liquid gold2-chemical analysis
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Transcript of liquid gold2-chemical analysis
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Liquid Gold: Chemical Assessment of Urine
Sharon S. Ehrmeyer, Ph. D.Dept. of Pathology and Lab Medicine
University of Wisconsin Medical School
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Presentation Overview
ReviewChemical (urine test strip) reactionsImportance of measurement Potential interferences with the reaction
Routine urinalysischemical assessment
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Chemical analysis
Multi-parameter urine test strips
Plastic strips with multiple pads permeated with reagentsChemical reactions are evaluated
– Visually through color comparisons
– With instrumentation
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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
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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
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Urine test strips parameters
Specific gravitypHLeukocytesNitriteProtein
GlucoseKetonesUrobilinogenBilirubinBlood
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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
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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)
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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.
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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
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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
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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
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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)
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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)
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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
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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
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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)
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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
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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
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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
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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
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Bilirubin - Hemoglobin catabolism
Bilirubin is transported by albumin to the liver
Bilirubin is continually formed from breakdown of hemoglobin in the reticuloendothelial system
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Hemoglobin Catabolism
RES cellsFree Hemoglobin
HEME
Bilirubin
Ironaa pool
liver
Gut flora Urobilinogen
Biliary Tract
feces
Urinary Urobilinogen
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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
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Bilirubin
bilirubin glucuronide + diazonium salt
azobilirubin compound (pink to red-violet color)
Lowest detectable amount - 0.5 mg/dL
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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
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Bilirubinuria
Causes Bilirubin
Prehepatic (hemolytic)
Negative (water insoluble)
Hepatic (hepatitis cirrhosis, etc.)
Positive
Posthepatic (obstruction)
Positive
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Hemoglobin Catabolism
RES cellsFree Hemoglobin
HEME
Bilirubin
Ironaa pool
liver
Gut flora Urobilinogen
Biliary Tract
feces
Urinary Urobilinogen
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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
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Urobilinogen
acidurobilinogen + azo-reagent red azo dye
azo-reagent = methoxybenzene-diazonium-tetrafluoroborate
Absence of urobilinogen (i.e., obstructions) cannot be detected
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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
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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
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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
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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
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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