5. MUST to KNOW in Clinical Microscopy

Nephron Urethra Urine formation (order) PCT ADH Urine composition 60g

Clearance tests

Creatinine clearance

Tubular Reabsorption Concentration tests Fishberg test (Old) Mosenthal test (Old) Specific Gravity (New) Osmolarity

PAH test PSP test Midstream/Catheterized Suprapubic aspiration

MUST TO KNOW IN CLINICAL MICROSCOPY URINALYSIS Basic structural unit of kidney 1M/kidney F: 3-4 cm M: 20 cm Glomerulus  Bowman’s capsule  PCT  Loop of Henle  DCT  CD 65% of reabsorption Regulate H2O reabsorption in DCT and CD 95-97% H2O 3-5% solids TS in 24 hrs 35g: Organic = Urea (major) 25g: Inorganic = Cl (#1) > Na+ > K+ Glomerular Filtration Evaluate glomerular filtration 1. Urea clearance 2. Creatinine clearance = most common 3. Inulin clearance = gold standard 4. Beta2-microglobulin 5. Radioisotopes Formula: Cc = U x V x 1.73 P A Normal values: M = 107-139 mL/min F = 87-107 mL/min Tubular Reabsorption 1st function to be affected in renal disease Evaluate tubular reabsorption Patient is deprived of fluid for 24hrs then measure urine SG (SG ≥ 1.026) Compare day and night urine in terms of volume and SG Influenced by # and density of particles in a solution Influenced by # of particles in a solution Principle: Freezing point depression - 1 Osm or 1000 mOsm/kg of H2O will lower the FP of H2O (0’C) by 1.86’C - FP = Osm Example: Determine Osm in mOsm/kg Temp. = -0.90’C Solution: 1000 mOsm/kg = _ _x____ -1.86’C -0.90’C x = 484 mOsm/kg Tubular Secretion and Renal Blood Flow p-aminohippuric acid Phensulfonphthalein test Obsolete, results are hard to interpret Methods of Collection Urine culture Anaerobic urine culture lec.mt 04 |Page | 134

3 glass technique

Pediatric specimen Drug Specimen Collection

Occasional/Single/Random 24 hr 12 hr Afternoon (2PM-4PM) 4 hr 1st morning Fasting/2nd morning Decreased Clarity Glucose Ketones Bilirubin Urobilinogen RBC/WBC Increased pH Bacteria Odor Nitrite Differentiate contamination from true infection Refrigeration Formalin Boric acid

For detection of prostatic infection 1. 1st portion of voided urine 2. Middle portion of voided urine: Serves as control for kidney and bladder infection -If (+), result for #3 is considered invalid 3. Urine after prostatic massage Compare WBC and Bacteria of specimen 1 and 3 Prostatic infection: 1 < 3 (10x) Wee bag Chain of custody: step by step documentation of handling and testing of legal specimen Required amount: 30-45 mL Temperature (urine): 32.5-35.7’C (w/in 4 mins) Blueing agent  Toilet bowl (to prevent adulteration) Types of Urine Specimen Routine Qualitative UA 1st voided urine  discarded w/ preservative Ex. 8AM  8AM Ex. 8AM  8PM Addis count: measure of formed elements in the urine using hemacytometer Urobilinogen (alkaline tide) Nitrite determination (1st morning/4 hr) NO3  NO2 = (+) UTI Pregnancy test (hCG) Ideal specimen for routine UA Most concentrated and most acidic = preservation of cells and casts Glucose determination 2nd voided urine after a period of fasting Changes in Unpreserved Urine Bacterial multiplication Precipitation of AU/AP Glycolysis Volatilization Photooxidation Oxidized to urobilin Disintegrate in alkaline urine Urea ---(Urease)---> NH3 Multiplication Urea ---(Urease)---> NH3 Bacterial multiplication Contamination:  Bacteria True infection:  Bacteria and WBCs Preservation 2-8’C  SG (hydrometer/urinometer) Precipitate AU/AP Addis count Urine culture lec.mt 04 |Page | 135

Sodium fluoride Sodium benzoate/ Benzoic acid Saccomanno’s fixative

Volume

Routine UA Polyuria Oliguria Anuria Nocturia Urine color Urine pigments

Normal Abnormal Colorless/Pale yellow Amber Orange Yellow-green Yellow-brown Green Blue-green Cloudy/Smoky red Clear red

Bacteriostatic to contaminants Glucose Substitute for sodium fluoride 50% ethanol + carbowax Cytology (50mL urine) Physical Examination of Urine NV: 24 hr = 600-1200 mL Ave (24 hr) = 1200-1500 mL Night: Day ratio = 1:2 to 1:3 Vol = 10-15 mL (Ave: 12 mL) -15 mL: for urinometry -physical, chemical, microscopic exam  Urine volume -Diabetes Mellitus:  vol,  SG -Diabetes Insipidus:  vol,  SG Calculus/kidney tumors Dehydration Complete cessation of urine flow >500mL w/ SG Biliverdin Pseudomonas aeruginosa Clorets, methylene blue, phenol Indican (blue): Hartnup disease or Blue diaper syndrome Hematuria (intact RBCs) Hemoglobin, Myoglobin lec.mt 04 |Page | 136

Red/Purple/Burgundy red/ purplish red/Portwine Brown/black

Cola-colored Yellow Red to brown Orange-red (acid) Bright orange-red (acid) Bright yellow Nubecula Bilifuscin (Dipyrrole) Clear Hazy Cloudy Turbid Milky Bacteria Chyluria Squamous epithelial cells Radiographic contrast media Vaginal cream Acidic urine Alkaline urine Soluble w/ heat Soluble w/ dilute acetic acid Insoluble in dilute acetic acid Soluble in ether

Porphyria (Lead poisoning: normal color) Methemoglobin (acid urine) Homogentisic acid: Alkaptonuria -Urine darkens after a period of standing -(-) Homogentisic acid oxidase Urine Color Changes w/ Commonly Used Drugs Levodopa (Tx: Parkinsonism) Red  Brown (alkaline) Mepacrine/Atabrine (Tx: Malaria, Giardiasis) Metronidazole/Flagyl (Tx: Trichomoniasis, Amoebiasis, Giardiasis) Methyldopa/Aldomet (Antihypertensive) Phenazopyridine/pyridium (Tx: UTI) Rifampin (Tx: TB) = all body fluids are red Riboflavin (Multivitamins) Faint cloud in urine after a period of standing WBCs, epithelial cells and mucus Hemoglobin Köln = unstable Red-brown urine Clarity/Transparency/Turbidity Transparent, no visible particulates Few particulates, print easily seen through urine Many particulates, print blurred through urine Print cannot be seen through urine May precipitate or clot Uniform turbidity NOT cleared by acidification or filtration Lymph fluid in urine Filariasis  females  SG by refractometer (>1.040) Rgt strip: not affected by RCM Tx: Candida Pseudochyluria Laboratory Correlations in Urine Turbidity AU RCM AP Carbonates AU Uric acid RBCs AP Carbonates WBCs Yeasts Spermatozoa Bacteria Lipids Lymph fluid Chyle

lec.mt 04 |Page | 137

SG

Refractometer (TS meter)

Urinometer

Rf < U by 0.002 Urinometer calibration Isosthenuria Hyposthenuria Hypersthenuria Aromatic/Odorless Ammoniacal Fruity, sweet Rotten fish/Galunggong Sweaty feet Mousy Cabbage Caramelized sugar, curry Bleach Sulfur

Principle (Rgt Strip) Other info. Harmonic Oscillation Densitometry Normal

Specific Gravity Density of solution compared w/ density of similar volume of distilled H2O at a similar temperature NV = 1.003-1.035 (random) SG 1.010 Urine Odor Normal Urea ---(Urease)---> NH3 Ex. UTI (Proteus:  urease) DM (Ketones) Trimethylaminuria Isovaleric acidemia Phenylketonuria Methionine malabsorption MSUD Contamination Cystine disorder Chemical Examination of Urine Specific Gravity pKa dissociation constant  concentration =  H+ Indicator: Bromthymol blue = () Blue  Green  Yellow () Not affected by glucose, protein and RCM Frequency of soundwave entering a solution will change in proportion to the density (SG) of the solution -Yellow IRIS (Automated): International Remote Imaging System pH Random = 4.5-8.0 1st morning = 5.0-6.0 pH 9.0 = Unpreserved urine lec.mt 04 |Page | 138

Acid urine

Alkaline urine

Principle (Rgt Strip)

Protein

Normal value Pre-renal proteinuria

Renal proteinuria

Post-renal proteinuria Principle (Rgt Strip)

Sulfosalicylic acid precipitation test

DM (Ketone bodies) Starvation (Ketone bodies) High protein diet Cranberry juice (Tx: UTI) After meal Vomiting Renal tubular acidosis: inability to produce an acid urine Vegetarian diet Old specimen: Urea ---(Urease)---> NH3 Double indicator system (Methyl red & Bromthymol blue) MR + H+ ------------> BTB – H+ pH 4.0-6.0 pH 6.0-9.0 (Red-Yellow) (Yellow-Blue) Protein White foam Analyte indicative of renal disease 1. Albumin: major serum protein found in urine 2. Serum and tubular microglobulins 3. Tamm-Horsfall protein (Uromodulin) 4. Proteins derived from prostatic and vaginal secretions < 10 mg/dL or < 100 mg/24 hrs < 150 mg/24 hrs (Henry) Prior to reaching the kidney 1. Intravascular hemolysis (Hgb) 2. Muscle injury (Mgb) 3. Severe inflammation (APRs) 4. Multiple myeloma: proliferation of Ig-producing plasma cells (BJP) Bence-Jones protein: Ig light chains (Identical: κ-κ, λ-λ) -ID: Serum IEP -Urine: precipitates at 40-60’C and dissolves at 100’C -Seen in MM, macroglobulinemia, malignant lymphoma Glomerular and tubular disorders 1. Diabetic nephropathy - Glomerular filtration  Renal failure -Microalbuminuria: proteinuria not detected by routine rgt strip -Micral test: a strip employing Ab enzyme conjugate that binds albumin (EIA) 2. Orthostatic/Postural/Cadet: proteinuria when standing due to pressure to renal veins -1st morning urine: (-) protein -2 hrs after standing: (+) protein 3. Fanconi’s syndrome Lower UTI/inflammation Protein (Sorensen’s) error of indicators Indicator + Protein ---(buffered at pH 3.0)---> (-) Yellow (+)[] Green  Blue [] Sensitive to albumin Indicators: a. Tetrabromphenol blue b. Tetrachlorophenol tetrabromosulfonphthalein Cold precipitation test that reacts equally to all types of protein If (-) rgt strip, (+) SSA = presence of other proteins lec.mt 04 |Page | 139

Negative Trace 1+ 2+ 3+ 4+ Glucose

Other substances in urine

Hyperglycemia associated Glycosuria

Renal associated Glycosuria Principle (Rgt Strip)

Copper Reduction test (Clinitest)

CSF protein = frequently tested -Det: TCA (preferred) and SSA SSA Reactions (Protein) No increase in turbidity < 6 mg/dL Distinct turbidity 6-30 mg/dL Noticeable turbidity w/ no granulation 30-100 mg/dL Turbidity w/ granulation but no flocculation 100-200 mg/dL Turbidity w/ granulation and flocculation 200-400 mg/dL Clumps of protein > 400 mg/dL Glucose Most frequently tested in urine Threshold substance Renal threshold = 160-180 mg/dL -Plasma concentration of a substance at w/c tubular reabsorption stops and  amount of substance in the urine ID: TLC 1. Fructose (Levulose):  fruits, honey syrup 2. Galactose: infants (Galactosemia: enzyme deficiencies) -Galactose-1-uridyltransferase deficiency -Galactokinase deficiency 3. Lactose -During lactation -Towards the end of pregnancy -Patient on strict milk diet -(+) Rubner’s test (Lead acetate) 4. Pentose -Xylose, arabinose -Xylulose:Benign pentosuria 5. Sucrose - Intestinal disorders -Nonreducing sugar -(-) Copper reduction test  Blood glucose,  Urine glucose 1. DM 2. Cushing’s syndrome/disease =  cortisol 3. Pheochromocytoma =  catecholamines 4. Acromegaly =  GH 5. Hyperthyroidism = T3/T4 N-Blood glucose, impaired tubular reabsorption of glucose 1. Fanconi’s syndrome: defective tubular reabsorption of glucose and amino acids Double sequential enzyme reaction: -Glucose oxidase -Peroxidase Chromogen: -KI (Brown) -Tetramethylbenzidine (Blue) Blue tablet Relies on the ability of glucose and other substances to reduce CuSO4 to Cu2O in the presence of alkali and heat CuSO4 (Blue) -------------> Cu2O (Brick red) lec.mt 04 |Page | 140

Pass through phenomenon (-) Glucose oxidase (+) Clinitest 1+ Glucose oxidase (-) Clinitest 4+ Glucose oxidase (-) Clinitest Ketones

Significance

Principle (Rgt Strip) Acetest

Hematuria

Hemoglobinuria Myoglobinuria Hgb vs. Mgb

Hemolytic anemia Principle (Rgt Strip) Hgb/Mgb Hematuria Extravascular lysis

Occurs if >2 g/dL sugar is present in urine Blue  Green  Yellow  Brick red  Blue (Pass through) To prevent, use 2 gtts urine (instead of 5 gtts) + 10 gtts H2O + Clinitest (+) Nonglucose reducing substance True glucosuria Small amount of glucose present False (+) Possible oxidizing agent interference on reagent strip Ketones Result from increased fat metabolism due to inability to metabolize CHO 78% BHA = major ketone but not detected 20% AA/Diacetic acid = parent ketone 2% Acetone Diabetic acidosis Insulin dosage monitoring Starvation Vomiting Legal’s test (Sodium nitroprusside reaction) AAA + Sodium nitroprusside --------------> (+) Purple (Acetone) (Glycine) Sodium nitroprusside Glycine Disodium phosphate Lactose Blood Cloudy red urine (Intact RBCs) Renal calculi GN Strenuous exercise Anticoagulants Clear red urine Intravascular hemolysis Clear red urine Rhabdomyolysis 1. Plasma examination -Hgb: Red/pink plasma, haptoglobin -Mgb: Pale yellow,  CK,  Aldolase 2. Blondheim’s test (Ammonium SO4): Precipitates Hgb Urine + 2.8g NH4SO4 (80% Satd.) ---(Filter/Centrifuge)---> Supernatant Supernatant: Red = Myoglobin = (+) Rgt strip Clear w/ red ppt. = Hemoglobin = (-) Rgt strip 1 hr post transfusion urine = Hgb Week after = Hemosiderin Pseudoperoxidase activity of hemoglobin Chromogen: TMB [(-) Yellow/(+){}Green  Blue {}] H2O2 + Chromogen ---(Heme)---> Oxidized chromogen + H2O Uniform green/blue Speckled/spotted  Unconjugated bilirubin  Urine and fecal urobilinogen lec.mt 04 |Page | 141

Intravascular lysis Bilirubin Significance Principle (Rgt Strip)

Ictotest

Urobilinogen Principle (Rgt Strip) Watson-Schwartz test

Hoesch test (Inverse Ehrlich reaction)

Hemolytic (ex. SCD) Hepatic Obstructive Nitrite

Principle (Rgt strip)

Significance Leukocyte esterase

(+) Hemoglobinuria  Haptoglobin and Hemopexin Bilirubin Amber urine w/ yellow foam Early indication of liver disease Hepatitis Cirrhosis Bile duct obstruction Diazo Reaction: Bilirubin diglucuronide + Diazo salt ---------> (+) Tan or pink purple Reagents: 2,4-Dichloroaniline diazonium salt 2,6-Dichlorobenzene diazonium salt (+) Blue to purple Reagents: p-nitrobenzene-diazonium-p-toluenesulfonate SSA Na2HCO3 Urobilinogen Afternoon specimen: 2PM-4PM (alkaline tide) Small amount in normal urine ( (+) Cherry red For differentiating urobilinogen and porphobilinogen from other Ehrlich’s reactive compounds 1. Urobilinogen: soluble to both chloroform and butanol 2. Porphobilinogen: insoluble to both chloroform and butanol 3. Other Ehrlich’s reactive compounds: soluble to butanol only Rapid screening test for urine porphobilinogen (≥2mg/dL) Hoesch reagent: Ehrlich reagent in 6M (6N) HCl 2 gtts urine + Hoesch rgt -------------> (+) Red Urine Bilirubin and Urobilinogen in Jaundice Urine Bilirubin Urobilinogen  /N/  /N (never report as negative) Nitrite 1st morning/4 hr urine Detection of bacteriuria (+) NO3: 100,000 orgs./mL UTI: ≥100,000 or ≥1 x 105 CFU/mL Greiss reaction: NO3 + Sulfanilamide + p-arsanilic acid + tetrahydrobenzoquinoline ------------> (+) Uniform pink Pink spots/edges = (-) Leukocytes UTI/inflammation Screening of urine culture specimen (+) Neutro/Eo/Baso/Mono (-) Lympho Maybe (+): Histiocytes, Trichomonas lec.mt 04 |Page | 142

Principle (Rgt strip)

30 seconds 40 seconds 45 seconds 60 seconds

120 seconds Vitamin C (Ascorbic acid)

SG pH Protein

Glucose

Ketones

Indoxyl carbonic acid ester + Diazonium salt ---(LE)---> Indoxyl + Acid indoxyl ----------> (+) Purple Strip can detect even lysed WBCs Reading Time (Reagent Strips) Glucose Bilirubin Ketones SG “PPBUN” pH Protein Blood Urobilinogen Nitrite Leukocytes 11th reagent pad Reducing property False (-) rgt strip: “BB LNG” -Blood -Bilirubin -Leukocytes -Nitrite -Glucose Rgt: Phosphomolybdate Phosphomolybdate + Vitamin C (≥5 mg/dL) --------> (+) Molybdenum blue

Sources of Error/Interference (Reagent Strips) False-positive False-negative High concentrations of protein Highly alkaline urines (>6.5) No known interfering substance Runover from adjacent pads Old specimens Highly buffered alkaline urine Proteins other than albumin Pigmented specimens, phenazopyridine Quarternary ammonium compounds (detergents) Antiseptics, chlorhexidine Loss of buffer from prolonged exposure of the reagent strip to the specimen High specific gravity Contamination by oxidizing agents and High levels of ascorbic acid detergents High levels of ketones High specific gravity Low temperatures Improperly preserved specimens Phthalein dyes Improperly preserved specimens Highly pigmented red urine Levodopa Medications containing free sulfhydryl grps

lec.mt 04 |Page | 143

Blood

Bilirubin Urobilinogen

Nitrite

Leukocytes

False-positive Strong oxidizing agents Bacterial peroxidases Menstrual contamination

Highly pigmented urines, phenazopyridine Indican (intestinal disorders) Metabolites of Lodine Porphobilinogen Indican p-aminosalicylic acid Sulfonamides Methyldopa Procaine Chlorpromazine Highly pigmented urine Improperly preserved specimens Highly pigmented urine

Strong oxidizing agents Highly pigmented urine, nitrofurantoin

Phase-contrast microscopy

Polarizing microscopy Interference contrast microscopy Sternheimer-Malbin Toluidine blue (Supravital) Lipid stains: ORO and Sudan III Gram stain Hansel stain Prussian blue stain

False-negative High specific gravity/crenated cells Formalin Captopril High concentration of nitrite Ascorbic acid >25 mg/dL Unmixed specimens Specimen exposure to light Ascorbic acid >25 mg/dL High concentrations of nitrite Old specimens Preservation in formalin

Nonreductase-containing bacteria Insufficient contact time between bacteria and nitrate Lack of urinary nitrate Large quantities of bacteria converting nitrite to nitrogen High concentrations of ascorbic acid High specific gravity High concentrations of protein, glucose, oxalic acid, ascorbic acid, gentamicin, cephalosporins, tetracyclines

Microscopic Exam of Urine Visualization of elements w/ low refractive indices: -Hyaline casts -Mixed cellular casts -Mucous threads -Trichomonas ID of cholesterol in OFB, FC and crystals Produces 3D microscopy-image and layer-by-layer imaging of a specimen 1. Hoffman microscope: modulation contrast microscope 2. Nomarski microscope: differential interference contrast microscope Crystal violet and safranin Nucleus and cytoplasm ID: WBCs, ECs, casts Enhances nuclear detail Differentiates WBCs and RTE Stain TG and neutral fats orange red ID: free fat droplets and lipid-containing cells and casts Differentiates Gram (+) and Gram (-) bacteria ID: bacterial casts Eosin Y and Methylene blue ID: Eosinophils Stains structures containing iron lec.mt 04 |Page | 144

RBCs

WBCs

Addis count

Squamous epithelial cells

Transitional epithelial cells (Urothelial cells) Renal tubular epithelial cells

Oval fat body Bubble cells Yeast T. vaginalis

ID: yellow-brown granules of hemosiderin in cells and casts Sediment Constituents NV = 0-2 or 0-3/hpf Hypertonic: crenated, shrink Hypotonic: Ghost cells, swell, hemolyzed Dysmorphic: glomerular membrane damage, w/ projections, fragmented Sources of error: -Yeasts -Oil droplets -Air bubbles -CaOx crystals ♫ Remedy: add 2% acetic acid -RBCs: lysed -Other cells: intact NV = 0-5 or 0-8/hpf Glitter cells (Hypotonic urine) -Granules swell -Brownian movement >1% eosinophils: significant - Drug-induced allergic reaction - Inflammation of renal interstitium Quantitative measure of formed elements of urine using hemacytometer Specimen: 12 hr urine Preservative: Formalin NV: a. RBCs: 0-500,000/12 hr urine b. WBCs: 0-1,800,000/12 hr urine c. Hyaline Casts: 0-5000/ hr urine Largest cell in the urine sediment From linings of vagina, female urethra and lower male urethra ♫ Variation: Clue cells: -EC w/c are studded w/ bacteria (bacterial vaginosis) -Whiff/Sniff test: vaginal discharge + 10% KOH  Fishy amine-like odor -Culture: G. vaginalis = HBT medium Spherical, polyhedral, or caudate w/ centrally located nucleus Derived from the linings of the renal pelvis, ureter, urinary bladder, male urethra (upper portion) Not clinically significant in small numbers Rectangular, polyhedral, cuboidal or columnar w/ an eccentriac nucleus, possibly bilirubin stained or hemosiderin laden From nephron: -PCT: rectangular, columnar/convoluted -DCT: round/oval >2 RTE/hpf: tubular injury Lipid containing RTE cells Lipiduria (Ex. nephrotic syndrome) Cholesterol: Maltese cross RTE cells w/ nonlipid containing vacuoles Acute tubular necrosis C. albicans (DM, vaginal moniliasis) Flagellate w/ jerky motility lec.mt 04 |Page | 145

S. haematobium E. vermicularis Casts (Cylindruria)

Hyaline casts

RBC casts WBC casts

Bacterial casts Epithelial cell casts Coarse/Fine granular casts

Fatty casts Waxy casts

Broad casts

Sediment preparation

RCF

Pingpong disease “Hematuria” Specimen: 24 hr unpreserved urine Most common fecal contaminant Formed in the DCT and CD ♫ Tamm-Horsfall protein (Uromodulin) -Major constituent -Glycoprotein secreted by RTE cells of DCT and CD NV = 0-2/lpf Beginning of all types of casts (prototype cast) a. Physiologic: - Strenuous exercise (HC, GC, RC) - Heat b. Pathologic: - GN - PN - CHF Bleeding w/in the nephron a. GN b. Strenuous exercise (HC, GC, RC) Inflammation w/in the nephron Differentiates upper UTI (pyelonephritis, w/ cast) from lower UTI (cystitis, no cast) To differentiate from EC cast: 1. Phase contrast microscopy 2. Supravital stain Seen in: -PN -AIN Pyelonephritis Renal tubular damage Advanced tubular destruction Formed from the disintegration of cellular cast GN PN Strenuous exercise (HC, GC, RC) Nephrotic syndrome: lipiduria Not stained by Sternheimer-Malbin Final degenerative form of all types of casts Stasis of renal flow Chronic renal failure Brittle, highly refractile, w/ jagged ends “Renal failure casts” Extreme urine stasis Widening and destruction of tubular walls Any type of cast can be broad Urine  Centrifuge: 400 RCF for 5 mins  Decant  Remaining: 0.5mL/1.0mL Urine sediment: 20μL (0.02 mL) -10 lpf -10 hpf -Reduced light 1.118 x 10-5 x radius (cm) x (rpm)2 lec.mt 04 |Page | 146

Amorphous Urates (Normal) (pH: acid) Uric Acid

Calcium Oxalate (Normal) (pH: acid/alkaline/neutral)

Amorphous Phosphates (Normal) (pH: alkaline/neutral) Ammonium Biurate (Normal) (pH: alkaline) Triple Phosphate (Normal) (pH: alkaline) Calcium Phosphate (Normal) (pH: alkaline/neutral)

Calcium Carbonate (Normal) (pH: alkaline) Cystine (Abnormal) (pH: acid) Cholesterol (Abnormal) (pH: acid)

Tyrosine (Abnormal) (pH: acid/neutral) Leucine (Abnormal) (pH: acid/neutral) Bilirubin (Abnormal)

Urine Crystals Yellow-brown granules Pink sediment (Uroerythrin) Mistaken as cystine crystals Rhombic, wedge, rosette, hexagonal, four-sided plate (whetstone) Lemon-shaped (Henry)  Lesch-Nyhan syndrome: orange sands in diaper  Gout  Chemotherapy 1. Weddelite = dihydrate -Envelope/pyramidal 2. Whewellite = monohydrate -Oval, dumbbell -Ethylene glycol poisoning (antifreeze agent) Most renal stones consist of CaOx White precipitate Granular appearance  After meal (alkaline tide) Yellow-brown Thorny apples Old specimen: due to the presence of urea-splitting bacteria A.k.a. Magnesium ammonium phosphate Coffin lid, “Struvite”, staghorn appearance Presence of urea-splitting bacteria Colorless, flat rectangular plates or thin prisms often in rosette formation Rosettes may resemble sulfonamides -To differentiate: CaPO4 dissolves in acetic acid 1. Calcium Phosphate = Apatite 2. Basic Calcium Phosphate = Hydroxyapatite 3. Calcium Hydrogen Phosphate = Brushite Small and colorless Dumbbell or spherical shapes Acetic acid: (+) Effervescence Colorless hexagonal plates Cystinuria Rectangular plate w/ notch in one or more corners Staircase pattern Lipiduria (Nephrotic syndrome) Resemble crystals of RCM, to differentiate a. Patient history b. Correlate w/ other UA results c. RCM: SG by refractometer ≥1.040 Colorless to yellow needles Liver disease (more common) (+) Nitroso-naphthol Yellow-brown spheres w/ concentric circles and radial striations Liver disease Clumped needles or granules w/ yellow color (+) Diazo reaction lec.mt 04 |Page | 147

(pH: acid) Sulfonamide (Abnormal) (pH: acid/neutral)

Ampicillin (Abnormal) (pH: acid/neutral)

Liver disease Colorless to yellow brown Deposits in nephrons Tubular damage Needles, sheaves of wheat, rosette (res. CaPO4 rosette) ♫ Lignin test: Newspaper = urine + 25% HCl  (+) Yellow orange color Massive doses Colorless needles

Uric Acid Cystine Color Yellow brown Colorless Solubility in NH3 Soluble Soluble Solubility in dilute HCl Insoluble Soluble Birefringence (Polarizing microscope) + Cyanide-Nitroprusside test + Urinary Sediment Artifacts 1. Starch granules (gloves): -Most common -Maltese cross (O, FC, S) -Dimpled center 2. Oil droplets 3. Air bubbles 4. Pollen grains = spheres w/ concentric circles 5. Hair and fibers = (+) Birefringence (polarizing microscope) 6. Fecal contaminants Cystitis

Urethritis

Glomerulonephritis Pyelonephritis Acute Interstitial Nephritis Renal carcinoma Nephrotic syndrome

Renal Diseases Inflammation of urinary bladder Infection WBCs, RBCs, bacteria NO CAST Inflammation of urethra WBCs, RBCs Usually NO BACTERIA on routine UA a. Male: GS of urethral exudates [Gram (-) diplococcic] b. Female: pelvic exam for vaginitis and cervicitis Inflammation of the glomerulus Immune-mediated RBCs, WBCs, RBC CASTS, WBC casts, hyaline and granular casts Infection of renal tubules WBCs, RBCs, bacteria, RBC casts, WBC CASTS, hyaline and granular casts Infection of the renal interstitium RBCs, WBCs, WBC casts, NO BACTERIA 1’ = RCC 2’ = Transitional CC RBCs and WBCs Massive proteinuria and lipiduria a. Serum (Chemistry) - Albumin, alpha1, beta and gamma globulins - alpha2 (AMG) b. Urine (CM) - Albumin, alpha1, beta and gamma globulins lec.mt 04 |Page | 148

Telescoped sediments

UTI

Rapidly progressive (Crescentic) GN Goodpasture syndrome Wegener’s granulomatosis Henoch-Schönlein purpura Membranous GN Membranoproliferative GN Chronic GN IgA nephropathy (Berger’s disease) Nephrotic syndrome Minimal change disease (Lipoid nephrosis) FSGS Alport syndrome Diabetic Nephropathy (Kimmelstiel-Wilson disease) Acute tubular necrosis Fanconi syndrome Nephrogenic DI Neurogenic DI Renal glucosuria Cystitis Acute PN Chronic PN

Aminoaciduria

-(-) alpha2 (AMG) -Oval fat bodies, fatty and waxy casts Simultaneous appearance of the elements of acute/chronic GN and nephrotic syndrome  Cells and Casts a. Lupus nephritis b. SBE E. coli = 90% cases of UTI S. saprophyticus = UTI among sexually active young females G. vaginalis = bacterial vaginosis S. pyogenes = AGN and ARF Viridans Streptococci = SBE Deposition of immune complex from systemic immune disorders on the glomerular membrane Attachment of cytotoxic antibody to glomerular and alveolar basement membrane Antineutrophilic cytoplasmic autoantibody Occurse in children following viral respiratory infection Decrease in platelets disrupts vascular integrity Thickening of the glomerular membrane following IgG immune complex deposition Cellular proliferation affecting the capillary walls or the glomerular basement membrane Marked decrease in renal function resulting from glomerular damage precipitated by other renal disorders Deposition of IgA on the glomerular membrane Disruption of the electrical charges that produce tightly fitting podocyte barrier Disruption of the podocytes occurring primarily in children following allergic reaction and immunization Disruption of podocytes in certain areas of glomeruli associated w/ heroin and analgesic abuse and AIDS Lamellated and thinning of glomerular basement membrane Most common cause of ESRD Microalbuminuria Damage to the renal tubules caused by ischemia or toxic agents Generalized defect in renal tubular reabsorption in the PCT Inability of the renal tubules to respond to ADH Inability of the hypothalamus to produce ADH Inability of the renal tubules to reabsorb glucose Ascending bacterial infection of the bladder Infection of the renal tubules and interstitium Recurrent infection of the renal tubules and interstitium Visicoureteral reflux: most common cause -Reflux of urine from the bladder back into the ureters Screening for Metabolic Disorders 1. Overflow type  AA in blood  AA in urine Ex. PKU, alkaptonuria, MSUD lec.mt 04 |Page | 149

2. Renal type N-AA in blood Impaired tubular reabsorption of AA Ex. Cystinuria (COLA), Fanconi’s syndrome Phenylalanine-Tyrosine Disorders Phenylalanine

(-)

PAH

PKU

Phenylpyruvic acid

Tyrosinemia

Tyrosyluria:

Tyrosine Tyrosine transaminase

(-)

p-Hydroxyphenylpyruvic acid p-Hydroxyphenylpyruvic acid oxidase

Homogentisic acid

p-OHPPA

(-)

Homogentisic acid oxidase

Maleylacetoacetic acid

p-OHPLA Alkaptonuria Homogentisic acid

Fumarylacetoacetic acid Fumaric acid and Acetoacetic acid

Phenylketonuria

Tyrosyluria

Alkaptonuria

Melanuria

MSUD Organic acidemias

Indicanuria

Severe mental retardation Mousy odor (-) PAH Screen: FeCl3  (+) Blue-green Confirm: Guthrie test (Bacterial inhibition) -B. subtilis -Inhibitor: Beta2-thienylalanine (neutralized by phenylalanine) -Growth = (+) PKU -No growth = (-) PKU Rancid butter odor (-) Tyrosine transaminase and p-OHPPA oxidase Screen: FeCl3  (+) Transient green Confirm: Nitroso-naphthol  (+) Orange-red Urine darkens after a period of standing (-) Homogentisic acid oxidase  Homogentisic acid in blood and urine FeCl3  (+) Transient blue Clinitest/Benedict’s  (+) Yellow ppt. Overproliferation of melanocytes FeCl3  Gray or black ppt. Ehrlich’s  Red Branched-Chain Amino Acid Disorders Accumulation of leucine, isoleucine and valine in blood and urine 2,4-DNPH  (+) Yellow turbidity/ppt. 1. Isovaleric acidemia = sweaty feet 2. Propionic acidemia 3. Methylmalonic acidemia Tryptophan Disorders Intestinal disorder lec.mt 04 |Page | 150

Argentaffinoma

Cystinuria (Renal type)

Cystinosis Homocystinuria Brand’s modification of Legal’s nitroprusside MPS Clinical significance

CTAB Lesch-Nyhan disease D-ALA Porphobilinogen Uroporphyrinogen

Coproporphyrinogen Protoporphyrinogen Protoporphyrin IX Heme Porphyrias

Blue color Hartnup disease: “Blue diaper syndrome” Obermayer’s test: FeCl3  (+) Violet w/ chloroform Carcinoid tumor involving argentaffin cells  5-HIAA: metabolite of serotonin FeCl3  (+) Blue-green (PKU) Nitrosonaphthol  (+) Violet w/ HNO3 Be sure patient should avoid banana, pineapple, tomatoes (serotonin-rich) Cystine Disorders Defect in renal tubular transport of: -Cystine (least soluble  urine) -Ornithine -Lysine -Arginine Inborn error of metabolism Cystine deposits in many areas of the body Cyanide-nitroprusside  (+) Red-purple Defect in the metabolism of homocystine Silver nitroprusside  (+) Red-purple Rxn: Cyanide-nitroprusside  (+) Red-purple Mucopolysaccharide Disorders Dermatan SO4 Keratan SO4 Heparan SO4 Alder-Reilly syndrome Hurler syndrome = MPS  cornea of the eye Hunter syndrome = Sex-linked recessive Sanfilippo syndrome = Mental retardation only (+) White turbidity Purine Disorders  Urinary uric acid crystals Porphyrias Glycine + Succinyl CoA ----(ALA synthetase)----> D-ALA D-ALA ----(ALA synthetase)----> Porphobilinogen Lead poisoning: inhibits ALA synthase Porphobilinogen -----(Uroporphyrinogen synthase/ Uroporphyrinogen cosynthase)----> Uroporphyrinogen Acute intermittent porphyria: (-) Uroporphyrinogen synthase Congenital erythropoietic porphyria: (-) Uroporphyrinogen cosynthase Uroporphyrinogen ---------(Uroporphyrinogen decarboxylase)---------------> Coproporphyrinogen Porphyria cutanea tarda: (-) Uroporphyrinogen decarboxylase Coproporphyrinogen ---------(Coproporphyrinogen oxidase)-------------------> Protoporphyrinogen Hereditary coproporphyria: (-) Coproporphyrinogen oxidase Protoporphyrinogen ---(Protoporphyrinogen oxidase)---> Protoporphyrin IX Variegate porphyria: (-) Protoporphyrinogen oxidase Protoporphyrin IX + Fe2+ ----(Ferrocheletase)--------------> Heme Lead poisoning: inhibits Ferrocheletase Vampire disease Disorders of porphyrin metabolism lec.mt 04 |Page | 151

Screening tests (porphyria)

Specimens

Lead poisoning (+) White Ring (+) Violet (+) White turbidity/ cloudiness

(+) coagulum (24 hrs) (+) coagulum (72’C for 5mins) Benedict’s Seliwanoff’s Rubner’s Bial Orcinol Tauber’s Others

Frommer’s Rothera’s Lange Acetest/Ketostix Gerhardt’s Gmelin Smith

1. Ehrlich reaction = (+) D-ALA and porphobilinogen 2. Fluorescence at 550-600nm = Uro/Copro/Protoporphyrin = (+) Red/pink/violet = (-) Blue 3. Free Erythrocyte Protoporphyrin (FEP) = CDC recommended test for Lead poisoning Urine: red/purple/portwine (normal: Lead poisoning) Stool Blood Bile RBC inclusion coarse basophilic stippling Qualitative Tests for Protein Heller’s Robert’s Spiegler’s Biuret (Albumin) Heat and acetic acid SSA Purdy’s Potassium ferrocyanide Picric acid Kingsbury-Clark (Rgt: SSA) Esbach’s -Rgt: Picric acid + Citric acid Tsuchiya’s Kwilecki’s -Rgt: Esbach’s + 10% FeCl3 Qualitative Tests for Sugars Reducing substances Rgt: Resorcinol Fructose  (+) Red Rgt: Lead acetate, NH3 H2O Lactose  (+) Bright red w/ red ppt. Glucose  (+) Red color w/ yellow ppt. Pentose  (+) Green Pentose  (+) Green Osazone or phenylhydrazine (Kowarsky) Nylander’s Moore Heller Borchardt’s Qualitative Tests for Ketones Acetone  (+) Purplish red ring Acetone & AAA  (+) Purple ring Acetone & AAA  (+) Purple ring Acetone  (+) Purple AAA  Bordeaux red Qualitative Tests for Bile Pigments Bile  (+) Play of colors Bile  (+) Emerald green lec.mt 04 |Page | 152

Harrison’s spot Ictotest Wallace and Diamond Schlesinger Benzidine Guiac Ortho-toluidine FeCl3 (Screening) Thomahlen Blackberg & Wanger Fantus Mercurimetric titration (Schales & Schales) Sulkowitch

Bile  (+) Blue to green Bile  (+) Blue to purple mat Rgt: PDAB Urobilinogen  (+) Cherry red Rgt: Lugol’s iodine, Alc. Zinc acetate Urobilin  (+) Greenish fluorescence Qualitative Tests for Hemoglobin (+) Green-blue (+) Blue (+) Blue Qualitative Tests for Melanin (+) Black (after 24 hrs) (+) Dark green or blue color (fresh urine) (+) Brown to black ppt. (24 hr urine) Qualitative Tests for Chloride (+) Reddish ppt (+) Blue-violet colored complex Qualitative Test for Calcium (+) Precipitation Renal Function Tests

Test for Glomerular filtration Test for Tubular reabsorption

Fishberg test Test for Tubular Secretion and Renal Blood flow Tests for NPN BCR (BUN: Crea Ratio)

Biohazard Symbol

PPE

Clearance Concentration tests -Fishberg (old) -Mosenthal (old) -SG (new) -Osmolality (new) Patient deprived of fluid for 24 hrs = SG ≥1.026 Patient deprived of fluid for 12 hrs = SG ≥1.022 PAH PSP 1. Urea: Urease, DAM (NV = 6-17 g/24 hr urine) 2. Creatinine: Jaffe (NV = 1-2 g/24 hr urine) 3. Uric acid: Uricase, PTA (NV = 0.25-0.75 g/24 hr urine) a. NV = 10:1 -BUN: 90% excreted, 10% reabsorbed -Crea: 99% excreted, 1% reabsorbed b. Renal disease: Normal ratio  BUN,  Crea c. Pre- and Post-renal disease:  Ratio  BUN, N-crea Other Topics 4 circles Top = Source Left = Host Right = Transmission Gloves Fluid-resistant gowns Eye and face shields Plexiglas countertop shields lec.mt 04 |Page | 153

Disinfection of sink Sodium hypochlorite soln. Chemical spills on skin Always add acid  water Handwashing

Hazard Classification (Yellow = Reactivity)

Hazard Classification (White = Specific Hazard)

Hazard Classification (Blue = Health)

Hazard Classification (Red = Flammability)

Types of Fire

In case of Fire

Fire extinguisher

Urinalysis Hippocrates Frederik Dekkers Thomas Bryant Thomas Addis Richard Bright UA (CLSI/NCCLS)

1:5 or 1:10 dilution of sodium hypochlorite (daily) Effective for 1 month Flush the area w/ water for at least 15 mins  seek medical attention Do not neutralize chemicals To avoid the possibility of sudden splashing caused by the rapid generation of heat in some chemical reactions Best way to break the chain of infection Clean between finger for at least 15 seconds Downward Sing “Happy Birthday” 0 = Stable 1 = Unstable if heated 2 = Violent chemical change 3 = Shock and heat may deteriorate 4 = May deteriorate OXY = Oxidizer ACID = Acid ALK = Alkali COR = Corrosive W = Use no water  = Radiation 0 = Normal material 1 = Slightly hazardous 2 = Hazardous 3 = Extreme danger 4 = Deadly 0 = Will not burn 1 = Above 200’F 2 = Below 200’F 3 = Below 100’F 4 = Below 73’C A = Wood, paper, clothing (ordinary combustibles) B = Flammable liquids C = Electrical equipment D = Flammable metals E = Detonation (Arsenal) R = Rescue A = Activate the alarm C = Contain the fire E = Extinguish P = Pull the pin A = Aim at the base of the fire S = Squeeze handles S = Sweep nozzle side to side Actually the beginning of laboratory medicine Uroscopy Discovered albuminuria by boiling urine Pisse prophets (charlatans) Examination of urinary sediment Introduced the concept of UA as part of doctor’s routine patient examination Defined as the testing of urine with procedures commonly performed in an expeditious, reliable, accurate, safe and cost-effective manner lec.mt 04 |Page | 154

Care of Reagent Strips

QC (Reagent Strips)

Resolution (Microscope) Parfocal (Microscope) Centering and Köhler illumination (condenser) Camel-hair brush Quality assessment

1. Store w/ dessicant in an opaque, tightly closed container 2. Store below 30’C. Do not freeze 3. Do not expose to volatile fumes 4. Do not use past the expiration date 5. Do not use if chemical pads become discolored 6. Removed strips immediately prior to use 1. Test open bottles of reagent strips w/ known positive and negative controls every 24 hr (some: at the beginning of each shift) 2. Resolve control results that are out of range by further testing 3. Test reagents used in backup tests w/ positive and negative controls 4. Perform positive and negative controls on new reagents and newly opened bottles of reagent strips 5. Record all control results and reagent lot numbers Ability to visualize fine details Ability of the lens to distinguish two small objects that are a specific distance apart Require only minimum adjustment when switching among objectives Provide optimal viewing of the illuminated field

Removes dust coating the optical surface of the microscope Overall process of guaranteeing quality patient care and is regulated throughout the total testing system Quality system Refers to all of the laboratory’s policies, processes, procedures, and resources needed to achieve quality testing Accreditation agencies JCAHO (Joint Commission on the Accreditation of Healthcare Organizations) CAP (College of American Pathologists) AABB (American Association of Blood Banks) AOA (American Osteopathic Association) ASHI (American Association of Histocompatibility and Immunogenetics) COLA (Commission on Laboratory Assessment) NCCLS (National Committee New: CLSI (Clinical and Laboratory Standards Institute) for Clinical Laboratory Standards) Policy for Handling 1. Do NOT assume any information about the specimen or patient Mislabeled Specimens 2. Do NOT relabel an incorrectly labeled specimen 3. Do NOT discard the specimen until investigation is complete 4. Leave specimen EXACTLY as you receive it; put in the refrigeration for preservation until errors can be resolved 5. Notify floor, nursing station, doctor’s office, etc. of problem and why it must be corrected for analysis to continue 6. Identify problem on specimen requisition with date, time and your initials 7. Make person responsible for specimen collection participate in solution of problem(s). Any action taken should be documented on the requisition slip 8. Report all mislabeled specimens to the quality assurance board Preanalytical Factors Test requests Patient preparation Specimen collection, handling and storage Analytical Factors Reagents Instrumentation and equipment Testing procedure QC lec.mt 04 |Page | 155

None Rare Few Moderate Many Casts (lpf) RBCs (hpf) WBCs (hpf) Preanalytical

Analytical

Postanalytical

TQM CQI PDCA PDSA

Preventive maintentance Access to procedure manuals Competency of personnel performing the tests Microscopic Quantitations EC (lpf) Crystals (hpf) Bacteria (hpf) Mucous threads 0 0 0 0-5 0-2 0-10 0-1 5-20 2-5 10-50 1-3 20-100 5-20 50-200 3-10 >100 >20 >200 >10 None = 0 Numerical ranges = 0-2/2-5/5-10/>10 None = 0 Numerical ranges = 0-2/2-5/5-10/10-25/25-50/50-100/>100 None = 0 Numerical ranges = 0-2/2-5/5-10/10-25/25-50/50-100/>100 Quality Assurance Errors Patient misidentification Wrong test ordered Incorrect urine specimen type collected Insufficient urine volume Delayed transport of urine to the laboratory Incorrect storage or preservation of urine Sample misidentification Erroneous instrument calibration Reagent deterioration Poor testing technique Instrument malfunction Interfering substances present Misinterpretation of quality control data Patient misidentification Poor handwriting Transcription error Poor quality of instrument printer Failure to send report Failure to call critical values Inability to identify interfering substances Based on a team concept involving personnel at all levels working together to achieve a final outcome of customer satisfaction through implementation Improving patient outcomes by providing continual quality care in a constantly changing health-care environment Plan-Do-Check-Act Plan-Do-Study-Act

lec.mt 04 |Page | 156

CSF

Production Functions CSF glucose Brain Meninges (Sing. Meninx)

Layers Arachnoid villi/ granulations Choroid plexuses CSF Total Volume

Blood brain barrier

Circumventricular organs

Specimen collection

Precautions

OTHER BODY FLUIDS Cerebrospinal Fluid 1st noted by Cotugno Not an ultrafiltrate of plasma Na+, Cl-, Mg2+: CSF than in plasma K+, Total Ca2+: CSF than in plasma 3rd major body fluid Filtration Active transport secretion Supply nutrients  nervous tissue Remove metabolic waste Provide mechanical barrier 60-70% of blood glucose 1,500g (Henry) Three layers: 1. Dura Mater = outermost - Meningeal layer next to the bone 2. Arachnoid mater (Arachnoidea) = spider web ♫ Subarachnoid space: where the CSF is flowing 3. Pia Mater = innermost Skin  Skull  Dura Mater  Arachnoid mater  Subarachnoid space  Pia mater  Brain Reabsorbs CSF If it can’t absorb CSF, CSF accumulates  Hydrocephalus Produce CSF at approximately 20 mL/hr Adults: 5th ed. = 90-150 mL 4th ed = 140-170 mL Neonates = 10-60 mL Between brain and blood Functions: 1. Protects brain from organisms 2. Shields brain from hormones and neurotransmitters 3. Maintains homeostasis for brain Regions of the brain where BBB is weak 1. Pineal gland: melatonin, associated w/ circadian rhythms 2. Neurohypophysis (posterior pituitary): ADH, oxytocin 3. Area postrema: vomiting center of the brain 4. Subfornical organ 5. Vascular organ of the lamina terminalis 6. Median eminence Lumbar tap: routine (collected by physician) -Bet. 3rd, 4th and 5th lumbar vertebrae -Fetal position Cisternal puncture: suboccipital region Ventricular puncture: infants w/ open fontanels Measurement of intracranial pressure Prevent infection (povidone iodine) Prevent damage  neural tissue

lec.mt 04 |Page | 157

Collection

Note

Low volume specimen Crystal clear Turbidity/cloudiness

Oily Hemolyzed/Bloody Clotted, pellicle Xanthochromia

Causes of Visible CSF Xanthochromia

CSF Protein

Distribution of Blood Clot Xanthochromia (+) Clot (-) Blood (+) Weblike pellicle (+) D-dimer Recent hemorrhage

3 Sterile Tubes 1. Chemistry/Serology (Frozen) 2. Microbiology (Room temp) 3. Hematology (Refrigerated) [4. Microbiology] Do not use glass tubes (Henry): Cells will adhere to glass surface producing erroneous low counts on Tube 3 Excess CSF ---(DO NOT)--> Discard Left-over supernatant  Chemistry/Serology CSF specimen  STAT If STAT not possible, specimens are stored Collected on 1 tube Microbiology  Hematology  Chemistry/Serology CSF Appearance Normal WBC > 200μL RBC > 400μL (+) Microorganisms RCM Aspirated fat  Protein (>45 but 150mg/dL Merthiolate contamination Carotenoids (orange) Melanin (brownish): meningeal metabolic melanoma Collection of CSF 2-5 days after traumatic tap In normal neonates: because of immature BBB NV = 15-45mg/dL >45 but 150mg/dL (xanthochromic) Intracranial Hemorrhage vs. Traumatic Tap Traumatic Tap Intracranial/Cerebral Hemorrhage Uneven (Tube 1 > 2 > 3) Even (Tube 1 = 2 = 3) + (Plasma Fibrinogen) + Meningitis Froin syndrome Blockage of CSF circulation After overnight refrigeration (12-24 hrs) TB meningitis Indicates formation of fibrin at a hemorrhage site Clear supernatant lec.mt 04 |Page | 158

Erythrophagocytosis/ Hemosiderin granules CSF Cell count WBC count

Total CSF cell count RBC count Clarity/Appearance Slightly Hazy Hazy Slightly Cloudy Cloudy/Slightly Bloody Bloody/Turbid CSF WBC Count Correction Differential Count Methods for specimen concentration Cytocentrifugation

CSF Differential count

To examine a bloody fluid for xanthochromia: Microhematocrit tube ---(Centrifuge)---> Examine supernatant against white BG Indicates intracranial hemorrhage Done immediately WBCs and RBCs lyse w/in 1 hr Refrigerate if cannot be processed immediately Routinely performed on CSF Diluting fluid: 3% Acetic acid NV (adults) = 0-5 WBC/μL NV (neonate) = 30 mononuclear WBCs/μL Vol. of 1 square = 0.1 μL Formula (Improved Neubauer counting chamber): WBC count = No. of cells x Dilution factor No. of sq. ctd x vol. of 1 sq. -For diluted and undiluted specimen -Neubauer counting chamber = No small RBC square (Improved NCC: w/ small RBC squares) -Four large squares (corner) and central large square on both sides of the hemocytometer Methylene blue = stains WBCs for better differentiation WBC ct + RBC ct Diluting fluid: 0.85% (0.9%) NSS RBC count = Not counted Used for the correction of CSF WBC count and CSF protein count when a traumatic tap has occurred Dilution Amount of Sample Amount of Diluent 1:10 30 μL 270 μL 1:20 30 μL 570 μL 1:100 30 μL 2970 μL 1:200 30 μL 5970 μL 1:10,000 0.1 mL of 1:100 dilution 9.9 mL WBC (added) = WBCBlood x RBCCSF or RBCBlood PBS (normal) = -1 WBC/700 RBCs (CSF) Performed on stained smears Specimen should be concentrated Sedimentation Filtration Centrifugation Cytocentrifugation Fluid  Conical chamber Cells are forced into a monolayer w/in a 6mm diameter circle on the slide Addition of albumin:  cell yield/recovery  cellular distortion Neonates (0-2 mos.) 50-90% monocytes 5-35% lymphocytes 0-8% neutrophils Children (2 mos.-18 y.o.) -Not yet established lec.mt 04 |Page | 159

Normal Adult Neonates Pleocytosis Lymphocytes Monocytes Neutrophils nRBCs (metarubricytes) Eosinophils

Plasma cells Macrophages Nonpathologically significant cells Malignant cells

Biweekly basis Monthly basis CSF protein

Adults (>18 y.o.) 40-80% lymphocytes 15-45% monocytes 0-6% neutrophils Cells in the CSF Lymphocytes Monocytes Neutrophils (occasional) Lymphocytes > Monocytes (70:30) Monocytes > Lymphocytes  no. of normal cells  amount of WBCs in any body fluid  viral, TB, fungal meningitis  multiple sclerosis  bacterial meningitis, cerebral hemorrhage  early cases of viral, TB, fungal meningitis BM contamination  helminthic parasitic infection  fungal infection (C. immitis)  medications and shunts Protozoans do not induce eosinophilia Charcot-Leyden crystals (E. histolytica)  eosinophils concentrate at the intestines and are degraded  multiple sclerosis  lymphocytic reactions (+) RBCs (+) RCM Choroidal cells: EC (choroid plexus) Ependymal cells Spindle-shaped cells: arachnoid Hematologic: -lymphoblasts -myeloblasts -monoblasts Nonhematologic -Astrocytomas -Retinoblastomas -Medulloblastomas QC of CSF and Other Body Fluid Cell Count All diluents ---(check for)---> Contamination Speed of cytocentrifuge should be checked w/ a tachometer CSF Protein Most frequently tested chemical test NV: Adults = 15-45 mg/dL Infants = 150 mg/dL Premature = 500 mg/dL Albumin = majority α-globulins = Haptoglobin β-globulins = Tau transferrin (major) γ-globulins = IgG (major), IgA (small amount) IgM, fibrinogen, β-LPP = not found in normal CSF lec.mt 04 |Page | 160

Transthyretin (prealbumin) Tau Transferrin  CSF protein  CSF protein

Correction for traumatic tap Measurement of Total CSF protein

CSF IgG index  CSF IgG CSF/Serum Albumin index

IgG index

Oligoclonal bands

Oligoclonal bands (not seen in serum)

Oligoclonal bands in serum but not in CSF

TRANSports THYroxine and RETINol 2nd most prevalent CHO-deficient β2-transferrin Seen in CSF and not in serum Electrophoresis: method of choice when determining if a fluid is actually CSF Damage: BBB (most common) Production of Ig in CNS (multiple sclerosis) CSF leakage Recent puncture Rapid CSF production Water intoxication If blood Hct and serum protein are normal: = -1 mg/dL protein/1,200 RBCs 1. Turbidimetric -Principle: precipitation of protein a. TCA = precipitates albumin and globulins (preferred) b. SSA = precipitates albumin, add Na2SO4 to precipitate globulins 2. Dye-binding technique -Principle: protein error of indicators -Coomassie Brilliant blue G250 (red) -Beer’s law -Protein binds to dye = red to blue In MS:  CNS IgG CNS IgG must be differentiated from serum IgG (damage to BBB) Damage to BBB Active production w/in CNS (MS) _CSF Albumin (mg/dL)_ Serum Albumin (g/dL) a. Index 0.70 = indicative of IgG production w/in CNS (MS) b. Index 35 mg/dL >25 35 mg/dL

Produces serum and CSF banding Symptoms: -Abnormal sensations: numbness, tingling, pain, burning, itching -Mood swings Protein component of the lipid-protein complex that insulate the nerve fibers Monitor the progress of MS Identify individuals w/ MS who do not show oligoclonal bands (approx. 10%) In patients w/ dementia Suggests CJD – caused by prions “Mad cow disease” in cattle Live proteins Infectious protein Spongiform encephalopathy (CJD)  CSF levels of microtubule associated Tau protein and decreased levels of βAP42 have been shown to significantly increase the accuracy of Alzheimer’s disease Meningitis Neonates – 1 month 1 month – 5 years old 5 – 29 years old >29 years old Infants Elderly Immunocompromised patients CSF Glucose NV = 60-70% of the plasma glucose concentration A plasma glucose must also be run for comparison Diagnostic significance: - values - CSF glucose values = result of  plasma glucose Bacterial, TB, fungal meningitis Viral meningitis CSF Lactate Frequently used to monitor severe head injuries False elevations: Xanthochromia/hemolysis (RBC contains lactate) Normal lactate Bacterial meningitis Fungal and TB meningitis Viral meningitis CSF Enzymes Serum: LD 2 > 1 > 3 > 4 > 5 [MI: LD 1 > 2 > 3 > 4 > 5] CSF (normal): LD 1 > 2 > 3 > 4 > 5 Neurologic abnormalities: LD 2 > 1 Bacterial meningitis: LD 5 > 4 > 3 > 2 > 1  Postcardiac arrest (poor prognosis) 5 days  abstinence =  volume,  motility Important: 1st portion of ejaculate 1. Masturbation: best 2. Coitus interruptus (withdrawal method) 3. Common condom collection -Condoms for sperm collection: a. Silastic (Silicone rubber) b. Polyurethane condoms: called the Male Factor Pak 4. Aspiration of semen from the vaginal vault after coitus 5. Specimen should be delivered in the lab w/in 1 hr (RT’) Specimen kept at 37’C awaiting analysis For artificial insemination, it can be preserved in frozen state and stored at -85’C (seminal banks) Fresh specimen is clotted Semen Analysis 30-60 mins If not yet liquefied after 2 hrs, use α-chymotrypsin Color = grayish white Volume = 2-5 mL Viscosity = pour in droplets pH = 7.2-8.0 Sperm concentration = >20,000,000/mL Sperm count = >40,000,000/ejaculate Motility = >50% w/in 1 hr Motility quality = >2.0 or a, b, c after 1 hr WBCs = 1,000,000/mL = inflammation : incomplete collection/infertility : prolonged abstinence Prolonged abstinence Medication Urine contaminationi Infection ( WBCs) (+) RBCs 0 (watery) 4 (gel-like) Too basic = infection Too acidic =  prostatic fluid lec.mt 04 |Page | 164

Sperm concentration

Diluting fluid: 1. Cold H2O 2. Formalin 3. NaHCO3 4. 0.5% in chlorazene 5. 1% formalin in 3% trisodium citrate 1:20 = mechanical positive displacement pipette Counting chamber 1. Neubauer counting chamber = diluted specimen (WHO recommended) 2. Makler chamber = undiluted w/ heating processes Purpose of Dilution To immobilize the sperm Det. sperm conc. (Short-cut) 1. 5 RBC squares # sperms counted x 1,000,000 = sperms in million/mL 2. 2 WBC squares # sperms counted x 100,000 = sperms in million/mL Sperm count Sperm concentration x volume of specimen Motility quality (20/hpf) 4.0 (a) = Rapid motility 3.0 (b) = Slower speed, some lateral movement 2.0 (b) = Slow forward progression + lateral movement 1.0 (c) = No forward progression 0 (d) = No movement at all CASA Computer-Associated Semen Analysis -Sperm concentration -Sperm velocity and trajectory Sperm morphology At least 200 sperms evaluated 1. Routine criteria = >30% normal morphology 2. Kruger’s strict criteria = >14% normal morphology -measure head, neck, tail using micrometer Head morphology Poor ovum penetration abnormalities Tail abnormality Poor motility Sperm head Oval, approximately 5 μm x 3 μm wide Tail 45 μm long Midpiece Contains mitochondria Connects head and tail Acrosomal cap Ovum penetration Covers approximately 2/3 of sperm nucleus and ½ of the head Tapered head Varicocele -Common cause of male sterility -Hardening of veins that drains the testes Stains Giemsa Papanicolau = method of choice Wright’s Fructose test If sperm count is low Rgt: Resorcinol End-color: Orange-red Specimens should be tested in 2 hrs or frozen  neutral-α-glucosidase Epididymis disorder Florence test Choline (+) Dark brown rhombic crystals Barbiero’s test Spermine (+) Yellow leaf-like crystals lec.mt 04 |Page | 165

Spinbarkeit test Sim Huhner test Bloom’s/Eosin-Nigrosin/ Sperm viability test

Decreased motility w/ clumping Normal seminalysis w/ continued infertility MAR Immunobead test Hamster egg penetration Cervical mucus penetration Hypo-osmotic swelling To determine whether semen is present Aspermia Azospermia Necrospermia Oligospermia

Tenacity of mucus Post-coital test Test for the ability of sperm cells to penetrate the cervical mucosa If N-sperm count but  motility Living sperm cells = bluish white Dead = red NV = 75% living sperms (25% dead) (+) Male antisperm antibodies Blood testis barrier  disrupted Clumps of sperm (+) Female antisperm antibodies Mixed agglutination reaction Detect IgG antibodies Detect IgG, IgA, IgM Demonstrate area of the sperm the autoantibodies are affecting Sperm incubated w/ species non-specific hamster egg Observed sperm penetration ability Test for membrane integrity and viability of sperm Microscopic exam for sperm cells Enhance w/ xylene Examine  Phase microscope Seminal glycoprotein p30: specific method No ejaculate Absence of sperm cells Immotile/dead sperm cells  sperm cells

Synovial Fluid Synovial Fluid Arthrocentesis Synoviocytes Specimen collection

Normal Values

Latin: “Egg” Diarthroses/joints Method of collections Phagocytic cells Secrete hyaluronic acid Fluid  Syringe (heparin) Micro: 3-5 mL in sterile tube, add 25 U heparin/mL fluid Hema: 3-5 mL, add 25 U heparin/mL fluid -Do not use crystalline EDTA but liquid EDTA may be accepted Chem: 3-5 mL in red top and observe for clotting NaF: glucose analysis Volume = 2.0 = mature fetal lungs Disadvantage: can’t be done on specimen contaminated w/ meconium Surfactant for alveolar stability Produced by type II alveolar pneumocytes (stored as lamellar bodies = about the size of platelets) Detect the presence of phosphatidyl glycerol Advantage: not affected by blood or meconium Amniotic fluid + 95% ethanol  shake for 15 secs  stand (15 mins) (+) Continuous line of bubbles Can be done bedside Creatinine ≥2.0 mg/dL (36 weeks/9 months) Gastric Fluid Analysis Pernicious anemia Peptic ulcer Zollinger-Ellison disease Produces HCl and intrinsic factor Produced by chief cells Pepsinogen ---(HCl)---> Pepsin Produced by G-cells Stimulates parietal cells to produce HCl  gastrin Gastric acid hypersecretion Adenoma of the islets of Langerhans (pancreas) = produce gastrin “Dangerous” anemia BAO/MAO = 0 Collect gastric juice for 1 hr 1. Levine tube = nose 2. Rehfuss tube = mouth Total gastric secretion during unstimulated fasting state Total acid secreted in the hour after stimulation 1. Pentagastrin = most preferred 2. Histamine lec.mt 04 |Page | 170

Test Meals Yeast cells in Gastric fluid Free HCl

Total Acidity

Combined HCl (bound to proteins) Euchlorhydria Hyperchlorhydria Hypochlorhydria Achlorhydria Diagnex tubeless test

Lactic acid

Creatorrhea

Celiac disease Steatorrhea

3. Histalog (Betazole) 4. Alcohols 5. Insulin = assess vagotomy procedure -SHAM feeding = sandwich 1. Ewald’s meal = bread and tea/H2O 2. Boa’s = oatmeal 3. Riegel’s = mashed potato and beef steak Fermentation in stomach because large amounts of food have been retained Quantitative Tests for Gastric Acidity (Topfer’s) Titration: NaOH pH indicator: Dimethylaminoazobenzol (+) Canary yellow NV = 25-50O Titration: NaOH pH indicator: phenolphthalein (+) Faint pink NV = 50-75O Titration: NaOH pH indicator: sodium alizarin (+) Violet NV = 10-15O Normal free HCl  free HCl Ex. peptic ulcer  free HCl Ex. carcinoma of the stomach (-) free HCl Ex. pernicious anemia [BAO/MAO = 0] Specimen: Urine Principle: -Azure blue is given by mouth -Presence of azure blue in urine indicates presence of free HCl in stomach Indicative of advanced gastric cancer 1. Modified Uffelmann’s -Rgt: FeCl3 and phenol -(+) Yellow 2. Strauss -Rgt: FeCl3 and ether -(+) Yellow 3. Kelling’s -Rgt: FeCl3 -(+) Yellow Fecalysis Undigested muscle fibers in feces Stain: Eosin a. Completely digested: no striations b. Partially digested: striation in one direction c. Undigested: striations in both directions >10 undigested muscle fibers = bile duct obstruction, cystic fibrosis Most common cause of malabsorption in developed countries  fat in stool G. lamblia lec.mt 04 |Page | 171

Melena Red Black

Green Gray Mucus/RBCs

Rice watery Pea soup Flattened/ribbon-like Bulky/frothy Butter-like Scybalous/Goat droppings Type 1 Type 2 Type 3 Type 4 Type 5 Type 6 Type 7 Occult blood Guaiac Benzidine O-toluidine Principle False (-) False (+)

APT test

Rotten egg odor Black tarry feces Upper GI bleeding Stool Color Lower GIT bleeding Tx: Rifampin Upper GIT bleeding Iron ingestion Bismuth (antacids) Charcoal Biliverdin Vegetables Bile duct obstruction Dysentery Colitis Malignancies Consistency Variations Cholera Typhoid fever Spastic colitis Bile duct obstruction Pancreatic disorders Abundant fats Cystic fibrosis Constipation Bristol Stool Chort Separate hard lumps, like nuts (hard to pass) Sausage-shaped but lumpy Sausage, w/ cracks on surface Sausage, snake, smooth surface Soft blobs w/ clear cut edge Fluffy w/ ragged ends (mushy) Watery Occult Blood “Hidden” Screening for colorectal cancer Significant: >2.5 mL blood/150g stool Least sensitive but the most preferred Most sensitive Pseudoperoxidase activity of Hgb (+) Blue Vitamin C Turnips, broccoli, cauliflower, banana, apple, melon, horseradish (to avoid, 3 days free) Aspirin, aspilet (promote GIT bleeding; to avoid, 7 days free) Red meat (to avoid, should be 3 days free) Distinguish fetal blood and maternal blood in an infant’s stool Specimen: infant stool/vomitus Rgt: 1% NaOH HbF: alkali-resistant Maternal Hgb: not alkali-resistant lec.mt 04 |Page | 172

Diarrhea Secretory diarrhea Osmotic diarrhea Intestinal hypermotility

Fecal enzymes

Fecal CHO

Fecal Leukocytes

Methylene blue Lactoferrin Latex agglutination test  amounts of striated fibers Fecal fats Qualitative Fecal fat test Split fat stain

Quantitative Fecal fat test

(+) Pink = HbF (-) Yellow brown = Maternal Hgb Acute: 4 weeks  solute secreted by the intestine Endotoxins  amounts of osmotically active solutes in the lumen (maldigestion)  CHO in stool Secretory and osmotic diarrhea Laxatives Emotions/stress Cardiovascular drugs 1. Trypsin = X-ray paper = Trypsin deficiency (CF): inability to digest gelatin on the X-ray paper 2. Chymotrypsin 3. Elastase I = pancreas specificity Stool pH = 7.0-8.0 pH 5.5 = CHO disorders (lactose intolerance) Clinitest: >0.5 g/dL = CHO intolerance Follow up tests: a. D-xylose: malabsorption b. Lactose intolerance test: maldigestion Primarily neutrophils  Neutrophils: -Salmonella -Shigella -Campylobacter -Yersinia -EIEC (-) Fecal leukocytes: -Parasites -Viruses -S. aureus -Vibrio spp For wet preparation Faster procedure than Wright’s and GS (for dry smears) but may be more difficult to interpret Sensitive in refrigerated and frozen specimens Biliary obstruction Gastrocolic fistulas NV = 1-6 g/day Sudan III = most routinely used Sudan IV Oil red O Free fatty acids and fatty acids from hydrolysis of soaps and neutral fats NV = 100 droplets (