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CLINICAL MICROSCOPY
1 Urine 53%
1.1 Anatomy and physiology of the kidney, formation of urine 5%
1.2 Macroscopic examination 10%
1.3 Chemical analyses 18%
1.4 Microscopic examination 15%
1.5 Pregnancy testing 2%
1.6 Renal calculi 3%
2 Feces 3%
3 Other body fluids 21%
3.1 CSF 5%
3.2 Seminal fluid 5%
3.3 Amniotic fluid 3%
3.4 Gastric fluid and duodenal content 2%
3.5 Sputum and bronchial washings 2%
3.6 Synovial fluid 2%
3.7 Peritoneal, pleural and pericardial fluids 2%
4 Collection, preservation and handling of specimens 10%
5 Microscope, automation and other instruments 5%
6 Quality assurance and laboratory safety 8%
TOTAL 100%
CLINICAL MICROSCOPY NOTES URINALYSIS
PHYSICAL EXAMINATION OF URINE
I. VOLUMENormal range (24o): 600 to 2000 mLAverage volume: 1200 to 1500 mLNight:day ratio________
1.PolyuriaDiuresis (Inc urine volume)
Increased fluid intakeDiuretic medicationDiuretic drinks (coffee, tea, alcohol)NervousnessDiabetes mellitusDiabetes insipidus
2.Oliguria Calculus or tumor of the kidneyDehydration
3.Anuria Complete obstruction (stones, carcinomas)Toxic agents
4.Nocturia > 500 mL with sp. gr. less than 1.018
II. COLORRoughly indicates the degree of hydration, and should correlate with urine sp. gr.
Pigments:
1. Urochrome
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2. Uroerythrin
3. Urobilin
Normal: Colorless to deep yellow
ColorlessPale yellow
Recent fluid consumptionPolyuriaDiabetes mellitusDiabetes insipidus
Dark yellowAmberOrange
Conc specimenBilirubinAcriflavinePyridiumNitrofurantoinPhenindione
Yellow – greenYellow - brown
Bilirubin oxidized to biliverdin
GreenBlue-green
Pseudomonas infectionCloretsIndicanMethylene bluePhenol
PinkRed
RBCsHemoglobinMyoglobin (25 mg/dL)PorphyrinBeets RifampinMenstrual contamination
BrownBlack
RBCs oxidized to methgbHomogentisic acidMelanin or melanogenMethyldopa or levodopaMetronidazole (Flagyl)
URINE COLOR CHANGES WITH COMMONLY USED DRUGSDrug Color
Alcohol, ethyl Pale, diuresis
Anthraquinone laxatives (senna, cascara) Reddish, alkaline; yellow-brown, acid
Chlorzoxazone (Paraflex) (muscle relaxant) Red
Deferoxamine mesylate (Desferal) (chelates iron) Red
Ethoxazene (Serenium) (urinary analgesic) Orange, red
Fluorescein sodium (given IV) Yellow
Furazolidone (Furoxone) (Tricofuron) (an antibacterial, antipro-tozoal nitrofuran)
Brown
Indigo carmine dye (renal function, cytoscopy) Blue
Iron sorbitol (Jectofer) (possibly other iron compounds forming iron sulfide in urine)
Brown on standing
Levodopoa (L-dopa) (for parkinsonism) Red then brown, alkaline
Mepacrine (Atabrine) (antimalarial) (intestinal worms, Giardia) Yellow
Methacarbamol (Robaxin) (muscle relaxant) Green-brown
Methyldopa (Aldomet) (antihypertensive) Darken; if oxidizing agents present, red to brown
Methylene blue (used to delineate fistulas) Blue, blue-green
Metronidazole (Flagyl) ( for Trichomonas infection, amebiasis, Giardia)
Darkening, reddish brown
Nitrofurantoin (Furadantin) (antibacterial) Brown-yellow
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Drug Color
Phenazopyridine (Pyridium) (urinary analgesic), also com-pounded with sulfonamides (Azo Gantrisin, etc.)
Orange-red, acid pH
Phenindione (Hedulin) (anticoagulant) (important to distinguish from hematuria)
Orange, alkaline; color disappears on acidifying
Phenol poisoning Brown; oxidized to quinines (green)
Phenolphthalein (purgative) Red-purple, alkaline pH
Phenolsulfonphthalein (also sulfobromophthalein) Pink-red, alkaline pH
Rifampin (Rifadin, Rimactane) (tuberculosis therapy) Bright orange-red
Riboflavin (multivitamins) Bright yellow
Sulfasalazine (Azulfidine) (for ulcerative colitis) Orange-yellow, alkaline pH
IV. CLARITY/TRANSPARENCY/TURBIDITY
TERMINOLOGYClear – transparent, no visible particulates
Hazy – few particulates, print easily seen through urine
Cloudy – many particulates, print blurred through urine
Turbid – print cannot be seen through urine
Milky – may ppt or clot
PATHOLOGIC CAUSES OF TURBIDITY1.RBCs2.WBCs3.Bacteria4.Yeast5.Nonsquamous epit cells6.Abnormal crystals7.Lymph fluid8.Lipids
NONPATHOLOGIC CAUSES OF TURBIDITY1.Squamous epit cells2.Mucus3.Amorphous crystals4.Semen, spermatozoa5.Fecal contamination6.Radiographic contrast media7.Talcum powder8.Vaginal cream
LABORATORY CORRELATIONS IN URINE TURBIDITYAcidic urine Amorphous urates, radiographic contrast media
Alkaline urine Amorphous phosphates, carbonates
Soluble with heat Amorphous urates, uric acid crystals
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
APPEARANCE AND COLOR OF URINEAppearance Cause Remarks
Colorless Very dilute urine Polyuria, D. insipidus
Cloudy Phosphates, carbonates Sol in dilute acetic acidUrates, uric acid Dissoves at 60C and in alkaliLeukocytes Insol in dilute acetic acidRed cells (“smoky”) Lyse in dilute acetic acidBacteria, yeasts Insol in dilute acetic acidSpermatozoa Insol in dilute acetic acidProstatic fluid
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Appearance Cause Remarks
Mucin, mucous threads May be flocculentCalculi, “gravel” Phosphates, oxalatesClumps, pus, tissueFecal contamination Rectovesical fistula
Milky Radiographic dye In acid urineMany neutrophil (pyuria) Insol in dilute acetic acidFatLipiduria, opalescentChyluria, milky
Nephrosis, crush injury, sol in etherLymphatic obstruction, sol in ether
Emulsified paraffin Vaginal creams
Yellow Acriflavine Green fluorescence
Yellow-orange Conc urine Dehydration, feverUrobilin in excess No yellow foamBilirubin Yellow foam, if sufficient bilirubin
Yellow-green Bilirubin-biliverdin Yellow foam
Yellow-brown Bilirubin-biliverdin “Beer” brown, yellow foam
Red Hemoglobin Pos. rgt strip for bldErythrocytes Pos. rgt strip for bldMyoglobin Pos. rgt strip for bldPorphyrin May be colorlessFuscin, aniline dye Foods, candyBeets Yellow alkaline, geneticMenstrual contam Clots, mucus
Red-purple Porphyrins May be colorless
Red-brown ErythrocytesHgb on standingMethemoglobin Acid pHMyoglobin Muscle injuryBilifuscin (dipyrrole) Result of unstable hemoglobin
Brown-black Methemoglobin Blood, acid pHHomogentisic acid On standing, alkaline; alkaptonuriaMelanin On standing, rare
Blue-green Indicans Small intestine infectionsPseudomonas infectionsChlorophyll Mouth deodorants
V. SPECIFIC GRAVITYDensity of solution compared with density of similar volume of dist water at a similar temperature
Influenced by number and size of particles in solution
DETERMINATION1.Refractormetry (TS meter)
Indirect mtd based on RI
Compensated to temp (15-38oC)
Requires corrections for glucose and proteino 1 g/dL Glucose ________o 1 g/dL Protein ________
Calibrationo Distilled water ________o 5% NaCl ________
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o 9% Sucrose ________
2.Urinometry Requires temp correction
0.001 must be subtracted from the reading every 3oC that the sp temp is below the urinometer calibration temp0.001 must be added from the reading every 3oC that the sp temp is above the urinometer calibration temp
Require corrections for glucose and protein
3.Reagent strip Prin. pKa change of a polyelectrolyte
Rgt sensitive to number of ions in the urine specimen; indicator changes color in relation to ionic concentration
Manufacturers recommend adding 0.005 to sp gr reading when pH is 6.5 or higher due to interference with the bromthymol blue indicator
Urine Specific Gravity Reagent Strip SummaryReagents Mutistix: Poly (methyl vinyl ether/maleic anhydride) bromthymol blue
Chemstrip: Ethyleneglycol-Bis (aminoethylether) bromthymol blueSensitivity 1.000-1.030
Interference False-positive: High concentration of proteinFalse-negative: Highly alkaline urines (>6.5)
4.Harmonic oscillation densitometry Frequency of sound wave entering a solution will change in proportion to the density of the solution
Summary of Urine Specific Gravity MeasurementsMethod Principle
Urinometry Density
Refractometry Refractive index
Harmonic oscillation densitometry Density
Reagent strip pKa change of a polyelectrolyte
VI. pHNormal: pH 4.5 to 8.0 (random)
ACID URINE EmphysemaDiabetes mellitusStarvationDehydrationDiarrheaPresence of acid-producing bacteria (E.coli)High protein dietCranberry juiceMedications (methenamine mandelate [Mandelamine], fosfomycin tromethamine)
ALKALINE URINEHyperventilationVomitingRenal tubular acidosisPresence of urease-producing bacteriaVegetarian dietOld specimens
REAGENT STRIP Prin: Double indicator system
Methyl redBromthymol blue
pH Reagent Strip SummaryReagents Methyl red, bromthymol blue
Sensitivity pH 5 - 9
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Sources of error/interference No known interfering subsRunover from adjacent padsOld specimens
Correlations with other tests NitriteLeukocytesMicroscopic
VII. ODORNormal: aromatic or odorless
1.Ammoniacal ____________________________
2.Fruity,sweet ____________________________
3.Rotting fish ____________________________
4.Rancid butter ____________________________
5.Sweaty feet ____________________________
6.Mousy odor ____________________________
7.Cabbage odor ____________________________
8.Maple syrup odor ___________________________(Caramelized sugar, curry)
9.Bleach ____________________________
CHEMICAL EXAMINATION OF URINEI. PROTEIN
Normal: <10 mg/dL or 100 mg/24o (Henry 150 mg/24o)Albumin – major serum protein found in urine
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PRE-RENAL PROTEINURIAIntravascular hemolysisMuscle injurySevere infection and inflammationMultiple myeloma
RENAL PROTEINURIA: GLOMERULAR DISORDERSImmune complex disordersAmyloidosisToxic agentsDiabetic nephropathy
MICRAL TEST Principle: Enzyme immunoassaySensitivity: 0 – 10 mg/dLReagents: Gold-labeled ab, B-galactosidase, chlorophenol red galactosideInterference: False negative: dilute urine
Strenuous exerciseDehydrationHypertensionPre-eclampsiaOrthostatic or postural proteinuria
RENAL PROTEINURIA: TUBULAR DISORDERSFanconi’s syndromeToxic agents/heavy metalsSevere viral infections
POST-RENAL PROTEINURIALower UTI/inflammationsInjury/traumaMenstrual contaminationProstatic fluid/spermatozoaVaginal secretions
REAGENT STRIPPrin: Protein error of indicators
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Tetrabromphenol blue (indicator)Tetrachlorophenol tetrabromosulfonphthalein (indicator)
Protein Reagent Strip SummaryReagents Multistix: Tetrabromphenol blue
Chemstrip: 3’, 3” 5’, 5” tetrachlorophenol, 3, 4, 5, 6-tetrabromosulfonphthaleinSensitivity Multistix: 15-30 mg/dL albumin
Chemstrip: 6 mg/dL albuminSources of error/interference False-positive:
Highly buffered alkaline urinePigmented specimens, phenozopyridineQuaternary ammonium compounds (detergents)Antiseptics, chlorhexidineLoss of buffer from prolonged exposure of the reagent strip to the specimenHigh specific gravity
False-negative: proteins other than albuminCorrelations with other tests Blood
NitriteLeukocytesMicroscopic
SULFOSALICYLIC ACID PRECIPITATION TESTCold precipitation test that reacts equally ith all forms of protein
Grade Turbidity Protein range (mg/dl)
Negative No increase in turbidity <6
Trace Noticeable turbidity 6-30
1+ Distinct turbidity with no granulation 30-100
2+ Turbidity with granulation, no flocculation 100-200
3+ Turbidity with granulation and flocculation 200-400
4+ Clumps of protein >400
II. GLUCOSERenal threshold: 160 to 180 mg/dLOther sugars in urine
FructoseGalactoseLactosePentose
CLINICAL SIGNIFICANCE OF URINE GLUCOSE
HYPERGLYCEMIA ASSOCIATEDDiabetes mellitus PancreatitisPancreatic cancer AcromegalyCushing’s syndrome HyperthyroidismPheochromocytoma StressCentral nervous system damage Gestational diabetes
RENAL ASSOCIATEDFanconi’s syndrome PregnancyAdvanced renal disease
REAGENT STRIPPrin: Double sequential enzyme reaction
Glucose oxidase and peroxidaseChromogen
O-toluidine (pink to purple)Potassium iodide (blue to brown)Aminopropryl-Carbazol (yellow to orange-brown)
Glucose Reagent Strip SummaryReagents Multistix: Glucose oxidase, peroxidase, potassium iodide
Chemstrip: Glucose oxidase, peroxidase, tetramethylbenzidineSensitivity Multistix: 75 – 125 mg/dL
Chemstrip: 40 mg/dL
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Interference False-positive: Contamination by oxidizing agents and detergentsFalse-negative:
High levels of ascorbic acidHigh levels of ketonesHigh specific gravityLow temperaturesImproperly preserved specimens
Correlations with other tests Ketones
COPPER REDUCTION TEST (Clinitest)Test relies on the ability of glucose and other substances to reduce copper sulfate to cuprous oxide in the presence of al-
kali and heatA color change progressing from a negative blue (CuSO4) through green, yellow and orange/red (Cu2O) occurs when the
reaction takes placeTablets contain copper sulfate, sodium carbonate, sodium citrate, and sodium hydroxide
Pass-through phenomenon may occur if >2 g/dL sugar present in urine
GLUCOSE OXIDASE AND CLINITEST REACTIONSGLUCOSE OXIDASE CLINITEST INTERPRETATION
Negative Positive Nonglucose reducing substance presentPossible interfering substance for reagent strip
1+ positive Negative Small amount of glucose present
4+ positive Negative Possible oxidizing agent interference on reagent strip
III. KETONESResults from INCREASED FAT METABOLISM due to inability to metabolize carbohydrate, as occurs in DM, increased loss of carbohydrate from vomiting, and inadequate intake of carbohydrate associated with starvation and malabsorption
78% BHA20% AAA/diacetic acid2% Acetone
SignificanceDiabetes acidosisInsulin dosage monitoringStarvationMalabsorption/pancreatic disordersStrenuous exerciseVomitingInborn error of amino acid metabolism
REAGENT STRIPPrin: Sodium nitroprusside reaction
Ketone Reagent Strip SummaryReagents Sodium nitroprusside
Glycine (Chemstrip)Sensitivity Multistix: 5 – 10 mg/dL acetoacetic acid
Chemstrip: 9 mg/dL acetoacetic acid, 70 mg/dL acetoneInterference False-positive:
Phthalein dyesHighly pigmented red urineLevodopaMedications containing free sulfhydryl groups
False-negative:Improperly preserved specimens
Correlations with other tests Glucose
ACETESTSodium nitroprusside, glycine, disodium phosphate and lactose
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IV. BLOODHematuria
Renal calculi, glomerulonephritis, pyelonephritis, tumors, trauma, exposure to toxic chemicals, anticoagualants, strenuous exercise
HemoglobinuriaTransfusion reactions, hemolytic anemias, severe burns, infections/malaria, strenuous exercise/red blood cell trauma
MyoglobinuriaMuscular trauma/crush syndromes, prolonged coma, convulsions, muscle-wasting diseases, alcoholism/over-dose, drug abuse, extensive exertion
HEMOGLOBINURIA VS MYOGLOBINURIA1. Plasma examination
HemoglobinMyoglobin
2. Blondheim’s test (Ammonium sulfate)HemoglobinMyoglobin
REAGENT STRIPPrin: Pseudoperoxidase activity of hemoglobin
Tetramethylbenzidine (chromogen)
Blood Reagent Strip SummaryReagents Multistix: Diisopropylbenzene dehydroperoxide tetramethylbenzidine
Chemstrip: 2,5-dimethyl-2,5-dihydroperoxide tetramethylbenzidineSensitivity Multistix: 5-20 RBCs/μL, 0.015-0.062 mg/dL hemoglobin
Chemstrip: 5 RBCs/μL, hemoglobin corresponding to 10 RBCs/μLInterference False-positive:
Strong oxidizing agentsBacterial peroxidasesMenstrual contamination
False-negative:High specific gravity/crenated cellsFormalinCaptoprilHigh conc. of nitriteAscorbic acid >25 mg/dLUnmixed specimens
Correlations with other tests ProteinMicroscopic
V. BILIRUBINEarly indication of liver diseaseSignificance:
HepatitisCirrhosisBiliary obstruction (gallstones, carcinoma)
REAGENT STRIPPrin: Diazo reaction
2,4-dichloroaniline diazonium salt2,6-dichlorobenzene-diazonium-tetrafluoroborate
Bilirubin Reagent Strip SummaryReagents Multistix: 2,4-dichloroaniline diazonium salt
Chemstrip: 2,6-dichlorobenzene-diazonium-tetrafluoroborateSensitivity Multistix: 0.4-0.8 mg/dL bilirubin
Chemstrip: 0.5 mg/dL bilirubin
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Interference False-positive:Highly pigmented urines, phenazopyridineIndican (intestinal disorders)Metabolites of Lodine
False-negative:Specimen exposure to lightAscorbic acid >25 mg/dLHigh concentrations of nitrite
Correlations with other tests Urobilinogen
ICTOTESTPositive: Blue to purple color
p-nitrobenzene-diazonium-p-toluenesulfonateSSASodium bicarbonate
VI. UROBILINOGENBile pigment that result from hgb degradationSmall amt in normal urine
<1 mg/dL or Ehrlich unit
REAGENT STRIPEhrlich’s reaction
Urobilinogen Reagent Strip SummaryReagents Multistix: PDAB
Chemstrip: 4-methoxybenzene-diazonium tetrafluoroborateSensitivity Multistix: 0.2 mg/dL urobilinogen
Chemstrip: 0.4 mg/dL urobilinogen
Interference MultistixFalse-positive:
PorphobilinogenIndicanp-aminoslicylic acidSulfonamidesMethyldopaProcaineChlorpromazineHighly pigmented urine
False-negativeOld specimensPreservation in formalin
ChemstripFalse-positive:
Highly pigmented urineFalse-negative:
Old specimensPreservation in formalinHigh concentrations of nitrate
Correlations with other tests Bilirubin
WATSON SCHWARTZ TEST For differentiating urobilinogen and porphobilinogen
Urobilinogen Porphobilinogen Other Ehrlich-Reactive Substances
Chloroform ExtractionUrine (top layer)Chloroform (bottom layer)
ColorlessRed
RedColorless
RedColorless
Butanol ExtractionButanol (top layer)Urine (bottom layer)
RedColorless
ColorlessRed
Red Colorless
Urine Bilirubin and Urobilinogen in Jaundice
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Urine Bilirubin Urine Urobilinogen
Hemolytic disease Negative +++
Liver damage + or - ++
Bile duct obstruction +++ Normal
HOESCH TESTRapid screening test for urine porphobilinogen (≥ 2mg/dL)
Hoesch reagent (Ehrlich rgt dissolved in 6M HCl)
VII. NITRITEDetection of bacteriuria
REAGENT STRIPPrin: Greiss reactionPositive nitrite corresponds to 100,000 organisms/mL
Nitrite Reagent Strip SummaryReagents Multistix: p-arsanilic acid
tetrahydronezo(h)quinolin-3-olChemstrip: Sulfanilamide 3-hydroxy-1,2,3,4-tetrahydro-7,8 benzoquinoline
Sensitivity Multistix: 0.06-0.1 mg/dL nitrite ionChemstrip: 0.05 mg/dL nitrite ion
Interference False-positive:Improperly preserved specimensHighly pigmented urine
False-negative:Nonreductase-containing bacteriaInsufficient contact time between bacteria and urinary nitrateLack of urinary nitrateLarge quantities of bacteria converting nitrite to nitrogenHigh concentrations of ascorbic acidHigh specific gravity
Correlations with other tests Protein LeukocytesMicroscopic
VIII. LEUKOCYTESignificance:
UTI/InflammationScreening of urine culture specimens
REAGENT STRIPPrin: Leukocyte esterase
Leukocyte Esterase Reagent Strip SummaryReagents Multistix: Derivatized pyerole amino acid ester, diazonium salt
Chemstrip: Indoxylcarbonic acid ester, diazonium saltSensitivity Multistix: 5-15 WBC/hpf
Chemstrip: 10-25 WBC/hpfInterference False-positive:
Strong oxidizing agentsHighly pigmented urine, nitrofurantoin
False-negative:High concentrations of protein, glucose, oxalic acid, ascorbic acid. gentamicin, cephalosporins, tetracyclines
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Correlations with other tests
ProteinNitriteMicroscopic
MICROSCOPIC EXAMINATION OF URINE
MICROSCOPIC TECHNIQUESTechnique Function
Bright-field microscopy Used for routine urinalysis
Phase-contrast mi-croscopy
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 microscopy Aids in identification of Treponema pallidum
Fluorescence mi-croscopy
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
SEDIMENT STAIN Stain Action Function
Sternheimer-Malbin Crystal violet and safraninDelineates structure and contrasting colors of the nucleus and cytoplasm
Identifies WBCs, epithelial cells, and casts
Toluidine blue Enhances nuclear detail Differentiates WBCs and renal tubular ep-ithelial cells
Lipid stains: Oil Red O and Sudan III Stains triglycerides and neutral fats orange-red
Identifies free fat droplets and lipid-contain-ing cells and casts
Gram stain Differentiates gram-positive and gram-nega-tive bacteria
Identifies bacterial casts
Hansel stain Methylene blue and eosin Y stain eosinophilic granules
Identifies urinary eosinophils
Prussian blue stain Stains structures containing iron Identifies yellow-brown granules of hemosiderin in cells and casts
SEDIMENT CONSTITUENTS
CELLS
1. RBCsNon-nucleated biconcave disksCrenated in hypertonic urineGhost cells in hypotonic urineDysmorphic with glomerular membrane damage
2. WBCsLarger than red blood cellsGranulated, multilobed neutrophilsGlitter cells in hypotonic urine
Eosinophils > 1% is considered significant
Mononuclear cells: lymphocytes, monocytes, macrophages and histiocytes
3. EPITHELIAL CELLSA. SQUAMOUS EPIT CELLS
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Largest cell in the sediment with abundant, irregular cytoplasm and prominent nuclei
B. TRANSITIONAL EPIT CELLS (UROTHELIAL)Spherical, polyhedral, or caudate with centrally located nucleus
C. RENAL TUBULAR EPIT CELLSRectangular, polyhedral, cuboidal, or columnar with an eccentric nucleus, possibly bilirubin stained or hemosiderin laden
Presence of > 2 RTE/hpf indicates _____________
Oval fat bodies ___________________________
Bubble cells______________________________
4. BACTERIA5. YEAST6. PARASITE7. SPERMATOZOA8. MUCUS
CASTSFormed in the distal convoluted tubule and collecting ductMajor constituent: Tamm Horsfall protein
1. HYALINE CASTSGlomerulonephritisPyelonephritisChronic renal diseaseCongestive heart failureStress and exercise
2. RBC CASTSGlomerulonephritisStrenuous exercise
3. WBC CASTSPyelonephritisAcute interstitial nephritis
4. BACTERIAL CASTSPyelonephritis
5. EPITHELIAL CELL CASTSRenal tubular damage
6. COARSE/FINE GRANULAR CASTSGlomerulonephritisPyelonephritisStress and exercise
7. FATTY CASTSNephrotic syndromeToxic tubular necrosisDiabetes mellitusCrush injuries
8. WAXY CASTSStasis of urine flowChronic renal failure
9. BROAD CASTSExtreme urine stasisRenal failure
CRYSTALS
NORMAL CRYSTALS
A. ACIDIC URINE
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1. Amorphous urateMic: yellow-brown granulesPink sediment (uroerythrin)
2. Uric acidRhombic, wedge, rosette, hexagonal, fousided plate (whetstone)↑Lesch-Nyhan, gout, leukemic pts receiving chemotherapy
3. Calcium oxalateEnveloped/pyramidal, oval↑Food high in oxalic/ascorbic acid (tomato, asparagus)Ethylene glycol poisoning
B. ALKALINE URINE
1. Amorphous phosphateGranular appearanceWhite ppt
2. Ammonium biurateYellow-brown, thorny applesOld specimen
3. Triple phosphateMagnesium ammonium phosphateCoffin lid
4. Calcium phosphateColorless, flat rectangular plates or thin prisms often in rosette formationDissolve in dilute acetic acid
5. Calcium carbonateSmall and colorless, with dumbbell or spherical shapesFormation of gas after addition of acetic acid
ABNORMAL CRYSTALS (Acid, neutral urine)1. Cystine
Colorless hexagonal platesCystinuria
2. CholesterolRectangular plate with notch in one or more corners, staircase patternLipiduria - nephrotic syndrome
3. TyrosineColorless to yellow needles
4. LeucineYellow-brown spheres with concentric circles and radial striations
5. BilirubinClumped needles or granules with yellow color
URINARY SEDIMENTS ARTIFACTS1. Starch granules2. Oil droplets3. Air bubbles4. Pollen grains5. Hair and fibers6. Fecal contamination
Qualitative Tests for Protein
Heller’s Conc. HNO3
Robert’s Sat. MgSO4.7H2O
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White ring at the zone of conatctSpiegler’s HgCl2NaClSuccinic acidDist. H2O
Biuret 10% NaOH/KOH Violet for albumin Rose for albuminoses and peptones
Heat and acetic acid 5-10% Acetic acid
SSA Exton’s qualitative rgt. Na2SO4
SSA Dist. H2O
White turbidity/cloudiness
Purdy’s Sat. NaCl
Potassium ferrocyanide 5-10% Potassium ferrocyanide
Picric acid Picric acid soln.
Quantitative Tests for Protein
Esbach’s Esbach’s rgt. 1 g picric acid 2 g citric acid
24o – read height of coagulum
Kwilecki’s Esbach’s rgt.10% FeCl3
72oC for 5 minutes – read height of coagu-lum
Tsuchiya’s PTA crystals95% alcoholConc. HCl
Same as Esbach’s
Kingsbury-Clark SSA Degree of turbidity is measured by compari-son with standard turbidities
Biuret Uses the same principle as that used for serum protein which depends upon the pres-ence of peptide linkages in protein
Sugars
Benedict’s Benedict’s rgt. Copper sulfate Sodium carbonate Sodium citrate buffer
Reducing substancesGreen-orange-red
Osazone or Phenylhydrazine (Kowarsky)
PhenylhydrazineSodium acetate
Glucose, fructose, lactose & pentoseCrystalline needles
Nylander’s Rochelle saltBismuth subnitrateNaOHKOH
Glucose & other reducing subsBrown to black color
Moore Heller 10% KOH Glucose & other reducing subsCanary yellow to black
Borchardt’sSeliwanoffResorcinol-HCl
25% HClResorcinol
FructoseRed color
Rubner’s Lead acetateAmmonia H2O
LactoseBrick red color w/red ppt
GlucoseRed color w/yellow ppt
Bial Orcinol HCl10% FeCl3
Green soln
Tauber’s Benzidine in glacial acetic acid Cherry red
Ketones
Frommer’s KOH10% salicyl aldehyde
AcetonePurplish red ring
Rothera’s Sodium nitroprussideAmmonium sulafate
Acetone & acetoacetic acidRose or purple ring
Lange Glacial acetic acidSodium nitroprussideAmmonia H2O
Acetone & acetoacetic acidPurple ring
AcetestKetostix
Aminoacetic acidSodium nitroprussideDisodium phosphateLactose
Acetone & acetoacetic acidPurple color
Gerhardt’s 10% FeCl3 Acetoacetic acidBordeaux red color
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BILE PIGMENTS( B i l i r u b i n , u r o b i l i n o g e n & u r o b i l i n )
Gmelin 10% BaCl2.HNO3 Bile pigmentsPlay of colors
Smith Alc. tincture of iodine Bile pigmentsEmerald green
Harrison’s spot 10% BaCl2Fouchet’s rgt. 10% FeCl3 TCA
Bile pigmentsBlue to green color
Ictotest Ictotest tablets Bile pigmentsBlue to purple mat
Wallace and Diamond Ehrlich rgt (PDAB) UrobilinogenCherry red color
Schlesinger Lugol’s iodineAlc. soln. of zinc acetate
UrobilinGreenish fluorescence
HEMOGLOBIN
Benzidine Benzidine powder in glacial acetic acid3% H2O2
Green-blue
Guiac 10% HAC95% alcoholGuiac powder
Blue
Orth-toluidine Ortho-toluidineH2O2
Blue
MELANIN
Screening test 10% FeCl3(Urine allowed to stand for 24o)
Urine will turn brown to black
Thormahlen(Fresh urine)
Sodium nitroprusside40% NaOH33% Acetic acid
Dark green or blue color
Blackberg & Wanger(24-hr urine)
Potassium persulfateMethyl alcoholEther5% NaOH
Brown to black ppt
CHLORIDE
Fantus AgNO3
K2CrO4
Reddish ppt.
Mercurimetric titrationSchales & Schales
Titrated with mercuric nitrateDiphenylcarbazone indicator
Blue-violet colored complex
CALCIUM
Sulkowitch Oxalic acidAmmonium oxalateGlacial acetic acidDistilled H2O
Precipitation
AMNIOTIC FLUID
Primary function of AF is to provide a protective cushion for the fetus and allow movement
The amount of amniotic fluid increases throughout pregnancy, reaching a peak of approximately 1 L during the third trimester, and then gradually decreases prior to delivery.
DURING THE FIRST TRIMESTER, the approximately 35 mL of amniotic fluid is derived primarily from the maternal circula-tion. During the latter third to half of pregnancy, the fetus secretes a volume of lung liquid necessary to expand the lungs with growth. During each episode of fetal breathing movement, secreted lung liquid enters the amniotic fluid, as evidenced by lung surfactants that serve as an index of fetal lung maturity.AFTER THE FIRST TRIMESTER, fetal urine is the major contributor to the amniotic fluid volume. At the time that fetal urine production occurs, fetal swallowing of the amniotic fluid begins and regu-lates the increase in fluid from the fetal urine.
Increased AF _________________________________
Decreased AF ________________________________
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Collection of amniotic fluid ____________________Maximum of 30 mL of AF is collected in sterile syringes
Second trimester amniocentesis _________________
Third trimester amniocentesis ___________________
Differentiation between AF and maternal urineAnalyte Amniotic Fluid Maternal Urine
Amniotic Fluid ColorColor Significance
Colorless Normal
Blood-streaked Traumatic tap, abdominal trauma, intra-amniotic hemorrhage
Yellow Hemolytic disease of the newborn (bilirubin)
Dark green Meconium
Dark red-brown Death
Tests for Hemolytic Disease of the Newborn
_____________________________________
_____________________________________
____________________________________
Tests for Neural Tube Defects
Screening test ________________________________
________________________________
Confirmatory test
_______________________________
_______________________________
Tests for Fetal Lung Maturity
Lecithin-sphingomyelin ratio________________________________________
________________________________________
________________________________________
Amniostat-FLM
________________________________________
________________________________________
________________________________________
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Foam test
________________________________________
________________________________________
________________________________________
Microviscosity
________________________________________
________________________________________
Lamellar body count
________________________________________
________________________________________
Optical Density 650 nm
________________________________________
________________________________________
Tests for Fetal Well-Being and MaturityTest Normal Values at Term Significance
Bilirubin scan ΔA450 > 0.025 Hemolytic disease of the newborn
Alpha-fetoprotein <2.0 MoM Neural tube disorders
Lecithin-sphingomyelin ratio ≥2.0 Fetal lung maturity
Amniostat-fetal lung maturity Positive Fetal lung maturity/ phosphotidyl glycerol
Foam stability index ≥47 Fetal lung maturity
Microviscosity ≥55 mg/g Fetal lung maturity
Optical density 650 nm ≥0.150 Fetal lung maturity
Lamellar body count ≥32,000/μL Fetal lung maturity
CEREBROSPINAL FLUID
Third major body fluid
Functionso Supply nutrients to nervous tissueo Remove metabolic wasteso Mechanical barrier to cushion the brain and spinal cord against trauma
Approximately 20 mL of fluid is produced every hour in the choroids plexuses and reabsorbed by the arachnoid villi
Total volume
o Adult: _________________________
o Neonates: _____________________
Collection ____ _______________________________
o First tube _______________________
o Second tube ____________________
o Third tube ______________________
Appearance
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Crystal clear _______________________________
Cloudy, turbid, milky
_________________________________________
_________________________________________
_________________________________________
_________
Xanthochromic
_________________________________________
_________________________________________
_________________________________________
________
Bloody_________________________________________________
_________________________________________________
__________________________________
Traumatic Tap Intracranial Hemorrhage
Dist. of blood
Clot formation
Supernatant
Erythrophages
Oily – radiographic contrast media
Clotted – protein, clotting factors
CELL COUNT
Performed immediatelyo WBCS and RBCs will begin to lyse within 1 houro 40% WBCs disintegrating within 2 hours
Normalo Adult ___________________________o Neonate ________________________
Calculation
Cells/uL = Number of cells counted x dilution Number of sq. counted x vol. of 1 sq.
1
Dilution
Slightly hazy
Hazy
Slightly cloudy
Slightly bloodyCloudyBloodyTurbid
RED BLOOD CELL COUNT_________________________________________________
_________________________________________________
__________________________________
WHITE BLOOD CELL COUNT
Diluent __________________________________
DIFFERENTIAL COUNT ON CSF
Performed on a stained smear
Specimen be concentrated prior to the preparation of smearo _______________________________
o _______________________________
o _______________________________
o _______________________________
Normal cells in CSF
o _______________________________
o _______________________________
Adult ____________________
Neonate _________________
Increased in number of normal cells in CSF
_______________________________
Lymphocytes NormalViral, tubercular, fungal meningitisMultiple sclerosis
Monocytes NormalViral, tubercular, fungal meningitisMultiple sclerosis
Neutrophils Bacterial meningitisEarly cases of viral, tubercular and fungal meningitisCerebral hemorrhage
Macrophages RBCs in spinal fluidContrast media
Blasts Acute leukemia
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Plasma cells Multiple sclerosisLymphocyte reactions
Ependymal, choroidal, and spindle-shaped cells
Diagnostic procedures
Malignant cells Metastatic carcinomasPrimary CNS carcinoma
CHEMISTRY TESTS
I. PROTEIN
NV (TP) ______________________________________
Elevated results: meningitis: hemorrhage, primary CNS tumors, multiple sclerosis, Guillain-Barrè syndrome, neu-rosyphilis, polyneuritis, myxedema, Cushing disease, connective tissue disease, diabetes, uremia
Decreased results: CSF leakage/trauma, recent puncture, rapid CSF production, water intoxication
Major CSF protein __________________
2nd prevalent __________________
Alpha globulin __________________
Beta globulin __________________
Gamma globulin __________________
NOT FOUND IN NORMAL CSF (3)
METHODS (TP)
1. Turbidimetric________________________________
________________________________
________________________________
2. Dye-binding________________________________
________________________________
ELECTROPHORESIS Detection of oligoclonal bands
Presence of 2 or more oligoclonal bands in CSF not present in serum, valuable for diagnosis of_____________________________________
Other: encephalitis, neurosyphilis, Guillan-Barre syndrome, and neoplastic disorders
MBP Monitor the course of MS
GLUCOSE
NV: ________________________________________
Decreased in: ______________________________
______________________________
______________________________
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Normal in ____________________________
LACTATENV: _______________________________________
Increased in:_____________________________
_____________________________
_____________________________
GLUTAMINENV: _______________________________________
Increased in:__________________________________________________________
MICROBIOLOGY TESTS
MENINGITIS
Bacterial Viral Tubercular Fungal
WBC
Protein
Glucose
Lactate
SEROLOGIC TESTING
Latex agglutination and ELISA for detection of bacterial antigensVDRL – neurosyphilis (recommended by CDC)
SEMINAL FLUID
Reasons for AnalysisFertility testingPostvasectomy semen analysisForensic analyses
Physiology
Semen is composed of four fractions that are contributed
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by:
1. ________________________ ___________
2. ________________________ ___________
3. ________________________ ___________
4. ________________________ ___________
Collection
Abstinence for _________________________________
Analysis should be done after liquefaction
Specimen awaiting analysis should be kept at 37oC
Semen Analysis
AppearanceGray-white, translucent
Inc white turbidity
Red coloration
Yellow coloration
Volume
NV:
Increased volume
Decreased volume
Viscosity
Normal: Pour in droplets
Increased viscosity
pH
NV:
Increased pH
Decreased pH
Sperm Concentration
NV:
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1. Improved Neubauer Counting Chamber
2. Makler Counting Chamber
Sperm Count
NV:
Calculation:
Sperm Motility
Evaluated in approximately __________
NV:
Sperm Motility GradingGrade WHO Criteria
4.0 a
3.0 b
2.0 b
1.0 c
0 d
Source: Urinalysis and Body Fluids, 5th edition by Strasinger and Di Lorenzo, p203
Sperm Morphology
Routine criteria:
Kruger’s strict criteria:
Normal Values for Semen AnalysisVolume 2 – 5 mL
Viscosity Pour in droplets
pH 7.2 – 8.0
Sperm concentration >20 million/mL
Sperm count >40 million/ejaculate
Motility >50% within 1 hr
Quality >2.0 or a,b,c
Morphology >30% normal forms (routine criteria)>14% normal forms (strict criteria)
Round cells <1.0 million/mL
Source: Urinalysis and Body Fluids, 5th edition by Strasinger and Di Lorenzo, p201
Sperm Viability
______________________________________
______________________________________
______________________________________
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Seminal Fluid Fructose
___________________________________________
_________________________________
Antisperm Antibodies
Detected in semen, cervical mucosa or serum
1. Mixed Agglutination Reaction (MAR)
Detects presence of IgG antibodies
Semen sample + AHG + latex particles or
treated RBCs coated with IgG
Normal: <10% motile sperm attached to the particles
2. Immunobead TestDetect the presence of IgG, IgM and IgA antibodies and will demonstrate what area of the sperm (head, neck, tail) the autoantibodies are affectingNormal: presence of beads on less than 20% of the sperm
Chemical Testing
Fructose ≥ 13 µmol/ejaculateNeutral α-glucosidase ≥ 20 mU/ejaculateZinc ≥ 2.4 µmol/ejaculateCitric acid ≥ 52 µmol/ejaculateACP ≥ 200 Units/ejaculate
Microbial Testing
>1 million WBCs/mL _______________________
Routine aerobic and anaerobic cultures and tests for C. trachomatis, M. hominis and U. urealyticum
Postvasectomy Semen Analysis
____________________________________________
____________________________________________
_____________________________
SYNOVIAL FLUID
Viscous fluid in cavities of movable joints o Lubricates jointso Reduce friction between boneso Provides nutrient to the articular cartilageo Lessen shock of joint compression occurring during activities such as walking or jogging
Collection
Method of collection
Volume
Distributed into the following test tubes
Heparinized tube
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Heparin or EDTA tube
Nonanticoagulated tube
Sodium fluoride tube
Appearance
Clear and pale yellow
Deeper yellow
Greenish tinge
Red
Milky
Viscosity
String test
Ropes or mucin clot test
Reagent:
Good
Fair
Low
Poor
Cell Counts
Diluting fluid:
RBCs <2000 cells/uL
WBCs <200 cells/uL
Differential Count
Monocytes and macrophages 65%
Neutrophils <20%
Lymphocytes <15%
Cells and Inclusions in Synovial Fluid
Cell/Inclusion Description Significance
Neutrophil PMN Bacterial sepsisCrystal-induced inflammation
Lymphocyte Mononuclear leukocyte Nonseptic inflammation
Macrophage Large mononuclear leukocyte, may be vacuolated
NormalViral infections
Synovial lining cell Similar to macrophage, may be multinucle-ated, res. mesothelial cell
Normal
LE cell Neutrophil containing ingested round body LE
Reiter cell Vacuolated macrophage with ingested neutrophils
Reiter’s syndromeNonspecific inflammation
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Cell/Inclusion Description Significance
RA cell Neutrophil with dark cytoplasmic granules containing immune complexes
RAImmunologic inflammation
Cartilage cells Large multinucleated cells Osteoarthritis
Rice bodies Macroscopic: res. polished riceMicroscopic: show collagen and fibrin
TB, septic and RA
Fat droplets Refractile intracellular and extracellular globulesStain with Sudan dyes
Traumatic injury
Hemosiderin Inclusions within clusters of synovial cells Pigmented villonodular synovitis
Crystal IdentificationCauses of crystal formationo Metabolic disorderso Decreased renal excretion that produce elevated blood levels of crystallizing chemicalso Degeneration of cartilage and boneso Injection of medications (corticosteroid)
A. Monosodium urateB. Calcium pyrophosphateC. Hydroxyapatite (calcium phosphate)D. CholesterolE. Corticosteroids
Fluid is examined unstained under polarized and COMPENSATED POLARIZED LIGHT for detection of MSU and CPPD crys-tals
Chemistry Tests
Glucose
Lactate
Protein
UA
Microbiology testsCommon organisms that infect synovial fluid Staphylococcus Streptococcus Haemophilus Neissreria gonorrhoeaeRoutine bacterial cultures should always include enrichment medium such as CAP
Serologic TestsAutoantibody detection: SLE and RAAntibody detection: Lyme disease
Joint DisordersGroup Classification Pathologic Significance Laboratory Findings
I. Noninflammatory Degenerative joint disorders Clear, yellow fluidGood viscosityWBCs <1000 μLNeutrophils <30%Normal glucose (similar to blood glucose)
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Group Classification Pathologic Significance Laboratory Findings
II. Inflammatory Immunologic disorders, rheumatoid arthritis, lupus erythematosus, sclerederma, polymyositis, ankylosing spondylitis, rheumatic fever, and Lyme arthritis
Crystal-induced gout and pseudogout
Immunologic origin:Cloudy, yellow fluidPoor viscosityWBCs 2000-75000 μLNeutrophils >50%Decreased glucose levelPossible autoantibodies present
Crystal-induced origin:Cloudy or milky fluidLow viscosityWBCs up to 100, 000 μLNeutrophils <70%Decreased glucose levelCrystals present
III. Septic Microbial infection Cloudy, yellow-green fluidVariable viscosityWBCs 50,000-100,000 μLNeutrophils >75%Decreased glucose levelPositive culture and Gram stain
IV. Hemorrhagic Traumatic injuryCoagulation deficiencies
Cloudy, red fluidLow viscosityWBCs equal to bloodNeutrophils equal to bloodNormal glucose levelRBCs present
Source: Urinalysis and Body Fluids, 5th edition by Strasinger and Di Lorenzo, p212
TRANSUDATE AND EXUDATES
Differentiation of Transudates and Exudates
Transudate Exudate
Appearance Clear Cloudy
Fluid:serum protein ratio <0.5 >0.5
Fluid:serum LD ratio <0.6 >0.6
White blood cell count <1000/μL >1000/μL
Spontaneous clotting No Possible
Pleural fluid cholesterol <45-60 mg/dL >45-60 mg/dL
Pleural fluid:serum cholesterol ratio <0.3 >0.3
Pleural fluid:bilirubin ratio <0.6 >0.6
Serum-ascites albumin gradient >1.1 <1.1
EXAMINATION OF FECES
Detection of pathogenic bacteria and parasites
Early detection of gastrointestinal bleeding, liver and biliary duct disorders, maldigestion syndromes and inflammation
Normal: 100 to 200 g of stool passed per day
Steatorrhea Mushy, foul smelling gray stool that floats on water
Constipation Small, firm, spherical masses of stool (scybala)
Spastic bowelRectal narrowing or stricture
Narrow, ribbon-like stool
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Blood from lower gutBeets
Red
Bleeding from upper GITBismuth, iron, charcoal
Black
Spinach and other green vegetables or calomel, or presence of biliverdin
Green
Presence of mucus in stool is abnormal and should be reportedSpastic constipation or mucous colitis Translucent gelatinous mucus clinging to the surface of formed
stoolNeoplasm or inflammatory process of the rectal canal Bloody mucus clinging to fecal mass
Ulcerative colitis, bacillary dysentery, ulcerating diverticulitis and intestinal tuberculosis
Mucus associated with pus and blood
Villous adenoma of the colon Copious quantity of mucus (3 or 4 L in 24 hours)
Patients with chronic ulcerative colitis and chronic bacillary dysentery frequently pass large quantities of pus with the stool
MACROSCOPIC STOOL CHARACTERISTICSBlack Upper gastrointestinal bleeding, iron therapy, charcoal, bismuth (antacids)
Red Low gastrointestinal bleeding, beets and food coloring, rifampin
Pale yellow, white, gray Bile-duct obstruction, barium sulfate
Green Biliverdin/oral antibiotics, green vegetables
Bulky/frothy Bile-duct obstruction, pancreatic disorders
Ribbon-like Intestinal constriction
Mucus/blood-streaked mucus Colitis, dysentery, malignancy, constipation
MICROSCOPIC EXAMINATION OF FECES
FatSudan III, Sudan IV or Oil Red O stainStool suspension + 95% ethanol + Sudan IIINeutral fats appear as large orange or red droplets
≥ 60/hpf (steatorrhea)Meat fiber Stool + 10% alcohol solution of eosin
Leukocytes Stool + Loeffler methylene blue
TESTS FOR FECAL OCCULT BLOOD
Determination of peroxidase and pseudoperoxidase activity of red blood cells including hemoglobin
Indicators include guaiac, orthotoluidine, orthodinisidne and benzidine
TESTS FOR STEATORRHEA
Screening tests Microscopic examination of feces for fat globules
Determination of serum carotenoid
Definitive test Fecal fat determination
Titrimetric method (Van de Kamer) Definitive diagnosis of steatorrhea
TESTS FOR REDUCING SUBSTANCES IN FECES
Stool suspension + Clinitest tablet
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Normal: ≤0.25 g/dL
Suspicious: 0.25g to 0.5 g/dL
Abnormal: >0.5 g/dL
FECAL SREENING TESTS
TEST METHODOLOGY/PRINCIPLE/INTERPRETATIONExam.for neutrophils Microscopic count of neutrophils in smear stained with methylene blue, Gram stain or
Wright’s stain Three per hpf indicates condition affecting intestinal wall
Quali. fecal fats Microscopic examination of direct smear with Sudan III – 60 large orange-red droplets indi-cates malabsorption
Microscopic examination of smear heated with acetic acid and Sudan III – 100 orange-red droplets measuring 6-75 µm indicates malabsorption
Occult blood Pseudoperoxidase activity of hemoglobin liberates oxygen from hydrogen peroxide to oxidize guaiac reagent
Blue color indicates gastrointestinal bleedingAPT test Addition of sodium hydroxide to hemoglobin-containing emulsion determines presence of mater-
nal or fetal blood Pink color indicates presence of fetal blood
Trypsin Emulsified specimen placed on x-ray paper determines ability to digest gelatin
Inability to digest gelatin indicates lack of trypsinClinitest Addition of Clinitest tablet to emulsified stool detects presence of reducing substances
Reaction of 0.5 g/dL reducing substances suggests carbohydrate intolerance