liquid gold2-chemical analysis

This information has been deemed accurate at time of release-10/04

Liquid Gold: Chemical Assessment of Urine

Sharon S. Ehrmeyer, Ph. D.Dept. of Pathology and Lab Medicine

University of Wisconsin Medical School


Presentation Overview

ReviewChemical (urine test strip) reactionsImportance of measurement Potential interferences with the reaction

Routine urinalysischemical assessment


Chemical analysis

Multi-parameter urine test strips

Plastic strips with multiple pads permeated with reagentsChemical reactions are evaluated

– Visually through color comparisons

– With instrumentation


Chemical analysis

Follow manufacturer’s directions for use and storage of urine test stripsKeep containers tightly closed

Only remove strips as needed

Follow additional test protocols established by test siteMaintain positive patient identification throughout the testing process including result reportingAdhere to all safety policies


Chemical analysisRemove strip; close containerImmerse strip into a well-mixed, uncentrifuged urine aliquot and quickly remove

Draw edge of strip along rim Blot side of strip on absorbent paper

Compare color of each pad to those on strip container

Timing for reactions varies with manufacturersConcentration correspondsto color change of pad


Urine test strips parameters

Specific gravitypHLeukocytesNitriteProtein

GlucoseKetonesUrobilinogenBilirubinBlood


Specific gravityReaction

In the presence of cations, protons are released by a complexing agent (on the pad) to produce a color changeBromthymol blue changes from blue to blue-green to yellow

Detects only ionic solutes not total solute content

Better reflects the kidneys’ concentrating and secreting ability

In comparison to other methodsProteins > 100 – 500 mg/dL may elevate valuesGlucose and urea > 1% may decrease values


pH

Kidneys, lungs and blood buffers maintain pHUrine the pH of a first morning sample typically is between 5.0 - 6.0 pH units

protein diet produces more acid urinevegetarian diet produces more alkaline urine

pH values <4.5 and >9.0 are physiologically impossible

Old urine (elevated pH)


pH

Reaction -- based on double indicatorsystem

Bromthymol blue and methyl red– pH 5.0 - 9.0

No known interferences

Correlate pH with other urine findingsmicroscopic examination– type of crystals, dissolution of urine elements, etc.


Leukocytesleukocyte esterase diazonium salt

ester indoxyl purple color

Granulocytic leukocytes release esterase Strip method detects leukocyte esterase from both intact and lysed cells

Screening used as an indicator of a urinary tract infection (UTI)Correlate with nitrite, protein and microscopic findings

– may have + reaction with no granulocytes on the microscopic


LeukocytesPositive reaction with ~5 - 15 granulocytes (WBCs)/hpf

result is read as either + or -negative result does not rule out an increase in WBCs

False positivesoxidizing agents mistakenly added to collection containerleukocyte esterase from other sources– large numbers of eosinophils– microbial peroxidases

drugs or foodstuffs that can color the pad

False negativescheck manufacturer’s product insert


Nitriteacid

aromatic amine + nitrite diazonium salt

aciddiazonium salt + aromatic compd red-violet azo dye

Screening for urinary tract infections (UTI)

Most frequent UTI bacteria reduce nitrate to nitrite

E. Coli, Klebsiella, Proteus, Staphylococcus, Psuedomonas

nitrate normally present in urine


NitriteStrip reaction is read as either + or -Preferable specimen

fresh, clean catchfirst morning or specimen collected after urine has incubated in bladder for a minimum of 4 hoursno preservative added

False negativesbacteria that do not reduce nitrate to nitrite– suspected samples view microscopically and/or culture

diet lacking vegetables; minimal nitrate in urinetoo short of incubation time in bladder for conversionhigh levels of ascorbic acid (> 25 mg/dL)


Protein

Strip measurement based on protein error of pH indicators

pH held constant by buffer on padproteins cause certain dyes (indicators) to change color– tetrabromphenol blue, etc.– color change of indicator equated to protein present

pH 3.0indicator + protein indicator (H+ released)(yellow) (light green/green)


Protein

Test strip measurementmeasures primarily albuminmay have negative reactions with globulins, myoglobin, hemoglobin, Bence Jones, mucoproteinssources of error– highly alkaline urine– contamination (basic) override buffering capacity – strips left in urine too long and buffer is removed

correlate with other findings– albumin often positive when RBCs, WBCs, bacteria, and

casts


Protein

Proteinuria – increased amount of protein in the urine

Often first indicator of renal disease– Also seen with other conditions

Normal urine contains up to 150 mg of protein/dayOriginates from glomerular filtrate (GF) and urinary tract–95-99% of protein in GF is reabsorbed


Glucose

GlucosuriaGlucose normally in GF is reabsorbed by proximal tubules–threshold limited (plasma glucose 160 - 180

mg/dL)

Pre-renal causes (hyperglycemia)–diabetes mellitus, hyperthyroidism, liver

problems, pancreatic disease, CNS disease, etc.

Renal causes (defective tubular reabsorption)


GlucoseTest strips detect glucose only

glucose oxidase

glucose + 02 gluconic acid + H202

peroxidaseH202 + chromogen oxidized chromogen + H20

(yellow) (green)

Amounts <40 -100 mg/dL are not detected


Glucose

False positivesstrong oxidizing agents (i.e., bleach)contaminating peroxides

False negativesrarely occur when ascorbic acid >100 mg/dLimproperly stored (old) urine (bacteria uses glucose)

Correlate with ketones and specific gravityketones associated with diabetespale urine with high specific gravity


KetonesKetone bodies

acetoacetate, acetone and β-hydroxybutyrate

When carbohydrates are not available, liver mitochondria begin active ketogenesis

provide energy to brain, heart, skeletal muscles and kidneys

Ketonuriainability to use carbohydrates (diabetes mellitus)Starvation, dietsloss of carbohydrates, digestive disorders


KetonesMeasurement

alkalineacetoacetate + sodium + glycine violet colorand acetone nitroprusside

False positivesCompounds having free-sulfhydryl groupsHighly pigmented urines

False negativesold urines -- breakdown of acetoacetate and acetoneimproper handling of strips

– exposure of strips to moisture, heat or light

Correlate with glucose


Bilirubin - Hemoglobin catabolism

Bilirubin is transported by albumin to the liver

Bilirubin is continually formed from breakdown of hemoglobin in the reticuloendothelial system


Hemoglobin Catabolism

RES cellsFree Hemoglobin

HEME

Bilirubin

Ironaa pool

liver

Gut flora Urobilinogen

Biliary Tract

feces

Urinary Urobilinogen


Bilirubin breakdown

Bilirubin-albumin complex is largedoes not pass through glomerulusindirect or unconjugated bilirubin is water insoluble

In liver, hepatocytes conjugate bilirubindirect or conjugated bilirubin is water soluble

Normally all conjugated bilirubin is excreted into bile canuliculi and ultimately into the bile duct

Once in the small intestine, intestinal flora convert bilirubin to urobilinogen


Bilirubin

bilirubin glucuronide + diazonium salt

azobilirubin compound (pink to red-violet color)

Lowest detectable amount - 0.5 mg/dL


Bilirubin

False positivesred-colored urine (drugs)drugs that react with the diazonium salt

False negativeslarge amounts of ascorbic acidelevated nitrites (UTI)specimen improperly stored– bilirubin is destroyed by exposure to light

Correlate with urobilinogen


Bilirubinuria

Causes Bilirubin

Prehepatic (hemolytic)

Negative (water insoluble)

Hepatic (hepatitis cirrhosis, etc.)

Positive

Posthepatic (obstruction)

Positive


Hemoglobin Catabolism

RES cellsFree Hemoglobin

HEME

Bilirubin

Ironaa pool

liver

Gut flora Urobilinogen

Biliary Tract

feces

Urinary Urobilinogen


Urobilinogen

Intestinal flora convert bilirubin to urobilinogen20-50% of urobilinogen is reabsorbed and enters the blood stream and is filtered by the glomerulusnormally present in urine (< 1.0 mg/dL)

Increased valuesincreased production of bilirubin (prehepatic causes)reabsorption prevented by liver disease

Decreased valuesbilirubin is not excreted into the small intestine suppressed intestinal flora from antibiotics


Urobilinogen

acidurobilinogen + azo-reagent red azo dye

azo-reagent = methoxybenzene-diazonium-tetrafluoroborate

Absence of urobilinogen (i.e., obstructions) cannot be detected


Urobilinogen

False positiveshighly colored urines

False negativesold, improperly stored specimens–photo-oxidized to urobilin–labile in acid urine

some preservatives (formalin)nitrites (>5 mg/dL)

Correlate with bilirubin


Bilirubinuria and Urobilinogenuria

Causes Bilirubin Urobilinogen

Prehepatic (hemolytic)

Negative (water insoluble)

Increased

Hepatic (hepatitis cirrhosis, etc.)

Positive Normal to increased

Posthepatic (obstruction)

Positive Decreased to absent


Hematuria and Hemoglobinuria

Hematuria -- abnormal amount of RBCs in urine

Blood can enter the urinary system anywhere– from glomerulus to urethra

– collection contaminant

Hemoglobinuria -- presence of hemoglobin in urineRBCs lyse releasing hemoglobin in alkaline and dilute urine

hemoglobin passing through glomerulus is rare– bound to haptoglobin forming a large complex


Blood

Based on pseudoperoxidase activity of hemoglobin

Hb; myoglobinH202 + chromogen oxidized chromogen + H20

(yellow) (green)

dotted pattern -- RBCs lyse on padhomogenous color change results from just Hbstrips detect intact RBCs, hemoglobin and myoglobin


Blood

Sensitivity -- ~5 - 10 RBCs/ µLFalse positives

contamination -- menstrual, hemorrhoidalmicrobial peroxidasesstrong oxidizing agents

False negativeshigh specific gravity

CHEMSTRIP® has no interference with Vc

Correlate with color, protein and microscopic

liquid gold2-chemical analysis

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