Clinical Microscopy Analysis of Urine and Other Body Fluids PDF

CLINICAL MICROSCOPY Analysis of Urine and Other Body Fluids College of Allied Medical Professions A.Y. 2024 - 2025 Reference Textbook: Urinalysis and Body Fluids by Susan K. Strasinger and Marjorie Di Lorenzo  REVISED MATRIX/ TABLE OF SPECIFICATIONS...

CLINICAL MICROSCOPY Analysis of Urine and Other Body Fluids College of Allied Medical Professions A.Y. 2024 - 2025 Reference Textbook: Urinalysis and Body Fluids by Susan K. Strasinger and Marjorie Di Lorenzo  REVISED MATRIX/ TABLE OF SPECIFICATIONS CLINICAL MICROSCOPY (10%) URINE 53% Anatomy and Physiology of the Kidney 5% Macroscopic Examination 10% Chemical Analyses 18% Microscopic Examination 15% Pregnancy Testing 2% Renal Calculi 3% FECES 3% OTHER BODY FLUIDS 21% CSF 5% Seminal Fluid 5% Amniotic Fluid 3% Gastric Fluid and Duodenal Content 2% Sputum and Bronchial Washings 2% Synovial fluid 2% Peritoneal, Pleural and Pericardial Fluids 2% COLLECTION, PRESERVATION AND HANDLING OF SPECIMENS 10% MICROSCOPE, AUTOMATION, OTHER INSTRUMENTS 5% QUALITY ASSURANCE AND LABORATORY SAFETY 5% TOTAL 100%  Safety in the Clinical Laboratory  TYPES OF SAFETY HAZARDS TYPE SOURCE Biological Infectious agents Sharps Needles, lancets, broken glass Chemical Preservatives and reagents Radioactive Equipment and radioisotopes Electrical Ungrounded or wet equipment; frayed cords Fire/ Explosive Bunsen burners, organic chemicals Physical Wet floors, heavy boxes, patients The CHAIN OF INFECTION requires a continuous link between: SOURCE METHOD OF TRANSMISSION SUSCEPTIBLE HOST CDC Guidelines for Hand Hygiene Use DETERGENT SOAP or ANTIMICROBIAL SOAP when: ❑ Hands are visibly soiled ❑ Before eating ❑ After using the comfort room Use ALCOHOL-BASED HAND RUB when: ❑ Hands are NOT visibly soiled ❑ Before having direct contact ❑ After contact with a patient’s INTACT skin ❑ Before wearing and removing gloves When moving from contaminated to clean body site ❑ After contact with inanimate objects CONTAINER WASTE Black Dry, Non-infectious Green Wet, Non-infectious Yellow Infectious/ Pathologic Red Sharps and Pressurized Container Orange Radioactive  Notes to Remember ❑ URINE may be discarded by pouring it into the LABORATORY SINK. ❑ The sink must be flushed with water and disinfected daily with Sodium hypochlorite. Empty urine containers can be discarded as ❑ NON-BIOLOGICALLY HAZARDOUS WASTE. UNIVERSAL PRECAUTIONS VS STANDARD PRECAUTIONS ❑ Universal precautions are applicable to all patients, while standard precautions provide an additional layer of protection for those at higher risk of transmitting certain infections. HAND CONTACT is the 1° method of infection transmission. HANDWASHING: ❑ Thorough cleaning must last for 15 seconds (Strasinger 5th ed.) Must last for 20 seconds (Strasinger, 6th ed.) ❑ Rinse hands in a DOWNWARD position ❑ Sing HAPPY BIRTHDAY TWICE  Donning and Doffing of PPE: Donning: Doffing: 1. Gown 1. Gloves 2. Mask/Respirator 2. Goggles/Face 3. Goggles/Face shield shield 4. Gloves 3. Gown 4. Mask/Respirator What’s the last step? DISPOSAL OF SHARP OBJECTS: ❑Puncture-resistant containers labeled with biohazard symbol BEST and MOST COMMONLY USED DISINFECTANT in the LAB: ❑10% Na hypochlorite (1 part Na hypochlorite in 9 parts H2O) ❑ Kills HIV in 2 minutes and HBV in 10 minutes ❑ Effective for 1 month if stored in plastic containers and protected from light NFPA Hazardous Materials Classification  Degree of Hazards 0- No hazard 1- Slight hazard 2- Moderate hazard 3- Serious hazard 4- Extreme hazard Notes to Remember ❑BEST FIRST AID when skin contact with chemical occurs: Flush the area with water for at least 15 minutes, then seek medical attention. ❑DO NOT neutralize chemicals that come in contact with the skin. ▪ Always add ACID to WATER! ▪ ACID to WATER! ❑Pipetting by mouth is UNACCEPTABLE in the lab. ❑ Equipment should NOT be operated with wet hands. ❑ All electrical equipment must be grounded with three-pronged plugs. When accident involving electrical shock occurs: ❑ Electrical source must be removed IMMEDIATELY ❑ NEVER touch the person or equipment involved. ❑ Turn off the circuit breaker, unplug the equipment or move the equipment using a non- conductive object.  Types of Fires and Fire Extinguishers FIRE EXTINGUISHING EXTINGUISHER TYPE MATERIAL Type A Ordinary combustibles Water, dry chemical or (wood, paper, clothing) loaded steam Type B Flammable organic Dry chemical, carbon chemicals (grease, oil, dioxide, halon or foam paints, gasoline) Type C Electrical equipment and Dry chemical, carbon motor switches dioxide, or halon Type D Combustible metals Sand, dry powder, Metal X Type E Arsenal fire(detonation) Allowed to burn out Type K Grease, oil, fats Liquid designed to cool the fire and prevent splashing  WHEN A FIRE IS DISCOVERED RACE R RESCUE anyone in immediate danger A activate the institutional fire ALARM system C CONTAIN, close all doors to potentially affected areas E Attempt to EXTINGUISH the fire, if possible, exit/ EVACUATE the area WHEN OPERATING A FIRE EXTINGUISHER PASS P PULL pin A AIM at the base of the fire PASS S SQUEEZE trigger me! S SWEEP nozzle side to side  Notes to Remember OSHA – Occupational Safety and Health Administration NFPA – National Fire Protection Association MSDS – Material Safety Data Sheet PPE – Personal Protective Equipment CDC-Centers for Disease Control and Prevention UP-Universal Precautions CA - cancer antigen NMP-22 - nuclear matrix protein CD - cluster of differentiation/designation URINE ❑ Ultrafiltrate of plasma & liquid tissue biopsy of the urinary tract ❑ Urea – major ORGANIC component ❑ Chloride – major INORGANIC component (Cl>Na>K) ❑ NaCl – PRINCIPAL SALT (15g/24hr) ❑ 600-2000 mL of urine is excreted per day ❑ Day:Night urine ratio: 2-3:1 Types of Urine Specimen 1. Random/Single/ ❑ For routine urinalysis Occasional 2. 1st morning/ ❑ Ideal screening specimen; 8-hour ❑ Optimal for preservation of specimen cells and casts 3. 2-hr ❑ For glucose monitoring Postprandial ❑ Fasting 4. Glucose ❑ Optional, correlated with GTT tolerance test 5. Midstream ❑ For bacterial culture and clean catch routine urinalysis 6. Pediatric ❑ Wee bags may be used Specimen 7. Catheterized ❑ For bacterial culture 8. Suprapubic ❑ Only specimen for both aerobic and anaerobic bacterial culture 9. Prostatitis For the diagnosis of prostatic specimen infection 10. Timed Urine Specimen ❑ Quantitative chemical tests ❑ For solutes exhibiting diurnal variations 2-4 PM/ Afternoon ❑ For urobilinogen specimen determination 4-hour urine ❑ For nitrite determination 12-hour urine ❑ For Addis count 24-hour urine For quantitative analysis 11. Drug Test Specimen ❑ Used for drug testing ❑ Must be 30 – 45mL (in a 60 mL container) ❑ Must be 32.5 – 37.7 °C when tested within 4 minutes CHAIN OF CUSTODY (COC): the process that provides documentation of proper sample identification from the time of collection to the receipt of lab results.  Notes to Remember Specimen should placed in a sterile, wide- mouthed disposable container with screw- cap lids and proper label Container capacity: 50 mL (UA); 60 mL (DT) Required volume for routine UA: 10-15 mL Specimen must be tested within 2 hours, if not, must be refrigerated or added with preservative LABEL !  Discuss the changes to the analytes if the urine is not processed within 2 hours  RENAL FUNCTION RENAL/ EXCRETORY SYSTEM ❑Consists of the kidneys, ureters, urinary bladder and urethra KIDNEYS- main organ of excretory system NEPHRON ❑ Basic structural and functional unit of the kidney ❑ Approx. 1-1.5 M are present in each kidney Kidneys receive approx. 25% of cardiac output Renal blood flow: 1200 mL/minute Renal plasma flow: 600-700 mL/minute URINE FORMATION Glomerulus RENAL FUNCTIONS i. Renal Blood Flow PCT ii. Glomerular Filtration iii. Tubular Reabsorption iv.Tubular Secretion Loop of Henle DCT Collecting Ducts ❑ Two types of nephrons: Cortical (85%) and juxtamedullary (15%) ❑ Podocytes are found in the VISCERAL PART of Bowman's capsule. Podocytes are also responsible for the glomerulus “shield of negativity” ❑ Hydrostatic pressure PROMOTES glomerular filtration while ONCOTIC pressure prevents it.  Increased BP = afferent arterioles constrict and efferent arterioles dilate Decreased BP = afferent arterioles dilate and efferent arterioles constrict Juxtaglomerular apparatus is composed of – Macula densa cells: afferent arteriole, monitors Na++ – JG cells: DCT, secretes renin 65-80% of tubular reabsorption occurs in the PCT HCO3 is reabsorbed in the form of CO2 TESTS FOR GLOMERULAR FILTRATION “CLEARANCE TESTS” A. Endogenous Clearance Test Measures a substance that is already present inside the body. ❑Urea ❑Creatinine ❑Beta-2-microglobulin ❑Cystatin C 1. Urea Clearance ❑ Earliest glomerular filtration test ❑ Normal value have to be readjusted that is why it is NOT recommended for laboratory applications. ❑ In advanced renal failure, urea clearance approaches unity with GFR, and is a better predictor of GFR than creatinine clearance 2. Creatinine Clearance ❑ MOST COMMONLY USED ❑ NOT a reliable method for patient with muscle wasting disease ❑ Specimen of Choice: 24-hour urine sample ❑ Cimetidine, Gentamicin, Cephalosporins: drug of choice to inhibit creatine secretion, thus causing the serum levels falsely low ❑Greatest source of error: Improperly timed urine sample ❑Formula: C = UV/P C= Creatinine clearance U= urine creatinine in mg/dl V= volume of urine in ml/minute P= plasma creatinine in mg/dl ❑ Formula: C = UV/P C= Creatinine clearance U= urine creatinine in mg/dl V= volume of urine in ml/minute P= plasma creatinine in mg/dl ❑ Given: Urine creatinine = 120 mg/dL Urine Volume = 1 mL/minute Plasma creatinine = 1.0 mg/dL ❑Given: ❑Urine Creatinine= 180 mg/dl ❑Plasma Creatinine= 2.0 mg/dl ❑Urine volume= 2500 ml/24 hours 2500 mL x 1 hr = 2500 mL = 1.74 ml/min 24 hrs 60 min 1440 min SOLVE ! Corrected Creatinine Clearance Normal values vary in size and muscle mass of a person. Normal body surface area= 1.73 m2 The laboratory receives a 24-hour urine sample from a 26 year-old (6’4” or a body surface area = 2.34 m2) Specimen volume = 800 ml/24 hrs Plasma Creatinine = 1.2 mg/dl Urine Creatinine = 150 mg/dl C = UV/P V = 800 mL x 1 Hr = 800 mL = 0.56 mL/min 24 Hr 60 mins 1440 mins C= UV/P C= 150 mg/dL x 0.56 = 84 C= 84/1.2 mg/dL C= 70 mL/min Creatinine Clearance x Normal body SA Corrected Creatinine Clearance = Given surface area  Creatinine Clearance x Normal body SA Corrected Creatinine Clearance = Given surface area 70 mL/min x 1.73 m2 Corrected Creatinine Clearance = 2.34 m2 Corrected Creatinine Clearance = 51.75 mL/min Normal value (Men) = 107 – 139 mL/min Normal value (Women) = 87 – 107 mL/min  SAMPLE PROBLEM ❑ The laboratory receives a 24-hour urine sample from a 24 year-old ❑ (5’4” or a body surface area = 1.75 m2) ❑ Specimen volume= 3000 mL/24 hrs ❑ Plasma Creatinine = 2.4 mg/dL ❑ Urine Creatinine = 300 mg/dL Solution: ❑ COCKCROFT AND GAULT FORMULA GENERAL EQUATION: Creatinine Clearance = (140 – age) x Weight in Kg 72 x serum creatinine (mg/dL) Example: ❑ Age = 50 ❑ Weight = 90 kgs ❑ Serum Creatinine = 1.1 mg/dL ❑ If FEMALE: MULTIPLY by 0.85 Solution:  SAMPLE PROBLEM ❑ Age = 73 Y.O. Female Patient ❑ Weight = 120 kgs ❑ Serum Creatinine = 0.35 mg/dL Solution:  3. Cystatin C ❑ Small protein (MW 13,359 D) produced at a constant rate by all nucleated cells ❑ Uses SERUM as sample ❑ It is readily filtered by the glomerulus and reabsorbed and broken down by the renal tubular cells. Therefore, no cystatin C is secreted by the tubules ❑ For pediatric patients, persons with diabetes, the elderly, and critically ill patients.  4. Beta2-microglobulin ❑ Constituent of MHC Class I molecule that dissociates from it at a constant rate and removed from plasma by glomerular filtration ❑ NOT reliable in patients who have a history of immunologic disorders or malignancy A. Exogenous Clearance Test Requires a substance to be infused inside the body. ❑ Inulin ❑ Radioisotopes 1. Inulin Clearance ❑ Inulin is a polymer of fructose; an extremely stable substance that is NOT reabsorbed nor secreted by the tubules. ❑ GOLD STANDARD and original REFERENCE METHOD 2. Radionucleotides ❑ E.g. 125I-iothalamate ❑ Provides a method for determining glomerular filtration through the plasma disappearance of the radioactive material and enables visualization of the filtration in one or both kidneys ❑ Specimen: PLASMA TESTS FOR TUBULAR REABSORPTION TUBULAR REABSORPTION ❑ FIRST function to be affected in renal disease ACTIVE TRANSPORT - requires a carrier protein PCT ALH DCT and PCT Glucose Chloride Sodium Amino acids (aldosterone) Salts Sodium (angiotensin II) PASSIVE TRANSPORT - requires a concentration gradient In all parts EXCEPT the ALH Water In PCT and ALH Urea In ALH Sodium 1. Fishberg ❑Patient is deprived of fluid for 24 hrs then SG is measured (> 1.026) ❑ Diabetes Insipidus ▪ Urine:Serum (1:1) 2. Mosenthal ❑ Volume and SG of day and night urine are compared 3. Specific Gravity ❑ Influenced by both number & density of particles in the solution; affected by HMW substance (x-ray dye, mannitol) 4. Osmolality ❑ Influenced ONLY by the number of particles ❑ Measured by Freezing point osmometers ❑ Freezing point osmometer: ❑ 1 mol or 1000 mOsm of a nonionizing substance dissolved in 1 kg of water is known to lower the freezing point 1.86°C. ❑ Vapor pressure osmometer: ❑ Measures the dew point ❑ Urine:Serum Osmolality ❑ Normal: 1:1, ❑ After a period of dehydration 3:1  TUBULAR SECRETION ❑ Elimination of wastes not filtered by the glomerulus (MW >70,000 D) ❑ Blood pH: 7.35 – 7.45 TESTS FOR TUBULAR SECRETION 1. p-aminohippuric acid test (PAH) ❑ Most common ❑ Reference method for measurement of RPF 2. Phenosulfonpthalein (PSP) ❑ Obsolete 3. Titratable acidity and Urinary Ammonia ❑ Measures kidney's ability to secrete ammonia ❑ Normal: Approximately 70 mEq/day of acid is excreted in the form of titratable acid, hydrogen phosphate ions, or ammonium ions ❑ Renal tubular acidosis  Routine Urinalysis Physical Exam of Urine 1. Urine Volume ❑ Normal daily urine output (1200 – 1500 ml) 600-2000 ml is considered normal ❑ Polyuria: >2,500 ml/day Excessive fluid intake, diuretics, nervousness, DM ❑ Oliguria: 500 mL at night with SG of < 1.018 Increased water intake at night ❑ Anuria: complete cessation Mercury poisoning, renal stones, renal tumors 2. Urine Color ❑ Varies depending on hydration, normal metabolic functions, physical activity, diet, or pathologic conditions ❑ Three pigments: urochrome (yellow), uroerythrin (pink), urobilin (orange- brown) ❑ Should correlate with urine SG Normal: colorless – deep yellow ❑ Most common abnormal: red To determine urine color, under a good light source LOOK DOWN through the container against a white background Most abnormal: _____________ \ \ 1. Colorless, pale Normal yellow, yellow, dark yellow (amber) 2. Orange Bilirubin (yellow foam when shaken) 3. Yellow-orange Phenazopyridine (yellow foam when shaken) Phenindione 4. Yellow-green Bilirubin oxidized to biliverdin 5. Green Pseudomonas infection 6. Blue-green Breath deodorizers, indicanuria 7. Red/ Pink RBCs, hemoglobin, myoglobin 8. Brown/ Black Malignant melanoma, methemoglobin 3. Urine Clarity REPORTING DESCRIPTION OF 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 May precipitate or clotted  Notes to Remember ❑ Freshly voided urine is usually CLEAR. ❑ Assess using a WELL-MIXED urine viewed through a clear container against a white background with black print (newspaper print). ❑ FOAM FORMATION White: protein Yellow: bilirubin Thick Orange: phenazopyridine (pyridium)  CAUSES OF URINE TURBIDITY NON-PATHOLOGIC PATHOLOGIC Squamous epithelial cells Red blood cells Mucus White blood cells Amorphous phosphates, Bacteria carbonates, urates Semen, spermatozoa Yeast Fecal contamination Nonsquamous epithelial cells Radiographic contrast Abnormal crystals media Talcum powder Lymph fluid Vaginal creams Lipids  Notes to Remember ❑ Chyluria: obstruction to lymph flow and rupture of lymphatic vessels into the renal pelvis, ureters, bladder, or urethra, filariasis (prevailing cause) ❑ Pseudochyluria: paraffin-based vaginal creams ❑ Lipiduria: nephrotic syndrome, skeletal trauma due to exposure of yellow marrow ❑ Bacterial growth: uniform opalescence NOT removed by acidification or filtration ❑ Leukocytes: white cloud that remains after acidification ❑ Uric acid and urates: white, pink, or orange cloud in ACID urine that dissolved at __________ ❑ Phosphates, ammonium urates, carbonates: white cloud in ALKALINE urine 4. Urine Odor ODOR CAUSE Aromatic Normal Bleach Contamination Foul, ammoniacal Urinary Tract Infection (UTI) Fruity, sweet Ketones Mousy Phenylketonuria (PKU) Maple Syrup, Maple Syrup Urine Disease Curry, (MSUD) Caramelized sugar Distinct Onion, garlic, or asparagus Sweaty Feet Isovaleric acidemia, glutaric acidemia  ODOR CAUSE Rancid butter Tyrosinemia Cabbage, hops Methionine malabsorption Rotting Fish Trimethyl aminuria Sulfur Cystine disorder Cat urine Hydroxymethylglutaric acid Tomcat urine Multiple carboxylase deficiency Odorless Acute Tubular Necrosis Swimming pool Hawkinsinuria Menthol-like Phenol-containing medications 5. Urine Specific Gravity ❑ Density of a solution compared with the density of a similar volume of dist. water at a similar temp ❑ Normal random spx: 1.002 to 1.035 ❑ Most random spx: 1.015 to 1.025 ❑ 1.010 – SG of GF ❑ 1.010 - Hypersthenuric A. Urinometer ❑ Consists of a weighted float attached to a scale that has been calibrated in terms of urine SG ❑ Calibrated to read 1.000 in distilled water at a particular temperature ❑ POTASSIUM SULFATE SOLUTION-used for calibration of the urinometer ❑ SG = 1.015 Disadvantages: ❑ Requires large volume of spx: 10 – 15 ml ❑ Requires temperature, glucose and protein corrections ❑ Subtract 0.001 for every 3° below 20 °C ❑ Add 0.001 for every 3° above 20 °C ❑ 1g/dL GLUCOSE = subtract 0.004 in SG reading ❑ 1g/dL PROTEIN = subtract 0.003 in SG reading B. Refractometer/ TS Meter ❑ Measures the refractive index ❑ ADVANTAGES Small volume requirement: 1 -2 drops NO NEED for temperature correction ❑ DISADVANTAGE Glucose and protein corrections are required  Calibrating Solutions Distilled H2O 1.000 3% NaCl 1.015 + 0.001 5% NaCl 1.022 ± 0.001 9% Sucrose 1.034 ± 0.001 ❑ 1g/dL GLUCOSE = subtract 0.004 in SG reading ❑ 1g/dL PROTEIN = subtract 0.003 in SG reading C. Harmonic Oscillation Densitometry ❑ Principle: The FREQUENCY OF A SOUND WAVE entering to the solution that is proportional to the density of the urine. D. Falling drop method ❑ Direct method, more accurate than refractometer, more precise than urinometer E. Reagent Strip ❑ Based on the change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium  Chemical Exam of Urine ❑ Makes use of reagent strip pads (dipsticks) impregnated with chemical reagents for analyte detection DIPSTICK or REAGENT STRIP METHOD ❑ Dipping the strip completely but briefly ( 400 mg/dL B. Trichloroacetic acid (TCA) Precipitation Test ❑Precipitates GLOBULINS more than albumin ❑Preferred method when sample is CSF  4. Glucose ❑ MOST FREQUENT CHEMICAL ANALYSIS DONE IN URINE Reagent strip method: GLUCOSE SENSITIVE ONLY ❑ Multi Stix: Potassium iodide as chromogen (green to brown) ❑ Chemstrip: Tetramethylbenzidine as chromogen (yellow to green)  False negative False positive Ketones Oxidizing agents High specific gravity Low temperatures Improperly preserved spx Ascorbic acid  Chemical Tests for Glucose A. COPPER REDUCTION TEST ❑ Relies on the ability of reducing sugars (e.g. glucose) to reduce CuSO4 to Cuprous oxide in the presence of heat and alkali ❑ NOT a confirmatory test for glucose; performed to detect increased levels of galactose and other non-glucose reducing sugars  CLINICAL SIGNIFICANCE: Glycosuria: SUGARS in urine Glucosuria: GLUCOSE in urine CLINITEST: ❑ Performed when results of glucose in dipstick test is trace ❑ 5 Drops of urine + 10 Drops of water + tablet ❑Components: copper sulfate, sodium carbonate and sodium citrate, _____________ ❑ Pass through phenomenon  OTHER SUGARS Fructose Inherited enzyme Seliwanoff deficiency or (Resorcinol and parenteral fructose HCl) feeding Berchardt and Barford's Galactose Galactosemia Lactose Found among Rubner’s lactating moms Pentose Increased fruit sugars, Bial’s drug therapies, in benign pentosuria, Sucrose Ingestion of large amounts of sucrose  Reporting of Benedict’s Test GRADING COLOR NEGATIVE Clear BLUE TRACE Bluish green 1+ Green w/ green or yellow ppt 2+ Yellow to green w/ yellow ppt 3+ Yellow-orange w/ yellow-orange ppt 4+ Reddish-yellow w/ BRICK RED ppt Glucose Oxidase and Clinitest Reactions Glucose Clinitest Interpretation Oxidase (strip) 1+ Negative Small amount of glucose present 4+ Negative Possible oxidizing agent interference on reagent strip Negative Positive Non-glucose reducing substance present Possible interference on reagent strip 5. Ketones ❑ INTERMEDIATE end products of fat metabolism caused by: Inability to metabolize carbohydrates (DM) Increased loss of carbohydrates (vomiting) Inadequate intake of carbohydrates (starvation or malabsorption) Reagent Strip ❑ Principle: Sodium nitroprusside reaction ❑ MOST SENSITIVE TO ACETOACETIC ACID ❑ Addition of glycine will make the strip sensitive to acetone ❑ NEVER sensitive to b-hydroxybutyric acid Sensitivity: 5-10 mg/dl THE 3 KETONE BODIES: 1. Acetoacetate ❑ PARENT ketone (_____) 2. Beta-hydroxybutyric acid ❑MOST ABUNDANT (_____) 3. Acetone (____) False positive False negative Phthalein dyes Improperly preserved Highly pigmented red urine specimen Levodopa Medications containing free sulfhydryl groups ACETEST TABLET TEST ❑ Done if the result in the strip is > trace ❑ Reagents: sodium nitroprusside, glycine, disodium phosphate (alkali buffer), lactose (for better color differentiation) ❑ May be used to detect ketones in plasma and other body fluids 6. Blood ❑ Reagent Strip Principle: Pseudoperoxidase activity of hemoglobin ❑ Based on the liberation of oxygen from peroxide in the reagent strip by the peroxidase-like activity of heme in free hemoglobin, lysed erythrocytes, or myoglobin.  Test Hematuria Hemoglobinuria Myoglobinuria Macroscopic Cloudy red Clear red urine Clear red urine urine Reagent strip (+) with (+) (+) speckled pattern Microscopic Intact No intact RBCs No intact RBCs RBCs Additional test: Red precipitate No Blondheim’s Clear precipitate test supernatant Red (ammonium (-) for reagent supernatant sulfate) strip (+) for reagent strip Clinical Significance of a Positive Reaction for Blood Hematuria Hemoglobinuria Myoglobinuria Renal calculi Transfusion reactions Muscular trauma Glomerulonephritis Crush injuries Pyelonephritis Hemolytic anemias Prolonged coma Tumors Severe burns Convulsions Trauma Infections Muscle-wasting disease Exposure to toxic Strenuous exercise/RBC Alcoholism chemicals trauma Anticoagulants Drug abuse Strenuous exercise Brown recluse spider Cholesterol-lowering bites medications 7. Bilirubin ❑ Principle: Diazo reaction ❑ The degradation product of hemoglobin ❑ 2 forms: Conjugated & unconjugated bilirubin ❑ Multistix: 2,4-dichloroaniline diazonium salt ❑ Chemstrip: 2,6- dichlorobenzenediazonium salt ICTOTEST TABLET: ❑ More specific and sensitive to less reactive bilirubin ❑ (+) blue to purple Reagents: p-nitrobenzene diazonium p-toluene, sulfosalicylic acid (SSA), and sodium bicarbonate  False positive False negative Highly pigmented urine Specimen exposure to light Indican Ascorbic acid Metabolites of Lodine High concentrations of nitrite Condition Urine urobilinogen Urine bilirubin Hemolytic disease +++ - Liver damage ++ +/- Bile duct obstruction -/+ +++  SERUM URINE DISEASE BILIRUBIN BILIRUBIN Hemolytic Increased Negative Disease Hepatic Disease B1 Increased Negative B2 Positive Positive Obstruction Increased Positive (partial) Obstruction Increased Positive (complete) 8. Urobilinogen ❑ Principle: Ehrlich reaction ❑ Formed from hydrolysis B2 in the colon by the resident flora (stercobilinogen and mesobilirubinogen are other forms of bilirubin formed) ❑ Must be processed immediately ❑Excretion is increased following a meal ❑ 50%: Reabsorbed into the portal circulation ❑ Vast majority of remaining urobilinogen is excreted in feces as colored urobilin or stercobilin after further hydrolysis ❑ Elevated results in non-hospitalized (1%) and hospitalized (9%) population are mainly due to constipation Reagent strip: Multistix: p-dimethylaminobenzaldehyde Chemstrip: 4-methoxybenzenediazonium- tetrafluoroborate False negative False positive Old specimen Highly pigmented urine Formalin High concentrations of nitrite  A. Watson-Schwartz Test ❑ Differentiates urobilinogen, porphobilinogen and Ehrlich's reactive compounds. Other Ehrlich Urobilinogen Porphobilinogen Reactive Substances Chloroform Extraction: Urine (Top) Colorless Red ……. Red ……. Chloroform (Bottom) Red Colorless Colorless Butanol Extraction: Urine (Bottom) Red ……. Colorless Red ……. Butanol (Top) Colorless Red Colorless B. Hoesch Screening Test ❑ RAPID SCREENING TEST for porphobilinogen ❑ Hoesch reagent: Ehrlich’s reagent dissolved in 6M HCl (the acid inhibits urobilinogen) ❑ (+) top solution appears RED in color 9. Nitrite Principle: Greiss reaction CLINICAL SIGNIFICANCE: ❑ SCREENING TEST FOR URINARY TRACT INFECTIONS Multistix: p-arsanilic acid Chem strip: Sulfanilamide SPECIMENS: 1. 4-hour urine ❑ Time it takes for Gram (-) bacteria (Enterobacteriaceae) to convert nitrate to nitrite 2. First morning urine: ❑ BEST SPECIMEN for NITRITE TESTING  False negative False positive Nonreductase-containing Improperly bacteria preserved Insufficient contact time specimens between bacteria and nitrate Lack of urinary nitrate Highly pigmented urine Large quantities of bacteria converting nitrite to nitrogen Presence of antibiotics High concentrations of ascorbic acid High specific gravity 10. Leukocyte Esterase Principle: Leukocyte esterase reaction Found in the primary/azurophilic granules of all WBC's except lymphocyte CLINICAL SIGNIFICANCE: ❑ Detection of inflammation in the kidney or GUT and/or UTI ❑ Bacterial or nonbacterial UTI Reagent strip: ❑ Multistix: Derivatized pyrrole amino acid ester and Diazonium salt ❑ Chemstrip: Indoxylcarbonic acid ester and Diazonium salt ❑ Esterases produced by leukocyte catalyze an enzymatic reaction on the strip resulting in a color change ❑ (+) for LE: Granulocytes, Monocytes, Trichomonas, and Histiocytes  False Negative False Positive High concentrations of Strong oxidizing agents protein Glucose Formalin Oxalic acid Highly pigmented urine Ascorbic acid Nitrofurantoin Gentamicin Cephalosporins Tetracyclines Inaccurate timing