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 side Keeping the strip flat, read results at the appropriate times by comparing the color to the appropriate color on the chart provided.
Chemical Exam of Urine Sources of error (& preventions) Testing cold specimens would result in a slowing down of reactions; test specimens when fresh or bring them to RT before testing Inadequate mixing of specimen could result in false reduced or negative reactions to blood and leukocyte tests; mix specimens well before dipping Over-dipping of reagent strip will result in leaching of reagents out of pads; briefly, but completely dip the reagent strip into the urine
Chemical Exam of Urine Inadequate blotting & Failure to keep strip horizontal will 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 tests over development of reagent pad colors leading to falsely increased results; follow manufacturer’s recommendations
Chemical Exam of Urine Inadequate light misinterpretation of results; use good lighting Mis-using color chart misinterpretation 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.
Chemical Exam of Urine Handling and Storage Keep strips in original container, stored at RT Protect from moisture and volatile fumes Use before expiration date Do not touch reagent pad areas
Chemical Exam of Urine Quality 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.
Urine Glucose Testing Normal : no glucose detected Clinical significance of abnormal results (Glucosuria) Plasma glucose level exceeds renal threshold (160-189 mg/dL) Diabetes mellitus Renal tubular dysfunction Filtered glucose not being reabsorbed in tubules
Urine Glucose Testing Dipstick Testing Method Glucose initiates reaction Coupled reaction Glucose 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. Chromogens Potassium iodide or Tetramethylbenzindine
Urine Glucose Testing Sensitivity @ 50-100 mg (compared to Clinitest’s 250) SO- Can have a positive dipstick but a neg Clinitest Specificity - is specific for glucose only. not affected by other sugars or reducing substances.
Urine Glucose Testing Interfering substances High specific gravity and high pH may depress color. Ascorbic acid-false neg Bleach or peroxide may give false positive
Urine Bilirubin Testing Normal : no bilirubin detected Clinical 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
Urine Bilirubin Testing Prehepatic / Hemolytic jaundice Excessive hemolysis of RBC; beyond what the liver can process Type of bilirubin? Is bilirubin found in the urine? YES/NO? Explain.
Urine Bilirubin Testing Prehepatic / Hemolytic jaundice Type of bilirubin? – indirect, insoluble, unconjugated Is bilirubin found in the urine? – No, the bilirubin is not water soluble
Urine Bilirubin Testing Hepatic jaundice Liver’s cells malfunctioning Ie. viral hepatitis, cirrhosis etc. Both (direct) bilirubin and urobilinogen found in urine.
Urine Bilirubin Testing Post hepatic (regurgative or obstructive) hepatitis Obstruction to outflow of bile – some type of blockage Gall stones Tumor Edema Conjugated bilirubin backed up into blood (Bilirubinuria) and passes into urine
Urine Bilirubin Testing Testing method Urine dipsticks for bilirubin – a diazo reaction Impregnated with stabilized diazotized 2,4 dichloraniline Color goes from buff to brown also shades of pink – violet If urine is strongly colored, look for change in pad color after dipping. Use Ictotest for backup.
Urine Bilirubin Testing Interfering substances Medication metabolites, pigments and indican may obscure readings False negatives due to aged specimens, especially those exposed to light and oxidation.
Urine Ketone Testing Ketone Bodies Origin - not normally present Products of fat catabolism - breakdown of fat into CO2 and H2O What are the 3 ketone bodies?
Urine Ketone Testing Acetone 2%. -Acetone is volatile, & excreted primarily through the lungs Diacetic Acid (Acetoacetic) the first formed, 20 % of the total the form detected by most ketone test procedures Beta hydroxybutyric Acid majority formed, but not detected by routine tests. Only Hart’s test, an old ‘wet chemical’ test will detect this one.
Urine Ketone Testing Definitions Ketonuria - ketones in the urine Ketonemia - ketones in the blood Ketosis - 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
Urine Ketone Testing Clinical significance Health – formed in liver and completely metabolized Disease – excessive formation and accumulation Disturbance of carbohydrate metabolism when 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, diabetes Starvation Vomiting and diarrhea in children Van Gierke's Disease – glycogen storage disease High fat diet
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 color Can be used on urine or blood
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.
Urine Specific Gravity Testing The specific gravity will always be greater than 1.000 and will increase as more materials are dissolved in the urine. The value changes throughout the day depending on fluid intake.
Urine Specific Gravity Testing Specific gravity between 1.002 & 1.035 on a random sample is normal if kidney function is normal. Specific gravity in Bowman’s capsule fluid is @ 1.007 Any reading below this indicates hydration Any reading above this indicates some degree of dehydration
Urine Specific Gravity Testing Again dilute urine will have values less than 1.010. Fixed specific gravity = 1.010; isothenuria Diabetes insipidus End-stage renal disease And concentrated urine will have values usually over 1.020. Usually due to dehydration and can be seen in well population as well as sick.
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
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
Urine Specific Gravity Testing Specific gravity > 1.035 (refractometer) Could have very high glucose levels Could contain radiographic dye
Urine Specific Gravity Testing Testing Polyelectrolytes, pH indicator (bromthymol blue measures the pH change), and alkaline buffer
Urine Specific Gravity Testing Interfering substances False 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
Urine Blood Testing Normally not found in urine Hemoglobinuria – free hemoglobin in urine Circulating free hemoglobin normally picked up by haptoglobin preventing loss in urine When serum levels of hemoglobin > 100 mg/dL threshold is exceeded Hematuria – RBCs in the urine Trauma / irritation of renal organs
Urine Blood Testing ‘Blood’ test detects Free Hemoglobin RBCs – get lysed on the pad & their hemoglobin reacts Myoglobin – muscle hemoglobin Principle based on the peroxidase-like activity of the heme portion of the molecule
Urine Blood Testing Sensitivity – can detect at levels of 5-10 cells/uL Interfering substances Ascorbic acid Nitrates Oxidizing agents (ie bleach) Contaminate blood (menstrual)
Urine pH Testing Normal: kidneys capable of 4.5 – 8.0 Factors effecting pH Diet – general & specific foods Time of day Metabolic disorders Drugs / medications Dipstick capable: 4.5 – 9.0
Urine pH Testing Test method Dipstick indicators – methyl red and bromthymol blue Range 4.5-9.0 Caution – other chemicals on dipstick can effect pH reading
Urine Protein Testing Normally not found in measurable amounts on dipstick (<150 mg/dL /day) Permeability of glomerulus Damage to glom capularies Changes in glom blood flow Albumin excretions may be increased temporarily due to exercise, uti, and acute illness with fever. Dipstick results of >@ 1+ (30mg/dL) would equal to @ 500 mg/dL (clinical proteinuria)
Urine Protein Testing Only albumin detectable by dipstick Sensitivity (@15-30 ml/dL)
Urine Protein Testing New testing for microalbumin & creatinine Results: Protein 20-200 mg/dL (30-300 mg/dL /24 hr) Creatinine 10-300 mg/dL Albumin/creatinine ratio Normally albumin in the urine is less than 30 mg/ gram creatinine
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 end Citrate Buffer – maintains pH 3 -quite acid
Urine Protein Testing Sources of error Sensitive only to albumin Urine with strong / unusual color makes reading difficult Highly alkaline or buffered urine will neutralize acid buffer and lead to increased erroneous results. Urine container contamination would interfere
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.
Urine Urobilinogen Testing Normally found in small amounts, especially in early afternoon Increased amounts may indicate liver disease or be seen as result of hemolytic disorders Decreased amounts: If intestinal bacteria destroyed Liver doesn’t conjugate bilirubin Biliary obstruction – failure of bilirubin to reach small intestine
Urine Urobilinogen Testing Test principle based on Ehrilich’s reaction Para-dimethylaminobenzaldehyde = Ehrlich's reagent. Must protect specimen from light and test immediately
Urine Nitrate Testing Nitrate Detects presence of certain types of bacteria screening for presence of UTI. Certain species of bacteria convert nitrate (normal constituent of urine) to nitrite Escherichia - most common cause of UTI Klebsiella Proteus Pseudomonas Enterobacter Citrobacter
Urine Nitrate Testing Aromatic amine in reagent strip reacts with nitrite; producing a diazonium salt The diazonium salt reacts with sulfanilic acid and acetic acid to produce a pink azo dye
Urine Nitrate Testing Limitations reported as positive or negative Not all UTI causing bacteria convert nitrate to nitrite Haemophilus Staphylococcus Streptococcus
Urine Nitrate Testing Fresh 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.
Urine Leukocyte Testing Leukocyte esterase testing is another test used as a means of screening for urinary tract infection. Does not measure concentration of leukocytes Will detect presence of lysed leukocytes as well as intact WBCs
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.
Urine Leukocyte Testing Reaction interference False positives - oxidizing detergents False 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.