Clinical Microscopy Notes PDF

07/11/2024 CLINICAL URINE FORMATION AND MICROSCOPY KIDNEY DISEASES REVIEW FUNCTIONS OF THE KIDNEYS 1. Excrete waste p...

07/11/2024 CLINICAL URINE FORMATION AND MICROSCOPY KIDNEY DISEASES REVIEW FUNCTIONS OF THE KIDNEYS 1. Excrete waste products of metabolism 2. Regulate acid-base balance 3. Regulate electrolyte balance 4. Regulate blood pressure URINALYSIS 5. Regulate red cell production(erythropoiesis) Urine Formation Nephron – functional unit of the kidney 1- 1.5 millions of nephrons in each kidney a. Cortical – cortex ; 85 % of nephrons -removal of waste products -reabsorption of nutrients b. Juxtamedullary – loop of henle to the medulla - concentration of urine Glomerulus- PCT- Loop of Henle – DCT Collecting Ducts – ureter – bladder-urethra - out 1  07/11/2024 Urine Formation 2. Glomerular Filtration 1. Renal Blood Flow 2. Glomerular Filtration 3. Tubular Reabsorption Factors that influence filtration process 4. Tubular Secretion A. cellular structure 3 layers: capillary wall memb., basement I. Renal Blood Flow ( based on ave memb., visceral epith.(podocytes) body surface of 1.73 m2) B. hydrostatic pressure 25 % of CO – ( 1, 200 ml /min) C. oncotic pressure Renal plasma flow – 600- 700 ml/min D. renin-angiotensin-aldosterone system Renin-angiotensin-aldosterone system 3. Tubular Reabsorption A. active transport – substance combines w/carrier Juxta glomerular appratus – juxtaglomerular cells & macula densa protein - electrochemical energy transfers the substance - Renin reacts with blood-borne substrate“Angiotensinogen” across the cell membrane to produce “Angiotensin I” Ex. Glucose, amino acids, salts, chloride, Sodium - Angiotensin I in the presence of ACE in the lungs is converted into “Angiontensin II” B. Passive transport- - Angiontensin II corrects Renal Blood Flow by: – differences in their concentration or electrical potential on opposite sides of the membrane  vasoconstriction of renal arterioles  stimulates PCT reabsorption of sodium  release of “aldosterone” (Na retaining hormone) from – Ex. Water, urea, sodium the adrenal cortex 2  07/11/2024 SUBSTANCE LOCATION 4. Tubular Secretion Functions: ACTIVE TRANSPORT GLUCOSE, AMINO ACIDS PCT (+CARRIER CHON) AND SALTS A. elimination of waste products not filtered by the glomerulus B. regulation of the acid-base balance CHLORIDE A Loop of henle SODIUM PCT & DCT Renal tubular acidosis- alkaline urine due to metabolic (controlled by aldosterone) acidosis PASSIVE WATER PCT, D Loop of Henle Normal blood pH is 7.4 TRANSPORT CD To maintain this blood pH a buffer (HCO3)/ Bicarbonate is needed (DIFFERENCE IN to eliminate excess acid formed by diet and body metabolism. CONC. “GRADIENT”) UREA PCT & Acid secreted a day = 70meq/day in the form of either A Loop of Henle - titrable acid (H+) - hydrogen phosphate SODIUM A Loop of henle - ammonium ions Glomerular Filtration Tests- RENAL FUNCTION TESTS Clearance Tests 1. Glomerular Filtration Tests 1. Urea clearance- 40 % is reabsorbed by A. Clearance Test – measures the rate at which the the tubules kidneys are able to remove a filterable substance 2. Creatinine clearance- routinely used; from the blood some is secreted by the tubules - uses 24 hour urine - affected by heavy meat diet - not reliable in muscle wasting dses Formula – UV 3. Inulin clearance – polymer of fructose not C ___ x 1.73 secreted nor reabsorbed ___ 4. beta2 microglobulin ( EIA using plasma) Normal Values: P A  Creatinine Clearance: 5. radioisotopes  Female – 75 - 112 ml/min 6. Cystatin C - small protein prod by nucleated  Male – 85 - 125 ml/min cells 3  07/11/2024 CALCULATED GLOMERULAR FILTRATION Tubular Reabsorption Tests ESTIMATES 1. Sp. Gr. 2. Fishberg Test – patients are deprived of fluids for Cockroft & Gault 24 hrs prior to measuring the specific gravity Variables – age, sex, weight in kg 3. Mosenthal Test – compares the volume and specific gravity of day and night samples Ccr = (140 –age)(wt in kg) 4. osmolarity 72 X serum crea in mg/dl Specific gravity depends on the number of particles Female – multiply by 0.85 present in the solution and the density of these particles osmolarity is affected by the number of particles present Composition of urine Tubular Secretion and water Renal Blood Flow Tests 1. 2. analytes 2.1 Organic  urea 1. Phenosulphonaphthalein (PSP)  Creatinine,uric acid, ammonia, undetermined nitrogen 2. P aminohippuric tests (PAH Test) 2.2 Inorganic 3. Titrable Acidity and Urinary Ammonia  chloride  sodium, potassium  phosphorus, calcium, magnesium and iron 3. others: hormones, vitamins, medications, formed elements 4  07/11/2024 Specimen collection and Handling Preservation  Information on requisition form: Refrigeration 1. Actual date & time of specimen collection 2. Method of preservation (if any) Freezing 3. Time : receipt , the test was performed 4. Tests requested Chemical preservative 5. Patient identification - bactericidal - inhibits urease Specimen should be examined w/in 2 hrs - preserves formed elements No ideal preservative URINE PRESERVATION Changes in Unpreserved Urine Refrigeration – does not interfere w/ chem.rxns Thymol 1. increase in bacterial content – Glucose & sediments 2. increased turbidity Boric Acid 3. increased pH (alkaline) – protein & formed elements 4. increased nitrite – Preservative in urine C/S transport kit Formalin – excellent sediment preservative 5. decreased glucose Na flouride 6. decreased ketones – Prevents glycolysis, best for drug analysis 7. decreased bilirubin Phenol 8. decreased urobilinogen – odor change 9. disintegration of RBCs and casts (dilute Saccomanno’s fixative alkaline urine) – cellular elements; for cytologic studies 10. changes in color due to oxidation and  MINERAL ACIDS (HCL or H2SO4 - adrenalin, noradrenaline, vanillylmandelic acid, and steroids reduction of metabolites - 10 ml/ 24 hr specimen 5  07/11/2024 TYPES OF URINE SPECIMEN Types of urine specimen 1. Random specimen 1. Catheterized –for bacteriologic and culture determination 2. First morning- concentrated specimen Disadvantage: discomfort to the patient 3. Fasting (2nd morning) and risk of infecting the bladder. 4. Timed Specimen a 24 Hr sp- substances that vary; start w/ an empty 2. Midstream clean catch- bacterial culture/routine urinalysis bladder b. 12 Hr sp - usually used for Addis Count with formalin as preservative 3. Suprapubic aspiration - for bacterial culture and cytologic c. 2 Hour Post Prandial examination -primarily for “monitoring insulin therapy” in persons Disadvantage: discomfort to the patient and risk of with Diabetes Mellitus infecting the bladder. -usually compared with fasting specimens 4. 3-glass collection – prostatic infection d. Afternoon Specimen (2-4 pm) - used for “urobilinogen determination” Suprapubic aspiration Catheterization 6  07/11/2024 Routine Urinalysis DRUG SPECIMEN 4 Parts COLLECTION 1. Specimen evaluation Chain of custody 2. Physical examination Urine Temperature- 32.5 – 37.7 3. Chemical Examination 4. Sediment Examination(Microscopic) I. Specimen Evaluation 1. Proper labeling 2. Visible signs of contamination 3. Proper specimen for the requested test 4. Any transportation delays PHYSICAL EXAM OF COLOR URINE 1. Color Varies, metabolic function, physical activity, diet 2. Clarity N = Pale yellow → amber 3. Specific gravity Pigments in Urine: 1. urochrome – constantly produced ↑ hyperthyroid, fasting, exercise 1. Uroerythrin – pink → refrigerated A.U. 2. Urobilin – orange-brown 7  07/11/2024 COLOR OF URINE SIGNIFICANCE COLOR of URINE Colorless D. mellitus Dark yellow/amber/orange Pale yellow D. insipidus, -concentrated urine Over hydrated -bilirubin → foam test → yellow Orange Bilirubin -phenazopyridine (pyridium) orange Acriflavin (+) orange foam Red/pink/brown Pyridium amt, pH, length of contact Yellow-green/brown Bilirubin → biliverdin acid urine → brown Green Pseudomonas RBC, Hgb, myoglobin Clorets porphobilinogen → porphyrin → PORT WINE Methocarbamol Phenol Indican → purple URINE CLARITY COLOR OF URINE SIGNIFICANCE Pink RBC beets Clear -transparent Red Hgb rifampin Hazy -print easily seen Myoglobin Cloudy -print blurred Methemoglobin Brown Turbid -print can’t be seen Black Alkaptonuria Milky -may ppt Melanin Metronidazole 8  07/11/2024 Cause of Turbid urine Specific Gravity NON-PATHOLOGIC PATHOLOGIC Squamous epith cell RBC (500 RBC/mm3) Mucus WBC (200 WBC/mm3) Density of a solution compared w/ density Amorphous urates/phosphates Bacteria of d. H2O at similar vol & temp Semen Yeast Assesses the kidney’s ability to reabsorb Fecal contamination Non-squamous epithelial Radiographic contrast media cells Detects possible dehydration or Talcum, vaginal creams Abnormal crystals abnormalities in ADH secretion Lymph fluid Evaluates the concentrating & diluting Lipids abilities of the kidneys Specific Gravity Refractometer Refractive Index Small specimen is needed No temperature correction- temp. compensated bet 15C- 38C Corrections for glu & protein Calibrated with: – distilled water: 1.000 – 5% NaCl: 1.022 – 9% sucrose: 1.034 9  07/11/2024 Sp Gr. Reagent strip( Dipstick) Sp Gr : Harmonic Principle: pK change of polyelectrolytes Oscillation Densitometry (polyelectrolyte ionizes releasing H+ ions in sound wave frequency will change in proportion to the # of ions in the soln) proportion to the density of the solution Inc sp gr, Inc H+ released = low pH (acidic) Used in automated urinalysis (IRIS) Dec sp gr , Dec H+ released = high pH (alkaline) Indicator : bromthymol blue 1.000 (blue) alkaline –shades of green – 1.030 (yellow) acid Highly alkaline urine – false (-) result Hypersthenuria – increased SG (>1.010) Volume diarrhea, dehydration, decreased fluid intake, fever Average Daily Urine Output -1200 -1500 ml proteinuria/glucosuria, lipid nephrosis Random -600 -2000 ml x-ray contrast media/IV pyelogram (SG > 1.035) dextran administration Oliguria – (500 ml by an adult at night with specific Fruity & sweet diabetic acidosis gravity of 2000ml/24 hrs) Sweaty feet isosvaleric/glutaric Diabetes Mellitus Cabbage methionine malabsorption Diabetes Insipidus Bleach Diuretics, caffeine or alcohol consumption PARAMETER Read- PRINCIPLE REAGENTS (Multistix) Chemical Examination of ing time Urine Leukocyte 2 mins Granulocytic Derivatized pyerole Esterase esterase rxn AA ester Diazonium salt Nitrite 60s Greiss rxn p-arsanilic acid Specific Gravity Tetrahydrobenzo(h) Urobilinogen Leucocytes quinolin-3-ol Bilirubin Glucose Protein Ketone Nitrite Urobilinogen 60s Ehrlich rxn p-diethylamino Blood pH benzaldehyde Protein Timing Protein error of Tetrabromphenol not indicators blue critical pH Double indicator Methyl red, system bromthymol blue 11  07/11/2024 PARAMETER Read PRINCIPLE REAGENTS -ing (Multistix) URINE pH time Pseudoperoxidase Diisopropyl- KIDNEY- regulates acid-base balance Blood 60s activity of Hgb benzene Secretion of H+ in the form of ammonium ions, dehydroperoxide, tetramethyl- hydrogen phosphate and weak organic acids benzidine Reabsorption of bicarbonates in the PCT Specific Gravity 45s pKa change of Bromthymol blue polyelectrolytes Normal blood pH is 7.4 Ketones 40s Na nitroprusside rxn Na nitro-prusside To maintain this blood pH a buffer Bicarbonate is Bilirubin 30s Diazo rxn 2,4-dichloro-aniline needed to eliminate excess acid formed by diet and diazonium salt body metabolism. Glucose 30s Double sequential Glu oxidase pH = 4.6 – 8 (random) enzyme rxn Peroxidase K iodide 5.0-6.0 (1st morning) URINE pH URINE pH ACID URINE ALKALINE URINE NH4Cl, methionine, methenamine → ↑ protein ↑ fruits & vegetables acidify urine Cranberries Citrus *they dissolve PO4 & Ca carbonate Acid producing bacteria Less acidic after meal (E. coli) (alkaline tide) stones Starvation Renal tubular acidosis Na2HCO3, K citrate, acetazolamide → Dehydration Urease producing alkalinize urine bacteria *they dissolve cystine, Ca oxalate, uric Diarrhea hyperventilation acid 12  07/11/2024 Urine pH Methods: PROTEIN 1. Reagent strip : Methyl red & bromthymol blue (Double Indicator system) 150 mg of protein in 1 day 2-10 mg/dl average random Methyl red (red to yellow) pH 4- 6 Bromthymol blue ( yellow to blue) pH 6-9 Important renal marker orange → green → blue as pH ↑ Test should not detect less that 8-20 mg/dl albumin – major serum protein found in normal 2. pH electrode – pH meter w/ glass electrode urine 3. Titrable acidity of urine - Functional Proteinuria Transient Proteinuria 4g per day -↓Serum albumin Urine CHON : (+) day; (-) night -↑Lipid in blood & urine Exaggerated lordotic posture -Granular cast First voided urine -Fatty cast -Oval fat body (-) CHON 2 hrs standing or walking (+) CHON Moderate 1 to 4 g/day proteinuria Minimal 3-4 g/day 1-2 g/day by acetic acid (5-10%) (-) charge on GBM is ↓ 2. nitric acid Urine (+) large protein Urine (+) small proteins 3. SSA a2 macroglobulin a1 microglobulin 4. TCA B-lipoprotein b2 microglobulin  glomerulonephritis Fanconi, cystinosis False (-) Highly alkaline urine 14  07/11/2024 PROTEIN Reagent Strip “Protein error of indicators” Quantitative Protein Indicators change in color in the presence of protein ( 1. Precipitation pH is constant). SSA +TCA ( nephelometer photometer) Indicator – tetrabromphenol blue 2. Colorimetric (-) yellow TCA- Biuret (+) green then blue 3. Dye binding (Coomasie brilliant blue, false (+) – highly alkaline urine Ponceau S) high sp gr quaternary ammonium compounds Quantitative Protein PROTEIN Bence Jones (Multiple Myeloma) protein electrophoresis MICROALBUMINURIA -amido black stain, coomasie – Cant be detected by brilliant blue rgt strip -single sharp peak at globulin area – Diabetic nepropathy – Hypertensive -ppt 40-60 C, dissolve at 100 C Micral Test MM- proliferative disorder of the immunoglobulin- producing plasma cells w/ high levels of BJP in serum 15  07/11/2024 GLUCOSE in Urine Glucose Detectable amt of glucose → glucosuria Renal threshold 160mg/ dl to 180mg/dl Renal Associated Glucosuria : Hyperglycemia associated: 1. Fanconi’s syndrome 1.DM 6. hyperthyroidism 2. Advanced Renal Disease 2.Pancreatitis 7. pheochromocytoma 3.Pancreatic Ca 8. CNS damage 3. Osteomalacia 4.Acromegaly 9. stress 5.Cushing’s syndrome 10. gestational diabetes 4. Pregnancy GLUCOSE Copper Reduction Test for Reagent Strip Sugar in Urine specific for glucose only Young pediatric patient “Double sequential enzyme” (Oxidase & Reducing substance (except sucrose) peroxidase) Neonatal life – first 2 wks False (+): oxidizing cleaning agent (+) glu, gal, fruc, lac False (-): Vit C, upon standing, high sp gr Normal pregnancy (+) lactose Benedicts and Clinitest 16  07/11/2024 1. Benedict’s Test 2. Clinitest Copper Sulfate Prin: Copper Reduction 2 drops Urine NaOH 5 drops Water Na carbonate Citric Acid Store in dry, away from sunlight Neg Blue Normal: spotted bluish white tablet Trace Green w/o ppt Dark blue to brown tablet – not good (discard) 1+ green w/yellow ppt Rgt: CuSO4, NaOH, Sodium carbonate, sodium citrate 2+ yellow green w/yellow ppt (+) rxn: blue to orange/red 3+ muddy orange w/yellow ppt Pass through phenomenon 4+ orange to red ppt Correlation of Reagent Strip and SSA results: (+) RgtStrip; (-) SSA = albumin present (+) Rgt Strip; (+) SSA = proteinuria (-) Rgt Strip; (+) SSA = BJP, globulins, etc Correlations of Glucose Oxidase & Benedict’s Glucose Oxidase Benedict’s Interpretation (+) (+) glucose (-) (+) non-glucose reducing sugar 17  07/11/2024 Ketones Clinical Significance of urine Acetone 2% ketones Acetoacetic acid(Diacetic acid) 20% Diabetic acidosis Beta-hydroxybutyric acid 78% Insulin dosage monitoring Starvation Products of incomplete fat metabolism Excessive carbohydrate loss Malabsorption/pancreatic disorders Defect in CHO metabolism or inadequate CHO in Strenuous exercise the diet – body compensates by metabolizing increasing amt of FA, when this inc is large , KB Vomiting appear in urine Tests for Ketones Test for Ketones 3. REAGENT STRIP Nitroprusside (Na nitroferricyanide) 1. Rothera mtd (Acetone + AA) (+) AA - purple - Na nitroprusside - red to purple ring 4. CHEMSTRIP 2. Ferric Chloride (Gerhardts) (+) AA only Na nitroferricyanide + glycine AA + acetone - bordeaux red 18  07/11/2024 Test for Ketones HEMATURIA 5. NITROPRUSSIDE TABLET TEST Renal calculi (ACETEST) Used if urine has interfering color IgA nephropathy Tablet – sensitive to humidity Glomerulonephritis Acetest tab: Na nitroprusside, glycine WB, plasma, urine Pyelonephritis 1 tab + white piece of paper + 1 drop specimen Trauma – Urine 30 sec, serum plasma 2 min, WB 10 min – Lavender to deep purple Tumors Excessive exercise HEMOGLOBINURIA HEMOSIDERIN Free Hgb in urine Free Hemoglobin catabolized to ferritin & (+) intravascular hemolysis hemosiderin Check Plasma → pink (+) 2-3 days after acute hemolytic episode Transfusion rxn Yellow brown granules Hemolytic anemia Free or in epithelial cell or cast Strenuous exercise 19  07/11/2024 MYOGLOBINURIA Rgt Strip for Blood Acute destruction of muscle fiber after trauma Rgt Strip (+) in Hgb, Myoglobin and Red brown pigment Marathon, karate, hematuria Pt (+) muscle tenderness *well mixed urine URINE Prin. Pseudoperoxidase activity of Red brown urine ( cola drink) (+) Hgb test (+) protein hemoglobin Few RBC SERUM clear, ↑ CK, aldolase Normal haptoglobin Rgt Strip for Blood Hemoglobin vs. Myoglobin Uses tetramethylbenzidine Blondheim Ammonium sulfate Test Yellow(neg) to Green to green-blue (pos) 5 ml urine + ammonium sulfate Speckled green – intact RBCs Centrifuge false (-) Myoglobin – colored supernatant ↑ urine SG → RBCs don’t lyse Hemoglobin – colorless supernatant Ascorbic acid - false (-) Hypochlorite (bleach) - false (+) Microbial peroxidase - false (+) 20  07/11/2024 URINE Bilirubin & Bilirubin Urobilinogen in Jaundice Clinical Significance: Urine Urine Bilirubin urobilinogen 1. Hepatitis Obstructive +++ Normal 2. Cirrhosis Jaundice 3. Other liver cirrhosis Liver Damage + 0r - ++ 4. Biliary obstruction(gallstones, CA) Hemolytic Neg +++ jaundice Rgt Strip for Bilirubin Confirmatory Bilirubin Test Diazo Rxn : ICTOTEST - Bilirubin + p-nitrobenzene diazonium p-toluene Coupling reaction of bilirubin w/ sulfonate diazonium salt in acid solution + result (blue & purple) (+) result : OTHER TESTS FOR BILIRUBIN: Multistix – buff to tan 1. Gmelin 2. Smith Chemstrip – pink to violet 3. Foam 4. Fouchet 21  07/11/2024 Urobilinogen Urobilinogen bilirubin → intestine → urobilinogen Clinical Significance: → ½ feces - urobilin 1. Early detection of liver disease → ½ back to the liver → small amt – 2. Liver diseases kidney 3. Hemolytic disorders Urobilinogen is Colorless & labile → urobilin (colored) ↑ in alkaline urine/ ↓ in acid Watson Schwartz Differentiation test Urobilinogen Urobilinogen – soluble in both chloroform & butanol REAGENT STRIP – Red chloroform & butanol layers Ehrlich aldehyde reaction Porphobilinogen – insoluble in both chloroform & Formation of red azo dye butanol MULTISTIX - Colorless chloroform & butanol layers P-dimethylamino benzaldehyde(PDAB) → reddish brown color Not specific to urobilinogen: porphobilinogen,Sulfonamide, procaine, 5HIAA, indole, methyldopa 22  07/11/2024 Indirect Test for UTI Hoesch Test NITRITE Reduce nitrate → nitrite Screening test for porphobilinogen E. coli, Klebsiella, Enterobacter, Proteus, Hoesch Rgt: Ehrlich Rgt in 6 M HCl Staphylococci 1st morning, mid-stream catch + result - red Method: depends on the conversion of Urobilinogen – inhibited by the highly acidic nitrate to nitrite pH Requires overnight bladder incubation (min. 4 hrs) (+) result → do culture Indirect Test for UTI Indirect Test for UTI Leukocyte Esterase Nitrite Test NITRITE – Greiss Rxn Human neutrophil primary granule has esterolytic activity Multistix: p-arsanilic acid forms diazonium salt → pink Sensitivity : 5-15 wbcs/hpf False (-): Non reductase containing bacteria (+) = either intact or lyzed ascorbic acid lack of nitrate in the diet insufficient contact time 23  07/11/2024 Leukocyte Esterase Ascorbic Acid RGT STRIP Neutrophil esterases catalyses the hydrolysis of ester Gives false (-) reaction in the ff rgt strip MULTISTIX tests Derivatized pyerole AA ester, diazonium salt 1. Glucose purple intensity is proportional to the # of WBC 2. Blood Vit C- false (-) 3. Bilirubin Contamination w/ vaginal fluid - (+) result 4. Nitite Trichomonas, eosinophil - (+) result 5. Leukocyte esterase MICROSCOPIC EXAMINATION OF URINE Procedure Examination of Urine Sediment Sample: 1.Place 10-15 well mixed urine in a test tube – Fresh 2.Centrifuge for 5 mins at 1,500-2500rpm or – Cells & casts begin to deteriorate w/in 2 hrs 400 RCF – Refrigeration: prevents lysis 3.Decant (vol of sediment = 0.5 to 1 ml) – ↑ precipitation of amorphous 4.Place a drop on a slide(0.02 ml), cover w/ cover slip 5.Examine under LPO then HPO 24  07/11/2024 MICROSCOPIC QUANTITATIONS Examination of Urine 10 fields Epith cells /LPF Sediment None : 0 Rare : 0-5 Few : 5-20 BRIGHTFIELD MICROSCOPY – Subdued light - translucent structures in urine: Moderate 20-100 Many : > 100 hyaline cast & mucus threads Casts/LPF = None 0-2, 2-5, 5-10, > 10 PHASE CONTRAST MICROSCOPY – Good for translucent formed elements – it hardens the outline of transparent structure RBCs/WBCs/HPF = None ,0-2,2-5,5-10,10- 25, 25-50,50-100, >100 (TNTC) POLARIZED MICROSCOPY – Identification of crystals and lipids Crystals/HPF = None: 0, Rare: 0-2 , Few 2-5 – (+) Maltese cross Moderate 5-20, Many :>20 RBC Staining Methods Pale, biconcave disc, 7 um Not fresh specimen: 1.Sternheimer- Malbin – delineates structures and – colorless circle “shadow cell” contrasting colors of nucleus & cytoplasm Crenate in hypertonic urine - small rough -uses Crystal violet & Safranin Swell & lyse in hypotonic urine – only membrane “ghost cell” 2. Toluidine blue –enhances nuclear detail Oil droplets: differ in size & more refractile 3. Oil Red O and Sudan III – fats Yeast: budding, add acetic acid 4. Hansel - eosinophils RBC 0-2/hpf from any part of the urinary tract - uses Methylene blue & Eosin Y C/S 5. Prussian blue – hemosiderin Glomerulonephritis Trauma Systemic and renal diseases 25  07/11/2024 Dysmorphic RBC Leukocyte NEUTROPHIL RBC with protrusion or Major, granular sphere 12 um, fragmentation multilobate nucleus, may confuse to RTC Dilute HOAc enhances nuclear renal glomerular detail bleeding Dilute or hypotonic urine, neutrophils swell, & their cytoplasmic granules show Brownian movement → glitter cells WBC lysis: alkaline & hypotonic urine Pyuria N = < 5 WBC/hpf Eosinophil UT Infection or inflammation Pyelonephritis, cystitis, prostatitis Not normally seen in urine Acute urethral syndrome (dysuria-pyuria) Hansel secretion stain (MB in EY) If w/ WBC cast → renal origin Seen in Tubulointerstitial disease Hypersensitivity to drugs (Penicillin) 26  07/11/2024 Epithelial Cell TRANSITIONAL (UROEPITHELIAL) CELL Pelvis → lower 3rd of urethra Round, pear shape, central round nucleus, occl binucleate SQUAMOUS EPITHELIAL CELL Most frequently seen EC normal urine Least significant, distal 1/3 urethra Few in normal U/A Large, flat, abundant cytoplasm, small round central nuclei, pink (+) large clumps = transitional cell CA Vagina, vulva Clue cells - SEC w/ Gardnerella vaginalis Renal Tubular Epithelial Cell Lipid in RTEC Most significant type of epith cell in urine RTE from PCT & DCT – occur singly(14-60u)- OVAL FAT BODIES oblong or egg shaped cells w/ coarse granules Tubular cell absorbed lipoproteins nephrotic syndrome RTE from collecting ducts(12 -20 u) cuboidal or polygonal, w/ slightly eccentric nucleus ↑ Chole (+) Maltese cross TAG, ORO, Sudan III C/S ↑ Acute tubular necrosis,tubular damage drug & Bubble Cells – RTE w/ non lipid filled heavy metal poisoning vacuoles 27  07/11/2024 Pigment CAST Formed w/in the lumen of DCT & CD Heme pigment absorbed into cell = Formed when protein precipitate & gel in the lumen hemosiderin Cylindrical w/ parallel sides and rounded ends Iron laden cell → urine sediment Sole site: kidney Yellow brown Tamm-Horsfall – Matrix of all casts (+) Prussian blue – Meshwork traps cells CAST ↑ CAST formation Appearance & width depends on the tubule: pH Broadcast – dilated tubule, stasis (CRF) Ionic conc Thin cast – swollen interstitium tissue, Obstruction & stasis Tail & tapering → disintegrating → cylindroid Proteinuria - ↑ albumin & globulin Normal = few casts Strenuous exercise, (+) protein Plasma CHON combine with Tamm Horsfall 28  07/11/2024 HYALINE CAST WAXY CAST CRF, become denser → waxy  Most frequent ↑ R.I. Entirely Tamm Horsfall Brittle → cracks translucent Tubular inflammation & degeneration > 0-2/ lpf Waxy – final phase of dissolution of fine granules C/S Congestive Heart Failure Tubular obstruction with prolonged stasis Glomerulonephritis Severe chronic renal failure Pyelonephritis Stress When broad → Renal Failure cast, tubular atrophy, Exercise stasis CELLULAR CAST CELLULAR CAST RBC CAST WBC CAST Cast matrix containing RBCs Bleeding in nephron Cast matrix containing WBCs Glomerular damage & protein - Glomerulonephritis and strenous Pyelonephritis, interstitial nephritis exercise 29  07/11/2024 GRANULAR CELLULAR CAST CAST FGC / CGC RENAL TUBULAR EPITHELIAL CELL CASTS Fairly common Hard to differentiate from WBC cast Pathologic: Most reliable is singular round nuclei 1. disintegration of cellular casts ATN, viral dses 2. Protein aggregates Nonpathologic condition: MIXED CELLULAR CASTS lysosomes excreted by tubular 2 distinct cell types present within a single cells cast c/s : glomerulonephritis, pyelonephritis FATTY CAST PIGMENTED CASTS. Protein matrix with oval fat bodies Hgb cast (blood cast): yellow to red - Maltese cross formation Myoglobin cast – red-brown Bilirubin – deep yellow brown Seen in Nephrotic syndrome 30  07/11/2024 MUCUS THREADS BROAD CAST Defined as having 2 to 6x diameter of Threadlike structures normal cast with low refractive index Tubular dilatation & stasis long ribbon-like CRF Poorly defined edges Pointed or splint ends CYLINDROIDS TELESCOPED SEDIMENT Resemble casts but have one end that tapers to a tail Elements of glomerulonephritis Elements of nephrotic syndrome RBCs, RBC casts,cellular casts, broad waxy casts,lipid droplets,oval fat bodies, fatty casts Collagen vascular disease(lupus nephritis) SBE 31  07/11/2024 MICROORGANISMS & PARASITES IN URINE TYPE DESCRIPTION N SIGNIFICANCE TYPE DESCRIPTION NORMAL SIGNIFICANCE Trichomonas Turnip shaped Neg infection of Bacteria Color: colorless Free of -contamination vaginalis flagellates Confused vagina and vulva Shape: Rods or bacteria in Rapidly multiply in with WBCs in females cocci Kidney & improperly stored Needs to be mobile Infection of Bladder specimen for identification urethra in males With increased WBCs, indicative of UTI Yeast colorless cells Negative Found in UTI, Enterobius Ova have one flat Neg Usually found in ovoid smooth cells especially patients vermicularis and one round side children and in with doubly with DM refractile walls with transparent fecal Immunosuppressed shell. Developing contamination Often show budding & pseudohyphae patient larvae can be seen Sometimes Skin or vaginal mistaken for RBCs infection Sperma Oval heads with in both Male: nocturnal Schistosoma Clear and colorless Neg Inhabits veins in tozoa long thin tails male & emission, hematobium ova, characteristic urinary bladder Female ejaculation and terminal spine urine disease of the genital organs Female: after coitus TYPE Description pH Solubility Significance Uric acid yellow-brown Acid Alkali- Associated with CRYSTALS different soluble, renal stones, - shapes, NaOH gout, diamond, -high purine rhombic metabolism Precipitation of urinary salt rosette - Lesch Nyhan lemon shaped syndrome Changes in pH, temp, conc whetstone Most ppt occur in ref & RT Calcium Colorless Acid Dilute In normal oxalate Envelope with or HCl- individuals after Most crystals are of limited clinical intersecting Neu soluble ingestion of diagonal lines tral Acetic oxalate rich food significance acid and large doses Monohydrate- insoluble of vitamin C Urine pH is important dunmbell - renal stones, Dihydrate- - ethylene glycol enveloped poisoning 32  07/11/2024 Type Description pH Solubility Significance Type Description pH Solubility Significance Hippuric yellow-brown Acid/ Soluble in diets high in fruits Triple Colorless Alkaline Soluble in Associated with acid to colorless Neutral water, and vegetables PO4 three to six dilute renal calculi, elongated alkali containing large (NH4-Mg sided prisms acetic enlarged prisms/plates quantities of PO4) coffin-lid shaped acid prostate, UTI with pyramidal Insoluble benzoic acid ends in acetic Found in acid normal urine Sodium yellow to Acid Soluble at Report as urate urate colorless 60oC crystals Amor- Colorless Alkaline Soluble in No clin. sig needle or phous granular patches neutral dil. acetic slender prisms No clin. sig PO4 with no definite acid in sheaves or shape Insoluble clusters at 60oC Calcium Colorless Alkaline Gas from No clin. sig Amor- brick-dust, Acid Soluble at No clin. sig carbonate small dumbbells acetic phous yellow brown 60oC and or spherical acid urates small granular alkali forms (efferves- precipitation Insoluble can be found in cence) in acetic granular masses Salts of acid or in pairs Na,Ca,K,Mg TYPE DESCRIPTION pH Solubility SIGNIFICANCE Calcium Colorless Alkaline Soluble in dil Associated with Phosphate long, thin prisms with neutral acetic acid renal calculi one pointed and Can be found in arranged as rosettes or normal urine clusters of needles Thin irregular plates that float on surface of urine Abnormal Crystals in Urine Ammonium yellow to brown Alkaline/ Soluble in Usually indicates Biurate Spherical bodies with Neutral acetic acid old urine long irregular spicules w/ heat “thorny apple” 33  07/11/2024 Type Solubility pH Solubility Significance ABNORMAL CRYSTALS IN URINE Cystine Colorless and Acid Soluble in -Cystinuria Type Description pH Solubility Significance refractile HCl, alkali, -Liverdse hexagonal with & NH4 -Kidney dse Chole- Colorless Acid Soluble in Excessive tissue equal and sterol Notched chloroform, breakdown unequal sides plates ether, Seen in nephritis Hot alcohol & nephritic Most often syndrome Leucine yellow to brown Acid Soluble in Severe liver found after Lipiduria, lipidemia spheroids with neutral hot alkali disease refrigeration and lymphatic radial concentric or alcohol obstruction due striations to neoplasms Highly refractile with oil-like Bilirubin yellow to acid Soluble in Obstructive jaundice appearance brown HOAc, HCl, Shape: NaOH, ether, Granules chloroform Tyrosine Colorless-yellow Acid Soluble in Severe liver or clusters with presence of Neutral alkali or disease and bilirubin heat tyrosinosis Sulfona brown to Acid Soluble in Patients taking highly refractile mides yellow neutral acetone sulfa drugs for UTI needles in needle-like sheaves or In bundles or clusters sheaves Stacks of wheat TYPE DESCRIPTION pH Solubility SIGNIFICANCE Ampicillin Colorless Acid Refrigerati Administration of Shape: elongated neutral long thin needles on forms bundles large doses RENAL CALCULI Radiograp hic media colorless Similar to acid Soluble in 10% NaOH Intravenous injection for – Urolithiasis cholesterol Also highly radiography Can appear up to – Nephrolithiasis birefringent 3 days after injection – Renal lithiasis – Asso. w/ renal colic, hematuria – Large stone may result to hydronephrosis Hemoside rin yellow to brown to red Acid/ alkaline Insoluble granules Associated with destruction of – Usually asymptomatic heavy large RBC granules Prussian blue stain for iron 34  07/11/2024 Stone Analysis STONE ANALYSIS II. CHEMICAL EXAMINATION SPOT PLATE TEST I. Physical Examination Stone Reagents Result 1. Size – sand , gravel , stone Uric Acid Na2CO3 ,UA rgt deep blue staghorn – calyces & pelvis smooth & round – bladder Oxalates HCL, MNO2 tiny bubbles 2. Appearance – Phosphates Ammonium molybdate yellow ppt Uric acid – brownish red, moderately hard Ca ox – dark color,very hard, rough surface Carbonates HCl effervescence Phosphate – pale , friable Cystine – yellow brown , greasy New techniques Patient management techniques Ph incompatible with crystallization of particular 1. Polarizing Microscopy chemicals 2. Radiographic diffraction Adequate hydration Dietary restrictions 3. X ray Crystallography Oxalates – avoid tea ,cocoa, coffee,cola - beans, rhubarb, spinach, 4. Infrared spectroscopy nuts, berries, citrus, vit. C 5. Electron Microscopy Uric Acid – avoid dietary intakes of purines - liver, dried beans, some fish, meat 35  07/11/2024 RENAL STONES RENAL STONES Chem. Comp % pH Causes Chem. Comp % pH Causes 1. Ca Ox 75 5.5-6.5 1. idiopathic 2. Magnessium 15 >7.0 infection with urea Ca phosphate hypercalciuria Ammonium splitting bacteria (Apatite) 2. bone dse Phosphate 3. primary hyper- thyroidism 3. Uric acid 10 200/ul 38  07/11/2024 CHARACTERISTICS CHARACTERISTICS XANTROCHROMIA Red-orange discoloration of CSF due to breakdown of hemoglobin OTHERS Takes about 4-5 hours after hemorrhage Pellicle formation Oxyhemoglobin Tuberculosis with protein > 1gm/dl Bilirubin Brown – melanin 24 hours of refrigeration Red-orange – rifampicin Orange- carotenoids Clot formation - increased fibrinogen RED COLOR OF CSF? HEMATOLOGY TRAUMATIC SUBARACHNOID CELL COUNT TAP BLEED 0-5 WBC / ul CLEARING + - TOTAL CELLS – WBC = RBC XANTHRO. - + ERYTHROPHAGIA - + HEMOSIDERIN - + D DIMER - + 39  07/11/2024 HEMATOLOGY HEMATOLOGY DILUTE ? 1st COUNT NO All cells in 10 large squares Clear This equals total cell count/ul (-) tyndall’s YES 2nd COUNT hazy Use 3 % acetic acid (dilute or rinse) (+) tyndall’s All cells in 10 large cells Use saline This equals WBC/ul HEMATOLOGY HEMATOLOGY CORRECTION DIFFERENTIAL COUNT WBC added = WBC (blood) x RBC (CSF) Centrifugation RBC (blood) Spin 5-10 minutes, transfer supernate to another tube, smear sediment, air dry, stain True WBC = WBC counted - WBC added Cytocentrifugation Add 0.1 ml of CSF and 1 drop of 30% albumin to conical chamber, spin. Filter paper absorbs fluid, cells forced onto 6 mm monolayer. Stain If WBC and RBC in blood are at reference range Subtract 1 WBC for every 700 RBC 40  07/11/2024 HEMATOLOGY MICROBIOLOGY 70% Lymphocytes Identification of organisms Can be concentrated by centrifugation 30% Monocytes Gram stain – most bacteria Acid fast stain - Tuberculosis Electron microscopy - viruses Increase in number or presence of any India ink – Cryptococcus other cell is significant PLEOCYTOSIS Culture for confirmation CHEMISTRY CHEMISTRY METHODS OF PROTEIN DETERMINATION PROTEIN TUBIDIMETRIC Less than 1% of plasma level TCA and SSA INCREASES IN INFLAMMATION Simple, inexpensive, no instruments, large volume (>.5) Increased permeability of BBB COLORIMETRIC Disintegrated cells and bacteria Lowry, CBB, Ponceau S, Biuret Increased antibodies Sensitive, smaller amounts IMMUNOLOGIC Expensive, smallest sample AUTOMATED 41  07/11/2024 CHEMISTRY CHEMISTRY CORRECTION FOR PROTEIN PROTEIN FRACTIONS If sample is bloody Albumin, pre albumin, transferrin (tau), IgG, IgA, ceruloplasmin and haptoglobin [S pro x (1-Hct) ] x CSF RBC / ul Determines if increase is due to Blood RBC / ul Passage from BBB (alb & glob) Neurologic dse (glob) CHEMISTRY CHEMISTRY FRACTIONATION TECHNIQUES IF oligoclonal band in gamma region Electrophoresis Serum and csf Radial immunodiffusion Leukemia, infection Nephelometry CSF only MS, encephalitis Guillan Barre 42  07/11/2024 CHEMISTRY CHEMISTRY PROTEIN INDICES CSF / serum albumin Index CSF albumin (mg/dl) GLUCOSE Serum albumin (g/dl) 60-70% of blood glucose level < 9 intact BBB Lags 30-50 mins behind blood level IgG index Decreased CSF IgG (mg/dl) / serum IgG (g/dl) CSF alb (mg/dl / serum alb (g/dl) Glycolysis by tissue >.77 IgG production in CSF Utilization by microorganisms CHEMISTRY SEROLOGY VDRL Syphilis LACTIC ACID Increases BAT C reactive Anaerobic glucose metabolism Protein Latex Hypoxia Bacteria high Xanthrochromia Agglutination C. neoformans Virus low GLUTAMINE Limulus Lysate Increases in severe liver disease Gram (-) bacteria LDH 43  07/11/2024 SEROLOGY CLINICAL CORRELATION SLIDEX PHADEBACT Character Bacterial Viral TB Fungal Meningitis kit for WBC ct     H influenza b Diff neutro lympho Lympho Lympho S pneumonia Mono Mono N meningitidis A, B, C Protein Marked  Mod. Marked  Mod  Sugar Marked  N  Norm - Lactate >35 N >25 >25 Other pellicle BLOOD BRAIN BARRIER CYTOCENTRIFUGE 44  07/11/2024 SEROUS FLUIDS-PHYSIOLOGY ultrafiltrate of plasma found bet. visceral & parietal memb. seen in closed cavities; pericardial, pleural, peritoneal SEROUS FLUIDS Effusion- accumulation of fluid Causes: 1. inc. hydrostatic pressure - CHF 2. dec. oncotic pressure - hypoproteinemia 1. inc. capillary permeability - infection & inflammation 1. Lymphatic obstruction- tumors FLUID COLLECTION ASPIRATION PROCEDURES Thoracentesis – pleural fluid Pericardiocentesis – pericardial fluid paracentesis Paracentesis – peritoneal fluid pericardiocentesis thoracentesis EDTA – cell count & diff count Heparinized – chemical serologic, microbial & cytologic analysis pH determination- place in ice paracentesis pericardiocentesis thoracentesis 45  07/11/2024 Lab differentiation Transudate Exudate TRANSUDATE VS. EXUDATE 1. Origin Non inflammatory Inflammatory Transudate Exudate 2. Clin. significance CHF,liver cirrhosis Infection, Nephrotic synd. malignancies Disruption of fluid Direct involvement and 3. transparency clear cloudy filtration and absorption injury to the 4. Sp gr 1.015 No direct involvement of membranes membranes 5. protein 3 g/dl 6. LDH 200IU CHF, liver cirrhosis Infection, inflammation 7. cholesterol 55mg/dl Hypoproteinemia, Malignancy,Infarction nephrotic syndrome 8. Cell count 1,000/ul Lab. Differentiation Transudate Exudate CORRELATION OF PF APPEARANCE & 9. Spontaneous clotting No Possible DISEASE 10. glucose Same as in < blood level Appearance Disease blood 11. PF:serum protein 0.5 Clear, pale yellow Normal 12. PF:serum LD 0.6 Turbid, white Microbial infection(TB) Bloody. Hemothorax 13. PF:serum cholesterol 0.3. hemorrhagic effusion 14. PF: serum 0.6 Milky Chylous effusion ( thoracic duct leakage) bilirubin Pseudo chylous (chronic inflammation) 15. serum-ascites alb >1.1 110 mg/dl 500 cells/ul to detect intra-abdominal bleeding > 50 % PMN Procedure: instillation of normal saline into the abdominal cavity, then after a short while the fluid is > 250 cells/ul absolute granulocyte count lavaged outside, presence of blood is determined Increased lactate RBC counts of greater then 100,000 cells/ uL is significant for bleeding 48  07/11/2024 PERITONEAL FLUID PERITONEAL FLUID LABORATORY TEST MICROBIOLOGY CEA GIT malignancy Gram stain Glucose Decreased in tubercular Culture peritonitis AFB stain Amylase High in pancreatitis, GIT Adenosine deaminase perforation CA 125 Ovarian malignancy Alkaline phosphatase GIT perforation BUN & Creatinine Ruptured bladder PSAMMOMA BODY PERICARDIAL FLUID PHYSIOLOGY A psammoma body in papillary carcinoma Usually small amount only: 10 to 15 ml of the thyroid. Lubricates the lining of the pericardium during contraction - psammoma body is a round collection of calcium, seen microscopically. - Greek word psammos meaning "sand." - Contains concentric striations of collagen-like material - Asso. w/ ovarian & thyroid malignancies 49  07/11/2024 PERICARDIAL FLUID PERICARDIAL FLUID APPEARANCE LABORATORY TESTS Clear, pale yellow Normal Increased Neutrophils Bacterial endocarditis Malignant cells Metastatic carcinoma Blood-streaked Infection & malignancy Carcinoembryonic Metastatic carcinoma Antigen (CEA) Grossly bloody Cardiac puncture (stab) Grams stain Bacterial endocarditis Milky Chylous and Acid-fast stain Tubercular effusion pseudochylous effusion Adenosine deaminase Tubercular effusion SWEAT ANALYSIS SWEAT LABORATORY TESTS Cystic fibrosis Methods done to stimulate sweating - is an inherited disease that causes sweat glands Gibson and cooke pilocarpine iontophoresis and other glands, especially those in the Use gauze pads, 75mg of sweat is needed pancreas and the air passages of the lungs, to Pilocarpine is given produce abnormally thick, cloggi