BFA - Ch.6 Chemical Exam of Urine

what is used to perform routine chemical tests on urine

reagent strips

consist of chemical-impregnated absorbent pads on a plastic strip

test performed for pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocyte esterase, and sp. grav

single and multitest strips available

what are the names 2 major types of reagent strips that are sold

Multistix and Chemstrip

some variations occur btwn the strips w/ regard to sensitivity, specificity, and interfering substances

• users should be familiar w/ the product limitations

reagent strips are also used w. automated instruments

how is a reagent strip read

color comparison charts are supplied by the manufacturer

several degrees of color are shown to provide semiquantitative readings

• neg, trace, 1+, 2+, 3+, and 4+

estimates of mg/dL are also provided for many test areas

reagent strip technqiue

dip strip briefly into well mixed specimen at rm temp

remove excess urine by touching edge of strip to container as strip is withdrawn

blot edge of strip on absorbent pad

wait specified amt of time

• leukocyte esterase is the longest at 2 min

compare color rxn to manufacturer’s chart under good lighting

hold strip horizontally when comparing colors

Improper technique errors for reagent strips

not mixing sample - RBCs and WBCs sink to the bottom of an unmixed specimen

too cold/warm - enzyme reactions on strip are based on rm temp. readings

long dip time - reagents will leach off a strip remaining in urine too long - dip briefly

urine amt - excess urine on the strip will cause runover of reagents among the pads

quality control of reagent strips

all negative controls should = negative

pos. control readings should agree w/ published control values by ± 1 color block

be aware of manufacturer stated limitations and interfering substances

relate chemical readings to each other and physical and microscopic readings

liquid and tablet backup tests are available for some rxns

must also have + and - controls performed whenever these tests are required:

• Protein: Sulfosalicylic Acid (SSA) Precipitation test

• Galactose: Clinitest

• Ketones: Acetest

• Bilirubin: Ictotest

what tests require positive and negative controls

• Protein: Sulfosalicylic Acid (SSA) Precipitation test

• Galactose: Clinitest

• Ketones: Acetest

• Bilirubin: Ictotest

Urine pH - reagent strips

test pad measures acidity and alkalinity

• pH sensitive dyes

kidneys are major regulator of acid-base balance

first morning specimen normally acidic (5-6)

postprandial specimen more alkaline

Ref range: 4.5-8

Normal fresh urine can’t reach pH 9

• indicated old bacteria contaminated urine

too acidic or alkaline may form crystals and stones that can block passageways

what can affect urine pH?

diet and medication regulation

• meat = acidic pH

• vegetables = alkaline pH

• medications for urinary tract infection → maintain an acid pH

Clinical significance of urine pH

1. respiratory or metabolic acidosis/ketosis

   1. breathing problems, obesity, airway blockage

2. respiratory or metabolic alkalosis

   1. hyperventilation, fever, sepsis, ventilators

3. defects in renal tubular secretion and reabsorption of acids and bases

   1. renal tubular acidosis

4. renal calculi formation

5. treatment of urinary tract infections

6. precipitation/identification of crystals

protein in urine

most indicative of renal disease

• proteinuria seen in early renal disease

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

low molecular weight serum proteins are filtered, while many are reabsorbed

• albumin is primary protein of concern

clinical significance of proteinuria (values and causes)

30 mg/dL or 300 mg/24 hours

Variety of causes (prerenal, renal, post renal)

prerenal proteinuria

conditions affecting the plasma, not the kidney

transient causes due to reabsorption overflow

• hemoglobin - intravascular hemolysis

• myoglobin - muscle injury

• infections/inflammation

Rarely seen on reagent strip b/c strip detects albumin

Prerenal proteinuria - Bence Jones Protein (BJP)

Multiple myeloma

• elevated immunoglobulin light chains (monoclonal globulin protein)

• body makes too many Abs, and they spill into the urine

requires confirmation by serum and urine immunoelectrophoresis

may not show on dipstick

Renal - Glomerular Proteinuria

damage to glomerular membrane which can impair selective filtration

• allows for serum protein (albumin) to pass thru membrane and into the urine

• Nephrotic syndrome - foamy urine

Inflammation

• Nephritic syndrome - includes blood along w/ protein

Increased pressure on the filtration mechanism

• hypertension

• strenuous exercise

• dehydration

• preeclampsia

nephrotic syndrome typical features

onset: insidious

edema: ++++

bp: normal

jugular venous pressure: normal/low

proteinuria: ++++

Hematuria: May/may not occur

RBC casts: absent

serum albumin: low

nephritic syndrome typical features

onset: abrupt

edema: ++

bp: raised

jugular venous pressure: raised

proteinuria: ++

Hematuria: +++

RBC casts: present

serum albumin: normal/slightly reduced

renal - glomerular proteinuria causes

glomerulonephritis

glomerulosclerosis

Diabetes mellitus

sickl disease transplants and infectious diseases

cancers

drugs

increased pressure on the filtration mechanism (hypertension, strenuous exercise, dehydration, preeclampsia in pregnancy)

Renal - Tubular proteinuria

normal - small proteins filtered then reabsorbed

tubular damage affects reabsorptive ability

• these proteins can no longer be absorbed

• acute tubular dysfunction (toxic substances, heavy metals, viral infections, systemic diseases, hemolytic disorders, pyelonephritis

amount of protein is less than 4 g/day

difference in affects to the amount of albumin found in urine for glomerular damage in the kidney vs. tubular damge

glomerular - albumin

tubular - little to no albumin

microalbuminuria

small amounts of albumin

1st sign of diabetic nephropathy (could appear 5 years before seeing positive urine protein)

• renal failure common in diabetics

• early screening for albumin levels that are lower than seen on routine reagent strip

• increased risk of cardiovascular disease

previously required 24 hr collection → reported mg/24 hr of albumin

random urine

postrenal proteinuria

protein added in the lower urinary and genitourinary tract

microbial infections causing inflammations and release of interstitial fluid protein

menstrual contamination or vaginal secretions

semen/ prostatic fluid

traumatic injury

principle of reagent strip reactions for protein

protein error of indicators

indicator dye release H+ if protein is present, causing a pH change and a change in color

pad maintained at pH 3.0 by a buffer

most sensitive to albumin → has more amino groups to accept H+ than other proteins

what is the color reaction that happens on the reagent strip for protein detection and what is it reported as

albumin + dye → albumin-dye complex + H+

color progresses thru green to blue

Report:

• Neg, trace, 1+, 2+, 3+, 4+ OR

• 30, 100, 300, 2000 mg/dL

• trace values are <30 mg/dL

what can interfere with protein detection on reagent strips

highly alkaline urine

• produces a rise in pH and color change unrelated to protein

false positives

• highly pigmented urine or high sp. grav

• ammonium compounds, detergents or antiseptics

false-negatives for proteins other than albumin

Sulfosalicyclic Acid (SSA) Confirmation

screening or confirmation for protein in urine

precipitates proteins w/o heat

use centrifuged specimen → removed extraneous contamination

compare against standards for 1+, 2+, 3+, 4+

Interferences:

• radiographic dye

• antibiotics

clinical significance of glucose in urine

diabetes mellitus

• renal threshold is 160-180 mg/dL

• higher blood sugar= glycosuria

gestational diabetes

• placental hormones block action of insulin

• crosses placenta

hormonal disorders → insulin

renal glycosuria

glucose reagent strip rxn

glucose oxidase rxn specific for glucose

• Glucose + O2 (glucose oxidase)→ gluconic acid + H2O2

• H2O2 + chromogen (peroxidase)→ oxidized chromogen (colored) + H2O peroxidase

specific to glucose and no other sugar

reporting:

• neg, trace, 1+, 2+, 3+, 4+

• 100-2000 mg/dL

what can interfere w/ glucose rxn on reagent strip

false positives

• peroxide or oxidizing detergents

False negatives

• enzymatic rxn interferences

  • ascorbic acid and strong reducing agents

  • high levels of ketons

  • high sp. grav

  • old specimens due to glycolysis - most common

clinitest procedure

not a specific test for glucose → tests for other reducing sugars (ex. galactose); confirmatory test

copper sulfate reacts w/ reducing substances to form cuprous oxide, which causes a color change

5 drops urine/ 10 drops diH2O

compare the color change to the color chart

ketones

three intermediate products of fat metabolism

• acetone

• acetoacetic acid

• beta-hydroxybutyric acid

both acetone and beta-hydroxybutyric acid are produced from acetoacetic acid

appear in urine when fat is broken down to glucose for energy

what is the clinical significance of ketones in urine?

diabetes mellitus

• inability to metabolize carbohydrate

• ketonuria shows inadequate insulin

vomiting

• creates loss of carbohydrates

starvation, malabsorption or dieting (fasting)

• decrease in carbohydrate intake

reagent strip reaction for ketons

primarily measures acetoacetic acid

• acetoacetic acid + Na nitroprusside → color change

Report:

• neg, small (1+), moderate (2+), large (3+)

• small (5,15), moderate (40), large (80, 160) mg/dL

what can interfere w/ ketone rxn on reagent strip

medications

abnormal urine color

only measures acetoacetate, not BHB/acetone

False-positives

• improper timing

False-negative

• old specimens

• bacterial breakdown of acetoacetic acid

Acetest procedure

measures ketones

tablet form

• sodium nitroprusside

• provides a better color

• slightly more sensitive

compare the tablet color w/ color chart

report as negative, small, moderate, or large

is the reagent strip or microscope more accurate for detecting blood?

the reagent strip

RBCs may lyse

what color urine does hematuria produce and what might it indicate?

intact RBCs→ causes a cloudy, red urine

causes:

• renal calculi

• glomerulonephritis

• pyelonephritis

• tumors

• trauma

• exposure to toxic chemicals

what color urine does hemoglobinuria produce and what might it indicate?

clear, red urine

causes:

• transfusion rxns

• hemolytic anemias

• severe burns

• infections/ malaria

• strenuous exercise/ RBC trauma

• brown recluse spider bites

hemosiderin

produces yellow-brown granules in sediment

• reabsorption of filtered hemoglobin

myoglobinuria

myoglobin → heme containing protein w/i muscle tissue

produces clear, red/brown urine

caused by:

• muscular trauma/ crush syndromes

• prolonged coma

• convulsions

• muscle-wasting diseases

• alcoholism/ overdose

• drug abuse

• cholesterol-lowering statin medications

clinical significance of hematuria

nephritis, trauma

clinical significance of hemoglobinuria

hemolysis

clinical significance of myoglobinuria

rhabdomyolysis, burns

reagent strip rxn for blood

lysing agent to lyse RBCs

• Blood +peroxide lysing agent = hemoglobin

• hemoglobin + indicator (bromphenol blue)= color change

intact RBCs show a speckled pattern

report:

• trace, small (1+), moderate (2+), large (3+)

sensitivity to 5 RBCs/ µL

reaction interferences for blood on reagent strip

false positive

• strong oxidizing agents

• bacterial peroxidases

• menstrual contamination

false-negative

• high sp. grav

• crenated cells

• unmixed specimens

• medications

• ascorbic acid >25 mg/dL

bilirubin

a degradation product of hemoglobin

correlates w/ urobilinogen and serum bilirubin

clinical significance of bilirubin in urine

bile duct obstruction

• bilirubin backs up into circulation

liver disease

• bilirubin leaks back into circulation from damaged liver

bilirubin reagent strip rxn

bilirubin + diazo salt → azobilirubin

report: neg, small (1+), moderate (2+), large (3+)

sensitivity to 0.40 mg/dL

bilirubin reagent strip rxn interferences

false positive

• urine pigments

false negative

• old specimens

• increased ascorbic acid and nitrite

• exposure to light

Ictotest

confirmatory for bilirubin

diazo salt reaction

more sensitive: .05-.10 mg/dL

may be requested for early disease

report pos or neg

Bilirubin metabolism

Heme —heme oxygenase→ biliverdin —bilverdin reductase→ unconjugated bilirubin —UDPGT→ conjugated bilirubin —intestinal bacteria→ urobilinogen

urobiligen can travel from intestine to kidney → urinary urobilinogen

urobilinogen

bilirubin in intestine converted to _____ and sterocobilinogen

____ is reabsorbed into circulation

• filtered by the glomerulus

always a small amount of ____ in the urine <1 mg/dL

clinical significance of urobilinogen in the body

early detection of liver disease

• liver disorders, hepatitis, cirrhosis, or carcinoma

hemolytic disorders

• excess bilirubin being converted to urobilinogen and increased urobilinogen recirculated to liver

• negative bilirubin and strong positive urobilinogen

bile duct obstruction

• no urobilinogen is seen in the urine

• strip will give normal result

what does high urine bilirubin and normal urobilinogen indicate

bile duct obstruction

what can a ± urine bilirubin paired with a moderately high (++) urobilinogen indicate?

liver damage

what does negative urine bilirubin paired with strong positive (+++) urobilinogen indicate?

hemolytic disease

reagent strip reaction for urobilinogen

different principles for multistix and chemstrip

most common - Ehrlich reagent

• urobilinogen + Ehrlich’s reagent —> colored complex

normal = 0.2-1 mg/dL, Abnormal= 2, 4, 8 mg/dL

rxn interference for urobilinogen - false positives for multistix

ehrlich reactive compounds

cold specimens

rxn interference for urobilinogen - false negatives for chemstrip

high nitrite interferences

rxn interference for urobilinogen -false results for both tests

due to urine colors

aged specimens'

timing of specimens

• urobilinogen is highest after meals

clinical significance of nitrite (disease states and uses)

gram negative bacteria most common

UTI

Cystitis (bladder infection)

pyelonephritis (kidney infection)

evaluation of antibiotic therapy

monitoring of patients at high risk for urinary tract infection

nitrite reagent strip rxn

Nitrite + p-arsenilic acid → diazo compound

diazo compound + tetrahydrobenzaquinolinol → colored complex

• tests ability of bacteria to reduce nitrate to nitrite

• Greiss rxn

• sensitive for 100,000 organisms/mL

• Results: negative/positive

• a true positive result should accompany a positive leukocyte esterase test

what can cause false negatives for nitrite test

other bacteria → skip nitrite stage to nitrogen gas

lack of urinary nitrate

presence of antibiotics

high conc. of ascorbic acid

high sp. grav

what can cause false positives for nitrite test

old specimens

• bacterial multiplication

highly pigmented urine

pink edges or spotting on reagent strip is considered negative

Leukocyte esterase (LE)

detection of leukocytes (WBC) - “Pyuria”

not considered a quantitative test

• perform microscopic evaluation is positive

detects presence of lysed leukocytes

• release of leukocyte esterase

clinical significance of leukocyte esterase

UTI

acute inflammation

renal calculus

also seen w/ Trichomonas, Chlamydia, yeast, and interstitial nephritis

reagent strip rxn of LE

esterase + acid ester → acid + active alcohol

active alcohol + diazonium salt → color change

• Report: trace, sm, mod, lg

• 15, 75, 125, 500 cells/ µL

false positive LE cause

strong oxidizing agents

highly pigmented urine

menstrual contamination

false negative LE causes

high conc. of protein, glucose, ascorbic acid, or antibiotics — reduces rxn sensitivity

leukocyte crenation from high sp. grav

• prevents release of esterase

ascorbic acid

vitamin C

common interference w/ different urine tests

oxidizes certain compounds - effects hydrogen peroxide and diazo salt rxns

blood, bilirubin, glucose, and nitrite

low levels → false negatives

_____ pad on dipsticks