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 <1.018 Pregnancy Roughly indicates the degree of hydration Should correlate w/ urine SG fluid intake: Dark urine, SG 1. Urochrome -Major pigment (yellow) -Production is directly proportional to metabolic rate - in thyrotoxicosis, fever, starvation 2. Uroerythrin -Pink pigment -May deposit in amorphous urates and uric acid crystals 3. Urobilin -Dark yellow/orange -Imparts an orange-brown color to a urine w/c is not fresh Urine Color Colorless to deep yellow Red/red brown (most common) Polyuria: DM/DI Bilirubin (yellow foam) [Protein: white foam, concentrated urine] Pyridium (Tx: UTI) -Yellow/orange foam -Orange and viscous Bilirubin ---(oxidized)---> 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 <1.003 = not a urine except DI Based on refractive index: RI = _light velocity in air_ light velocity in soln Compensated to temperature (15-38’C) Corrections: a. 1g/dL glucose: (-0.004) b. 1g/dL protein: (-0.003) Calibrations: a. Distilled H2O = 1.000 b. 5% NaCl = 1.022 ± 0.001 c. 9% Sucrose = 1.034 ± 0.001 Requires temperature correction a. 3’C calibration temperature (20’C) = (+0.001) b. 3’C calibration temperature (20’C) = (-0.001) Requires correction for glucose and protein (Rf/U) Refractometer reading is lower than the urinometer reading by 0.002 K2SO4 solution: 1L H2O + 20.29g K2SO4 SG = 1.015 SG = 1.010 (Glomerular filtrate) SG < 1.010 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 (<1 mg/dL or EU) Ehrlich’s reaction (PDAB): Urobilinogen + PDAB ----------> (+) 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) RCM RBCs Protein Clotting factors Pink: very slight amount of HbO2 Orange: heavy hemolysis Yellow: HbO2 Unconjugated bilirubin RBC degradation products: most common cause HbO2 from artifactual RBC lysis Bilirubin in jaundiced patients CSF protein >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 (cloudy/turbid) >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 <9 = Intact BBB b. Index ≥9 = BBB is damaged c. Index 100 = Complete damage to BBB _CSF IgG (mg/dL)_ Serum IgG (g/dL)_____ CSF/Serum Albumin index a. Index >0.70 = indicative of IgG production w/in CNS (MS) b. Index <0.70 = no active production w/in CNS ♫ Elevated IgG indices and presence of oligoclonal bands – complementary findings useful in the diagnosis of MS Not normally present in CSF Represents inflammation w/in the CNS Not pathognomonic for multiple sclerosis Encephalitis Guillain-Barré syndrome Neurosyphilis Neoplastic disorder Multiple sclerosis ♫ Single band should not be interpreted as positive Leukemia Lymphoma Viral infections ♫ Produces serum banding w/c can appear in the CSF because of BBB leakage or traumatic tap lec.mt 04 |Page | 161
HIV Multiple sclerosis Myelin basic protein Protein 14-3-3 Prions β-amyloid protein 42
S. agalactiae H. influenzae N. gonorrhoeae S. pneumoniae L. monocytogenes
CSF Glucose
CSF glucose N-CSF glucose CSF Lactate 10-22 mg/dL >35 mg/dL >25 <25 mg/dL LD
CK-BB CSF Glutamine >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) <17 mg/mL = recovery CSF Glutamine Product of ammonia and α-ketoglutarate NV = 8-18 mg/dL Requested for patients w/ coma of unknown origin Associated w/ some disturbances of consciousness lec.mt 04 |Page | 162
Reye’s syndrome Gram Stain
C. neoformans
Limulus lysate test
CSF serology PAM (Henry)
L. monocytogenes Interstitial cells of Leydig Seminiferous tubule Epididymis Prostate gland Seminal vesicles Vas deferens Bulbourethral (Cowper’s) gland Sertoli cells Spermatogenesis Round cells Seminal fluid (SV) Prostatic fluid
Life threatening Glutamine in children Degeneration of the liver blood clotting problems and bleeding Concentrated specimen = often very few organisms are present at the onset of disease Organisms: -S. pneumoniae -H. influenzae -E. coli -N. meningitidis -S. agalactiae (NB) -L. monocytogenes (NB) -C. neoformans India ink GS: Starburst pattern -seen more often than a (+) India ink Latex agglutination test Diagnosis of Gram (-) bacteria Reagent: -Blood cells (Amoebocytes) of horse-shoe crab (Limulus polyphemus) -Amoebocytes: contain copper complex that gives them blue color Principle: In the presence of endotoxin, amoebocytes (WBCs) will release lysate (protein) (+) Clumping/Clot formation All materials must be sterile (Tap water: endotoxin) Det. neurosyphilis VDRL: recommended by Centers for Disease Control and Prevention (CDC) FTA-ABS Naegleria fowleri Acanthamoeba spp. Acridine orange stain: useful to differentiate amoeba (brick-red) from leukocytes (bright green) The only Gram (+) that produces endotoxin (-) Limulus lysate test Seminal Fluid Secrete testosterone Outside the seminiferous tubules Site of spermatogenesis Stores and concentrates sperm Sperm maturation Zn3+, enzymes and proteins (coagulation and liquefaction) Fluid: fructose Transports sperm ejaculatory duct Secretes alkaline mucus neutralize prostatic and vaginal acidity Serve as nurse cells for developing sperm cells Inside the seminiferous tubules Spermatogonia 1’ Spermatocytes 2’ Spermatocytes Spermatids Sperm Either WBCs or spermatids Seminal Fluid Composition 60-70% 20-30% lec.mt 04 |Page | 163
Spermatozoa Bulbourethral gland ACP Zn3+ Fructose K+, citric acid, ascorbic acid Proteolytic enzymes Spermine and Choline Importance of Seminalysis
Sexual abstinence Methods of collection
Methods of preservation
Liquefaction time Normal values
Volume Yellowish semen White turbidity Red coloration Viscosity pH
5% 5% Chemical Composition of Seminal Fluid For liquefaction in prostatic disease Major nutrient of spermatozoa -Liquefaction and coagulation Inhibit growth of bacteria To investigate the causes of infertility in marriages To check the effectiveness of previous vasectomy In medico-legal cases, where paternity is being disclamed on the basis of male sterility 2-3 days and not >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 ♫ >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 = <3.5 mL Color = pale yellow Clarity = clear Viscosity = 4-6 cm long Crystals = none present Glucose = <10 mg/dL lower than the blood glucose Lactate = <250 mg/dL Total protein = <3 mg/dL Uric acid = equal to blood value lec.mt 04 |Page | 166
Turbidity Milky Rope’s/Mucin clot test
WBC
Differential count Very viscous specimen LE cell Reiter cells Ragocyte Cartilage cells Rice bodies Ground pepper appearance of synovial fluid Hemosiderin Cartilage cells Normal Abnormal
MSU CPP Group I: Non-inflammatory Group II: Inflammatory Group III: Septic
WBCs (+) Crystals Hyaluronate polymerization test 2-5% acetic acid Grading: -Good = solid clot -Fair = soft clot -Low = friable clot -Poor = no clot at all Cells in Synovial Fluid Total WBC count: most frequently tested STAT, otherwise, refrigerate Neubauer counting chamber Clear fluids = counted undiluted Diluting fluid = NSS If necessary to lyse RBCs: -Hypotonic saline (0.3%) -Saline w/ saponin Do not use WBC diluting fluid 65% = Monocytes & Macrophages <25% = Neutrophils <15% = Lymphocytes Add hyaluronidase 0.5 mL of fluid or 1 drop of 0.05% hyaluronidase in PO4 buffer/mL of fluid 37’C for 5 mins Neutrophil Vacuolated macrophage w/ ingested neutrophils Neutrophil w/ dark cytoplasmic granules Large multinucleated cells Macroscopically resembles polished rice Ochronotic shards = debris Metal and plastic joint prosthetic Pigmented villonodular synovitis Osteoarthritis Crystals No crystals Hydroxyapatite crystals Cholesterol Corticosteroid MSU = needle-like (gout) CPP = needle-like/rods (pseudogout) (-) Birefringence (yellow) on compensated (red) polarizing microscopy (+) Birefringence (blue) on compensated (red) polarizing microscopy Joint Disorders OA Traumatic arthritis Neuroarthropathy Immunologic: RA, SLE Crystal-induced: milky synovial fluid Yellow green Low viscosity >50,000 WBC/μL (highest) lec.mt 04 |Page | 167
Group IV: Hemorrhagic Serous fluids Effusion Transudate
Exudate
Specimen collection Rivalta’s test/Serosa mucin clot test Fluid: Serum LD ratio Fluid: Serum protein ratio Thoracentesis Normal appearance Turbid Bloody
Milky CA 15-3 and CA 549 CYFRA 21-1 (Cytokeratin Fragment) AMS ADA Mesothelial cells Glucose Lactate pH Pericardiocentesis
(+) Culture: CAP = Haemophilus, N. gonorrhoeae (gonococcal arthritis) S. aureus: predominant pathogen in adult joint infection B. burgdorferi: Lyme disease (Stage 3: Lyme arthritis) Sickle cell disease Glucose = Blood Serous Fluids Fluids that are formed between the parietal and visceral membrane Ultrafiltrate of plasma Provides lubrication Increase in serous fluid amount between the membranes Either transudate or exudates From disturbances of fluid production and regulation between serous membranes -Hypoproteinemia -Nephrotic syndrome -CHF Testing of transudates is not necessary Purulent fluid that forms in any body cavity as a result of inflammatory processes -Infection -Malignancies >100 mL is usually collected EDTA = cell counts and differential Heparin = chemistry, serology, microbiology, cytology Differentiates transudates from exudates Acetic acid + H2O + unknown fluid heavy ppt. = (+) exudate Most reliable differentiation between transudates and exudates (except peritoneal fluid: SAAG) Pleural Fluid Method of collection Clear/pale yellow WBCs (infection) Hemothorax (traumatic injury): = Pleural fluid Hct is >50% of WB Hct Chronic membrane disease/Hemorrhagic effusion: = Pleural fluid Hct < WB Hct Chylous (thoracic duct leakage) = TG, (+) Sudan III Pseudochylous (chronic inflammation) = cholesterol, (-) Sudan III Breast cancer Lung cancer Pancreatitis Esophageal rupture TB Malignancy TB infection bacterial infection in pneumonia in esophageal rupture Pericardial Fluid Method of collection lec.mt 04 |Page | 168
Normal volume Mesothelial cells Function Normal appearance Grossly bloody Milky Bacterial endocarditis Paracentesis Peritoneal lavage WBC <500/μL RBC >100,000/μL Serum Ascites Albumin Gradient (SAAG) Psammoma bodies (+) CA 125 (-) CEA Glucose ALP AMS BUN Creatinine Cystic fibrosis (mucoviscidosis)
Gibson and Cooke Pilocarpine Iontophoresis Sodium Chloride During 1st trimester After 1st trimester 3rd trimester Oligohydramnios
Hydramnios/ Polyhydramnios Amniocentesis Fern test
10-50 mL Produces pericardial fluid Reduces friction Clear/pale yellow Accidental cardiac puncture Misuse of anticoagulant medicatios Chylous/pseudochylous effusions >1000 WBCs/μL (% neutrophils) Peritoneal Fluid (Ascites) Method of collection Sensitive test for the detection of intra-abdominal bleeding Normal Blunt trauma cases Recommended to differentiate transudates from exudates a. >1.1 = Transudate b. <1.1 = Exudate Contains concentric striations of collagen materials Ovarian and thyroid malignancies Tumor of ovaries, fallopian tubes or endometrium TB peritonitis Intestinal perforation Pancreatitis GI perforation Ruptured bladder or accidental puncture of the bladder Sweat Test Autosomal recessive Pancreatic insufficiency Respiratory distress Intestinal obstruction -Bulky offensive greasy stools (butter-like) Sweat Cl- and Na+ = >70 mEq/L Borderline = 40 mEq/L Pilocarpine w/ mild current = stimulates sweat glands FEP, IEE Manual or automated titration Amniotic Fluid 35 mL: derived from maternal circulation/plasma Fetal urine Peak: 1L amniotic fluid amniotic fluid Urinary tract deformities Membrane leakage fetal swallow of urine amniotic fluid fetal swallow of urine Neural tube defects Method of collection 2nd trimester: assess genetic defects 3rd trimester: FLM or HDN Specimen: Vaginal fluid lec.mt 04 |Page | 169
Normal appearance Green Yellow Bloody/red Dark-brown/ Reddish-brown Assessment of HDN
Neural tube defects
L/S ratio Lecithin Surfactants Amniostat FLM Foam stability test (Foam/Shake test) Fetal age Clinical significance Parietal cells Pepsinogen Gastrin Zollinger-Ellison disease Pernicious anemia Specimen collection Basal Acid Output (BAO) Maximal Acid Output (MAO) Gastric stimulants
Glass slide air dry (+) Fern-like crystals (+) Amniotic fluid Clear Meconium Bilirubin (HDN) Trauma, abdominal trauma, intra-amniotic hemorrhage Fetal death OD at 450 nm OD reading at a Liley graph 1. Zone 1: observe fetus for stress 2. Zone 2: moderate disease 3. Zone 3: severe disease Ex. Spina bifida and Anencephaly Screening: AFP Confirmatory: Acetylcholinesterase Fetal Lung Maturity Reference method >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 Chronic: >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 (<4 μm) Slightly increased = 100 droplets (1-8 μm) Increased = 100 droplets (6-75 μm) Confirmative test for steatorrhea 3 day specimen lec.mt 04 |Page | 173
Duodenal fluid Secretin Pancreozymin Secretin test Pancreatic cancer Chronic pancreatitis
1st morning 24 hr Throat swab Tracheal aspirate Transparent White/yellow Gray Yellow-green Green Red or Bright red Anchovy sauce/rusty brown Rusty red Brown Black Prune juice Olive green/grass green Causes of blood-stained sputum Heart failure cells Odorless Foul/putrid
-intake of fat = 100g/day Prior to and during collection paint cans Van de Kamer titration = NaOH Duodenal Fluid Physiologically acidic pH stimulates mucosal cells to produce secretin Stimulates watery pancreatic secretions w/ bicarbonate Provokes enzyme production of pancreas (AMS, LPS) Secreted by mucosal cells (-) Pancreozymin = (-) AMS & LPS Most sensitive test for impaired pancreatic function Secretin is administered IV, then duodenal fluid HCO3- is tested volume N-HCO3N-AMS volume HCO3 AMS Sputum Most ideal Volume measurement: vol = bronchiectasis, lung abscess, edema, gangrene, TB vol = bronchial asthma, acute bronchitis, early pneumonia Pediatric patients Debilitated patients Sputum Color Normal Mucus only (+) Pus (+) Pus, EC TB, bronchiectasis P. aeruginosa (+) Bile Fresh blood (hemorrhage) TB, bronchiectasis Old blood Pneumonia Lobar pneumonia CHF Heavy smokers Anthracosis Inhalation of dust, dirt, carbon, charcoal Pneumonia Chronic lung cancer Chronic lung cancer a. Rusty, (+) pus = pneumococcal pneumonia b. Rusty, (-) pus = CHF c. Bright streaks in viscid sputum = K. pneumoniae d. Spurious hemoptysis = nosebleed Hemosiderin laden macrophages in the alveolar spaces Sputum Odor Normal Cavitary TB lec.mt 04 |Page | 174
Fruity Sweetish Mucoid Serous/frothy Mucopurulent Bronchial casts Cheesy masses Dittrich’s plugs
Lung stones/Pneumoliths/ Broncholiths Curschmann’s spirals Layer formation (3)
PAS (+) Macrophages PAS (+) rounded bodies that take silver stain Elastic fibers Charcot-Leyden crystals Creola bodies Fungi Parasites
Lung abscess Gangrene Advanced necrotizing tumors P. aeruginosa TB, Bronchiectasis Consistency Asthma and bronchitis Lung edema TB, bronchiectasis Macroscopic Structures in Sputum Made of fibrin Branching tree-like casts Lobar pneumonia Fragments of necrotic pulmonary tissue Pulmonary TB Pulmonary gangrene Grayish to yellowish material Size of pinhead Foul odor when crushed Cellular, fatty acids, fat globles, some bacteria Bronchiectasis Chronic bronchitis Bronchial asthma (3C’s) From calcified pulmonary tissue Histoplasmosis Mucoid threads that are twisted/coiled Bronchial asthma (3C’s) Bronchitis Bronchiectasis Lung abscess Gangrene Top = Frothy mucus Middle = opaque H2O material Bottom = pus, bacteria, tissues Microscopic Elements Alveolar proteinosis P. carinii TB From degeneration of eosinophils Bronchial asthma (3C’s) Clusters of columnar cells Bronchial asthma (3C’s) C. albicans C. neoformans (Torulosis) C. immitis 1. Migrating larva (Heart Lung) = “ASH” -Ascaris -Strongyloides -Hookworm 2. P. westermani 3. E. granulosus (Pulmonary hydatid disease) lec.mt 04 |Page | 175
Clinical significane Macrophages (56-80%) Lymphocytes (1-15%) Neutrophils (<3%) Eosinophils (<1 to 2%) hCG
Specimen Ascheim-Zondek Hogben Galli-Mainini Friedmann’s Frank-Bermann
4. E. gingivalis 5. T. tenax Bronchoalveolar Lavage P. jiroveci Most predominant interstitial lung disease, pulmonary lymphoma, nonbacterial infection cigarette smokers, bronchopneumonia, toxin exposure hypersensitivity reaction (asthma) Human Chorionic Gonadotropin Produced by cytotrophoblast cells of the placenta 1st trimester of pregnancy α-subunit = FSH, LH, TSH, hCG β-subunit = unique to hCG 1st morning urine Bioassays Female mice Subcutaneous injection (+) Corpora lutea, hemorrhagic follicles Female toad Lymph sac injection (+) Oogenesis Male frogs, male toads Subcutaneous injection (+) Spermatogenesis Mature virgin female rabbit Marginal ear vein injection Female rats Intraperitoneal injection (+) Ovarian hyperemia
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