Urinalysis (Macroscopic( Chemical Tests) ) Background. Ishihara Color Blindness Tests 12/22/2012. Mohammad Reza Bakhtiari DCLS, PhD

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1 Urinalysis (Macroscopic( Chemical Tests) ) Mohammad Reza Bakhtiari DCLS, PhD Background Routine chemical examination of urine has changed dramatically since the early days of urine testing, owing to the development of the reagent strip method for chemical analysis. Reagent strips currently yprovide a simple, rapid means for performing medically significant chemical analysis of urine, including ph, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, leukocytes, and specific gravity. Reagent strips consist of chemical-impregnated absorbent pads attached to a plastic strip. A colorproducing chemical reaction takes place when the absorbent pad comes in contact with urine. Ishihara Color Blindness Tests 1

2 Color Blindness Causes: Hereditary Prevalence: Caucasian: 10%, Asians: 5% Red-Green Color blindness Chrom. X Linked 95% Blue-Yellow Color blindness Chrom. 7 Linked 5% Some Other Inherited Diseases Cone Dystrophy Cone-rod Dystrophy Achromatopsia Blue cone monochromatism Retinitis pigmentosa Macular Degeneration Retinoblastoma Leber s congenital amaurosis Non Hereditary Shaken Baby Syndrome Trauma UV Damage Alzheimer s disease Diabetes mellitus Glaucoma Leukemia Liver disease Multiple sclerosis Parkinson s Disease Sickle cell anemia Chronic alcoholism Care of Reagent Strips 1. Store with desiccant in an opaque, tightly closed container. 2. Store below 30C; do not freeze. 3. Do not expose to volatile fumes. 4. Do not use past the expiration date. 5. Do not use if chemical pads become discolored. 6. Remove strips immediately prior to use. Improper technique can result in errors 1. Mix uncentrifuged specimen well. 2. Let refrigerated specimens warm to room temperature before testing. 3. Dip the strip completely, but briefly, into specimen. 4. Remove excess urine by withdrawing the strip against the rim of the container and by blotting the edge of the strip. 5. Compare reaction colors with the manufacturer s chart under a good light source at the specified time. 6. Perform backup tests when indicated. 7. Be alert for the presence of interfering substances. 8. Understand the principles and significance of the test, read package inserts. 9. Relate chemical findings to each other and to the physical and microscopic urinalysis results. 10. Reagent strips and color charts from different manufacturers are not interchangeable. 2

3 Quality Control 1. Test open bottles of reagent strips with known positive and negative controls every 24 hr. 2. Resolve control results that are out of range by further testing. 3. Test reagents used in backup tests with positive and negative controls. 4. Perform positive and negative controls on new reagents and newly opened bottles of reagent strips. 5. Record all control results and reagent lot numbers. Causes of Acid and Alkaline Urine A healthy individual usually produces a first morning specimen with a slightly acidic ph of 5.0 to 6.0; A more alkaline ph is found following meals (alkaline tide). The ph of normal random samples can range from 4.5 to 8.0. Consequently, no normal values are assigned to urinary ph, and it must be considered in conjunction with other patient information, such as: the acid-base content of the blood, the patient s renal function, the presence of a urinary tract infection, the patient s dietary intake, the age of the specimen Clinical Significance of Urine ph 1.Respiratory or metabolic acidosis/ketosis 2.Respiratory or metabolic alkalosis 3.Defects in renal tubular secretion and reabsorption of acids and bases renal tubular acidosis i Reabsorption of filtered bicarbonate. 4.Renal calculi formation 5.Treatment of urinary tract infections Excretion of secreted hydrogen ions combined with phosphate. 6.Precipitation/identification of crystals 7.Determination of unsatisfactory specimens Excretion of secreted hydrogen ions combined with ammonia produced by the tubules. 3

4 ph Indicator System A double-indicator system of methyl red (4-6) and bromthymol blue (6-9). Methyl red produces a color change from red to yellow, and bromthymol blue turns from yellow to blue. Therefore, in the ph range 5 to 9, one sees colors progressing from orange at ph 5 through yellow and green to a final deep blue at ph 9. Urine Protein Other proteins include: Small amounts of serum and tubular microglobulins, Tamm-Horsfall protein, Proteins from prostatic, seminal, and vaginal secretions. Urine Protein MECHANISMS OF PROTEINURIA 1- Overflow (Pre renal) Plasma cell dyscrasias Hemoglobin, myoglobin, The Acute Phase Reactants associated with infection and inflammation (Transient) 2-Altered glomerular permeability Increased glomerular filtration 3- Inadequate tubular reabsorption Tubulointerstitial disorders Exposure to toxic substances and heavy metals Severe viral infections Fanconi syndrome. 4- Increased tubular secretion Tamm-Horsfall proteinuria Reflux nephropathy, Obstructive uropathy, Some other tubulointerstitial disorders 4

5 Urine Protein Reagent Strips Principle of the protein error of indicators Proteins accept H+ from some indicators (In acid buffers) More Sensitive to Albumin Reagents Multistix: Tetrabromphenol blue Chemstrip: tetrachlorophenol tetrabromosulfophthalein Trace values are considered to be less than 30 mg/dl. Reporting of trace values may be a laboratory option. The specific gravity of the specimen should be considered because a trace protein in a dilute specimen is more significant than in a concentrated specimen. Summary of Protein Reagent Strip Reagents Multistix:Tetrabromphenol blue Chemstrip: 3, 3, 5, 5 tetrachlorophenol 3, 4, 5, 6-tetrabromosulfophthalein Sensitivity Multistix: mg/dl albumin Chemstrip: 6 mg/dl albumin Sources of error/ interference False-positive: Highly buffered alkaline urine Pigmented specimens, phenazopyridine Quaternary ammonium compounds (detergents) Antiseptics, chlorhexidine Loss of buffer from prolonged exposure of the reagent strip to the specimen High specific gravity False-negative: Proteins other than albumin Microalbuminuria Correlations with other tests Blood Nitrite Leukocytes Microscopic Sulfosalicylic Acid Precipitation Test Add 3 ml of 3% SSA reagent to 3 ml of centrifuged urine. Mix by inversion and observe for cloudiness. Grade the degree of turbidity 5

6 Urine Glucose Renal threshold for glucose is approximately 160 to 180 mg/dl. Blood glucose levels fluctuate, and a normal person may have glycosuria following a meal with a high glucose content. Therefore, the most informative glucose results are obtained from specimens collected under controlled conditions. Fasting prior to the collection of samples for screening tests is recommended. Hyperglycemia of nondiabetic origin is seen in a variety of disorders and also produces glycosuria (Exocrine Pancreas, Excess of Counter regulatory hormones, ). Glycosuria occurs in the absence of hyperglycemia when the reabsorption of glucose by the renal tubules is compromised ( renal glycosuria ). End-stage renal disease, cystinosis, and Fanconi syndrome. Glycosuria not associated with gestational diabetes is occasionally seen as a result of a temporary lowering of the renal threshold for glucose during pregnancy. Urine Glucose Urine Ketones Ketones=3 intermediate products of fat metabolism: acetone, acetoacetic acid, and betahydroxybutyric acid. Clinical reasons for increased fat metabolism include: the inability to metabolize carbohydrate, as occurs in diabetes mellitus increased loss of carbohydrate from vomiting. inadequate intake of carbohydrate associated with starvation and malabsorption. 78% 20% 2% 6

7 Urine Ketones 78% 20% 2% Urine Blood Most hematuria originate in the uro-genital tract Urine Blood 7

8 Urine Bilirubin Status Urine Bilirubin Urine Urobilinogen Status 8

9 Urine Urobilinogen Urine Nitrite The nitrite producing bacteria: Escherichia coli Enterococcus Proteus Staphylococci.. Leukocyte Esterase Infections caused by Trichomonas, Chlamydia, yeast, and inflammation of renal tissues (i.e., Interstitial Nephritis) produce Lukocyturia without Bacteriuria. Abacterial Leukocyturia can also occur in cases of renal tuberculosis. Lymphocytes, erythrocytes, bacteria, and renal tissue cells do not contain esterases 9

10 Leukocyte Esterase {tç~ çéâ yéü çéâü TààxÇà ÉÇ dâxáà ÉÇáR ár Pathologic and nonpathologic causes of abnormal urine dipstick findings Pediatr Clin N Am 53 (2006)

11 Causes of false positive and false negative results in urine dipstick testing Pediatr Clin N Am 53 (2006) Micral -Test Strips for Screening Microalbuminuria Principle: Sensitivity: Reagents: Interference: Enzyme immunoassay 0-10 mg/l Gold-labeled antibody B-galactosidase Chlorophenol red galactoside False negative: Dilute urine Easy-to-use on-site dipstick test with results in 60 seconds No 24-hour urine sample required Immunological assay with 94% sensitivity and 94% specificity of radioimmunoassay Positive detection to 20 mg/l albumin Semi-quantitative; detection levels at negative, 20 mg/l, 50 mg/l, and 100 mg/l Immunodip Strips for Microalbuminuria Screening Principle: Immunochromographics Sensitivity: mg/dl Reagents: Antibody coated blue latex particles Interference: False negative-dilute urine 11

12 Clinitest Microalbumin Strips/Multistix-Pro for Albumin:Creatinine Ratio Principle: Sensitive albumin tests related to creatinine concentration to correct for patient hydration Requires Clinitek Status analyzer Reagents: Albumin: diodo-dihydroxydinitrophenyl tetrabromosulfonphtalein Creatinine: copper sulfate, tetramethylbenzidine, diisopropylbenzenedihydroperoxide Sensitivity: Albumin: mg/l Creatinine: mg/dl, mmol/l Interference: Visibly bloody or abnormally colored urine Creatinine: Cimetidine-False Positive 12