ENHANCEMENT NOTES IN CLINICAL MICROSCOPY Prepared by: John Alvin O. Reyes, RMT URINALYSIS |._URINE COMPOSITION- 95% water, 5% solutes ORGANIC UREA- breakdown product of protein and amino acids, comprises haif of the total dissolved solids in the urine Creatinine | Uric acid INORGANIC Chiloride- major inorganic solute followed by; Sodium and potassium Urea and creatinine are useful in determi in higher concentration a fluid is urine because these substances are present urine as compared to other body fluids Il. URINE VOLUME Normal daily urine output is usually 1200ml to 1500mI A range of 600-2000 mi is stil considered normal OLIGURIA- decrease in urine output Infants: < tmi/kg/ hr children: < 0.5 mi/kg/hr Adults: <400 miiday Anuria: cessation of urine flow Nocturia: increased urinary excretion at night Polyuria: increase in daily urine volume (greater than 2.5Liday in adults and 2.5-3mL/kg/day in children) Diabetes mellitus Diabetes insipidus Cause Defect in production of function of insulin | Decrease production of function of leading to increased glucose antidiuretic homone, thus water is not concentration reabsorbed [Appearance Dilute Dilute Specific gravity High Low Il, SPECIMEN COLLECTION + Specimens must be collected in a clean, dry, leak-proof container + Containers should have a wide mouth and must be made out of clear material for observation of color and clarity + Recommended capacity: 50 ml, which allows; for 12 ml of specimen needed for microscopic analysis IV. SPECIMEN HANDLING + Following collection, specimens should be delivered to the laboratory promptly within 2 hours + Specimens that can't be tested within 2 hours should be refrigerated or have an appropriate chemical preservative. SPECIMEN PRESERVATION 1. The most routinely used method of preservation is refrigeration at 2°C-8°C, which decreases bacterial growth and metabolism 2. Refrigeration can increase the specific gravity, when measured by urinometer and precipitation of amorphous phosphates and urates can obscure the microscopic sediment analysis ANALYTE CHANGE CAUSE COLOR MODIFIED/DARKENED (Oxidation/Reduction of metabolites CLARITY DECREASED | Bacterial growth and multiplication ODOR INCREASED Bacterial multiplication or breakdown of urea to ammonia pH INCREASED Breakdown of ammonia by urease _ producing bacteria/ loss of CO2 [GLUCOSE DECREASED Glycolysis and bacterial use KETONES DECREASED [Volatilization and bacterial metabolism BILIRUBIN DECREASED Exposure to light! photooxidation to bilivergin UROBILINOGEN DECREASED (Oxidation to urobilin NITRITE INCREASED Multiplication of nitrite reducing bacteria RBC and WBC casts DECREASED. Disintegration in dilute alkaline urine BACTERIA INCREASED [Multipication URINALYSIS |PHYSICAL EXAMINATION 1. COLOR NORMAL URINE COLOR * Urine color which is normally various shades of yellow, can range from colorless to amber to orange, red, green, blue, brown or even black + The characteristic yellow color of the urine is caused by the presence of a pigment called urochrome. + Because urochrome production and excretion are constant, the intensity of the color of urine provides a crude indicator of urine concentration and body hydration ‘ABNORMAL COLOR ‘CAUSE, Dark yellow to amber ‘does not always signify a normal concentrated urine but can be caused by the presence of bilirubin Yellow green Yellow orange photo-oxidation of bilirubin phenazopyridine (pyridium) administration Cloudy red presence of red blood cells Brown ‘oxidation of hemoglobin to methemoglobin Fresh brown May indicate glomeruiar bleeding resulting from conversion of hemoglobin to methemoglobin. Clear red hemoglobinuria or myoglobinuria Port wine ‘oxidation of porphobilinogen to porphyrins Green Pseudomonas infection Blue Increased indican in the urine due to bacterial infection nN Black Presence of melanin which is an oxidation product of ‘metanogen, produced in excess when melanoma is present Homogentisic acid- a metabolite of phenylalanine, imparts a black color to alkaline urine from Hemoglobinuria= red urine + red plasma Myoglobinuria= red urine + clear plasma Non-pathogenic causes of red urine: - Menstrual contamination - Highly pigmented foods urine) + _ Ingestion of blackberries, - Genetically susceptible persons eating fresh beets (alkaline persons with alkaptonuria = oes [oe] orogens] [Meaeina) [esr isp ‘estes (Peo) [Fearne Il. CLARITY ‘* Refers to transparency! turbidity of a urine specimen + Terminology used to report include clear, hazy, cloudy, turbid and milky ‘+ Normal clarity- Freshly voided urine is usually clear. Precipitation of amorphous phosphates and carbonates may cause white cloudiness 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 | Miky | May precipitate or be clotted Urine Color and Clarity Procedure 1, Evaluate an adequate volume of specimen, 2. Use a well-mixed specimen, 3. View the urine through a clear container. 4. View the urine against a white background using adequate room lighting '5. Maintain adequate room lighting 6. Evaluate a consistent volume of urine \ NON-PATHOLOGIC TURBIDITY PATHOLOGIC TURBIDITY + Presence of squamous epithelial cells and mucus + Amorphous phosphates, carbonates, and urates precipitate in refrigerated specimens + Amorphous phosphate and carbonate: precipitate in alkaline urine + Amorphous urate= pink brick dust (uroerythin), acidic urine ite ‘Caused by: RBC WeC Bacteria Non-squamous epithelial cells Yeast ‘Abnormal crystals Lymph fluid and lipids LABORATORY CORRELATIONS OF URINE TURBIDITY Acidic Urine ‘Amorphous urates, Radiographic contrast media Alkaline Urine ‘Amorphous phosphates, carbonates Soluble with Heat ‘Amorphous urates, uric acid crystals URINALYSIS |Soluble in Dilute Acetic Acid | RBCs, Amorphous phosphates, carbonates Insoluble in Dilute Acetic Acid | WBCs, Bacteria, yeast, Spermatozoa Soluble in Ether Lipids Lymphatic fluid, chyle lll. SPECIFIC GRAVITY ‘+ Evaluation of the kidney's reabsorption ability can be performed by measuring the specific gravity of a specimen «The specific gravity of urine is a measure of the density of the dissolved chemicals in the specimen DIRECT METHODS I INDIRECT METHODS * Urinometer ‘© Refractometer * Harmonic oscillation densitometry ‘* Reagent strip A. URINOMETER 1. An adequate amount of urine (10 to 15ml) is poured into a proper size container. 2. The urinometer is added with a spinning motion. 3. The scale reading is then taken at the bottom of the urine meniscus CORRECTION FOR URINOMETER ‘For every 3°C that the specimen temperature is below the urinometer calibration temperature, 0.001 must be subtracted from the reading + 0.001 must be added for every 3°C that the specimen measures above the calibration temperature ‘+ For every gram of protein present, 0.003 must be subtracted from the specific gravity reading ‘+ For each gram of glucose, 0.004 must be subtracted from the specific gravity reading REFRACTOMETER * Determines the concentration of dissolved particles in a specimen by measuring the refractive index * REFRACTIVE INDEX- a comparison of the velocity of the light in air with the velocity of light in a solution CORRECTION: + Temperature corrections are not necessary (temperature is compensated between 15C to 38C) + Corrections for glucose and protein are still calculated CALIBRATION OF REFRACTOMETER STEPS IN REFRACTOMETER * Distilled H20: 1.000 1. Put one or two drops of sample on the prism. © 5% NaCl: 1.022 + 0.001 2. Close the daylight plate gently © 9% Sucrose: 1.034 + 0.001 3. The sample must spread all over the prism surface, 4. Look at the scale through the eyepiece. 5. Read the scale where the boundary line intercepts it. 6. Wipe the sample from the prism clean with a tissue paper and water. A. HARMONIC OSCILLATION DENSITOMETRY * Based on the principle that a sound wave entering a solution changes in proportion to the density of the solution. Shifts in harmonic oscillation are measured and relative density is calculated CLINICAL SIGNIFICANCE Isosthenuric urine specific gravity of 1.010 Hyposthenuric urine specific gravity below 1.010 pene | Hypersthenuric urine specific gravity above 1.010 | fefacomety Reactveinex weimen 10081095 Csnclty Cares in colgatve properties typi Sample is not urine 1.003 nae: Most random specimen 1.015 and 1.025 feagenttip pk hngesc pleco bys pst Radiographic contrast media | 1.035 IV. URINE ODOR ‘Normal urine has a characteristic aromatic odor ‘A long-standing urine specimen is ammoniacal due to the conversion of urea to ammonia +A patient with bacterial infection has an ammoniacal fresh urine sample ODOR CAUSE ‘Aromatic, faintly Normal urine Lack of odor ‘Acute renal failure with acute tubular necrosis ‘Ammoniacal ‘Old urine- improperly stored Pungent, fetid Urinary tract infection ‘Sweet, fruity Ketone production due to: Diabetes mellitus, starvation, dieting, mainuttition strenuous exercise, vomiting, diarrhea URINALYSIS |Unusual odo ‘Associated with amino acid disorder: Mousy Phenylketonuria Maple syrup Maple syrup urine disease Rancid Tyrosinemia Rotting) old fish Trimethylaminuria Cabbage, hops Methionine malabsorption ‘Sweaty feet Isovaletic academia Distinctive Ingested substances: asparagus, garlic, onions Mentholike Phenol containing medications Bleach ‘Adulteration of the specimen or container contamination CHEMICAL EXAMINATION REAGENT STRIPS REFLECTANCE PHOTOMETRY: Employed by automated reagent strip readers Reagenis: methyl red, bromthymol blue Sensitivity 59 Sources of error Tunover from adjacent pads, old specimens First moming specimen ph 5.0- 6.0 Random samples 458.0 Principle Double indicator system of methyl red and bromthymol Blue The colors progress ‘from orange at pH 5 through yellow and green toa final deep blue at pH 9 REACTION TIME 605 CLINICAL SIGNIFICANCE OF pH ‘ACID URINE EMPHYSEMA, DIABETES MELLITUS, STARVATION, DEHYDRATION, DIARRHEA PRESENCE OF ACID PRODUCING BACTERIA (E.COLI), HIGH PROTEIN DIET ‘CRANBERRY JUICE, MEDICATIONS (methenamine mandelate [Mandelamine], Fosfomycin |_| tromethamine) ne: - - a“ ALKALINE URINE | Hyperventilation, Vomiting, Renal tubular acidosis, Presence of urease producing bacteria, Vegetarian diet, Old specimens PROTEM MULTISTIX CHEMSTRIP ‘etrabromphenal blue Tetrachlorophenol, tetrabromeuffophthalein Sensitivity: 15-30 mg/l alburrin Sensitivity: 6 mg/dl albumin SOURCES OF ERROR FALSE POSITIVE: Highly buffered alkaline urine, Pigmented specimens, Phenazopyridne, Quaternary ‘ammonium compounds (detergents), Anti-septics, Loss of buffer, High specific gravity FALSE NEGATIVE | Protein other than albumin, Microalbuminuria BENCE JONES PROTEIN: coagulates at temperatures between 40°C and 60°C and dissolves when the temperature reaches 100°C. Therefore, a specimen that appears turbid between 40°C and 60°C and clear at 100°C cen be suspected of containing Bence Jones protein. PRINCIPLE PROTEIN ERROR OF INDICATORS ‘At pH level of 3, the incicator appears yellow In the absence of protein, however, as the protein concentration increases, the color progresses through various shades of green and finally blue REACTION TIME 605 CLINICAL SIGNIFICANCE. PRE RENAL RENAL POST RENAL INTRAVASCULAR HEMOLYSIS | GLOMERULAR: LOWER UTI ACUTE PHASE REACTANTS. ‘= IMMUNE COMPLEX DISORDERS | INJURY. MUSCLE INJURY + AMYLOIDOSIS MENSTRUAL CONTAMINATION MULTIPLE MYELOMA * DIABETIC NEPHROPATHY VAGINAL SECRETIONS = PRE-ECLAMPSIA PROSTATIC FLUID/ * ORTHOSTATIC PROTEINURIA SPERMATOZOA TUBULAR: FANCONI SYNDROME HEAVY METALS SEVERE VIRAL INFECTIONS ‘SULFOSALICYLIC ACID PRECIPITATION TEST ] URINALYSIS |PROCEDURE: ‘GRADING + Add 3 mL of 3% SSA reagent to 3 mL of ‘GRADE | TURBIDITY PROTEIN centrifuged urine, RANGE + Mix by inversion and observe for cloudiness. (mgidL) + Grade the degree of turbidity Negative | No increase in turbidity 6 FALSE POSITIVE | FALSE NEGATIVE Trace | Noticeable turbid 6-30 Radiographic dyes | Highly alkaline urine + Onn ubiatywihne [30-100 tolbutamide " fer eon. = metabolites 2 wifrnofoceuston | cephalosporins cs Turbidity with 200-400 penicillins granuiefloriand sulfonamides flocculation & lumps of protein 3400 MICROALBUMIN TESTING ‘A. MICRAL TEST B. IMMUNODIP C. TRADITIONAL (24H URINE a | ___SAMPLE) _ Principle: enzyme immunoassay | Principle: ‘SIGNIFICANT VALUES: Sensitivity: 0-10 ma/dl Immunochromographics + Albumin/24 hours= (30- Reagents: Gold-labeled ‘Sensitivity: 1.2-8.0 mg/dL 300mg/24h) antibody, B-galactosidase Reagents: Antibody coated blue | * Albumin excretion in Chlorophenol red galactoside | jatex particles ‘ugimin (AER)= 20-200 Interference: False negative: _| Interference: False negative- ugimin Dilute urine dilute urine GLUCOSE MULTISTIX CHEMSTRIP glucose oxidase, peroxidase, polasium lodide (green to | glucose oxidase, peroxidase, tetramethyibenzidine brown) (yellow to green) Sensitivity: 75-125 mgidl Sensitivity: 40 mg/dl INTERFERENC False positive ‘oxidizing agents and detergents False negative High levels of ascorbic acid, High level of ketone, High specific gravity, Low temperature, improperly preserved specimen PRINCIPLE Double sequential enzyme reaction REACTION TIME 30s COPPER REDUCTION TEST FOR GLUCOSE ‘+ PRINCIPLE: The test relies on the ability of glucose and other substances to reduce copper sulfate to copper oxide in the presence of alkali and heat ‘+ Acolor change progressing from a negative blue, through green, yellow and orangelred occurs when the reaction takes place CLINITEST TABLET COMPONENTS: Copper sulfate, sodium carbonate, sodium citrate and sodium hydroxide ‘SENSITIVITY 200 mglél. PRINCIPLE: Upon addition of the tablet to water and urine, heat is produced by the hydrolysis of sodium hydroxide and its reaction with sodium citrate, and carbon dioxide is released from the sodium carbonate to prevent room air from interfering with the reduction reaction SOURCES OF ERROR | “Pass through” high glucose levels the color produced passes through the orange’red stage and retums to a green-brown color, and if not observed, a high glucose level may be reported as negative ascorbic acid, certain drug metabolites, and antibiotics such as the cephalosporins ‘SIGNIFICANCE: > Commonly found reducing sugars: galactose, fructose, pentose, and lactose, of which galactose is the most significant. > Galactose in the urine of a newbom represents an “inborn error of metabolism” in which lack of the enzyme galactose-1-phosphate uridyl transferase prevents breakdown of ingested galactose and results in failure to thrive and other complications, including death. GLUCOSE OXIDASE | CLINITEST INTERPRETATION i+ Negative ‘Small amount of glucose present ae Negative Presence of oxidizing agent, possible interference with the reagent stip Negative Positive Non-glucose reducing substance present Possible interfering substance for reagent strip URINALYSIS |Hypergiycemia-associated CLINICAL SIGNIFICANCE Diabetes mellitus, Pancreatitis, Pancreatic cancer, Acromegaly, Cushing syndrome, Hyperthyroidism, Pheochromocytoma, CNS damage, Gestational diabetes Renal-associated Fanconi syndrome, ESRD, Cystinosis, Osteomelacia CHEMSTRIP | MULTISTIX 70 mg/dl; acetone sodium nitroprusside, glycine SENSITIVITY: 9 mg/dl; acetoacetic acid ‘SENSITIVITY: 5-10 mg/dl, acetoacatic acid INTERFERENCE False positive in dyes, highly pigmented red urine, Levodopa, Medications containing free False negative served specimens PRINCIPLE ‘SODIUM NITROPRUSSIDE REACTION A «= Acetoacetic (20%) acid in an alkaline medium reacts with sodium nitroprusside to produce a purple color + The test does not detect b-hydroxybutyric (78%) acid and only slightly sensitive to acetone (2%) when glycine is present REACTION TIME as ‘ACETEST TABLET * Provides sodium nitroprusside, glycine, disodium phosphate, and lactose + The addition of lactose is for bet 9 color differentiation + Acetest tablets are hygroscopic; ifthe specimen is not completely absorbed within 30 seconds, a new tablet should be used CLINICAL SIGNIFICANCE Diabetic acidosis, insulin dosage monitoring, Starvation, Malabsorption’ pancreatic disorders, Strenuous exercise, Vomiting, Inborn error of metabolism Blood. [Reagent | Tetramethylbenzidine * Principle Pseudoperoxidase activity of hemoglobin: Hemoglobin catalyzes a reaction between H202 and @ chromogen, which has a green blue color REACTION TIME | 60s INTERFERENCE False positive False negative menstrual contamination Strong oxidizing agents, bacterial peroxidase, High specific gravity/ crenated calls, formalin, captopril, high concentrations of nitrite, ascorbic acid > 25mgidl, unmixed specimen CLINICAL SIGNIFICANCE [HEMATURIA | HEMOGLOBINURIA [ MYOGLOBINURIA Renal calcull “Transfusion reactions Muscular trauma/ crush injury Glomerulonephritis Hemolytic anemias Prolonged coma Pyelonephrits Severe bums Convulsions Tumors. Infections/ malaria ‘Muscle wasting disease Trauma Strenuous exercise Alcoholism Exposure to toxic chemicals Brown recluse sider Cholesterol lowering statin Anticoagulants medications. Strenuous exercise: HEMOGLOBINURIA VERSUS MYOGLOBINURIA to sit for 5 minutes strip, BLONDHEIM’S TEST (AMMONIUM SULFATE PRECIPITATION TEST) + 2.89 of ammonium sulfate are added to 5 mL of centrifuged urine After mixing and allowing the specimen + The urine is filtered or centrifuged, and the supernatant is tested for a reaction for blood with a reagent HEMOGLOBIN MYOGLOBIN Precipitated by the ammonium sulfate, produces a red precipitate and a supernatant that tests negative for blood The Supernatant retains the red color and gives a positive reagent strip test for blood BILIRUBIN MULTISTIX CHEMSTRIP dichlroaniline diazonium salt dichlorobenzazne diazonium salt Sensitivity: 0.4-0.8 mg/dl bilirubin Sensitivity: 0.5 mg/el bilirubin (FALSE POSITIVE [FALSE NEGATIVE URINALYSIS |Highly pigmented urines, phenazopyridi ; ‘Specimen exposure to ight, ascorbic acid, high metabolites, iodine concentration of nitrite PRINCIPLE DIAZO REACTION: Bilirubin reacts with diazonium sait to produce an azodye, with colors ranging from increasing degrees of tan or pink to violet REACTION TIME 30s, CLINICAL SIGNIFICANCE Hepatitis, Cirmosis, Other liver disorders, Biliary obstruction sulphonamides, methyldopa, procaine, chlorpromazine, highly pigmented urine URINE BILIRUBIN URINE UROBILINOGEN Bile duct obstruction + Normal [Liver damage + or + Hemolytic disease Negative He UROBILINOGEN MULTISTIX CHEMSTRIP -dimethylaminobenzaidehyde methoxybenzene-diazonium-tetrafluoroborate Sensitivity: 0.2 mg/dl Sensitivity: 0.4 mi dl FALSE POSITIVE FALSE NEGATIVE orphobilinogen, indican, p-aminosalicylic acid, ld specimens, preservation in formalin PRINCIPLE ci shemstrip: uses, p-dab, to produce colors ranging from light to dark pink Multistix: 4-methoxybenzene-dlazonium-tetrafluoraborate, producing colors ranging from white to pink REACTION TIME 60s CLINICAL Early datacton of liver disease, Liver disorders, hepatii, cirhosis, carcinoma, SIGNIFICANCE. Hemolytic disorders WATSON-SCHWARTZ DI |FFERENTIATION TEST. + Chloroform sinks + Butanol floats + Urobilinogen is extracted by chloroform and butanol + Porphobilinogen is neither extracted by chloroform nor butanol + Other Ehrlich reactive compounds are extracted by butanol HOESCH SCREENING TEST FOR PORHOBIILINOGEN + Two drops of urine are added to 2 ml of hoesch reagent (ehrlich reagent in 6m HCI) + Appearance of a red color in the top portion of the solution indicates the presence of porphobilinogen Urobilinegen | Other Ehrich- | Porphobilinogen 0 Reactive ‘Substances Chloroform extraction Urine (op) __| Colorless Red Red f CChioroform | Red Colorless Colorless {bottom) ‘Butanol Extraction Butanol (Top) | Red Red Colorless Urine (Batiom) | Colorless Coleress Red trope Pophiengn Fe ttre Maltistix_ P-arsanilic acid Tetrahydrobenzo(h)-quinolin-3-ol Chemstrip ‘Sulfanilamide, hydroxytetrahydrobenzoquinoline PRINCPILE Bacterial reduction of nitrate to ritite, nitrite is detected by the Greiss reaction which gives a pink color if It Is positive Sensitivity ‘approximately 1%10° organism or more produces a positive result in most cases REACTION TIME 60s FALSE NEGATIVE Nor-reductase containing bacteta, Insufficient contact time, Lack of urinary nitrate Presence of bacteria that conver ritrite to nitrogen, Antibiotics, Ascorbic acid High specific gravity FALSE POSITIVE Improperly preserved specimen, Highly pigmented urine LEUNOCYTE ESTERASE REAGENTS ESTER, DIAZONIUM SALT PRINCIPLE HYDROLYSIS OF AN ACID ESTER REACTION TIME 1205 URINALYSIS |CLINICAL SIGNIFICANCE Bacterial and non bacterial UTI, Inflammation of the urinary tract, Screening of urine culture spacimens, Trichomonas, yeast, Chlamydia and renal inflammation cause leukocyturia without bacteriuria INTERFERENCE FALSE POSITIVE [FALSE NEGATIVE Sirong oxidizing agents Inaccurate ting Highly pigmented urine, nitrofurantoin High concentration of: Protein, glucose, oxalic acid, etc. SPECIFIC GRAVITY REAGENTS bromthymol blue Sensitivity 1,000-1.030 PRINCIPLE Change in pKa of a polyelectrolyte in an alkaline medium “The indicator changes from blue, through shades of green, to yellow CLINICAL “Monitoring patient hydration of dehydration, Loss of renal tubular concentrating abilly SIGNIFICANCE: Diabetes insipidus. Determination of unsatisfactory specimens due to low concentration INTERFERENCE py ™ FALSE POSITIVE | FALSE NEGATIVE high concentration of protein highly alkaline urine TEST PRINCIPLE REAGENT STRIP REACTION TIME GLUCOSE DOUBLE SEQUENTIAL ENZYME Potassium iodide (green-brown) 0s REACTION Tetramethylbenzidine (yellow-green) BILIRUBIN DIAZO REACTION Tan or pink to yellow 30s KETONES ‘SODIUM NITROPRUSSIDE Purple color 40s REACTION SPECIFICGRAVITY | pka change of polyelectrolyte From blue (1.000), through shades of 458 sateen, to yellow (1.030) pH Double indicator system Orange at pH 5 through yellow end green | 608 tofinal deep blue at pH @ PROTEIN PROTEIN ERROR OF Green to blue 0s INDICATORS BLOOD PSEUDOPEROXIDASE ACTIVITY | Hemogiobinimyogiobin- yellowto green to | 608, ‘OF Hb a strongly positive green-blue RBC- speckled pattern UROBILINOGEN. EHRLICH’S REACTION MULTISTIX: Light to dark pink 60s CHEMSTRIP: White to pink NITRITE GREISS REACTION PINK COLORED AZODYE 0s LeuKocyTE ESTERASE REACTION PURPLE AZODYE 1208 ESTERASE MICROSCOPIC EXAMINATION ADDIS COUNT RBC. WBC and Epithelial cells Hyaline casts (0-500,000 0-1,800,000 10-5000 |. SPECIMEN PREPARATION: + RBCs, WBCs and hyaline casts disintegrate rapidly in dilute alkaline urine + The midstream clean-catch specimen minimizes external contamination of the sediment + Urine samples are recommended to be collected in containers with a 50-mL capacity + The recommended container capacity for drug testing is 60 mL and 30-45 mL of sample is usually obtained + The standard amount of urine: 10-15 ml centrifuged in a conical tube + 12 ml is frequently used because multiparameter reagent strips are easily immersed in this volume + Centrifugation: 5 minutes at 400 RCF + All centrifuge tubes should be capped to prevent biohazardous aerosols SEDIMENT VOLUME AFTER CENTRIFUGATION: URINALYSIS |Recommended volume of urine to be transferred t. Coverslip: 22x22 mm. 0.5 ml and 1 ml are frequently used after decantation, 10 a slide: 20 uL REPORTING: > 100 Crystals, Epithelial cells: Rare, few, moderate or m Casts: reported as the average per LPF following exat RBC and WBC: Reported as the average per number per 10 HPFs or 0- ation of 10 fields or range of 0-2, 2-5, 5-10, >10 2-5, 5-10, 10-25, 25-50, 50-100, any! 1+, 2+, 3+, 4+ Il_ MICROSCOPIC TECHNIQUES Technique Function Bright-field microscop) Used for routine urinalysis Phase-contrast microscopy Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads, and Trichomonas Polarizing microscopy ‘Aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals Dark-field microscop) ‘Aids in identification of Treponema pallidum Fluorescence microscopy ‘Allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye Interference contrast Produces a three-dimensional microscopy-image and layer-by-layer imaging of a specimen ‘A__Modulation contrast microscope |B. Differential interference contrast microscope I CELLS IN THE URINARY SEDIMENT Hoffman Nomarski 1. RBC ‘Smooth non-nucleated biconcave disks Dysmorphic- glomerular damage Crenated- hypertonic urine Ghost cell- hypotonic urine ‘Approximately 7mm in diameter Reported as the average number seen per 10 hpts Source of error: oil droplets, yeast cells 2. WHITE BLOOD CELLS * Larger than RBCs, granulated, muttilobed neutrophils measuring an average of about 12 mm Glitter cells in hypotonic urine ‘Source of error: RTE cells Reporting: number per 10/hpfs SIGNIFICANCE: UTI, inflammation of urinary system ‘SQUAMOUS EPITHELIAL CELLS Appearance: largest cell in the sediment, with abundant irregular cytoplasm and prominent nuclei Source of error: folded cells may resemble casts Reporting: rare (0-5), few (5-20), moderate (20-100), or many per LPF (>100) Significance: normal cellular sloughing 5. RTE CELLS ‘+ Appearance: rectangular, columnar, round, oval or cuboidal with an eccentric nucleus Source of error: spherical transitional cells, granular casts Reporting: ave. Number per 10 HPF Significance: presence of more than two RTE per HPF indicates tubular injury ‘4. TRANSITIONAL CELLS ‘Appearance: appear in several forms, including spherical, polyhedral and caudate Source of error: spherical forms resemble RTE cells Reporting: rare, few, moderate, many per HPF Significance: Increase in abnormal forms may indicate malignancy or viral infection 6. OVAL FAT BODIES ‘+ Appearance: highly refractile RTE cells ‘+ Source of error: confirm with fat stains (orange: red droplets) and polarized microscopy (maltese-cross formation) + Reporting: ave. Number per hpf Significance: Nephrotic syndrome, diabetes mellitus, trauma, lipid storage disease 7 BUBBLE CELL RTE cells containing large, nonlipi¢-filled vacuoles BACTERIA YEAST TRICHOMONAS: ‘Appearance ‘Small, spherical rod- | Small, oval, _refractile | Pear-shaped shaped structures strucutures with buds | Motile, flagellated or mycelia Reporting Rare (0-10), Few (10-50) | Few, moderate, or many | Few, moderate, or many moderate (50-200), or | per HPF per HPF many (>200) per HPF ‘SPERMATOZOA MUCUS ‘APPEARANCE ‘Tapered oval head with long tail Single or clumped ‘SOURCE OF ERROR None Hyaline casts REPORTING Present, based on lab protocol | Rare (0-1), few (1-3), moderate (3-10), many (10) per LPF URINALYSIS |IV. CAST COMPOSITION AND FORMATION The major constituent of casts is uromodulin * Other proteins: albumin and immunoglobulins are also incorporated into the cast matrix ‘+ Uromodulin protein is found in both normal and abnormal urine and is a major constituent of mucus * Broad casts may result from tubular distension or, in the case of extreme urine stasis, from formation in the collecting ducts. + CYLINDROIDS- Formation of casts at the junction of the ascending loop of Henle and the distal convoluted tubule may produce structures with a tapered end. CYLINDRURIA- The presence of urinary casts, TELESCOPED SEDIMENT- simultaneous occurrence of elements of glomerulonephritis and those of nephrotic syndrome in the same specimen. Include red cells, red cell casts, cellular casts, broad waxy casts, lipid droplets, oval fat bodies, and fatty casts * ORDER OF CAST DEGENERATION: Cellular cast— coarse granular cast— fine granular cast—> waxy cast HYALINE CAST APPEARANCE: Colorless homogenous matrix, consists almost entirely of uromodulin SIGNIFICANCE: Normal= 0-2 Increased in: glomerulonephritis, pyelonephritis,chronic renal disease, congestive heart failure, exercise RBC CAST ‘APPEARANCE: Orange-red color, cast matrix containing RBCs SIGNIFICANCE: Glomerulonephritis, strenuous exercise, associated with proteinuria and dysmorphic RBCs WBC CAST ‘APPEARANCE: Cast mattix containing WBC, granular, multfobed SIGNIFICANCE: Pyelonephritis, Acute interstitial nephritis, a primary marker for distinguishing pyelonephritis (upper UTI) from cystitis (lower UTI), may accompany RBCs on glomerulonephritis BACTERIAL CAST "APPEARANCE: Bacilli bound to protein matrix, may be pure bacterial or mixed with WBCs SIGNIFICANCE: Pyelonephritis, EPITHELIAL CELL CAST | APPEARANCE: RTE cells attached to protein matrix SIGNIFICANCE: Advanced tubular destruction, associated with heavy ‘metal and chemical or drug-induced toxicity, viral infections, and allograft rejection, bilirubin-stained RTE cells are seen in cases of hepatitis GRANULAR CAST APPEARANCE: Coarse and fine granules in a cast matrix MIXED CELLULAR CAST | APPEARANCE: casts containing multiple cell types SIGNIFICANCE: Glometulonephritis, pyelonephritis, stress and exercise, granules may represent disintegration of cellular casts and tubule cells, SIGNIFICANCE: RBC and WBC casts in glomerulonephritis WBC and RTE cell casts, or WBC and bacterial casts in pyelonephritis. WAXY CAST Highly refractile cast with jagged edges, stains dark pink I stai SIGNIFICANCE: extreme urine stasis, indicating chronic renal failure FATTY CAST ‘APPEARANCE: Highly refractile and may contain fat droplets and oval fat bodies BROAD CAST/RENAL | APPEARANCE: Wider than normal cast FAILURE CAST SIGNIFICANCE: Extreme urine stasis, renal failure, indicates destruction attached to a protein matrix. cholesterol demonstrates characteristic Maltese cross formations under polarized light, and triglycerides and neutral fats stain orange. SIGNIFICANCE: Nephrotic syndrome, Crush injuries, DM, Tubular necrosis (widening) of the tubular walls. V. URINARY CRYSTALS REPORTING: Normal crystal: rare (0-2), few (2-5), moderate (5-20), or many (>20) per HPF Abnormal crystals: averaged and reported per LPF A. NORMAL CRYSTALS SEEN IN ACIDIC URINE The most common erystals seen in the urine are amorphous urates, urie acid, acid urates and sodium urates Amorphous urates appear as yellow brown granules, produce a pink sediment in refrigerated specimen Amorphous urates are found in acidic urine with a ph of greater than 5.5, whereas uric acid can appear when the ph is lower URIC ACID Seen in a variety of shapes, including rhombic, four-sided flat plates (whetstones), lemon-shaped, wedges and rosettes, URINALYSIS |Usually appear as yellow-brown, but may be colorless and have a six-sided shape (hexagonal uric acid crystals are highly birefringent as compared to cystine crystals) + Seen patients with leukemia who are receiving chemotherapy, in patients with Lesch-Nyhan syndrome and sometimes, in patients with gout CALCIUM OXALATE © Frequently seen in ac urine, but they can be found in neutral or even rarely in alkaline urine Dihydrate/ WEDDELLITE ‘| Monohydrate crystals/ WEWELLITE colorless, octahedral, or enveloped shaped __| oval or dumbbell shaped, ethylene glycol poisoning, figure of eight hour glass + The finding of CaOx in fresh urine may be related to formation of renal calculi, because the majority of renal calculi (75%) are composed of calcium oxalate or calcium phosphate B. NORMAL CRYSTALS SEEN IN ALKALINE URINE 1, AMORPHOUS PHOSPHATES * Granular in appearance similar to amorphous urates + When present in large quantities following specimen refrigeration, they cause a white precipitate that does not dissolve on warming + They can be differentiated from amorphous urates by the color of the sediment and the urine pH 2. TRIPLE PHOSPHATE, + AKA ammonium magnesium phosphate + Frequently resembles a coffin lid, birefringent under polarized light + Calcium phosphate is a common constituent of renal calculi 3. MAGNESIUM AMMONIUM PHOSPHATE ‘+ Forms stones at an alkaline pH * May form when there is infection with Proteus spp. that causes alkalization of urine ‘+ May become large forming casts, showing staghorns 4, CALCIUM CARBONATE CRYSTALS ‘+ Small and colorless with dumbbell or spherical shapes 5. CALCIUM PHOSPHATE! APATITE HYDROXYAPATITE ‘+ Flat rectangular plates or thin prisms. 6. AMMONIUM BIURATE ‘+ Exhibit the characteristic yellow-brown color of the urate crystal seen in acidic urine ‘+ Frequently described as thorny apples because of their appearance as spicule-covered spheres ¢. ABNORMAL URINE CRYSTALS 1. CYSTINE CRYSTALS Appear as colorless hexagonal plates + Found in urine of persons who inherit a metabolic disorder that prevents reabsorption of cysteine by the renal tubules (cystinuria) © & + Weakly birefringent in polarized light © + Positive confirmation of cysteine crystals is made using cyanide-nitroprusside test 2. CHOLESTEROL CRYSTALS + Resemble a rectangular plate with a notch in or more comers as + Associated with disorders producing lipiduria, such as nephrotic syndrome, in A. ee conjunction with fatty casts and oval fat bodies ’ 3. RADIOGRAPHIC DYE CRYSTAL + Have a similar appearance to cholesterol crystals and are also highly birefringent + The specific gravity of a specimen containing radiographic contrast media is markedly elevated when measured by refractometer D. CRYSTALS ASSOCIATED WITH LIVER DISORDERS 4. TYROSINE CRYSTALS URINALYSIS |+ Appear as fine colorless to yellow needles that frequently form clumps or rosettes 2. LEUCINE CRYSTALS + Yellow brown spheres that demonstrate concentric circles and radial striations 3, BILIRUBIN CRYSTALS + Appear as clumped needles or granules with a characteris yellow color 4, SULFONAMIDE CRYSTALS * Inadequate patient hydration was and still is the primary cause of sutfonamide crystallization + Shapes most frequently encountered include needles, rhombics, whetstones, sheaves of wheat and rosettes with colors ranging from colorless to yellow-brown, 5. AMPICILLIN CRYSTALS + Observed following massive doses of this penicillin compound without adequate hydration + Appear as colorless needles that tend to from bundles when refrigerated NOTE: Trichomonas vaginalis- most frequently encountered parasite in the urine Enterobius vermicularis- most common fecal contaminant in the urine Acid-albumin and cetyltrimethylammonium bromide (CTAB)- a thick white turbidity forms in a urine that contains mucopolysaccharides. E, URINARY SEDIMENT ARTIFACTS 4, STARCH GRANULE + Highly refractile spheres, usually with a dimpled center + They resemble fat droplets when polarized, producing maltese cross formation 2, OIL DROPLETS AND BUBBLES + Highly refractile and may resemble RBCs 3, POLLEN GRAINS + Appear as spheres with a cell wall and occasional concentric circles 4. HAIR AND FIBERS + _ May be mistaken for casts, though they are usually much longer and more refractile 5. FECAL CONTAMINATION * May appear as plant or meat fibers or as brown amorphous material in a variety of sizes and shapes URINALYSIS |