7 Chemical Exam of Urine Reagent strip precautions and source of errors Normal dipstick procedure:Dip strip briefly, but completely into well mixed, room temperature urine sample.Withdraw strip, blot briefly on its sideKeeping the strip flat, read results at the appropriate times by comparing the color to the appropriate color on the chart provided.
8 Chemical Exam of Urine Sources of error (& preventions) Testing cold specimenswould result in a slowing down of reactions; test specimens when fresh or bring them to RT before testingInadequate mixing of specimencould result in false reduced or negative reactions to blood and leukocyte tests; mix specimens well before dippingOver-dipping of reagent stripwill result in leaching of reagents out of pads; briefly, but completely dip the reagent strip into the urine
9 Chemical Exam of Urine Improper timing of tests Inadequate blotting & Failure to keep strip horizontalwill result in over-run or mixing of reagents between the different reaction pads; blot excess urine off the strip and keep strip horizontal. If dipping from the tube, can run the side of the strip along the rim to remove excess urine.Improper timing of testsover development of reagent pad colors leading to falsely increased results; follow manufacturer’s recommendations
10 Chemical Exam of Urine Inadequate light Mis-using color chart misinterpretation of results; use good lightingMis-using color chartmisinterpretation of results; hold strip just over color chart and match colors as close as possible, consider use of back-up tests, if needed, especially if urine’s color masks reaction colors.
11 Chemical Exam of Urine Handling and Storage Keep strips in original container, stored at RTProtect from moisture and volatile fumesUse before expiration dateDo not touch reagent pad areas
12 Chemical Exam of UrineQuality Control - use appropriate, commercially prepared positive and negative controls.Use commercially prepared pos and neg controls, at least once per 24 hours, and anytime a new bottle is opened, or question of validity of results. Readings should agree with published results ± one color block.
13 Urine Glucose Testing Normal : no glucose detected Clinical significance of abnormal results (Glucosuria)Plasma glucose level exceeds renal threshold ( mg/dL)Diabetes mellitusRenal tubular dysfunctionFiltered glucose not being reabsorbed in tubules
14 Urine Glucose Testing Dipstick Testing Method Glucose initiates reactionCoupled reactionGlucose oxidase – oxidizes glucose to gluconic acid and concurrently reduces oxygen to hydrogen peroxide.Hydrogen peroxide in presence of the enzyme peroxidase will oxidize an indicator, giving a colored reaction.ChromogensPotassium iodide orTetramethylbenzindine
15 Urine Glucose Testing Sensitivity @ mg (compared to Clinitest’s 250) SO- Can have a positive dipstick but a neg ClinitestSpecificity - is specific for glucose only.not affected by other sugars or reducing substances.
16 Urine Glucose Testing Interfering substances High specific gravity and high pH may depress color.Ascorbic acid-false negBleach or peroxide may give false positive
17 Urine Bilirubin Testing Normal : no bilirubin detectedClinical significance of abnormal results (Bilirubinuria)Jaundice - Condition when serum bilirubin becomes greater than the liver can handle, and there is an abnormal collection of bilirubin in the tissues giving them a yellow color
18 Urine Bilirubin Testing Prehepatic / Hemolytic jaundiceExcessive hemolysis of RBC; beyond what the liver can processType of bilirubin?Is bilirubin found in the urine? YES/NO? Explain.
19 Urine Bilirubin Testing Prehepatic / Hemolytic jaundiceType of bilirubin? – indirect, insoluble, unconjugatedIs bilirubin found in the urine? – No, the bilirubin is not water soluble
20 Urine Bilirubin Testing Hepatic jaundiceLiver’s cells malfunctioningIe. viral hepatitis, cirrhosis etc.Both (direct) bilirubin and urobilinogen found in urine.
21 Urine Bilirubin Testing Post hepatic (regurgative or obstructive) hepatitisObstruction to outflow of bile – some type of blockageGall stonesTumorEdemaConjugated bilirubin backed up into blood (Bilirubinuria) and passes into urine
22 Urine Bilirubin Testing Testing methodUrine dipsticks for bilirubin – a diazo reactionImpregnated with stabilized diazotized 2,4 dichloranilineColor goes from buff to brown also shades of pink – violetIf urine is strongly colored, look for change in pad color after dipping. Use Ictotest for backup.
23 Urine Bilirubin Testing Interfering substancesMedication metabolites, pigments and indican may obscure readingsFalse negatives due to aged specimens, especially those exposed to light and oxidation.
24 Urine Ketone Testing Ketone Bodies Origin - not normally presentProducts of fat catabolism - breakdown of fat into CO2 and H2OWhat are the 3 ketone bodies?
25 Urine Ketone Testing Acetone Diacetic Acid (Acetoacetic) 2%. -Acetone is volatile, & excreted primarily through the lungsDiacetic Acid (Acetoacetic)the first formed,20 % of the totalthe form detected by most ketone test proceduresBeta hydroxybutyric Acidmajority formed, but not detected by routine tests.Only Hart’s test, an old ‘wet chemical’ test will detect this one.
26 Urine Ketone Testing Definitions Ketonuria - ketones in the urine Ketonemia - ketones in the bloodKetosis - disease state, when patient has increased amount of ketones.Acidosis - state when blood pH is decreased, an accumulation of acids; commonly occurs as a result of ketosis
27 Urine Ketone Testing Clinical significance High fat diet Health – formed in liver and completely metabolizedDisease – excessive formation and accumulationDisturbance of carbohydrate metabolismwhen there is a decrease of carbohydrate metabolism, then the body stores of fat must be metabolized to supply energy.As a result of this increased fat metabolism ketones will be found in the urine. Ex. low carbohydrate diets, diabetesStarvationVomiting and diarrhea in childrenVan Gierke's Disease – glycogen storage diseaseHigh fat diet
29 Urine Ketone Testing Testing most use nitroprusside detects diacetic acid and a small amount of acetone, but does not detect β-hydroxybutyric acid.Produces purple colorCan be used on urine or blood
30 Urine Specific Gravity Testing The specific gravity is a measure of the weight of urine compared to an equal amount of water. Specific gravity it proportional to urine osmolality which is a measure of concentration.
31 Urine Specific Gravity Testing The specific gravity will always be greater than and will increase as more materials are dissolved in the urine. The value changes throughout the day depending on fluid intake.
32 Urine Specific Gravity Testing Specific gravity between & on a random sample is normal if kidney function is normal.Specific gravity in Bowman’s capsule fluid 1.007Any reading below this indicates hydrationAny reading above this indicates some degree of dehydration
33 Urine Specific Gravity Testing Again dilute urine will have values less than Fixed specific gravity = 1.010; isothenuriaDiabetes insipidusEnd-stage renal diseaseAnd concentrated urine will have values usually overUsually due to dehydration and can be seen in well population as well as sick.
34 Urine Specific Gravity Testing Increased urine specific gravity may indicate / be seen in:* Dehydration* Diarrhea* Excessive sweating* Glucosuria* Heart failure (related to decreased blood flow to the kidneys)* Renal arterial stenosis* Syndrome of inappropriate antidiuretic hormone secretion (SIADH)* Vomiting* Water restriction
35 Urine Specific Gravity Testing Decreased urine specific gravity may indicate / be seen in:* Excessive fluid intake* Diabetes insipidus – central or nephrogenic* Renal failure (that is, loss of ability to reabsorb water)* Pyelonephritis
36 Urine Specific Gravity Testing Specific gravity > (refractometer)Could have very high glucose levelsCould contain radiographic dye
37 Urine Specific Gravity Testing Polyelectrolytes , pH indicator (bromthymol blue measures the pH change), and alkaline buffer
38 Urine Specific Gravity Testing Interfering substancesFalse elevation of results may be seen in samples with increased protein concentration.Some reports of reduced specific gravity results on alkaline specimens.Lipids may also effect results
39 Urine Blood Testing Normally not found in urine Hemoglobinuria – free hemoglobin in urineCirculating free hemoglobin normally picked up by haptoglobin preventing loss in urineWhen serum levels of hemoglobin > 100 mg/dL threshold is exceededHematuria – RBCs in the urineTrauma / irritation of renal organs
42 Urine Blood Testing ‘Blood’ test detects Free HemoglobinRBCs – get lysed on the pad & their hemoglobin reactsMyoglobin – muscle hemoglobinPrinciple based on the peroxidase-like activity of the heme portion of the molecule
43 Urine Blood TestingSensitivity – can detect at levels of 5-10 cells/uLInterfering substancesAscorbic acidNitratesOxidizing agents (ie bleach)Contaminate blood (menstrual)
44 Urine pH Testing Normal: kidneys capable of 4.5 – 8.0 Factors effecting pHDiet – general & specific foodsTime of dayMetabolic disordersDrugs / medicationsDipstick capable: 4.5 – 9.0
45 Urine pH Testing Test method Dipstick indicators – methyl red and bromthymol blueRangeCaution – other chemicals on dipstick can effect pH reading
46 Urine Protein TestingNormally not found in measurable amounts on dipstick (<150 mg/dL /day)Permeability of glomerulusDamage to glom capulariesChanges in glom blood flowAlbumin excretions may be increased temporarily due to exercise, uti, and acute illness with fever.Dipstick results of 1+ (30mg/dL) would equal 500 mg/dL (clinical proteinuria)
47 Urine Protein Testing Only albumin detectable by dipstick Sensitivity ml/dL)
48 Urine Protein Testing New testing for microalbumin & creatinine Results:Protein mg/dL ( mg/dL /24 hr)Creatinine mg/dLAlbumin/creatinine ratioNormally albumin in the urine is less than 30 mg/ gram creatinine
49 Urine Protein Testing Principle - Protein error of indicators at fixed pH, certain indicators show one color in the presence of protein and another in absence of protein - the “error” of the indicator.Indicator – tetrabromphenol blue - can be hard to read at the trace endCitrate Buffer – maintains pH 3 -quite acid
50 Urine Protein Testing Sources of error Sensitive only to albumin Urine with strong / unusual color makes reading difficultHighly alkaline or buffered urine will neutralize acid buffer and lead to increased erroneous results.Urine container contamination would interfere
51 Urine Protein Testing Urine back up test 3% sulfosalicylic acid Added to the supernatant to detect any kind of protein. Urine will turn cloudy if protein is present.
52 Urine Urobilinogen Testing Normally found in small amounts, especially in early afternoonIncreased amounts may indicate liver disease or be seen as result of hemolytic disordersDecreased amounts:If intestinal bacteria destroyedLiver doesn’t conjugate bilirubinBiliary obstruction – failure of bilirubin to reach small intestine
53 Urine Urobilinogen Testing Test principle based on Ehrilich’s reactionPara-dimethylaminobenzaldehyde = Ehrlich's reagent.Must protect specimen from light and test immediately
54 Urine Nitrate Testing Nitrate Detects presence of certain types of bacteriascreening for presence of UTI.Certain species of bacteria convert nitrate (normal constituent of urine) to nitriteEscherichia - most common cause of UTIKlebsiellaProteusPseudomonasEnterobacterCitrobacter
55 Urine Nitrate TestingAromatic amine in reagent strip reacts with nitrite; producing a diazonium saltThe diazonium salt reacts with sulfanilic acid and acetic acid to produce a pink azo dye
56 Urine Nitrate Testing Limitations reported as positive or negative Not all UTI causing bacteria convert nitrate to nitriteHaemophilusStaphylococcusStreptococcus
57 Urine Nitrate TestingFresh first morning specimen is preferred - besides being the most concentrated specimen, the urine has been in the bladder longer, allowing bacteria time and opportunity to convert the nitrates to nitrites.
58 Urine Leukocyte Testing Leukocyte esterase testing is another test used as a means of screening for urinary tract infection.Does not measure concentration of leukocytesWill detect presence of lysed leukocytes as well as intact WBCs
59 Urine Leukocyte Testing test principle:Leukocyte esterase, an enzyme present in granulocytes, hydrolyzes indoxylcarbonic acid esterase to produce indoxyl, which reacts with a diazonium salt to create a purple color usually in 2 min.
60 Urine Leukocyte Testing Reaction interferenceFalse positives - oxidizing detergentsFalse negatives - greatly increased glucose, protein, or specific gravity- increased sp gr could cause WBC to crenate preventing their releasing their esterase, So it is possible for the dipstick to be negative when there are WBCs present.