, gentamicin,
cephalosporins, tetracyclines
 inaccurate timing

CLINICAL SIGNIFICANCE
 bacterial and nonbacterial UTI
 inflammation of the urinary tract
 screening of urine culture specimens

SPECIFIC GRAVITY

 ↑ H+ = ↑ SG = ↓ pH

1.002-1.035 (normal)
VALUES <1.002 (not urine)
>1.035 (presence of
radiographic contrast sign)

pKa (dissociation constant) change

PRINCIPLE
of polyelectrolytes
Multistix: poly (methyl vinyl ether /
maleic anhydride bromothymol
blue
REAGENTS
Chemstrip: ethylene glycol
diaminoethyl ether tetra-acetic
acid, bromothymol blue
COLOR & TIME blue-green  yellow (120s)

INTERFERENCE
 False Positive:
 high concentrations of protein
 False Negative:
 highly alkaline urines (>6.5)

CLINICAL SIGNIFICANCE
 monitoring patient hydration and dehydration
 loss of renal tubular concentrating ability
 diabetes insipidus
 determination of unsatisfactory specimens
due to low concentration

College Of Medical Laboratory Science │Ranz Barron Bamba