MLS Review - AUBF - Urine Analysis

MEDICAL LABORATORY SCIENCE REVIEW ANALYSIS OF URINE AND OTHER BODY FLUIDS: URINE (MACROSCOPIC & CHEMICAL ANALYSIS) • • Green - pseudomonas infection Blue-green - clorets URINE CLARITY OUTLINE • Urine o Macroscopic Urine Examination o Changes in Unpreserved Urine o Chemical Urine Examination o Reagent Strip (10 + 1 Parameters) o Other Urine Chemical Test o Obsolete Urine Chemical Tests o Microalbumin Testing (Immunologic Test) • • • • • • Denotes the turbidity of the urine Clear - No visible particulates, transparent Hazy - Few particulates, print easily seen through urine Cloudy - Many particulates, print blurred through urine Turbid - Print cannot be seen through urine Milky - With precipitate or be clotted o Shalle be reported immediately • Non-Pathologic Cause of Urine Turbidity o Squamous epithelial cells ▪ Most common and normal finding in the urine ▪ Point of reference for microscopic examination ▪ Due to normal sloughing of skin ▪ Determines the cleanliness of urine collection • For correctness of midstream clean catch o Mucus o Amorphous phosphates, carbonates, urates o Semen, spermatozoa ▪ Normally found if a guy just finished release ▪ Not reported in routine urinalysis unless physician requested for testing fertility • Presence of spermatozoa in urine can be a pathologic finding in men (retrograde ejaculation - cause of male infertility due to sperm going to urinary bladder after release to the urethra) o Fecal contamination o Radiographic contrast media o Talcum powder o Vaginal creams Pathologic Cause of Urine Turbidity o RBCs o WBCs o Bacteria o Yeast o Non-squamous epithelial cells o Abnormal crystals - seen in acidic urine o Lymph fluid o Lipids URINE MACROSCOPIC URINE EXAMINATION • Physical Examination of Urine includes o Urine Color o Clarity o Specific Gravity ▪ Urinometer and refractometer was used before URINARY PIGMENTS • • • Urochrome - Pigment present in urine that gives a yellow color of urine (Thudichum) o In normal urine, there is slight yellowish colorization Uroerythrin - Pink pigment, is most evident in specimens that have been refrigerated, resulting in the precipitation of amorphous urates. Uroerythrin attaches to the urates, producing a pink color to the sediment o pH is acidic o white precipitate - result of ppt of amorphous phosphate (pH is alkaline) Urobilin - an oxidation product of the normal urinary constituent urobilinogen, imparts an orange-brown/amber color to urine that is not fresh o Caused by prolonged standing URINE COLOR WITH LABORATORY CORRELATION • Red / Port Wine Color - Oxidation of porphobilinogen to porphyrins • • Black o Melanoma - oxidation of melanogen (colorless pigment) to melanin (produce in excess) o Alkaptonuria - presence of homogentisic acid (metabolite phenylalanine) in the urine which imparts a black color to alkaline urine from person with inborn metabolism ▪ Upon standing in room temperature, urine changes to black color ▪ Alkaptonuria - inborn error of metabolism 1 o SPECIFIC GRAVITY • • • • • • Specific Gravity o Evaluation of urine concentration. o Random Urine Sample - SG 1.015-1.030 o Determine whether specimen concentration is adequate to ensure the accuracy of chemical tests. ▪ Diluted urine may cause problem in reporting ▪ Morning urine - most concentrated urine sample Isosthenuric - SG 1.010 Hyposthenuric - SG <1.010 Hypersthenuric - SG >1.010 Current Urine Specific Gravity Measurement o Refractometry ▪ Principle: Refractive Index ▪ Uses correction factors ▪ Junction between blue and white is read o Osmolality ▪ Principle: Changes in colligative properties by particle number o Reagent Strip ▪ Principle: pKa changes of polyelectrolyte by ions present Calibration of Manual Specific Gravity Measurement o Refractometer ▪ Temperature (No adjustment) ▪ for every 1 gram of Protein, subtract 0.003 ▪ for every 1 gram of Glucose, subtract 0.004 o Urinometer ▪ Temperature oC = 5/9 (F-32) ▪ for every 1 gram of Protein, subtract 0.003 ▪ for every 1 gram of Glucose, subtract 0.004 o Sample Problem: A specimen containing 1 g/dL protein and 1 g/dL glucose has a SG of 1.030. calculate the corrected reading. (Uses Refractometer) 1.030 - 0.003 = 1.027 1.027 - 0.004 = 1.023 Therefore, the corrected SG = 1.023 o Sample Problem: A specimen containing 2 g/dL protein and 2 g/dL glucose has a SG of 1.030. calculate the corrected reading. (Uses Refractometer) 1.030 - 0.006 = 1.024 1.024 - 0.008 = 1.016 MACROSCOPIC SCREENING & MICROSCOPIC CORRELATIONS • • • • • Color - Blood Clarity - Hematuria vs. Hemoglobinuria / Myoglobinuria; Pathologic & Non-pathologic cause of turbidity Blood - RBCs, RBC Casts Protein - Casts, Cells Nitrite - Bacteria, WBCs • • True UTI has bacteria present microscopically and accompanied by inc. nitrite, leukocyte esterase, WBC Leukocyte Esterase - WBCs, WBC Casts, Bacteria Glucose - Yeast o May be caused by C. albicans o Not immediately related with diabetes o o Gross hematuria - red colored urine and turbid urine with packed field RBC Microscopic hematuria - normal in color but there are intact RBCs when checked microscopically URINE ODOR • • • • • • • • • • • Not part of routine urinalysis Aromatic - normal Foul, Ammonia-like - bacterial decomposition, UTI Fruity, Sweet - ketones (DM, starvation, vomiting) Maple Syrup - MSUD Mousy - Phenylketonuria (PKU) Rancid - Tyrosinemia Sweaty Feet - Isovaleric Acidemia Cabbage - Methionine Malabsorption Bleach - Contamination Rotting-Fish - “Galunggong-like odor” - Trimethylaminuria CHANGES IN UNPRESERVED URINE Analyte Color Change Modified / Darkened Decreased Cause Oxidation or reduction of metabolites Clarity Bacterial growth & precipitation of amorphous material Glucose Glycolysis and bacterial use Ketones Volatilization and bacterial metabolism Bilirubin Exposure to light/photo oxidation to biliverdin Urobilinogen Oxidation to urobilin RBCs/ WBCs/ Disintegration in dilute alkaline Casts urine Trichomonas Loss of motility, death Odor Increased Bacterial multiplication causing breakdown of urea to ammonia pH Breakdown of urea to ammonia by urease-producing bacteria/loss of CO2 Nitrite Multiplication of nitratereducing bacteria Bacteria Multiplication • Refrigeration - most common preservation of urine • Urine must be processed within 2 hours after collection • Trichomonas - becomes WBC-like when not observed in a freshly collected sample • Odor - aromatic become ammoniacal over time 2 CHEMICAL URINE EXAMINATION • REAGENT STRIP TECHNIQUE • • Errors caused by Improper Technique o Unmixed Specimen (RBC and WBC sink at the bottom) o Leaching of reagents from the pads ▪ Caused by cutting strip in half o Run-over between chemicals on adjacent pad ▪ After dipping, pat the strip with an absorbent paper or tissue o Good light source ▪ Can be subjective among people due Timing for reaction o pH: immediate o LE: 120s o Rest of the parameters: read between 60s - 120s • • REAGENT STRIP (10 + 1 PARAMETERS) pH • • • • Principle: Double indicator system Reagent Strip Reaction: Double-indicator system of methyl red and bromthymol blue o Methyl Red → Red to Yellow (pH 4-6) o Bromthymol Blue → Yellow to Blue (pH 6-9) o Orange from pH 5 through yellow; green to final deep blue at pH 9 o Methyl red + H+ → bromthymol blue – H+ (Redorange → yellow) (green → blue) o Reagents: Methyl Red, Bromthymol Blue Sensitivity o Multistix: 5.0 to 8.5 in 0.5 increments o Chemstrip: 5.0 to 9.0 in 1.0 increments Interferences o No known interfering substances o Run-over from adjacent pads o Old specimens Correlations o Nitrite o Leukocytes o Microscopic ▪ Amorphous urates - acidic ▪ Amorphous phosphate - alkaline Causes of Acid & Alkaline Urine o Acid Urine ▪ Emphysema ▪ Diabetes mellitus ▪ Starvation ▪ Dehydration ▪ Diarrhea ▪ Presence of acid-producing bacteria (Escherichia coli) ▪ High-protein diet ▪ Cranberry juice ▪ Medications (methenamine mandelate [Mandelamine], fosfomycin tromethamine [Monurol]) o Alkaline Urine ▪ Hyperventilation ▪ Vomiting ▪ Renal tubular acidosis • Inability to produce acid urine due to an impaired production of ammonia ▪ Presence of urease producing bacteria ▪ Vegetarian diet ▪ Old specimen PROTEIN • • Principle: protein error of indicator Reagent Strip Reaction: protein error of indicators to produce a visible colorimetric reaction o Chemstrip (3',3",5',5"-tetrachlorophenol, 3,4,5,6tetrabromosulfonphthalein) / Multistix (tetrabromophenol blue) and an ACID BUFFER to maintain the pH at a constant level o pH 3.0 (appears YELLOW; in the absence of protein) o As protein concentration increases (various shades of GREEN to BLUE) o Trace Value: <30mg/dL o Negative, Trace, 1+, 2+, 3+, 4+ (30mg/dL, 100mg/dL, 300mg/dL, 2000mg/dL) o Indicator (Yellow) + Protein -pH 3.0→ Protein + H+ Indicator - H+ (Blue-Green) o o Reagents ▪ Multistix: Tetrabromophenol blue ▪ Chemstrip: 3’,3’’,5’,5’’-tetrachlorophenol 3,4,5,6tetrabromosulfophthalein Sensitivity ▪ Multistix: 15-30 mg/dL albumin ▪ Chemstrip: 6 mg/dL albumin (more sensitive) 3 o o Interferences ▪ False Positive • Highly buffered interference alkaline urine • Pigmented specimens, phenazopyridine • Quaternary ammonium compounds (detergents) • Antiseptics, chlorhexidine o If a person is allergic to iodine, use chlorhexidine • Loss of buffer from prolonged exposure of the strip to the specimen reagent • High specific gravity ▪ False Negative • Proteins other than albumin • Microalbuminuria Correlations ▪ Blood ▪ Nitrite ▪ Leukocytes ▪ Microscopic • • • GLUCOSE • • Principle: Double sequential Enzymatic Reaction Reagent Strip Reaction (Glucose Oxidase) o Glucose oxidase testing method by impregnating the testing area with a mixture of glucose oxidase, peroxidase, chromogen, and buffer to produce a double sequential enzyme reaction ▪ Oxidase as catalyst always have peroxidase with a biproduct of H2O2 ▪ H2O2 - oxidizing agent; oxidize chromogen with peroxidase ▪ If there is no glucose in the urine, there will be no glucose to be converted resulting to no change in color or negative reporting o Chromogens ▪ Multistix: potassium iodide (green to brown) ▪ Chemstrip: tetramethylbenzidine (yellow to green) ▪ Technically in reduced form (colorless) o Negative, Trace, 1+, 2+, 3+, 4+ o 100 mg/dL - 2 g/dL or 0.1% - 2% o SPECIFIC FOR GLUCOSE ONLY Glucose Oxidase o Glucose + O2 (air) → Gluconic Acid + H2O2 Peroxidase o H2O2 + Chromogen → Oxidized Colored Chromogen + H2O KETONES • • • • • • Reagents o Multistix: Glucose oxidase, Peroxidase, Potassium iodide o Chemstrip: Glucose oxidase, Peroxidase, Tetramethylbenzidine Sensitivity o Multistix: 75-125 mg/dL o Chemstrip: 40 mg/dL (more sensitive) Interferences o False Positive ▪ Contamination by oxidizing agents and detergents • There may be additional oxidizing agent o False Negative ▪ High levels of ascorbic acid ▪ High levels of ketones ▪ High specific gravity ▪ Low temperatures ▪ Improperly preserved specimens • May be due to glycolysis and bacterial use Correlations o Ketones o Protein Reagent Strip Reaction (Copper Reduction Test Clinitest) o Measures reducing sugars/substances ▪ Sucrose - non-reducing sugar o Can detect other reducing sugars besides glucose o Relies on the ability of glucose and other substances to reduce copper sulfate to cuprous oxide in the presence of alkali and heat o A color change progressing from a negative blue (CuSO4) through green, yellow, and orange/red (Cu2O) occurs when the reaction takes place ▪ Blue → green → red o “PASS-THROUGH” phenomenon ▪ There may be instances that the test will return to negative blue due to prolonged standing causing inaccurate reading. o Interferences of other reducing sugars (galactose, lactose, fructose, maltose, pentoses, ascorbic acid, certain drug metabolites, and antibiotics such as the cephalosporins.) Principle: Nitroprusside reaction Reagent Strip Reaction: Sodium nitroprusside (nitroferricyanide) reaction to measure ketones o Acetoacetic acid in an alkaline medium reacts with sodium nitroprusside to produce a purple color o Negative, Trace, small (1+), moderate (2+), large (3+), o Negative, trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), or large (80 to 160 mg/dL) o ACETEST TABLETS Ketones represents 3 immediate products of fat metabolism o Acetone (2%) - sodium nitroprusside (reagent strip) reaction is slightly sensitive to acetone in the presence of glycine o Acetoacetic acid (20%) - main component measured in the reagent strip reaction o Beta-hydroxybutyrate (78%) - not measured in sodium nitroprusside (reagent strip) 4 • • • • Reagents o Sodium nitroprusside o Glycine (Chemstrip) Sensitivity o Multistix: 5-10 mg/dL acetoacetic acid o Chemstrip: 9 mg/dL acetoacetic acid; 70 mg/dL acetone Interferences o False Positive ▪ Phthalein dyes ▪ Highly pigmented red urine ▪ Levodopa ▪ Medications containing free sulfhydryl groups o False Negative ▪ Improperly preserved specimens Correlations: Glucose • • BLOOD • • • • • • • • Principle: Pseudoperoxidase activity of Hgb Reagent Strip Reaction o Pseudoperoxidase activity of hemoglobin to catalyze a reaction between the heme component of both hemoglobin and myoglobin and the chromogen o tetramethylbenzidine to produce an oxidized chromogen, which has a green-blue color Reagents o Multistix: Diisopropylbenzene dihydroperoxide and 3,3’,5,5’- tetramethylbenzidine o Chemstrip: dimethyldihydroperoxyhexane and tetramethylbenzidine Speckled pattern reaction Sensitivity o Multistix: 5-10 RBCs/mL, 0.015-0.062 mg/dL Hgb o Chemstrip: 5 RBCs/mL, Hgb corresponding to 10 RBCs/mL Interferences o False-positive: ▪ Strong oxidizing agents ▪ Bacterial peroxidases ▪ Menstrual contamination o False-negative: ▪ High specific gravity/crenated cells ▪ Formalin ▪ Captopril ▪ High concentrations of nitrite ▪ Ascorbic acid greater than 25 mg/dL ▪ Unmixed specimens Correlations o Protein o Microscopic Hematuria o Pathologic: disorders of renal or genitourinary origin (trauma or damage) ▪ Eg. renal calculi, glomerular disease o Non-pathologic: strenuous exercise, menstruation o Reagent strip reaction: ▪ Speckled pattern on reagent strip pad ▪ Presence of intact RBCs (microscopic hematuria) Hemoglobinuria o Presence of hgb in urine o Result from the lysis of RBC produced in the urinary tract, particularly in dilute, alkaline urine o Intravascular hemolysis o Reagent strip reactions ▪ Uniform color ranging from a negative yellow through green to a strongly positive green-blue appears on the pad Myoglobinuria o Presence of myoglobin in urine (clear red-brown urine or coca-cola-like color) o Result of muscle destruction (rhabdomyolysis) o Myoglobin - heme-containing protein found in muscle tissue (heme portion is toxic to renal tubules → acute renal failure) o Reagent strip reaction: ▪ Uniform color ranging from a negative yellow through green to a strongly positive green-blue appears on the pad BILIRUBIN • • Principle: Diazo reaction Reagent Strip Reaction: Uses the diazo reaction o Bilirubin combines with 2,4-dichloroaniline diazonium salt or 2,6 dichlorobenzene- diazoniumtetrafluoroborate in an acid medium to produce an azodye, with colors ranging from increasing degrees of tan or pink to violet ▪ Purple - positive ▪ Tan/pink - negative o Negative, small, moderate, or large, or as negative, 1+, 2+, or 3+ o ICTOTEST TABLETS • Reagents o Multistix: 2,4-dichloroaniline diazonium salt o Chemstrip: 2,6-dichlorobenzenediazonium salt Sensitivity o Multistix: 0.4 - 0.8 mg/dL bilirubin o Chemstrip: 0.5 mg/dL bilirubin Interferences o False-positive: ▪ Highly pigmented urines, ▪ phenazopyridine ▪ Indican (intestinal disorders) ▪ Metabolites of Lodine o False-negative: ▪ Specimen exposure to light ▪ Ascorbic acid greater than 25 mg/dL ▪ High concentrations of nitrite Correlations: Urobilinogen • • • UROBILINOGEN • • Principle: Ehrlich’s reaction (M) / Diazo reaction © Reagent Strip Reaction o Multistix: uses Ehrlich’s aldehyde reaction, in which urobilinogen reacts with pdimethylaminobenzaldehyde (Ehrlich reagent) to produce colors ranging from light to dark pink/red 5 o Chemstrip: incorporates an azo-coupling (diazo) reaction using 4-methoxybenzenediazoniumtetrafluoroborate to react with urobilinogen, producing colors ranging from white to pink/red • • • • • • • Reagents o Multistix: p-dimethylaminobenzaldehyde o Chemstrip: 4-methoxybenzenediazoniumtetrafluoroborate Sensitivity o Multistix: 0.2 mg/dL urobilinogen o Chemstrip: 0.4 mg/dL urobilinogen Interferences o Multistix: ▪ False-positive: • Porphobilinogen • Indican • p-aminosalicylic acid • Sulfonamides • Methyldopa • Procaine • Chlorpromazine • Highly pigmented urine ▪ False-negative: • Old specimens • Preservation in formalin o Formalin - preservative for analyzing microscopic urinary sediments o Chemstrip: ▪ False-positive: • Highly pigmented urine ▪ False-negative: • Old specimens • Preservation in formalin • High concentrations of nitrite Correlations o Bilirubin • LEUKOCYTE ESTERASE • • Principle: Hydrolysis of Ester Reagent Strip Reaction o Uses the action of LE (leukocyte esterase) to catalyze the hydrolysis of an acid ester embedded on the reagent pad to produce an aromatic compound and acid o The aromatic compound then combines with a diazonium salt present on the pad to produce a purple azodye o LE reaction requires the longest time of all the reagent strip reactions (2 minutes) o Trace, small, moderate, and large or trace, 1+, 2+, and 3+ • Reagents o Multistix: Derivatized pyrrole amino acid ester, Diazonium salt o Chemstrip: Indoxylcarbonic acid ester, Diazonium salt Sensitivity o Multistix: 5 to 15 WBC/hpf o Chemstrip: 10 to 25 25 WBC/hpf Interferences o False-positive: ▪ Strong oxidizing agents ▪ Formalin ▪ Highly pigmented urine, nitrofurantoin o False-negative: ▪ High concentrations of protein, glucose, oxalic acid, ascorbic acid, gentamicin, cephalosporins, tetracyclines ▪ Inaccurate timing NITRITE • • Principle: Greiss reaction Reagent Strip Reaction o Nitrite at an acidic pH reacts with an aromatic amine (para-arsanilic acid or sulfanilamide) to form a diazonium compound that then reacts with tetrahydrobenzoquinolin (chromogen) compounds to produce a pink-colored azodye o Ability of certain bacteria to reduce nitrate, a normal constituent of urine, to nitrite, which does not normally appear in the urine Reagents o Multistix: p-arsanilic acid, Tetrahydrobenzo(h)quinolin-3-ol o Chemstrip: Sulfanilamide, hydroxytetrahydrobenzoquinoline Sensitivity o Multistix: 0.06 - 0.1 mg/d: nitrite ion o Chemstrip: 0.05 mg/dL nitrite ion Interferences o False-negative: ▪ Non-reductase-containing bacteria ▪ Insufficient contact time between bacteria and urinary nitrate (reading time = 60s) ▪ Lack of urinary nitrate ▪ Large quantities of bacteria converting nitrite to nitrogen ▪ Presence of antibiotics ▪ High concentrations of ascorbic acid ▪ High specific gravity o False-positive: ▪ Improperly preserved specimens ▪ Highly pigmented urine Correlations o Protein o Leukocytes o Microscopic • • 6 • Correlations o Protein o Nitrite o Microscopic • SPECIFIC GRAVITY • • Principle: Change in pKa of polyelectrolytes Reagent Strip Reaction o Based on the change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium o Incorporation of the indicator bromthymol blue on the reagent pad measures the change in pH. o As the specific gravity increases, the indicator changes from blue (1.000 [alkaline]), through shades of green, to yellow 1.030 [acid]) ▪ 1.000 = diluted samples/water ▪ Random urine sample = 1.015 - 1.030 Reporting: Benedict's Test Negative No change in color (Blue Solution) ± / T Trace Green opacity. No precipitate. + Positive Green solution with yellow precipitate ++ Positive Green to yellow solution with yellow precipitate +++ Positive Muddy orange solution with yellow precipitate ++++ Positive Orange to brick red precipitate SULFOSALICYLIC ACID PRECIPITATION (SSA) TEST • • • • • • • • Reagents o Multistix: Poly (methyl vinyl ether/maleic anhydride) bromthymol blue o Chemstrip: Ethylene glycol diaminoethyl ether tetraacetic acid, bromthymol blue Sensitivity: 1.000 to 1.030 Interferences o False-positive: High concentrations of protein o False-negative: Highly alkaline urines (greater than 6.5) Protein Principle: precipitation of urine protein by strong acid Cold precipitation test that reacts equally with all forms of protein. Sulfosalicylic Acid Precipitation Test 1. Add 3 mL of 3% SSA reagent to 3 mL of centrifuged urine. 2. Mix by inversion and observe for cloudiness. 3. Grade the degree of turbidity. Reporting: SSA Turbity Protein Range Grade Turbidity (mg/dL) Neg <6 No increase in turbidity Trace 6-30 Noticeable turbidity 1+ 30-100 Distinct turbidity, no granulation 2+ 100-200 Turbidity, granulation, no flocculation 3+ 200-400 Turbidity, granulation, flocculation 4+ >400 Clumps of protein ASCORBIC ACID (ASCORBATE) (11TH PARAMETER) • • • Principle: Action of Ascorbic Acid to reduce a dye in the impregnated pad Positive Result in reagent strip: can detect ascorbic acid (AA) concentration as low as 7.0mg/dL and consistently detect 20mg/dL of AA in 90% urine tested. Significance of AA in Reagent strip: high levels of AA cause false-negative result on glucose, blood (>25 mg/dL), bilirubin (>25 mg/dL), nitrite, leukocyte esterase o AA can interfere with most of the parameters OTHER URINE CHEMICAL TEST BENEDICT’S TEST • • • • Glucose (other reducing sugar) Principle: copper reduction *Reducing Sugars: All sugars are reducing, except for sucrose. WATSON-SCHWARTZ TEST • • • • • Classic test for differentiating Urobilinogen & Porphobilinogen Chloroform - always at the bottom layer Butanol - always at the top layer Red coloration = compound is soluble Yellow / no changes = compound is insoluble Analyte Urobilinogen Porphobilinogen Other Ehrlich Reacting Compounds Chloroform Soluble Insoluble Insoluble Butanol Soluble Insoluble soluble 7 MICROALBUMIN TESTING (IMMUNOLOGIC TEST) • • • • • • • Normally, urine contains protein (albumin) of >10 mg/dL or 100 mg/24 hrs Persistent microalbuminuria indicates a high probability of damage to kidney o Clinical diabetic nephropathy - person has microalbuminuria (>20 mg/L 2/3 morning specimens) Albuminuria - presence of albumin in urine (>200 mg/L) Microalbuminuria - albumin in urine (20-200 mg/L) o Normal value do not rule out renal disease There is a monoclonal antibodies embedded in the reagent strip pad that is highly specific for human albumin Albumin concentration of an average urine specimen should not exceed between 15 to 20 mg/L Micral-Test o Principle: Enzyme Immunoassay o Sensitivity: 0 to 10 mg/dL o Reagents: Gold-labeled antibody, B-galactosidase, Chlorophenol red galactoside o Interference: False-negative: Dilute urine OBSOLETE URINE CHEMICAL TESTS Test Heat & Acetic AcidNitric Acid Test Benedict Heat Precipitation Test Rothera Gunning Gerhadt Seliwanoff Rubner Gmelin Analyte Albumin / Protein End Result Turbidity Glucose (other reducing sugar) Bence Jones Protein Brick-red precipitate Ketones Levulose Lactose Bile Smith Ehrlich Schlesinger Obermayer Guaiac Ammonium Sulfate Thormalen Sulkowitch Fantus Urobilinogen Urobilin Indican Hemoglobin Melanin Calcium Chloride Turbidity (classic rxn = ppt at 60C dissolved at 100C) Red purple color Iodoform crystals (microscopic) Bordeaux red color Orange to red color Brick red precipitate Play of colors (yellow, red, green) Green ring Cherry red Fluorescent green Indigo blue to indigo red Blue color (junction) Precipitation of Hgb • ImmunoDip o Principle: Immunochromographics o Sensitivity: 1.2 to 8.0 mg/dL o Reagents: Antibody-coated blue latex particles o Interference: False-negative: Dilute urine Prussian blue color Turbidity White precipitate 8