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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

1

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:

1

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

1

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