Chemical Examination PDF

lOMoARcPSD|30338068 1 - Chemical examination Medical technology (Universidad de Sta. Isabel) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Kein Kendrick Denver Saulon ([email protected]) ...

lOMoARcPSD|30338068 1 - Chemical examination Medical technology (Universidad de Sta. Isabel) Scan to open on Studocu Studocu is not sponsored or endorsed by any college or university Downloaded by Kein Kendrick Denver Saulon ([email protected])  lOMoARcPSD|30338068 ANALYSIS OF URINE AND OTHER BODY FLUIDS CHEMICAL EXAMINATION OF URINE (Strasinger 5th Ed p. 53-75) READING URINE PRINCIPLE POSITIVE COLOR TIME PARAMITER 3O sec Glucose Double sequential enzyme reaction Green to brown Bilirubin Diazo Reaction Tan or pink to violet 40 sec Ketones Sodium nitroprusside reaction Purple 45 Specific Gravity pKa change of a polyelectrolyte Blue (SG 1.000 to Yellow (SG 1.030) 60 Protein Protein (Sorrensen’s) Error of indicators Blue pH Double indicator system Orange (pH 5.0) to Blue (pH 9.0)` Blood Psuedoperoxidase activity of Uniform green/blue hemoglobin (Hgb or Mb) Speckled/Spotted (intact RBCs) Urobilinogen Ehrlich reaction Red Nitrite Greiss reaction Uniform pink 120 Secs Leukocytes Leukocyte esterase Purple 180 secs Ascorbic acid Molybdenum blue Reagent strip technique a. Dip the reagent strip briefly (No longer than 1 sec) into a well-mixed uncentrifuged urine specimen at RT b. Remove excess urine by touching the edge of the strip to the container as the strip is withdrawn c. Blot the edge of the strip on a disposal absorbent pad. d. Wait the specified amount of time for the reaction to occur e. Compare the color reaction of the strip pads to the manufacturer’s color chart in good lighting Care of reagent strips a. Store with desiccant in an opaque, tightly closed container b. Store below 30’C (room temperature); do not freeze c. Do not expose to volatile fumes d. Do not use past the expiration date e. Do not use chemical pads become discolored f. Remove strips immediately prior to use. Automated Reagent Strip Readers Principle: Reflectance Photometry -Light reflection from the test pads decreases in proportion to the intensity of color produced by the concentration of the test substance I. Specific Gravity  Density of solution compared with density of similar volume of distilled water at a similar temperature  Influenced by number and size of particles in a solution Normal Sg (random) Isosthenuria When SG 1.040 Hypersthenuria Determination 1. URINOMETRY (URINOMETER/ HYDROMETER)  Calibration temperature: 20’C  Requires temperature correction  (- 0.001) for every 3’C that the Spx temp. is below the calibration temp.  (+ 0.001) for every 3’C that the Spx temp. is above the calibration temp.  Requires correction for glucose and protein  1g/dl Glucose = (- 0.004)  1 g/dl Protein = (- 0.003)  Urine volume required: 10-15ml  CALIBRATION -potassium sulfate solution  SG reading should be 1.015 JAN ETHAN V. LOVENDINO, RMT, MSPH AUBF-CLINICAL INSTRUCTOR (USI) Page 1 Downloaded by Kein Kendrick Denver Saulon ([email protected])  lOMoARcPSD|30338068 _____________________________________________________________________________________ 2. Refractometer (Refractometer/ TS meter)  Indirect method based on refractive index (RI) RI = Light velocity in air Light velocity in sol.  Compensated to temp. (15-38’C)  No need for temperature correction  Requires correction for glucose and protein 1g/dl Glucose = (- 0.004) 1 g/dl Protein = (- 0.003)  Calibration: Distilled water = 1.000 5% NaCl = 1.022 +/- 1 (1.021 or 1.023) 9% Sucrose = 1.034 +/- 1 (1.033 or 1.035) Steps in using the refractometer 1. Put 1 or 2 drops of sample on the prism. 2. Close the daylight plate gently 3. Sample must spread all over the prism surface 4. Look at the scale through the eyepiece 5. Read scale where the boundary line intercepts it. 6. Wipe the sample from the prism clean w/ tissue and water Specific gravity dilution  Specimens with very high SG reading can be diluted and retested  To obtain the actual SG, multiply the decimal portion of SG by the Dilution factor Example: Urine specimen diluted 1:4 has a reading of 1.014. what is the actual SG reading? 3. Reagent Strip Reaction for Specific Gravity (45 seconds) Principle Change in pKa (dissociation constant) of a Polyelectrolyte -the polyeletrolyte ionize, releasing hydrogen ions in proportion to the number of ions in the solution. Reagent is sensitive to the no. of ions in urine; indicator changes color in relation to ionic concentration Reagents Multistix = Poly (methyl vinyl ether/ maleic anhydride) bromthymol blue Chemstrip = ethyleneglycoldiaminoethylethertatraacetic acid bromthymol blue Interferences False (+) = high concentration of protein False (-) = highly alkaline urine (>6.5) Notes Add 0.005 to SG reading when pH is >6.5 due to interference with the bromthymol blue indicator Not affected by glucose, protein & radiographic dye 4. Harmonic oscillation densitometry Based on the frequency of a soundwave entering a solution changes in proportion the density of the solution. Ex: Yellow IRIS (International Remote Imaging System) Iris Diagnostic Models 300 and 500 workstations: 6ml= required urine volume  4 ml (of 6ml) =for IRIS slide-less microscope  2ml (of 6ml) = for IRIS mass gravity meter (for specific gravity determination- by using harmonic oscillation) II. pH  Important in the identification of crystal and determination of unsatisfactory specimens.  Normal pH: 4.0 – 8.0 Random = 1st morning = 5.0 – 6.0  pH of 9.0 = unpreserved urine and alkaline tide - prolonged storage of urine inside the body causes elevation in ammonia  alkaline tide occurs after meals due to withdrawal of hydrogen ions for the purpose of secretion of HCL - pH of blood = 7.35 – 7.45 JAN ETHAN V. LOVENDINO, RMT, MSPH AUBF-CLINICAL INSTRUCTOR (USI) Page 2 Downloaded by Kein Kendrick Denver Saulon ([email protected])  lOMoARcPSD|30338068 CAUSES OF ACID URINE CAUSES OF ALKALINE URINE Diabetes Mellitus Renal tubular acidosis (inability to produce acidic Starvation urine) High protein diet Vegetarian diet Cranberry juice After meal (alkaline tide) Emphysema, dehydration, diarrhea, presence of Vomiting (excretion of HCL) acid producing bacteria (E. coli), medications Old specimens (ammonium Biurate crystals), hyperventilation, presence of urease producing bacteria Reagent Strip Reaction for pH (60 seconds) Principle Double indicator system Reagents Methyl red, bromthymol blue Interference No known interfering substances Run over from adjacent pads, old specimens Correlations with other tests= Nitrite, Leukocytes, Microscopic III. Protein  Most indicative of renal disease  Protein causes white foam in urine when shaken; yellow foam; Bilirubin 1. Albumin 2. Other proteins  Major serum protein found in urine a. Serum & tubular microglobulins  Normal values; b. Tamm-Horsfall protein (a.k.a >10 mg/dL or >100mg/24 hrs Uromodulin c. Protein derived from prostatic and vaginal secretions PRE-RENAL (“Before”) or OVERFLOW PROTIENURIA  Caused by conditions that affect the plasma prior to its reaching the KIDNEY: a. Intravascular hemolysis = Hemoglobin b. Muscle injury = myoglobin c. Severe infection and inflammation = high APRs (Acute Phase Reactants) d. Multiple myeloma= proliferation of immunoglobin-producing plasma cells (Bence-Jones protein BJP= immunoglobin light chain  Test = serum electrophoresis, immunofixation electrophoresis  Urine= Precipitates at 40-60’C cloudy and dissolves at 100’C (clear) RENAL PROTIENURIA (“true renal disease”) A. Glomerular Proteinuria 1. Diabetic nephropathy  Decreased glomerular filtration  May lead to renal failure  Indicator: microalbuminuria = Proteinuria undetectable by routine reagent strip  Albumin Excretion Rate (AER)= in ug/min or un mg/24 hours o Normal AER = 0-20 ug/min o Microalbuminuria= 20-200 ug/min (or 30-300 mg/24 hrs) o Clinical Albuminuria= >200 ug/min Micral test  Test for microalbuminuria  A strip employing antibody-enzyme conjugate that binds albumin  Principle: Enzyme Immunoassay  Reagents:  Gold-labeled antibody  B-galactosidase  Chlorophenol red galactoside  Sensitivity: 0-10 mg/dl  Interference: dilute urine= False (-)  Positive color: pink  Negative color: white Effective time: _____________ JAN ETHAN V. LOVENDINO, RMT, MSPH AUBF-CLINICAL INSTRUCTOR (USI) Page 3 Downloaded by Kein Kendrick Denver Saulon ([email protected])  lOMoARcPSD|30338068 2. Orthostatic/Cadet/Postural proteinuria  Proteinuria when standing due to increased pressure to renal veins Orthostatic proteinuria Clinical Proteinuria First morning Negative Positive 2 hours after standing Positive Positive B. Tubular Proteinuria  Normally filtered albumin can no longer be reabsorbed 1. Fanconi’s syndrome 2. Toxic agents/ heavy metals 3. Severe viral infections POST-RENAL PROTIENURIA (“after”) 1. Lower UTI/ inflammations 2. Injury / trauma 3. Menstrual contamination 4. Prostatic fluid/Spermatozoa 5. Vaginal secretion Reagent Strip Reaction for Protien 60 Seconds Principle Protein (Sorrensen’s) Error of Indicator Reagents Multistix = Tetrabromphenol blue Chemstrip = tetrachlorophenol tetrabromosulfonphthalein Interferences False (+) = highly buffered alkaline urine, pigmented specimen, phenazopyridine, quaternary ammonium compounds (detergents) antiseptics, chlorhexidine, loss of buffer from prolonged exposure of the reagent strip to the specimen, high SG False (-) = Proteins other than albumin, microalbuminuria Notes: Indicator/Protein is Sensitive to albumin Correlates with other tests = Blood, nitrite, leukocytes, microscopic SULFOSALICYLIC ACID (SSA) PRECIPITATION TEST: A cold precipitation test that reacts equally with all forms of protein SSA procedure 3ml of 3% SSA + 3mL centrifuged urine = (+) Cloudiness SSA GRADING Grade Turbidity Protein range (mg/dl) Negative No increase in turbidity 400 IV. Glucose (Dextrose)  Most frequently tested in urine  Renal threshold= plasma concentration of a substance at which tubular reabsorption stops  Renal threshold= 160-180 mg/dL Other sugars in urine. 1. Fructose (Levulose) = high fruits, honey, fructose intolerance 2. Galactose = high during infants with galactosemia 3. Lactose (Glu+Gal) = high during pregnancy, lactation, strictly milk diet, lactose intolerance 4. Pentose= High benign essential pentosuria (xylose, arabinose) 5. Sucrose (Glu+fru) = high intestinal disorders, sucrose intolerance; (-) Copper reduction test [non-reducing sugar] Clinical Significance of Urine Glucose Hyperglycemia Renal Associated High : Blood glucose = High : urine glucose Low : Blood glucose = High : urine glucose Causes: Impaired tubular reabsorption of glucose  DM Cause:  Cushing’s syndrome (high cortisol)  Fanconi Syndrome :defective tubular JAN ETHAN V. LOVENDINO, RMT, MSPH AUBF-CLINICAL INSTRUCTOR (USI) Page 4 Downloaded by Kein Kendrick Denver Saulon ([email protected])  lOMoARcPSD|30338068  Pheochromocytoma (high catecholamine) reabsorption of glucose and amino acids  Acromegaly  Hyperthyroidism (high T3 and T4) Reagent strip reaction for glucose (30 seconds) Principle Double sequential enzyme reaction Reagents Multistix= glucose oxidase, Peroxidase, Potassium iodide (blue to green to brown) Chemstrips= Glucose oxidase, Peroxidase, tetramethybenzidine (yellow to green) Interferences: False (+) = Oxidizing agents, detergents False (-) = High levels of ascorbic acid, ketones, high SG. Low temp, improperly preserved specimen Notes: Sensitivity = 100 mg/dl Other chromogens: aminopropylcarbazole (yellow to orange-brown); o-toluidine (pink to purple) Correlations with other tests= ketones, protein Copper reduction test (Clinitest/Benedict’s test) Test Nonspecific test reducing sugars Principle Copper reduction False-positive Reducing Agents (Ascorbic Acid) False- negative Oxidizing Agents (Detergents) Clinitest procedure 5 gtts urine + 10 gtts H2O + Clinitest tablets Pass-through phenomenon  Occurs when > 2 g/dl sugar is present  Blue > Green > Yellow > Brick Red>>>>Blue or Green-brown  To prevent pass through, use 2 gtts urine The tablets contain:  CuSO4= main reacting agent  NaCo3= eliminates interfering O2  Na Citrate= for heat production  NaOH= for heat production Summary of glucose oxidase and Clinitest reactions Glucose Oxidase Clinitest Interpretation 1+ positive Negative Small amount of glucose 4+ positive Negative Oxidizing agent inference on reagent strip Negative Positive Non-glucose reducing substance Possible interfering substance for reagent strip “Sucrose” V. Ketones  Results from increased fat metabolism due to inability to metabolize carbohydrates  Seen in: o Type I DM (destroyed beta cells, no insulin) o Vomiting (no carbohydrates) o Starvation (fasting produces ketones) o Malabsorption Ketone bodies 78% Beta-hyrdoxybutyric acid= major ketone but not detected in reagent strips 20% Acetoacetic acid (AAA)/ Diacetic Acid= parent ketone (only substance detected among ketone bodies) 2% Acetone Reagent Strip for Ketone (40 seconds) Principle Sodium Nitroprusside Reaction Reagents Sodium nitroprusside, Glycine (chemstrip) Interference False (+) = Pthalein dyes, highly pigmented red urine, levodopa, medications containing sulfhydryl groups False (-) = Improperly preserved specimens Notes Acetone is detected only when glycine is present JAN ETHAN V. LOVENDINO, RMT, MSPH AUBF-CLINICAL INSTRUCTOR (USI) Page 5 Downloaded by Kein Kendrick Denver Saulon ([email protected])  lOMoARcPSD|30338068 Correlations with other test= Glucose VI. Blood Hematuria Hemoglobinuria Myoglobin Cloudy red urine Clear red urine Clear red (reddish-brown) urine Seen in: Seen in: Seen in:  Glomerulonephritis  Intravascular hemolysis  Rhabdomyolysis  Renal calculi, Tumors  Transfusion reaction  Muscular trauma, crush  Strenuous exercise,  Hemolytic anemia syndromes trauma  Severe burns  Extensive exertion  Brown recluse spider Heme portion of the myoglobin is bites toxic to the renal tubules! Hemoglobin vs. Myoglobin Test Hemoglobin Myoglobin 1. Plasma examination Pink-red plasma (hemolysis) Pale-yellow plasma Low haptoglobin CK-MM – muscle CK-MB- heart CK-BB- brain 2. Blondheim’s test (ammonium sulfate) Procedure: Urine +2.8NH4sulfate (805 satd) Low filter/centri Test supernatant for Blood with a reagent strip Reagent Strip for Blood (60 seconds) Principle Pseudoperosxidase activity of Hemoglobin Reagents Multistix= Diisoprophylbenzene dehydroperoxidase tetramethylbenzidine Chemstrips= Dimethyldihydroperxyhexane tetramethylbenzidine Interferences False (+) = Strong oxidizing agents, bacterial peroxidases, menstrual contamination False (-) =High SG. Creanated cells, formalin, captopril, high concentrations of nitrite, ascorbic acid (>25 mg/dl), unmixed specimens Notes Uniform green/blue color= Hemoglobin/myolglobin Speckled/Spootted= Hematuria (intact RBC) Correlations with other test= Protein, microscopic VII. Bilirubin  Conjugated bilirubin (CB)- water Ictotest (Tablet) soluble More sensitive than reagents strip w/ less interference  Early indication of liver desease Contains:  Amber urine w/ yellow foam  p-nitrobenzene-diazonium p- toluenesulfonate  Clinical significance- liver disorders  SSA  Hepatitis  Sodium carbonate  Cirrhosis  Boric Acid  Biliary obstruction (+) Blue to purple color (gallstones, carcinoma) JAN ETHAN V. LOVENDINO, RMT, MSPH AUBF-CLINICAL INSTRUCTOR (USI) Page 6 Downloaded by Kein Kendrick Denver Saulon ([email protected])  lOMoARcPSD|30338068 Reagent Strip for Bilirubin (30 Seconds) Principle Diazo Reaction Reagents Multistix= 2,4 dichloroaniline diazonium salt Chemstrips = 2,6 dicholorobenzene diazonium salt Interference False (+) = highly pigmented urines, phenazopyridine, indicant, metabolites of Lodine False (-)= Specimen exposure to light, high concentrations of nitrite , ascorbic acid (>25 mg/dl) Notes (+) Tan or Pink to Violet Correlations with other tests= Urobilinogen Hemoglobin Degredation JAN ETHAN V. LOVENDINO, RMT, MSPH AUBF-CLINICAL INSTRUCTOR (USI) Page 7 Downloaded by Kein Kendrick Denver Saulon ([email protected])  lOMoARcPSD|30338068 VIII. Urobilinogen  Bile pigment that result from hemoglobin degradation  NV=