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CLINICAL MICROSCOPY 1 Urine 1.1 Anatomy and physiology of the kidney, formation of urine 1.2 Macroscopic examination 1.3 Chemical analyses 1.4 Microscopic examination 1.5 Pregnancy testing 1.6 Renal calculi 2 Feces 3 Other body fluids 3.1 CSF 3.2 Seminal fluid 3.3 Amniotic fluid 3.4 Gastric fluid and duodenal content 3.5 Sputum and bronchial washings 3.6 Synovial fluid 3.7 Peritoneal, pleural and pericardial fluids 4 Collection, preservation and handling of specimens 5 Microscope, automation and other instruments 6 Quality assurance and laboratory safety TOTAL CLINICAL MICROSCOPY NOTES URINALYSIS PHYSICAL EXAMINATION OF URINE I. VOLUME Normal range (24o): 600 to 2000 mL Average volume: 1200 to 1500 mL Night:day ratio________ 5% 5% 3% 2% 2% 2% 2% 10% 5% 8% 100% 5% 10% 18% 15% 2% 3% 3% 21% 53%

1.Polyuria
Diuresis (Inc urine volume) Increased fluid intake Diuretic medication Diuretic drinks (coffee, tea, alcohol) Nervousness Diabetes mellitus Diabetes insipidus

2.Oliguria
Calculus or tumor of the kidney Dehydration

3.Anuria
Complete obstruction (stones, carcinomas) Toxic agents

4.Nocturia > 500 mL with sp. gr. less than 1.018

II. COLOR Roughly indicates the degree of hydration, and should correlate with urine sp. gr. Pigments: 1. Urochrome

2. Uroerythrin 3. Urobilin

Normal: Colorless to deep yellow Colorless Pale yellow Dark yellow Amber Orange Recent fluid consumption Polyuria Diabetes mellitus Diabetes insipidus Conc specimen Bilirubin Acriflavine Pyridium Nitrofurantoin Phenindione Bilirubin oxidized to biliverdin Pseudomonas infection Clorets Indican Methylene blue Phenol RBCs Hemoglobin Myoglobin (25 mg/dL) Porphyrin Beets Rifampin Menstrual contamination RBCs oxidized to methgb Homogentisic acid Melanin or melanogen Methyldopa or levodopa Metronidazole (Flagyl)

Yellow green Yellow - brown Green Blue-green

Pink Red

Brown Black

URINE COLOR CHANGES WITH COMMONLY USED DRUGS Drug Alcohol, ethyl Anthraquinone laxatives (senna, cascara) Chlorzoxazone (Paraflex) (muscle relaxant) Deferoxamine mesylate (Desferal) (chelates iron) Ethoxazene (Serenium) (urinary analgesic) Fluorescein sodium (given IV) Furazolidone (Furoxone) (Tricofuron) (an antibacterial, antiprotozoal nitrofuran) Indigo carmine dye (renal function, cytoscopy) Pale, diuresis

Color Reddish, alkaline; yellow-brown, acid Red Red Orange, red Yellow Brown Blue

Iron sorbitol (Jectofer) (possibly other iron compounds forming Brown on standing iron sulfide in urine) Levodopoa (L-dopa) (for parkinsonism) Red then brown, alkaline Mepacrine (Atabrine) (antimalarial) (intestinal worms, Giardia) Yellow Methacarbamol (Robaxin) (muscle relaxant) Methyldopa (Aldomet) (antihypertensive) Methylene blue (used to delineate fistulas) Metronidazole (Flagyl) ( for Trichomonas infection, amebiasis, Giardia) Nitrofurantoin (Furadantin) (antibacterial) Green-brown Darken; if oxidizing agents present, red to brown Blue, blue-green Darkening, reddish brown Brown-yellow

Drug

Color

Phenazopyridine (Pyridium) (urinary analgesic), also Orange-red, acid pH compounded with sulfonamides (Azo Gantrisin, etc.) Phenindione (Hedulin) (anticoagulant) (important to distinguish Orange, alkaline; color disappears on acidifying from hematuria) Phenol poisoning Brown; oxidized to quinines (green) Phenolphthalein (purgative) Phenolsulfonphthalein (also sulfobromophthalein) Rifampin (Rifadin, Rimactane) (tuberculosis therapy) Riboflavin (multivitamins) Sulfasalazine (Azulfidine) (for ulcerative colitis) Red-purple, alkaline pH Pink-red, alkaline pH Bright orange-red Bright yellow Orange-yellow, alkaline pH

IV. CLARITY/TRANSPARENCY/TURBIDITY TERMINOLOGY Clear 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 TURBIDITY 1.RBCs 2.WBCs 3.Bacteria 4.Yeast 5.Nonsquamous epit cells 6.Abnormal crystals 7.Lymph fluid 8.Lipids NONPATHOLOGIC CAUSES OF TURBIDITY 1.Squamous epit cells 2.Mucus 3.Amorphous crystals 4.Semen, spermatozoa 5.Fecal contamination 6.Radiographic contrast media 7.Talcum powder 8.Vaginal cream LABORATORY CORRELATIONS IN URINE TURBIDITY Acidic urine Alkaline urine Soluble with heat Soluble in dilute acetic acid Insoluble in dilute acetic acid Soluble in ether

Amorphous urates, radiographic contrast media Amorphous phosphates, carbonates Amorphous urates, uric acid crystals RBCs, amorphous phosphates, carbonates WBCs, bacteria, yeast, spermatozoa Lipids, lymphatic fluid, chyle

APPEARANCE AND COLOR OF URINE Appearance

Cause

Remarks

Colorless Cloudy

Very dilute urine Phosphates, carbonates Urates, uric acid Leukocytes Red cells (smoky) Bacteria, yeasts Spermatozoa

Polyuria, D. insipidus Sol in dilute acetic acid Dissoves at 60C and in alkali Insol in dilute acetic acid Lyse in dilute acetic acid Insol in dilute acetic acid Insol in dilute acetic acid

Appearance

Cause

Remarks

Milky

Prostatic fluid Mucin, mucous threads Calculi, gravel Clumps, pus, tissue Fecal contamination Radiographic dye Many neutrophil (pyuria) Fat Lipiduria, opalescent Chyluria, milky Emulsified paraffin Acriflavine Conc urine Urobilin in excess Bilirubin Bilirubin-biliverdin Bilirubin-biliverdin Hemoglobin Erythrocytes Myoglobin Porphyrin Fuscin, aniline dye Beets Menstrual contam Porphyrins Erythrocytes Hgb on standing Methemoglobin Myoglobin Bilifuscin (dipyrrole)

May be flocculent Phosphates, oxalates Rectovesical fistula In acid urine Insol in dilute acetic acid Nephrosis, crush injury, sol in ether Lymphatic obstruction, sol in ether Vaginal creams Green fluorescence Dehydration, fever No yellow foam Yellow foam, if sufficient bilirubin Yellow foam Beer brown, yellow foam Pos. rgt strip for bld Pos. rgt strip for bld Pos. rgt strip for bld May be colorless Foods, candy Yellow alkaline, genetic Clots, mucus May be colorless

Yellow Yellow-orange

Yellow-green Yellow-brown Red

Red-purple Red-brown

Acid pH Muscle injury Result of unstable hemoglobin Blood, acid pH On standing, alkaline; alkaptonuria On standing, rare Small intestine infections Mouth deodorants

Brown-black

Methemoglobin Homogentisic acid Melanin Indicans Pseudomonas infections Chlorophyll

Blue-green

V. SPECIFIC GRAVITY Density of solution compared with density of similar volume of dist water at a similar temperature Influenced by number and size of particles in solution DETERMINATION 1.Refractormetry (TS meter) Indirect mtd based on RI Compensated to temp (15-38oC) Requires corrections for glucose and protein o 1 g/dL Glucose ________ o 1 g/dL Protein ________

Calibration

o Distilled water o 5% NaCl 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 temp 0.001 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 Summary Reagents Mutistix: Poly (methyl vinyl ether/maleic anhydride) bromthymol blue Chemstrip: Ethyleneglycol-Bis (aminoethylether) bromthymol blue Sensitivity 1.000-1.030 Interference False-positive: High concentration of protein False-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 Measurements Method Urinometry Refractometry Harmonic oscillation densitometry Reagent strip Principle Density Refractive index Density pKa change of a polyelectrolyte

VI. pH Normal: pH 4.5 to 8.0 (random) ACID URINE Emphysema Diabetes mellitus Starvation Dehydration Diarrhea Presence of acid-producing bacteria (E.coli) High protein diet Cranberry juice Medications (methenamine mandelate [Mandelamine], fosfomycin tromethamine)

ALKALINE URINE Hyperventilation Vomiting Renal tubular acidosis Presence of urease-producing bacteria Vegetarian diet Old specimens REAGENT STRIP Prin: Double indicator system Methyl red Bromthymol blue pH Reagent Strip Summary

Reagents Sensitivity Sources of error/interference Correlations with other tests

Methyl red, bromthymol blue pH 5 - 9 No known interfering subs Runover from adjacent pads Old specimens Nitrite Leukocytes Microscopic

VII. ODOR Normal: 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 URINE I. PROTEIN Normal: <10 mg/dL or 100 mg/24o (Henry 150 mg/24o) Albumin major serum protein found in urine _______________________________________________________ _______________________________________________________ PRE-RENAL PROTEINURIA Intravascular hemolysis Muscle injury Severe infection and inflammation Multiple myeloma RENAL PROTEINURIA: GLOMERULAR DISORDERS Immune complex disorders Amyloidosis Toxic agents Diabetic nephropathy MICRAL TEST Principle: Enzyme immunoassay Sensitivity: 0 10 mg/dL Reagents: Gold-labeled ab, B-galactosidase, chlorophenol red galactoside Interference: False negative: dilute urine Strenuous exercise Dehydration Hypertension Pre-eclampsia Orthostatic or postural proteinuria RENAL PROTEINURIA: TUBULAR DISORDERS Fanconis syndrome Toxic agents/heavy metals Severe viral infections POST-RENAL PROTEINURIA Lower UTI/inflammations Injury/trauma Menstrual contamination Prostatic fluid/spermatozoa Vaginal secretions

REAGENT STRIP Prin: Protein error of indicators Tetrabromphenol blue (indicator) Tetrachlorophenol tetrabromosulfonphthalein (indicator) Protein Reagent Strip Summary Reagents Sensitivity Sources of error/interference Multistix: Tetrabromphenol blue Chemstrip: 3, 3 5, 5 tetrachlorophenol, 3, 4, 5, 6-tetrabromosulfonphthalein Multistix: 15-30 mg/dL albumin Chemstrip: 6 mg/dL albumin False-positive: Highly buffered alkaline urine Pigmented specimens, phenozopyridine Quaternary ammonium compounds (detergents) Antiseptics, chlorhexidine Loss of buffer from prolonged exposure of the reagent strip to the specimen High specific gravity False-negative: proteins other than albumin Blood Nitrite Leukocytes Microscopic

Correlations with other tests

SULFOSALICYLIC ACID PRECIPITATION TEST Cold precipitation test that reacts equally ith all forms of protein Grade Negative Trace 1+ 2+ 3+ 4+ No increase in turbidity Noticeable turbidity Distinct turbidity with no granulation Turbidity with granulation, no flocculation Turbidity with granulation and flocculation Clumps of protein Turbidity Protein range (mg/dl) <6 6-30 30-100 100-200 200-400 >400

II. GLUCOSE Renal threshold: 160 to 180 mg/dL Other sugars in urine Fructose Galactose Lactose Pentose CLINICAL SIGNIFICANCE OF URINE GLUCOSE HYPERGLYCEMIA ASSOCIATED Diabetes mellitus Pancreatic cancer Cushings syndrome Pheochromocytoma Central nervous system damage RENAL ASSOCIATED Fanconis syndrome Advanced renal disease Pancreatitis Acromegaly Hyperthyroidism Stress Gestational diabetes Pregnancy

REAGENT STRIP Prin: Double sequential enzyme reaction Glucose oxidase and peroxidase Chromogen O-toluidine (pink to purple) Potassium iodide (blue to brown) Aminopropryl-Carbazol (yellow to orange-brown)

Glucose Reagent Strip Summary Reagents Sensitivity Interference

Correlations with other tests

Multistix: Glucose oxidase, peroxidase, potassium iodide Chemstrip: Glucose oxidase, peroxidase, tetramethylbenzidine Multistix: 75 125 mg/dL Chemstrip: 40 mg/dL False-positive: Contamination by oxidizing agents and detergents False-negative: High levels of ascorbic acid High levels of ketones High specific gravity Low temperatures Improperly preserved specimens 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 alkali and heat A color change progressing from a negative blue (CuSO4) through green, yellow and orange/red (Cu2O) occurs when the reaction takes place Tablets 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 REACTIONS GLUCOSE OXIDASE CLINITEST Negative 1+ positive 4+ positive Positive Negative Negative INTERPRETATION

Nonglucose reducing substance present Possible interfering substance for reagent strip Small amount of glucose present Possible oxidizing agent interference on reagent strip

III. KETONES Results 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% BHA 20% AAA/diacetic acid 2% Acetone Significance Diabetes acidosis Insulin dosage monitoring Starvation Malabsorption/pancreatic disorders Strenuous exercise Vomiting Inborn error of amino acid metabolism REAGENT STRIP Prin: Sodium nitroprusside reaction Ketone Reagent Strip Summary Reagents Sensitivity Interference Sodium nitroprusside Glycine (Chemstrip) Multistix: 5 10 mg/dL acetoacetic acid Chemstrip: 9 mg/dL acetoacetic acid, 70 mg/dL acetone False-positive: Phthalein dyes Highly pigmented red urine Levodopa Medications containing free sulfhydryl groups False-negative: Improperly preserved specimens Glucose

Correlations with other tests

ACETEST

Sodium nitroprusside, glycine, disodium phosphate and lactose

IV. BLOOD Hematuria Renal calculi, glomerulonephritis, pyelonephritis, tumors, trauma, exposure to toxic chemicals, anticoagualants, strenuous exercise Hemoglobinuria Transfusion reactions, hemolytic anemias, severe burns, infections/malaria, strenuous exercise/red blood cell trauma Myoglobinuria Muscular trauma/crush syndromes, prolonged coma, convulsions, muscle-wasting diseases, alcoholism/overdose, drug abuse, extensive exertion HEMOGLOBINURIA VS MYOGLOBINURIA 1. Plasma examination Hemoglobin Myoglobin 2. Blondheims test (Ammonium sulfate) Hemoglobin Myoglobin REAGENT STRIP Prin: Pseudoperoxidase activity of hemoglobin Tetramethylbenzidine (chromogen) Blood Reagent Strip Summary Reagents Sensitivity Interference Multistix: Diisopropylbenzene dehydroperoxide tetramethylbenzidine Chemstrip: 2,5-dimethyl-2,5-dihydroperoxide tetramethylbenzidine Multistix: 5-20 RBCs/L, 0.015-0.062 mg/dL hemoglobin Chemstrip: 5 RBCs/L, hemoglobin corresponding to 10 RBCs/L False-positive: Strong oxidizing agents Bacterial peroxidases Menstrual contamination False-negative: High specific gravity/crenated cells Formalin Captopril High conc. of nitrite Ascorbic acid >25 mg/dL Unmixed specimens Protein Microscopic

Correlations with other tests

V. BILIRUBIN Early indication of liver disease Significance: Hepatitis Cirrhosis Biliary obstruction (gallstones, carcinoma) REAGENT STRIP Prin: Diazo reaction 2,4-dichloroaniline diazonium salt 2,6-dichlorobenzene-diazonium-tetrafluoroborate Bilirubin Reagent Strip Summary Reagents Sensitivity Multistix: 2,4-dichloroaniline diazonium salt Chemstrip: 2,6-dichlorobenzene-diazonium-tetrafluoroborate Multistix: 0.4-0.8 mg/dL bilirubin Chemstrip: 0.5 mg/dL bilirubin

Interference

Correlations with other tests

False-positive: Highly pigmented urines, phenazopyridine Indican (intestinal disorders) Metabolites of Lodine False-negative: Specimen exposure to light Ascorbic acid >25 mg/dL High concentrations of nitrite Urobilinogen

ICTOTEST Positive: Blue to purple color p-nitrobenzene-diazonium-p-toluenesulfonate SSA Sodium bicarbonate VI. UROBILINOGEN Bile pigment that result from hgb degradation Small amt in normal urine <1 mg/dL or Ehrlich unit REAGENT STRIP Ehrlichs reaction Urobilinogen Reagent Strip Summary Reagents Multistix: PDAB Chemstrip: 4-methoxybenzene-diazonium tetrafluoroborate Sensitivity Multistix: 0.2 mg/dL urobilinogen Chemstrip: 0.4 mg/dL urobilinogen Interference Multistix False-positive: Porphobilinogen Indican p-aminoslicylic acid Sulfonamides Methyldopa Procaine Chlorpromazine Highly pigmented urine False-negative Old specimens Preservation in formalin Chemstrip False-positive: Highly pigmented urine False-negative: Old specimens Preservation in formalin High concentrations of nitrate Correlations with other tests Bilirubin

WATSON SCHWARTZ TEST For differentiating urobilinogen and porphobilinogen Urobilinogen Porphobilinogen Chloroform Extraction Urine (top layer) Chloroform (bottom layer) Butanol Extraction Butanol (top layer) Urine (bottom layer) Colorless Red Red Colorless Red Colorless Colorless Red

Other Ehrlich-Reactive Substances Red Colorless Red Colorless

Urine Bilirubin and Urobilinogen in Jaundice

Urine Bilirubin Hemolytic disease Liver damage Bile duct obstruction Negative + or +++

Urine Urobilinogen +++ ++ Normal

HOESCH TEST Rapid screening test for urine porphobilinogen ( 2mg/dL) Hoesch reagent (Ehrlich rgt dissolved in 6M HCl) VII. NITRITE Detection of bacteriuria

REAGENT STRIP Prin: Greiss reaction Positive nitrite corresponds to 100,000 organisms/mL Nitrite Reagent Strip Summary Reagents Sensitivity Interference Multistix: p-arsanilic acid tetrahydronezo(h)quinolin-3-ol Chemstrip: Sulfanilamide 3-hydroxy-1,2,3,4-tetrahydro-7,8 benzoquinoline Multistix: 0.06-0.1 mg/dL nitrite ion Chemstrip: 0.05 mg/dL nitrite ion False-positive: Improperly preserved specimens Highly pigmented urine False-negative: Nonreductase-containing bacteria Insufficient contact time between bacteria and urinary nitrate Lack of urinary nitrate Large quantities of bacteria converting nitrite to nitrogen High concentrations of ascorbic acid High specific gravity

Correlations with other tests

Protein Leukocytes Microscopic

VIII. LEUKOCYTE Significance: UTI/Inflammation Screening of urine culture specimens REAGENT STRIP Prin: Leukocyte esterase Leukocyte Esterase Reagent Strip Summary Reagents Multistix: Derivatized pyerole amino acid ester, diazonium salt Chemstrip: Indoxylcarbonic acid ester, diazonium salt Sensitivity Multistix: 5-15 WBC/hpf Chemstrip: 10-25 WBC/hpf Interference False-positive: Strong oxidizing agents Highly pigmented urine, nitrofurantoin False-negative: High concentrations of protein, glucose, oxalic acid, ascorbic acid. gentamicin, cephalosporins, tetracyclines

Correlations with other tests

Protein Nitrite Microscopic

MICROSCOPIC EXAMINATION OF URINE MICROSCOPIC TECHNIQUES Technique Bright-field microscopy Used for routine urinalysis Phase-contrast microscopy Polarizing microscopy Dark-field microscopy Fluorescence microscopy Interference-contrast Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads and Trichomonas Aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals Aids in identification of Treponema pallidum Allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye Produces a three-dimensional microscopy-image and layer-by-layer imaging of a specimen Function

SEDIMENT STAIN Stain Sternheimer-Malbin

Action

Function

Crystal violet and safranin Identifies WBCs, epithelial cells, and casts Delineates structure and contrasting colors of the nucleus and cytoplasm Toluidine blue Enhances nuclear detail Differentiates WBCs and renal tubular epithelial cells Lipid stains: Oil Red O and Sudan III Stains triglycerides and neutral fats orange- Identifies free fat droplets and lipidred containing cells and casts Gram stain Differentiates gram-positive and gramIdentifies bacterial casts negative bacteria Hansel stain Methylene blue and eosin Y stain eosinophilic Identifies urinary eosinophils granules Prussian blue stain Stains structures containing iron Identifies yellow-brown granules of hemosiderin in cells and casts SEDIMENT CONSTITUENTS CELLS 1. RBCs Non-nucleated biconcave disks Crenated in hypertonic urine Ghost cells in hypotonic urine Dysmorphic with glomerular membrane damage 2. WBCs Larger than red blood cells Granulated, multilobed neutrophils Glitter cells in hypotonic urine Eosinophils > 1% is considered significant Mononuclear cells: lymphocytes, monocytes, macrophages and histiocytes 3. EPITHELIAL CELLS A. SQUAMOUS EPIT CELLS

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 CELLS Rectangular, 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. BACTERIA 5. YEAST 6. PARASITE 7. SPERMATOZOA 8. MUCUS CASTS Formed in the distal convoluted tubule and collecting duct Major constituent: Tamm Horsfall protein 1. HYALINE CASTS Glomerulonephritis Pyelonephritis Chronic renal disease Congestive heart failure Stress and exercise 2. RBC CASTS Glomerulonephritis Strenuous exercise 3. WBC CASTS Pyelonephritis Acute interstitial nephritis 4. BACTERIAL CASTS Pyelonephritis 5. EPITHELIAL CELL CASTS Renal tubular damage 6. COARSE/FINE GRANULAR CASTS Glomerulonephritis Pyelonephritis Stress and exercise 7. FATTY CASTS Nephrotic syndrome Toxic tubular necrosis Diabetes mellitus Crush injuries 8. WAXY CASTS Stasis of urine flow Chronic renal failure 9. BROAD CASTS Extreme urine stasis Renal failure CRYSTALS NORMAL CRYSTALS A. ACIDIC URINE

1. Amorphous urate Mic: yellow-brown granules Pink sediment (uroerythrin) 2. Uric acid Rhombic, wedge, rosette, hexagonal, fousided plate (whetstone) Lesch-Nyhan, gout, leukemic pts receiving chemotherapy 3. Calcium oxalate Enveloped/pyramidal, oval Food high in oxalic/ascorbic acid (tomato, asparagus) Ethylene glycol poisoning B. ALKALINE URINE 1. Amorphous phosphate Granular appearance White ppt 2. Ammonium biurate Yellow-brown, thorny apples Old specimen 3. Triple phosphate Magnesium ammonium phosphate Coffin lid 4. Calcium phosphate Colorless, flat rectangular plates or thin prisms often in rosette formation Dissolve in dilute acetic acid 5. Calcium carbonate Small and colorless, with dumbbell or spherical shapes Formation of gas after addition of acetic acid

ABNORMAL CRYSTALS (Acid, neutral urine) 1. Cystine Colorless hexagonal plates Cystinuria 2. Cholesterol Rectangular plate with notch in one or more corners, staircase pattern Lipiduria - nephrotic syndrome 3. Tyrosine Colorless to yellow needles 4. Leucine Yellow-brown spheres with concentric circles and radial striations 5. Bilirubin Clumped needles or granules with yellow color URINARY SEDIMENTS ARTIFACTS 1. Starch granules 2. Oil droplets 3. Air bubbles 4. Pollen grains 5. Hair and fibers 6. Fecal contamination Qualitative Tests for Protein Hellers Roberts Conc. HNO3 Sat. MgSO4.7H2O

Spieglers

Biuret Heat and acetic acid SSA

HgCl2 NaCl Succinic acid Dist. H2O 10% NaOH/KOH 5-10% Acetic acid Extons qualitative rgt. Na2SO4 SSA Dist. H2O Sat. NaCl 5-10% Potassium ferrocyanide Picric acid soln. Quantitative Tests for Protein Esbachs rgt. 1 g picric acid 2 g citric acid Esbachs rgt. 10% FeCl3 PTA crystals 95% alcohol Conc. HCl SSA

White ring at the zone of conatct

Violet for albumin Rose for albuminoses and peptones

White turbidity/cloudiness

Purdys Potassium ferrocyanide Picric acid Esbachs Kwileckis Tsuchiyas Kingsbury-Clark Biuret

24o read height of coagulum 72oC for 5 minutes read height of coagulum Same as Esbachs

Degree of turbidity is measured by comparison with standard turbidities Uses the same principle as that used for serum protein which depends upon the presence of peptide linkages in protein Sugars Benedicts rgt. Copper sulfate Sodium carbonate Sodium citrate buffer Phenylhydrazine Sodium acetate Rochelle salt Bismuth subnitrate NaOH KOH 10% KOH 25% HCl Resorcinol Lead acetate Ammonia H2O Reducing substances Green-orange-red Glucose, fructose, lactose & pentose Crystalline needles Glucose & other reducing subs Brown to black color Glucose & other reducing subs Canary yellow to black Fructose Red color Lactose Brick red color w/red ppt Glucose Red color w/yellow ppt Green soln Cherry red Acetone Purplish red ring Acetone & acetoacetic acid Rose or purple ring Acetone & acetoacetic acid Purple ring Acetone & acetoacetic acid Purple color

Benedicts

Osazone or Phenylhydrazine (Kowarsky) Nylanders

Moore Heller Borchardts Seliwanoff Resorcinol-HCl Rubners

Bial Orcinol Taubers Frommers Rotheras Lange Acetest Ketostix

HCl 10% FeCl3 Benzidine in glacial acetic acid Ketones KOH 10% salicyl aldehyde Sodium nitroprusside Ammonium sulafate Glacial acetic acid Sodium nitroprusside Ammonia H2O Aminoacetic acid Sodium nitroprusside Disodium phosphate Lactose 10% FeCl3

Gerhardts

Acetoacetic acid Bordeaux red color

Gmelin Smith Harrisons spot

Ictotest Wallace and Diamond Schlesinger

BILE PIGMENTS (Bilirubin, urobilinogen & urobilin) 10% BaCl2.HNO3 Bile pigments Play of colors Alc. tincture of iodine Bile pigments Emerald green 10% BaCl2 Bile pigments Fouchets rgt. Blue to green color 10% FeCl3 TCA Ictotest tablets Bile pigments Blue to purple mat Ehrlich rgt (PDAB) Urobilinogen Cherry red color Lugols iodine Urobilin Alc. soln. of zinc acetate Greenish fluorescence HEMOGLOBIN

Benzidine Guiac Orth-toluidine

Benzidine powder in glacial acetic acid 3% H2O2 10% HAC 95% alcohol Guiac powder Ortho-toluidine H2O2 MELANIN 10% FeCl3 (Urine allowed to stand for 24o) Sodium nitroprusside 40% NaOH 33% Acetic acid Potassium persulfate Methyl alcohol Ether 5% NaOH CHLORIDE AgNO3 K2CrO4 Titrated with mercuric nitrate Diphenylcarbazone indicator CALCIUM Oxalic acid Ammonium oxalate Glacial acetic acid Distilled H2O

Green-blue Blue Blue

Screening test Thormahlen (Fresh urine) Blackberg & Wanger (24-hr urine)

Urine will turn brown to black Dark green or blue color Brown to black ppt

Fantus Mercurimetric titration Schales & Schales Sulkowitch

Reddish ppt. Blue-violet colored complex

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 circulation. 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 regulates the increase in fluid from the fetal urine. Increased AF _________________________________ Decreased AF ________________________________

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 urine Analyte

Amniotic Fluid

Maternal Urine

Amniotic Fluid Color Color Colorless Blood-streaked Yellow Dark green Dark red-brown Normal Traumatic tap, abdominal trauma, intra-amniotic hemorrhage Hemolytic disease of the newborn (bilirubin) Meconium Death Significance

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

________________________________________ ________________________________________ ________________________________________ Foam test ________________________________________ ________________________________________ ________________________________________ Microviscosity ________________________________________ ________________________________________ Lamellar body count ________________________________________ ________________________________________ Optical Density 650 nm ________________________________________ ________________________________________ Tests for Fetal Well-Being and Maturity Test Bilirubin scan Alpha-fetoprotein Lecithin-sphingomyelin ratio Amniostat-fetal lung maturity Foam stability index Microviscosity Optical density 650 nm Lamellar body count Normal Values at Term Significance Hemolytic disease of the newborn Neural tube disorders Fetal lung maturity Fetal lung maturity/ phosphotidyl glycerol Fetal lung maturity Fetal lung maturity Fetal lung maturity Fetal lung maturity

A450 > 0.025 <2.0 MoM 2.0 Positive 47 55 mg/g 0.150 32,000/L

CEREBROSPINAL FLUID
Third major body fluid Functions o Supply nutrients to nervous tissue o Remove metabolic wastes o 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

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 immediately o WBCS and RBCs will begin to lyse within 1 hour o 40% WBCs disintegrating within 2 hours Normal o Adult ___________________________ o Neonate ________________________ Calculation Number of cells counted x dilution Number of sq. counted x vol. of 1 sq.

Cells/uL =

Dilution

Slightly hazy Hazy Slightly cloudy Slightly bloody Cloudy Bloody Turbid

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 smear o _______________________________

o _______________________________ o _______________________________ o _______________________________

Normal cells in CSF

o _______________________________ o _______________________________
Adult ____________________ Neonate _________________

Increased in number of normal cells in CSF _______________________________

Lymphocytes Monocytes Neutrophils Macrophages Blasts Plasma cells

Normal Viral, tubercular, fungal meningitis Multiple sclerosis Normal Viral, tubercular, fungal meningitis Multiple sclerosis Bacterial meningitis Early cases of viral, tubercular and fungal meningitis Cerebral hemorrhage RBCs in spinal fluid Contrast media Acute leukemia Multiple sclerosis Lymphocyte reactions

Ependymal, choroidal, and spindleshaped cells Malignant cells

Diagnostic procedures Metastatic carcinomas Primary CNS carcinoma

CHEMISTRY TESTS I. PROTEIN NV (TP) ______________________________________

Elevated results: meningitis: hemorrhage, primary CNS tumors, multiple sclerosis, Guillain-Barr syndrome, neurosyphilis, 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: ______________________________ ______________________________ ______________________________ Normal in ____________________________

LACTATE NV: _______________________________________

Increased in: _____________________________ _____________________________ _____________________________

GLUTAMINE NV: _______________________________________

Increased in: _____________________________ _____________________________

MICROBIOLOGY TESTS MENINGITIS Bacterial WBC Viral Tubercular Fungal

Protein Glucose Lactate

SEROLOGIC TESTING Latex agglutination and ELISA for detection of bacterial antigens VDRL neurosyphilis (recommended by CDC)

SEMINAL FLUID
Reasons for Analysis Fertility testing Postvasectomy semen analysis Forensic analyses Physiology Semen is composed of four fractions that are contributed by:

1. ________________________ 2. ________________________ 3. ________________________ 4. ________________________

___________ ___________ ___________ ___________

Collection Abstinence for _________________________________ Analysis should be done after liquefaction Specimen awaiting analysis should be kept at 37oC

Semen Analysis Appearance Gray-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. Improved Neubauer Counting Chamber

2. Makler Counting Chamber

Sperm Count NV: Calculation: Sperm Motility Evaluated in approximately __________ NV: Sperm Motility Grading Grade 4.0 3.0 2.0 1.0 0 a b b c d WHO Criteria

Source: Urinalysis and Body Fluids, 5th edition by Strasinger and Di Lorenzo, p203

Sperm Morphology Routine criteria: Krugers strict criteria:

Normal Values for Semen Analysis Volume Viscosity pH Sperm concentration Sperm count Motility Quality Morphology Round cells

2 5 mL Pour in droplets 7.2 8.0 >20 million/mL >40 million/ejaculate >50% within 1 hr >2.0 or a,b,c >30% normal forms (routine criteria) >14% normal forms (strict criteria) <1.0 million/mL

Source: Urinalysis and Body Fluids, 5th edition by Strasinger and Di Lorenzo, p201

Sperm Viability ______________________________________ ______________________________________ ______________________________________ 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 Test Detect the presence of IgG, IgM and IgA antibodies and will demonstrate what area of the sperm (head, neck, tail) the autoantibodies are affecting Normal: presence of beads on less than 20% of the sperm

Chemical Testing Fructose 13 mol/ejaculate Neutral -glucosidase 20 mU/ejaculate Zinc 2.4 mol/ejaculate Citric acid 52 mol/ejaculate ACP 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 joints o Reduce friction between bones o Provides nutrient to the articular cartilage o 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 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 Neutrophils Lymphocytes 65% <20% <15%

Cells and Inclusions in Synovial Fluid Cell/Inclusion Neutrophil Lymphocyte Macrophage Synovial lining cell LE cell Reiter cell PMN Mononuclear leukocyte Description Significance Bacterial sepsis Crystal-induced inflammation Nonseptic inflammation

Large mononuclear leukocyte, may be Normal vacuolated Viral infections Similar to macrophage, may be Normal multinucleated, res. mesothelial cell Neutrophil containing ingested round body LE Vacuolated macrophage with ingested neutrophils Reiters syndrome Nonspecific inflammation

Cell/Inclusion RA cell Cartilage cells Rice bodies Fat droplets Hemosiderin

Description

Significance

Neutrophil with dark cytoplasmic granules RA containing immune complexes Immunologic inflammation Large multinucleated cells Osteoarthritis Macroscopic: res. polished rice TB, septic and RA Microscopic: show collagen and fibrin Refractile intracellular and extracellular Traumatic injury globules Stain with Sudan dyes Inclusions within clusters of synovial cells Pigmented villonodular synovitis

Crystal Identification Causes of crystal formation o Metabolic disorders o Decreased renal excretion that produce elevated blood levels of crystallizing chemicals o Degeneration of cartilage and bones o Injection of medications (corticosteroid)

A. B. C. D. E.

Monosodium urate Calcium pyrophosphate Hydroxyapatite (calcium phosphate) Cholesterol Corticosteroids

Fluid is examined unstained under polarized and COMPENSATED POLARIZED LIGHT for detection of MSU and CPPD crystals Chemistry Tests Glucose Lactate Protein UA Microbiology tests Common organisms that infect synovial fluid Staphylococcus Streptococcus Haemophilus Neissreria gonorrhoeae Routine bacterial cultures should always include enrichment medium such as CAP Serologic Tests Autoantibody detection: SLE and RA Antibody detection: Lyme disease Joint Disorders Group Classification I. Noninflammatory Pathologic Significance Degenerative joint disorders Laboratory Findings Clear, yellow fluid Good viscosity WBCs <1000 L Neutrophils <30% Normal glucose (similar to blood glucose)

Group Classification II. Inflammatory

Pathologic Significance Immunologic disorders, rheumatoid arthritis, lupus erythematosus, sclerederma, polymyositis, ankylosing spondylitis, rheumatic fever, and Lyme arthritis

Laboratory Findings Immunologic origin: Cloudy, yellow fluid Poor viscosity WBCs 2000-75000 L Neutrophils >50% Decreased glucose level Possible autoantibodies present Crystal-induced origin: Cloudy or milky fluid Low viscosity WBCs up to 100, 000 L Neutrophils <70% Decreased glucose level Crystals present Cloudy, yellow-green fluid Variable viscosity WBCs 50,000-100,000 L Neutrophils >75% Decreased glucose level Positive culture and Gram stain Cloudy, red fluid Low viscosity WBCs equal to blood Neutrophils equal to blood Normal glucose level RBCs present

Crystal-induced gout and pseudogout

III. Septic

Microbial infection

IV. Hemorrhagic

Traumatic injury Coagulation deficiencies

Source: Urinalysis and Body Fluids, 5th edition by Strasinger and Di Lorenzo, p212

TRANSUDATE AND EXUDATES

Differentiation of Transudates and Exudates Transudate Appearance Fluid:serum protein ratio Fluid:serum LD ratio White blood cell count Spontaneous clotting Pleural fluid cholesterol Pleural fluid:serum cholesterol ratio Pleural fluid:bilirubin ratio Serum-ascites albumin gradient Clear <0.5 <0.6 <1000/L No <45-60 mg/dL <0.3 <0.6 >1.1 Cloudy >0.5 >0.6 >1000/L Possible >45-60 mg/dL >0.3 >0.6 <1.1 Exudate

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 Constipation Spastic bowel Rectal narrowing or stricture Mushy, foul smelling gray stool that floats on water Small, firm, spherical masses of stool (scybala) Narrow, ribbon-like stool

Blood from lower gut Red Beets Bleeding from upper GIT Black Bismuth, iron, charcoal Spinach and other green vegetables or calomel, or presence of Green biliverdin Presence of mucus in stool is abnormal and should be reported Spastic constipation or mucous colitis Translucent gelatinous mucus clinging to the surface of formed stool Neoplasm or inflammatory process of the rectal canal Bloody mucus clinging to fecal mass Ulcerative colitis, bacillary dysentery, ulcerating diverticulitis and Mucus associated with pus and blood intestinal tuberculosis 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 CHARACTERISTICS Black Upper gastrointestinal bleeding, iron therapy, charcoal, bismuth (antacids) Red Pale yellow, white, gray Green Bulky/frothy Ribbon-like Mucus/blood-streaked mucus Low gastrointestinal bleeding, beets and food coloring, rifampin Bile-duct obstruction, barium sulfate Biliverdin/oral antibiotics, green vegetables Bile-duct obstruction, pancreatic disorders Intestinal constriction Colitis, dysentery, malignancy, constipation

MICROSCOPIC EXAMINATION OF FECES Fat Sudan III, Sudan IV or Oil Red O stain Stool suspension + 95% ethanol + Sudan III Neutral 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

Normal: 0.25 g/dL Suspicious: 0.25g to 0.5 g/dL Abnormal: >0.5 g/dL

FECAL SREENING TESTS

TEST
Exam.for neutrophils stain

METHODOLOGY/PRINCIPLE/INTERPRETATION

Microscopic count of neutrophils in smear stained with methylene blue, Gram stain or Wrights Three per hpf indicates condition affecting intestinal wall Microscopic examination of direct smear with Sudan III 60 large orange-red droplets indicates Microscopic examination of smear heated with acetic acid and Sudan III 100 orange-red
malabsorption droplets measuring 6-75 m indicates malabsorption Pseudoperoxidase activity of hemoglobin liberates oxygen from hydrogen peroxide to oxidize guaiac reagent Blue color indicates gastrointestinal bleeding Addition of sodium hydroxide to hemoglobin-containing emulsion determines presence of maternal or fetal blood Pink color indicates presence of fetal blood Emulsified specimen placed on x-ray paper determines ability to digest gelatin Inability to digest gelatin indicates lack of trypsin Addition of Clinitest tablet to emulsified stool detects presence of reducing substances Reaction of 0.5 g/dL reducing substances suggests carbohydrate intolerance

Quali. fecal fats

Occult blood

APT test

Trypsin Clinitest