MUST TO KNOW Clinical Microscopy PDF

MUST TO KNOW IN CLINICAL MICROSCOPY URINALYSIS Nephron Basic structural unit of kidney 1M/kidney Urethra F: 3-4 cm M: 20 cm Urine formation (order) Glomerulus  Bow...

MUST TO KNOW IN CLINICAL MICROSCOPY URINALYSIS Nephron Basic structural unit of kidney 1M/kidney Urethra F: 3-4 cm M: 20 cm Urine formation (order) Glomerulus  Bowman’s capsule  PCT  Loop of Henle  DCT  CD PCT 65% of reabsorption ADH Regulate H2O reabsorption in DCT and CD Urine composition 95-97% H2O 3-5% solids 60g TS in 24 hrs 35g: Organic = Urea (major) 25g: Inorganic = Cl (#1) > Na+ > K+ Glomerular Filtration Clearance tests Evaluate glomerular filtration 1. Urea clearance 2. Creatinine clearance = most common 3. Inulin clearance = gold standard 4. Beta2-microglobulin 5. Radioisotopes Creatinine clearance Formula: Cc = U x V x 1.73 P A Normal values: M = 107-139 mL/min F = 87-107 mL/min Tubular Reabsorption Tubular Reabsorption 1st function to be affected in renal disease Concentration tests Evaluate tubular reabsorption Fishberg test (Old) Patient is deprived of fluid for 24hrs then measure urine SG (SG ≥ 1.026) Mosenthal test (Old) Compare day and night urine in terms of volume and SG Specific Gravity (New) Influenced by # and density of particles in a solution Osmolarity 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 PAH test p-aminohippuric acid PSP test Phensulfonphthalein test Obsolete, results are hard to interpret Methods of Collection Midstream/Catheterized Urine culture Suprapubic aspiration Anaerobic urine culture lec.mt 04 |Page | 134 3 glass technique 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) Pediatric specimen Wee bag Drug Specimen Collection 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 Occasional/Single/Random Routine Qualitative UA 24 hr 1st voided urine  discarded w/ preservative Ex. 8AM  8AM 12 hr Ex. 8AM  8PM Addis count: measure of formed elements in the urine using hemacytometer Afternoon (2PM-4PM) Urobilinogen (alkaline tide) 4 hr Nitrite determination (1st morning/4 hr) NO3  NO2 = (+) UTI 1st morning Pregnancy test (hCG) Ideal specimen for routine UA Most concentrated and most acidic = preservation of cells and casts Fasting/2nd morning Glucose determination 2nd voided urine after a period of fasting Changes in Unpreserved Urine Decreased Clarity Bacterial multiplication Precipitation of AU/AP Glucose Glycolysis Ketones Volatilization Bilirubin Photooxidation Urobilinogen Oxidized to urobilin RBC/WBC Disintegrate in alkaline urine Increased pH Urea ---(Urease)---> NH3 Bacteria Multiplication Odor Urea ---(Urease)---> NH3 Nitrite Bacterial multiplication Differentiate contamination Contamination:  Bacteria from true infection True infection:  Bacteria and WBCs Preservation Refrigeration 2-8’C  SG (hydrometer/urinometer) Precipitate AU/AP Formalin Addis count Boric acid Urine culture lec.mt 04 |Page | 135  Bacteriostatic to contaminants Sodium fluoride Glucose Sodium benzoate/ Substitute for sodium fluoride Benzoic acid Saccomanno’s fixative 50% ethanol + carbowax Cytology (50mL urine) Physical Examination of Urine Volume NV: 24 hr = 600-1200 mL Ave (24 hr) = 1200-1500 mL Night: Day ratio = 1:2 to 1:3 Routine UA Vol = 10-15 mL (Ave: 12 mL) -15 mL: for urinometry -physical, chemical, microscopic exam Polyuria  Urine volume -Diabetes Mellitus:  vol,  SG -Diabetes Insipidus:  vol,  SG Oliguria Calculus/kidney tumors Dehydration Anuria Complete cessation of urine flow Nocturia >500mL w/ SG Biliverdin Yellow-brown Green Pseudomonas aeruginosa Blue-green Clorets, methylene blue, phenol Indican (blue): Hartnup disease or Blue diaper syndrome Cloudy/Smoky red Hematuria (intact RBCs) Clear red Hemoglobin, Myoglobin lec.mt 04 |Page | 136 Red/Purple/Burgundy red/ Porphyria purplish red/Portwine (Lead poisoning: normal color) Brown/black Methemoglobin (acid urine) Homogentisic acid: Alkaptonuria -Urine darkens after a period of standing -(-) Homogentisic acid oxidase Urine Color Changes w/ Commonly Used Drugs Cola-colored Levodopa (Tx: Parkinsonism) Red  Brown (alkaline) Yellow Mepacrine/Atabrine (Tx: Malaria, Giardiasis) Red to brown Metronidazole/Flagyl (Tx: Trichomoniasis, Amoebiasis, Giardiasis) Methyldopa/Aldomet (Antihypertensive) Orange-red (acid) Phenazopyridine/pyridium (Tx: UTI) Bright orange-red (acid) Rifampin (Tx: TB) = all body fluids are red Bright yellow Riboflavin (Multivitamins) Nubecula Faint cloud in urine after a period of standing WBCs, epithelial cells and mucus Bilifuscin (Dipyrrole) Hemoglobin Köln = unstable Red-brown urine Clarity/Transparency/Turbidity Clear Transparent, no visible particulates Hazy Few particulates, print easily seen through urine Cloudy Many particulates, print blurred through urine Turbid Print cannot be seen through urine Milky May precipitate or clot Bacteria Uniform turbidity NOT cleared by acidification or filtration Chyluria Lymph fluid in urine Filariasis Squamous epithelial cells  females Radiographic contrast  SG by refractometer (>1.040) media Rgt strip: not affected by RCM Vaginal cream Tx: Candida Pseudochyluria Laboratory Correlations in Urine Turbidity Acidic urine AU RCM Alkaline urine AP Carbonates Soluble w/ heat AU Uric acid Soluble w/ dilute acetic RBCs acid AP Carbonates Insoluble in dilute acetic WBCs acid Yeasts Spermatozoa Bacteria Soluble in ether Lipids Lymph fluid Chyle lec.mt 04 |Page | 137  Specific Gravity SG 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 Aromatic/Odorless Normal Ammoniacal Urea ---(Urease)---> NH3 Ex. UTI (Proteus:  urease) Fruity, sweet DM (Ketones) Rotten fish/Galunggong Trimethylaminuria Sweaty feet Isovaleric acidemia Mousy Phenylketonuria Cabbage Methionine malabsorption Caramelized sugar, curry MSUD Bleach Contamination Sulfur Cystine disorder Chemical Examination of Urine Specific Gravity Principle (Rgt Strip) pKa dissociation constant  concentration =  H+ Indicator: Bromthymol blue = () Blue  Green  Yellow () Other info. Not affected by glucose, protein and RCM Harmonic Oscillation Frequency of soundwave entering a solution will change in proportion to the Densitometry density (SG) of the solution -Yellow IRIS (Automated): International Remote Imaging System pH Normal Random = 4.5-8.0 1st morning = 5.0-6.0 pH 9.0 = Unpreserved urine lec.mt 04 |Page | 138 Acid urine DM (Ketone bodies) Starvation (Ketone bodies) High protein diet Cranberry juice (Tx: UTI) Alkaline urine After meal Vomiting Renal tubular acidosis: inability to produce an acid urine Vegetarian diet Old specimen: Urea ---(Urease)---> NH3 Principle (Rgt Strip) 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 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 Normal value < 10 mg/dL or < 100 mg/24 hrs < 150 mg/24 hrs (Henry) Pre-renal proteinuria 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 Renal proteinuria 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 Post-renal proteinuria Lower UTI/inflammation Principle (Rgt Strip) 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 Sulfosalicylic acid Cold precipitation test that reacts equally to all types of protein precipitation test If (-) rgt strip, (+) SSA = presence of other proteins lec.mt 04 |Page | 139  CSF protein = frequently tested -Det: TCA (preferred) and SSA SSA Reactions (Protein) Negative No increase in turbidity < 6 mg/dL Trace Distinct turbidity 6-30 mg/dL 1+ Noticeable turbidity w/ no granulation 30-100 mg/dL 2+ Turbidity w/ granulation but no flocculation 100-200 mg/dL 3+ Turbidity w/ granulation and flocculation 200-400 mg/dL 4+ Clumps of protein > 400 mg/dL Glucose 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 Other substances in 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 Hyperglycemia associated  Blood glucose,  Urine glucose Glycosuria 1. DM 2. Cushing’s syndrome/disease =  cortisol 3. Pheochromocytoma =  catecholamines 4. Acromegaly =  GH 5. Hyperthyroidism = T3/T4 Renal associated Glycosuria N-Blood glucose, impaired tubular reabsorption of glucose 1. Fanconi’s syndrome: defective tubular reabsorption of glucose and amino acids Principle (Rgt Strip) Double sequential enzyme reaction: -Glucose oxidase -Peroxidase Chromogen: -KI (Brown) -Tetramethylbenzidine (Blue) Copper Reduction test Blue tablet (Clinitest) 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 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 (-) Glucose oxidase (+) Nonglucose reducing substance (+) Clinitest 1+ Glucose oxidase True glucosuria (-) Clinitest Small amount of glucose present 4+ Glucose oxidase False (+) (-) Clinitest Possible oxidizing agent interference on reagent strip Ketones 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 Significance Diabetic acidosis Insulin dosage monitoring Starvation Vomiting Principle (Rgt Strip) Legal’s test (Sodium nitroprusside reaction) AAA + Sodium nitroprusside --------------> (+) Purple (Acetone) (Glycine) Acetest Sodium nitroprusside Glycine Disodium phosphate Lactose Blood Hematuria Cloudy red urine (Intact RBCs) Renal calculi GN Strenuous exercise Anticoagulants Hemoglobinuria Clear red urine Intravascular hemolysis Myoglobinuria Clear red urine Rhabdomyolysis Hgb vs. Mgb 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 Hemolytic anemia 1 hr post transfusion urine = Hgb Week after = Hemosiderin Principle (Rgt Strip) Pseudoperoxidase activity of hemoglobin Chromogen: TMB [(-) Yellow/(+){}Green  Blue {}] H2O2 + Chromogen ---(Heme)---> Oxidized chromogen + H2O Hgb/Mgb Uniform green/blue Hematuria Speckled/spotted Extravascular lysis  Unconjugated bilirubin  Urine and fecal urobilinogen lec.mt 04 |Page | 141 Intravascular lysis (+) Hemoglobinuria  Haptoglobin and Hemopexin Bilirubin Bilirubin Amber urine w/ yellow foam Early indication of liver disease Significance Hepatitis Cirrhosis Bile duct obstruction Principle (Rgt Strip) Diazo Reaction: Bilirubin diglucuronide + Diazo salt ---------> (+) Tan or pink purple Reagents: 2,4-Dichloroaniline diazonium salt 2,6-Dichlorobenzene diazonium salt Ictotest (+) Blue to purple Reagents: p-nitrobenzene-diazonium-p-toluenesulfonate SSA Na2HCO3 Urobilinogen Urobilinogen Afternoon specimen: 2PM-4PM (alkaline tide) Small amount in normal urine ( (+) Cherry red Watson-Schwartz test 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 Hoesch test (Inverse Rapid screening test for urine porphobilinogen (≥2mg/dL) Ehrlich reaction) Hoesch reagent: Ehrlich reagent in 6M (6N) HCl 2 gtts urine + Hoesch rgt -------------> (+) Red Urine Bilirubin and Urobilinogen in Jaundice Urine Bilirubin Urobilinogen Hemolytic (ex. SCD) -  Hepatic /- N/ Obstructive  /N (never report as negative) Nitrite Nitrite 1st morning/4 hr urine Detection of bacteriuria (+) NO3: 100,000 orgs./mL UTI: ≥100,000 or ≥1 x 105 CFU/mL Principle (Rgt strip) Greiss reaction: NO3 + Sulfanilamide + p-arsanilic acid + tetrahydrobenzoquinoline ------------> (+) Uniform pink Pink spots/edges = (-) Leukocytes Significance UTI/inflammation Screening of urine culture specimen Leukocyte esterase (+) Neutro/Eo/Baso/Mono (-) Lympho Maybe (+): Histiocytes, Trichomonas lec.mt 04 |Page | 142 Principle (Rgt strip) Indoxyl carbonic acid ester + Diazonium salt ---(LE)---> Indoxyl + Acid indoxyl ----------> (+) Purple Strip can detect even lysed WBCs Reading Time (Reagent Strips) 30 seconds Glucose Bilirubin 40 seconds Ketones 45 seconds SG 60 seconds “PPBUN” pH Protein Blood Urobilinogen Nitrite 120 seconds Leukocytes Vitamin C (Ascorbic acid) 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 SG High concentrations of protein Highly alkaline urines (>6.5) pH No known interfering substance Runover from adjacent pads Old specimens Protein 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 Glucose Contamination by oxidizing agents and High levels of ascorbic acid detergents High levels of ketones High specific gravity Low temperatures Improperly preserved specimens Ketones Phthalein dyes Improperly preserved specimens Highly pigmented red urine Levodopa Medications containing free sulfhydryl grps lec.mt 04 |Page | 143  False-positive False-negative Blood Strong oxidizing agents High specific gravity/crenated cells Bacterial peroxidases Formalin Menstrual contamination Captopril High concentration of nitrite Ascorbic acid >25 mg/dL Unmixed specimens Bilirubin Highly pigmented urines, phenazopyridine Specimen exposure to light Indican (intestinal disorders) Ascorbic acid >25 mg/dL Metabolites of Lodine High concentrations of nitrite Urobilinogen Porphobilinogen Old specimens Indican Preservation in formalin p-aminosalicylic acid Sulfonamides Methyldopa Procaine Chlorpromazine Highly pigmented urine Nitrite Improperly preserved specimens Nonreductase-containing bacteria Highly pigmented urine 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 Leukocytes Strong oxidizing agents High concentrations of protein, glucose, oxalic Highly pigmented urine, nitrofurantoin acid, ascorbic acid, gentamicin, cephalosporins, tetracyclines Microscopic Exam of Urine Phase-contrast microscopy Visualization of elements w/ low refractive indices: -Hyaline casts -Mixed cellular casts -Mucous threads -Trichomonas Polarizing microscopy ID of cholesterol in OFB, FC and crystals Interference contrast Produces 3D microscopy-image and layer-by-layer imaging of a specimen microscopy 1. Hoffman microscope: modulation contrast microscope 2. Nomarski microscope: differential interference contrast microscope Sternheimer-Malbin Crystal violet and safranin Nucleus and cytoplasm ID: WBCs, ECs, casts Toluidine blue (Supravital) Enhances nuclear detail Differentiates WBCs and RTE Lipid stains: Stain TG and neutral fats orange red ORO and Sudan III ID: free fat droplets and lipid-containing cells and casts Gram stain Differentiates Gram (+) and Gram (-) bacteria ID: bacterial casts Hansel stain Eosin Y and Methylene blue ID: Eosinophils Prussian blue stain Stains structures containing iron lec.mt 04 |Page | 144  ID: yellow-brown granules of hemosiderin in cells and casts Sediment Constituents RBCs 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 WBCs 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 Addis count 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 Squamous epithelial cells 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 Transitional epithelial cells Spherical, polyhedral, or caudate w/ centrally located nucleus (Urothelial cells) Derived from the linings of the renal pelvis, ureter, urinary bladder, male urethra (upper portion) Not clinically significant in small numbers Renal tubular epithelial Rectangular, polyhedral, cuboidal or columnar w/ an eccentriac nucleus, cells possibly bilirubin stained or hemosiderin laden From nephron: -PCT: rectangular, columnar/convoluted -DCT: round/oval >2 RTE/hpf: tubular injury Oval fat body Lipid containing RTE cells Lipiduria (Ex. nephrotic syndrome) Cholesterol: Maltese cross Bubble cells RTE cells w/ nonlipid containing vacuoles Acute tubular necrosis Yeast C. albicans (DM, vaginal moniliasis) T. vaginalis Flagellate w/ jerky motility lec.mt 04 |Page | 145  Pingpong disease S. haematobium “Hematuria” Specimen: 24 hr unpreserved urine E. vermicularis Most common fecal contaminant Casts (Cylindruria) Formed in the DCT and CD ♫ Tamm-Horsfall protein (Uromodulin) -Major constituent -Glycoprotein secreted by RTE cells of DCT and CD Hyaline casts 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 RBC casts Bleeding w/in the nephron a. GN b. Strenuous exercise (HC, GC, RC) WBC casts 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 Bacterial casts Pyelonephritis Epithelial cell casts Renal tubular damage Advanced tubular destruction Coarse/Fine granular casts Formed from the disintegration of cellular cast GN PN Strenuous exercise (HC, GC, RC) Fatty casts Nephrotic syndrome: lipiduria Not stained by Sternheimer-Malbin Waxy casts Final degenerative form of all types of casts Stasis of renal flow Chronic renal failure Brittle, highly refractile, w/ jagged ends Broad casts “Renal failure casts” Extreme urine stasis Widening and destruction of tubular walls Any type of cast can be broad Sediment preparation 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 RCF 1.118 x 10-5 x radius (cm) x (rpm)2 lec.mt 04 |Page | 146  Urine Crystals Amorphous Urates Yellow-brown granules (Normal) Pink sediment (Uroerythrin) (pH: acid) Uric Acid Mistaken as cystine crystals Rhombic, wedge, rosette, hexagonal, four-sided plate (whetstone) Lemon-shaped (Henry)  Lesch-Nyhan syndrome: orange sands in diaper  Gout  Chemotherapy Calcium Oxalate 1. Weddelite = dihydrate (Normal) -Envelope/pyramidal (pH: acid/alkaline/neutral) 2. Whewellite = monohydrate -Oval, dumbbell -Ethylene glycol poisoning (antifreeze agent) Most renal stones consist of CaOx Amorphous Phosphates White precipitate (Normal) Granular appearance (pH: alkaline/neutral)  After meal (alkaline tide) Ammonium Biurate Yellow-brown (Normal) Thorny apples (pH: alkaline) Old specimen: due to the presence of urea-splitting bacteria Triple Phosphate A.k.a. Magnesium ammonium phosphate (Normal) Coffin lid, “Struvite”, staghorn appearance (pH: alkaline) Presence of urea-splitting bacteria Calcium Phosphate Colorless, flat rectangular plates or thin prisms often in rosette formation (Normal) Rosettes may resemble sulfonamides (pH: alkaline/neutral) -To differentiate: CaPO4 dissolves in acetic acid 1. Calcium Phosphate = Apatite 2. Basic Calcium Phosphate = Hydroxyapatite 3. Calcium Hydrogen Phosphate = Brushite Calcium Carbonate Small and colorless (Normal) Dumbbell or spherical shapes (pH: alkaline) Acetic acid: (+) Effervescence Cystine Colorless hexagonal plates (Abnormal) Cystinuria (pH: acid) Cholesterol Rectangular plate w/ notch in one or more corners (Abnormal) Staircase pattern (pH: acid) 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 Tyrosine Colorless to yellow needles (Abnormal) Liver disease (more common) (pH: acid/neutral) (+) Nitroso-naphthol Leucine Yellow-brown spheres w/ concentric circles and radial striations (Abnormal) Liver disease (pH: acid/neutral) Bilirubin Clumped needles or granules w/ yellow color (Abnormal) (+) Diazo reaction lec.mt 04 |Page | 147 (pH: acid) Liver disease Sulfonamide Colorless to yellow brown (Abnormal) Deposits in nephrons (pH: acid/neutral) Tubular damage Needles, sheaves of wheat, rosette (res. CaPO4 rosette) ♫ Lignin test: Newspaper = urine + 25% HCl  (+) Yellow orange color Ampicillin Massive doses (Abnormal) Colorless needles (pH: acid/neutral) 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 Renal Diseases Cystitis Inflammation of urinary bladder Infection WBCs, RBCs, bacteria NO CAST Urethritis 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 Glomerulonephritis Inflammation of the glomerulus Immune-mediated RBCs, WBCs, RBC CASTS, WBC casts, hyaline and granular casts Pyelonephritis Infection of renal tubules WBCs, RBCs, bacteria, RBC casts, WBC CASTS, hyaline and granular casts Acute Interstitial Nephritis Infection of the renal interstitium RBCs, WBCs, WBC casts, NO BACTERIA Renal carcinoma 1’ = RCC 2’ = Transitional CC RBCs and WBCs Nephrotic syndrome 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  -(-) alpha2 (AMG) -Oval fat bodies, fatty and waxy casts Telescoped sediments Simultaneous appearance of the elements of acute/chronic GN and nephrotic syndrome  Cells and Casts a. Lupus nephritis b. SBE UTI 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 Rapidly progressive Deposition of immune complex from systemic immune disorders on the (Crescentic) GN glomerular membrane Goodpasture syndrome Attachment of cytotoxic antibody to glomerular and alveolar basement membrane Wegener’s granulomatosis Antineutrophilic cytoplasmic autoantibody Henoch-Schönlein purpura Occurse in children following viral respiratory infection Decrease in platelets disrupts vascular integrity Membranous GN Thickening of the glomerular membrane following IgG immune complex deposition Membranoproliferative GN Cellular proliferation affecting the capillary walls or the glomerular basement membrane Chronic GN Marked decrease in renal function resulting from glomerular damage precipitated by other renal disorders IgA nephropathy Deposition of IgA on the glomerular membrane (Berger’s disease) Nephrotic syndrome Disruption of the electrical charges that produce tightly fitting podocyte barrier Minimal change disease Disruption of the podocytes occurring primarily in children following allergic (Lipoid nephrosis) reaction and immunization FSGS Disruption of podocytes in certain areas of glomeruli associated w/ heroin and analgesic abuse and AIDS Alport syndrome Lamellated and thinning of glomerular basement membrane Diabetic Nephropathy Most common cause of ESRD (Kimmelstiel-Wilson Microalbuminuria disease) Acute tubular necrosis Damage to the renal tubules caused by ischemia or toxic agents Fanconi syndrome Generalized defect in renal tubular reabsorption in the PCT Nephrogenic DI Inability of the renal tubules to respond to ADH Neurogenic DI Inability of the hypothalamus to produce ADH Renal glucosuria Inability of the renal tubules to reabsorb glucose Cystitis Ascending bacterial infection of the bladder Acute PN Infection of the renal tubules and interstitium Chronic PN 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 Aminoaciduria 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 Tyrosine Tyrosine transaminase (-) p-Hydroxyphenylpyruvic acid Tyrosinemia Tyrosyluria: p-Hydroxyphenylpyruvic acid oxidase p-OHPPA Homogentisic acid (-) p-OHPLA Homogentisic acid oxidase Alkaptonuria Maleylacetoacetic acid Homogentisic acid Fumarylacetoacetic acid Fumaric acid and Acetoacetic acid Phenylketonuria 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 Tyrosyluria Rancid butter odor (-) Tyrosine transaminase and p-OHPPA oxidase Screen: FeCl3  (+) Transient green Confirm: Nitroso-naphthol  (+) Orange-red Alkaptonuria Urine darkens after a period of standing (-) Homogentisic acid oxidase  Homogentisic acid in blood and urine FeCl3  (+) Transient blue Clinitest/Benedict’s  (+) Yellow ppt. Melanuria Overproliferation of melanocytes FeCl3  Gray or black ppt. Ehrlich’s  Red Branched-Chain Amino Acid Disorders MSUD Accumulation of leucine, isoleucine and valine in blood and urine 2,4-DNPH  (+) Yellow turbidity/ppt. Organic acidemias 1. Isovaleric acidemia = sweaty feet 2. Propionic acidemia 3. Methylmalonic acidemia Tryptophan Disorders Indicanuria Intestinal disorder lec.mt 04 |Page | 150  Blue color Hartnup disease: “Blue diaper syndrome” Obermayer’s test: FeCl3  (+) Violet w/ chloroform Argentaffinoma 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 Cystinuria (Renal type) Defect in renal tubular transport of: -Cystine (least soluble  urine) -Ornithine -Lysine -Arginine Cystinosis Inborn error of metabolism Cystine deposits in many areas of the body Cyanide-nitroprusside  (+) Red-purple Homocystinuria Defect in the metabolism of homocystine Silver nitroprusside  (+) Red-purple Brand’s modification of Rxn: Cyanide-nitroprusside  (+) Red-purple Legal’s nitroprusside Mucopolysaccharide Disorders MPS Dermatan SO4 Keratan SO4 Heparan SO4 Clinical significance Alder-Reilly syndrome Hurler syndrome = MPS  cornea of the eye Hunter syndrome = Sex-linked recessive Sanfilippo syndrome = Mental retardation only CTAB (+) White turbidity Purine Disorders Lesch-Nyhan disease  Urinary uric acid crystals Porphyrias D-ALA Glycine + Succinyl CoA ----(ALA synthetase)----> D-ALA Porphobilinogen D-ALA ----(ALA synthetase)----> Porphobilinogen Lead poisoning: inhibits ALA synthase Uroporphyrinogen Porphobilinogen -----(Uroporphyrinogen synthase/ Uroporphyrinogen cosynthase)----> Uroporphyrinogen Acute intermittent porphyria: (-) Uroporphyrinogen synthase Congenital erythropoietic porphyria: (-) Uroporphyrinogen cosynthase Coproporphyrinogen Uroporphyrinogen ---------(Uroporphyrinogen decarboxylase)---------------> Coproporphyrinogen Porphyria cutanea tarda: (-) Uroporphyrinogen decarboxylase Protoporphyrinogen Coproporphyrinogen ---------(Coproporphyrinogen oxidase)-------------------> Protoporphyrinogen Hereditary coproporphyria: (-) Coproporphyrinogen oxidase Protoporphyrin IX Protoporphyrinogen ---(Protoporphyrinogen oxidase)---> Protoporphyrin IX Variegate porphyria: (-) Protoporphyrinogen oxidase Heme Protoporphyrin IX + Fe2+ ----(Ferrocheletase)--------------> Heme Lead poisoning: inhibits Ferrocheletase Porphyrias Vampire disease Disorders of porphyrin metabolism lec.mt 04 |Page | 151 Screening tests (porphyria) 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 Specimens Urine: red/purple/portwine (normal: Lead poisoning) Stool Blood Bile Lead poisoning RBC inclusion coarse basophilic stippling Qualitative Tests for Protein (+) White Ring Heller’s Robert’s Spiegler’s (+) Violet Biuret (Albumin) (+) White turbidity/ Heat and acetic acid cloudiness SSA Purdy’s Potassium ferrocyanide Picric acid Kingsbury-Clark (Rgt: SSA) (+) coagulum (24 hrs) Esbach’s -Rgt: Picric acid + Citric acid Tsuchiya’s (+) coagulum Kwilecki’s (72’C for 5mins) -Rgt: Esbach’s + 10% FeCl3 Qualitative Tests for Sugars Benedict’s Reducing substances Seliwanoff’s Rgt: Resorcinol Fructose  (+) Red Rubner’s Rgt: Lead acetate, NH3 H2O Lactose  (+) Bright red w/ red ppt. Glucose  (+) Red color w/ yellow ppt. Bial Orcinol Pentose  (+) Green Tauber’s Pentose  (+) Green Others Osazone or phenylhydrazine (Kowarsky) Nylander’s Moore Heller Borchardt’s Qualitative Tests for Ketones Frommer’s Acetone  (+) Purplish red ring Rothera’s Acetone & AAA  (+) Purple ring Lange Acetone & AAA  (+) Purple ring Acetest/Ketostix Acetone  (+) Purple Gerhardt’s AAA  Bordeaux red Qualitative Tests for Bile Pigments Gmelin Bile  (+) Play of colors Smith Bile  (+) Emerald green lec.mt 04 |Page | 152 Harrison’s spot Bile  (+) Blue to green Ictotest Bile  (+) Blue to purple mat Wallace and Diamond Rgt: PDAB Urobilinogen  (+) Cherry red Schlesinger Rgt: Lugol’s iodine, Alc. Zinc acetate Urobilin  (+) Greenish fluorescence Qualitative Tests for Hemoglobin Benzidine (+) Green-blue Guiac (+) Blue Ortho-toluidine (+) Blue Qualitative Tests for Melanin FeCl3 (Screening) (+) Black (after 24 hrs) Thomahlen (+) Dark green or blue color (fresh urine) Blackberg & Wanger (+) Brown to black ppt. (24 hr urine) Qualitative Tests for Chloride Fantus (+) Reddish ppt Mercurimetric titration (+) Blue-violet colored complex (Schales & Schales) Qualitative Test for Calcium Sulkowitch (+) Precipitation Renal Function Tests Test for Glomerular Clearance filtration Test for Tubular Concentration tests reabsorption -Fishberg (old) -Mosenthal (old) -SG (new) -Osmolality (new) Fishberg test Patient deprived of fluid for 24 hrs = SG ≥1.026 Patient deprived of fluid for 12 hrs = SG ≥1.022 Test for Tubular Secretion PAH and Renal Blood flow PSP Tests for NPN 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) BCR (BUN: Crea Ratio) 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 Biohazard Symbol 4 circles Top = Source Left = Host Right = Transmission PPE Gloves Fluid-resistant gowns Eye and face shields Plexiglas countertop shields lec.mt 04 |Page | 153 Disinfection of sink 1:5 or 1:10 dilution of sodium hypochlorite (daily) Sodium hypochlorite soln. Effective for 1 month Chemical spills on skin Flush the area w/ water for at least 15 mins  seek medical attention Do not neutralize chemicals Always add acid  water To avoid the possibility of sudden splashing caused by the rapid generation of heat in some chemical reactions Handwashing Best way to break the chain of infection Clean between finger for at least 15 seconds Downward Sing “Happy Birthday” Hazard Classification 0 = Stable (Yellow = Reactivity) 1 = Unstable if heated 2 = Violent chemical change 3 = Shock and heat may deteriorate 4 = May deteriorate Hazard Classification OXY = Oxidizer (White = Specific Hazard) ACID = Acid ALK = Alkali COR = Corrosive W = Use no water  = Radiation Hazard Classification 0 = Normal material (Blue = Health) 1 = Slightly hazardous 2 = Hazardous 3 = Extreme danger 4 = Deadly Hazard Classification 0 = Will not burn (Red = Flammability) 1 = Above 200’F 2 = Below 200’F 3 = Below 100’F 4 = Below 73’C Types of Fire A = Wood, paper, clothing (ordinary combustibles) B = Flammable liquids C = Electrical equipment D = Flammable metals E = Detonation (Arsenal) In case of Fire R = Rescue A = Activate the alarm C = Contain the fire E = Extinguish Fire extinguisher P = Pull the pin A = Aim at the base of the fire S = Squeeze handles S = Sweep nozzle side to side Urinalysis Actually the beginning of laboratory medicine Hippocrates Uroscopy Frederik Dekkers Discovered albuminuria by boiling urine Thomas Bryant Pisse prophets (charlatans) Thomas Addis Examination of urinary sediment Richard Bright Introduced the concept of UA as part of doctor’s routine patient examination UA (CLSI/NCCLS) 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 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 QC (Reagent Strips) 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 Resolution (Microscope) Ability to visualize fine details Ability of the lens to distinguish two small objects that are a specific distance apart Parfocal (Microscope) Require only minimum adjustment when switching among objectives Centering and Köhler Provide optimal viewing of the illuminated field illumination (condenser) Camel-hair brush Removes dust coating the optical surface of the microscope Quality assessment 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  Preventive maintentance Access to procedure manuals Competency of personnel performing the tests Microscopic Quantitations EC (lpf) Crystals (hpf) Bacteria (hpf) Mucous threads None 0 0 0 - Rare 0-5 0-2 0-10 0-1 Few 5-20 2-5 10-50 1-3 Moderate 20-100 5-20 50-200 3-10 Many >100 >20 >200 >10 Casts (lpf) None = 0 Numerical ranges = 0-2/2-5/5-10/>10 RBCs (hpf) None = 0 Numerical ranges = 0-2/2-5/5-10/10-25/25-50/50-100/>100 WBCs (hpf) None = 0 Numerical ranges = 0-2/2-5/5-10/10-25/25-50/50-100/>100 Quality Assurance Errors Preanalytical 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 Analytical Sample misidentification Erroneous instrument calibration Reagent deterioration Poor testing technique Instrument malfunction Interfering substances present Misinterpretation of quality control data Postanalytical 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 TQM Based on a team concept involving personnel at all levels working together to achieve a final outcome of customer satisfaction through implementation CQI Improving patient outcomes by providing continual quality care in a constantly changing health-care environment PDCA Plan-Do-Check-Act PDSA Plan-Do-Study-Act lec.mt 04 |Page | 156  OTHER BODY FLUIDS Cerebrospinal Fluid CSF 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 Production Filtration Active transport secretion Functions Supply nutrients  nervous tissue Remove metabolic waste Provide mechanical barrier CSF glucose 60-70% of blood glucose Brain 1,500g (Henry) Meninges (Sing. Meninx) 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 Layers Skin  Skull  Dura Mater  Arachnoid mater  Subarachnoid space  Pia mater  Brain Arachnoid villi/ Reabsorbs CSF granulations If it can’t absorb CSF, CSF accumulates  Hydrocephalus Choroid plexuses Produce CSF at approximately 20 mL/hr CSF Total Volume Adults: 5th ed. = 90-150 mL 4th ed = 140-170 mL Neonates = 10-60 mL Blood brain barrier Between brain and blood Functions: 1. Protects brain from organisms 2. Shields brain from hormones and neurotransmitters 3. Maintains homeostasis for brain Circumventricular organs 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 Specimen collection 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 Precautions Measurement of intracranial pressure Prevent infection (povidone iodine) Prevent damage  neural tissue lec.mt 04 |Page | 157 Collection 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 Note Excess CSF ---(DO NOT)--> Discard Left-over supernatant  Chemistry/Serology CSF specimen  STAT If STAT not possible, specimens are stored Low volume specimen Collected on 1 tube Microbiology  Hematology  Chemistry/Serology CSF Appearance Crystal clear Normal Turbidity/cloudiness 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 CSF Protein NV = 15-45mg/dL >45 but 150mg/dL (xanthochromic) Intracranial Hemorrhage vs. Traumatic Tap Traumatic Tap Intracranial/Cerebral Hemorrhage Distribution of Blood Uneven (Tube 1 > 2 > 3) Even (Tube 1 = 2 = 3) Clot + (Plasma Fibrinogen) - Xanthochromia - + (+) Clot Meningitis (-) Blood Froin syndrome Blockage of CSF circulation (+) Weblike pellicle After overnight refrigeration (12-24 hrs) TB meningitis (+) D-dimer Indicates formation of fibrin at a hemorrhage site Recent hemorrhage Clear supernatant lec.mt 04 |Page | 158  To examine a bloody fluid for xanthochromia: Microhematocrit tube ---(Centrifuge)---> Examine supernatant against white BG Erythrophagocytosis/ Indicates intracranial hemorrhage Hemosiderin granules CSF Cell count Done immediately WBCs and RBCs lyse w/in 1 hr Refrigerate if cannot be processed immediately WBC count 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 Total CSF cell count WBC ct + RBC ct Diluting fluid: 0.85% (0.9%) NSS RBC count = Not counted RBC count Used for the correction of CSF WBC count and CSF protein count when a traumatic tap has occurred Clarity/Appearance Dilution Amount of Sample Amount of Diluent Slightly Hazy 1:10 30 μL 270 μL Hazy 1:20 30 μL 570 μL Slightly Cloudy 1:100 30 μL 2970 μL Cloudy/Slightly Bloody 1:200 30 μL 5970 μL Bloody/Turbid 1:10,000 0.1 mL of 1:100 dilution 9.9 mL CSF WBC Count Correction WBC (added) = WBCBlood x RBCCSF or RBCBlood PBS (normal) = -1 WBC/700 RBCs (CSF) Differential Count Performed on stained smears Specimen should be concentrated Methods for specimen Sedimentation concentration Filtration Centrifugation Cytocentrifugation 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 CSF Differential count 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  Adults (>18 y.o.) 40-80% lymphocytes 15-45% monocytes 0-6% neutrophils Cells in the CSF Normal Lymphocytes Monocytes Neutrophils (occasional) Adult Lymphocytes > Monocytes (70:30) Neonates Monocytes > Lymphocytes Pleocytosis  no. of normal cells  amount of WBCs in any body fluid Lymphocytes  viral, TB, fungal meningitis Monocytes  multiple sclerosis Neutrophils  bacterial meningitis, cerebral hemorrhage  early cases of viral, TB, fungal meningitis nRBCs (metarubricytes) BM contamination Eosinophils  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 Plasma cells  multiple sclerosis  lymphocytic reactions Macrophages (+) RBCs (+) RCM Nonpathologically Choroidal cells: EC (choroid plexus) significant cells Ependymal cells Spindle-shaped cells: arachnoid Malignant cells Hematologic: -lymphoblasts -myeloblasts -monoblasts Nonhematologic -Astrocytomas -Retinoblastomas -Medulloblastomas QC of CSF and Other Body Fluid Cell Count Biweekly basis All diluents ---(check for)---> Contamination Monthly basis Speed of cytocentrifuge should be checked w/ a tachometer CSF Protein 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) TRANSports THYroxine and RETINol 2nd most prevalent Tau Transferrin CHO-deficient β2-transferrin Seen in CSF and not in serum Electrophoresis: method of choice when determining if a fluid is actually CSF  CSF protein Damage: BBB (most common) Production of Ig in CNS (multiple sclerosis)  CSF protein CSF leakage Recent puncture Rapid CSF production Water intoxication Correction for traumatic If blood Hct and serum protein are normal: tap = -1 mg/dL protein/1,200 RBCs Measurement of Total CSF 1. Turbidimetric protein -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 CSF IgG index In MS:  CNS IgG CNS IgG must be differentiated from serum IgG (damage to BBB)  CSF IgG Damage to BBB Active production w/in CNS (MS) CSF/Serum Albumin index _CSF Albumin (mg/dL)_ Serum Albumin (g/dL) a. Index 0.70 = indicative of IgG production w/in CNS (MS) b. Index 29 years old L. monocytogenes Infants Elderly Immunocompromised patients CSF Glucose 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  CSF glucose Bacterial, TB, fungal meningitis N-CSF glucose Viral meningitis CSF Lactate CSF Lactate Frequently used to monitor severe head injuries False elevations: Xanthochromia/hemolysis (RBC contains lactate) 10-22 mg/dL Normal lactate >35 mg/dL Bacterial meningitis >25 Fungal and TB meningitis 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 CK-BB  Postcardiac arrest (poor prognosis) 35 mg/dL Associated w/ some disturbances of consciousness lec.mt 04 |Page | 162 Reye’s syndrome Life threatening  Glutamine in children Degeneration of the liver  blood clotting problems and bleeding Gram Stain 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 C. neoformans India ink GS: Starburst pattern -seen more often than a (+) India ink Latex agglutination test Limulus lysate 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) CSF serology Det. neurosyphilis VDRL: recommended by Centers for Disease Control and Prevention (CDC) FTA-ABS PAM (Henry) Naegleria fowleri Acanthamoeba spp. Acridine orange stain: useful to differentiate amoeba (brick-red) from leukocytes (bright green) L. monocytogenes The only Gram (+) that produces endotoxin (-) Limulus lysate test Seminal Fluid Interstitial cells of Leydig Secrete testosterone Outside the seminiferous tubules Seminiferous tubule Site of spermatogenesis Epididymis Stores and concentrates sperm Sperm maturation Prostate gland Zn3+, enzymes and proteins (coagulation and liquefaction) Seminal vesicles Fluid:  fructose Vas deferens Transports sperm  ejaculatory duct Bulbourethral (Cowper’s) Secretes alkaline mucus  neutralize prostatic and vaginal acidity gland Sertoli cells Serve as nurse cells for developing sperm cells Inside the seminiferous tubules Spermatogenesis Spermatogonia  1’ Spermatocytes  2’ Spermatocytes  Spermatids  Sperm Round cells Either WBCs or spermatids Seminal Fluid Composition Seminal fluid (SV) 60-70% Prostatic fluid 20-30% lec.mt 04 |Page | 163 Spermatozoa 5% Bulbourethral gland 5% Chemical Composition of Seminal Fluid ACP For liquefaction Zn3+  in prostatic disease Fructose Major nutrient of spermatozoa K+, citric acid, ascorbic acid -- Proteolytic enzymes Liquefaction and coagulation Spermine and Choline Inhibit growth of bacteria Importance of Seminalysis 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 Sexual abstinence 2-3 days and not >5 days  abstinence =  volume,  motility Methods of collection 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’) Methods of preservation 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 Liquefaction time 30-60 mins If not yet liquefied after 2 hrs, use α-chymotrypsin Normal values 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 Volume : incomplete collection/infertility : prolonged abstinence Yellowish semen Prolonged abstinence Medication Urine contaminationi  White turbidity Infection ( WBCs) Red coloration (+) RBCs Viscosity 0 (watery) 4 (gel-like) pH 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 Tenacity of mucus Sim Huhner test Post-coital test Test for the ability of sperm cells to penetrate the cervical mucosa Bloom’s/Eosin-Nigrosin/ If N-sperm count but  motility Sperm viability test Living sperm cells = bluish white Dead = red NV = 75% living sperms (25% dead) Decreased motility w/ (+) Male antisperm antibodies clumping Blood testis barrier  disrupted Clumps of sperm Normal seminalysis w/ (+) Female antisperm antibodies continued infertility MAR Mixed agglutination reaction Detect IgG antibodies Immunobead test Detect IgG, IgA, IgM Demonstrate area of the sperm the autoantibodies are affecting Hamster egg penetration Sperm incubated w/ species non-specific hamster egg Cervical mucus penetration Observed sperm penetration ability Hypo-osmotic swelling Test for membrane integrity and viability of sperm To determine whether Microscopic exam for sperm cells semen is present Enhance w/ xylene Examine  Phase microscope Seminal glycoprotein p30: specific method Aspermia No ejaculate Azospermia Absence of sperm cells Necrospermia Immotile/dead sperm cells Oligospermia  sperm cells Synovial Fluid Synovial Latin: “Egg” Fluid Diarthroses/joints Arthrocentesis Method of collections Synoviocytes Phagocytic cells Secrete hyaluronic acid Specimen collection 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 Normal Values Volume = 2.0 = mature fetal lungs Disadvantage: can’t be done on specimen contaminated w/ meconium Lecithin Surfactant for alveolar stability Surfactants Produced by type II alveolar pneumocytes (stored as lamellar bodies = about the size of platelets) Amniostat FLM Detect the presence of phosphatidyl glycerol Advantage: not affected by blood or meconium Foam stability test Amniotic fluid + 95% ethanol  shake for 15 secs  stand (15 mins) (Foam/Shake test) (+) Continuous line of bubbles Can be done bedside Fetal age Creatinine ≥2.0 mg/dL (36 weeks/9 months) Gastric Fluid Analysis Clinical significance Pernicious anemia Peptic ulcer Zollinger-Ellison disease Parietal cells Produces HCl and intrinsic factor Pepsinogen Produced by chief cells Pepsinogen ---(HCl)---> Pepsin Gastrin Produced by G-cells Stimulates parietal cells to produce HCl Zollinger-Ellison disease  gastrin Gastric acid hypersecretion Adenoma of the islets of Langerhans (pancreas) = produce gastrin Pernicious anemia “Dangerous” anemia BAO/MAO = 0 Specimen collection Collect gastric juice for 1 hr 1. Levine tube = nose 2. Rehfuss tube = mouth Basal Acid Output (BAO) Total gastric secretion during unstimulated fasting state Maximal Acid Output Total acid secreted in the hour after stimulation (MAO) Gastric stimulants 1. Pentagastrin = most preferred 2. Histamine lec.mt 04 |Page | 170  3. Histalog (Betazole) 4. Alcohols 5. Insulin = assess vagotomy procedure -SHAM feeding = sandwich Test Meals 1. Ewald’s meal = bread and tea/H2O 2. Boa’s = oatmeal 3. Riegel’s = mashed potato and beef steak Yeast cells in Gastric fluid Fermentation in stomach because large amounts of food have been retained Quantitative Tests for Gastric Acidity (Topfer’s) Free HCl Titration: NaOH pH indicator: Dimethylaminoazobenzol (+) Canary yellow NV = 25-50O Total Acidity Titration: NaOH pH indicator: phenolphthalein (+) Faint pink NV = 50-75O Combined HCl Titration: NaOH (bound to proteins) pH indicator: sodium alizarin (+) Violet NV = 10-15O Euchlorhydr