MLS Review - AUBF - Microscopic Urine Analysis

MEDICAL LABORATORY SCIENCE REVIEW ANALYSIS OF URINE AND OTHER BODY FLUIDS: MICROSCOPIC URINE ANALYSIS SEDIMENT EXAMINATION TECHNIQUE • OUTLINE • Microscopic Urine Analysis o Pre-Examination Processing o Sediment Examination Technique o Urinary Sediments o Urinary Casts o Normal Urinary Crystals o Abnormal Urinary Crystals MICROSCOPIC URINE ANALYSIS PRE-EXAMINATION PROCESSING • • • • • • • Specimen Preparation o Specimen should be examined while fresh or adequately preserved o Immediate examination is needed due to components (cells, crystals, parasites) that disintegrate during prolonged standing o Refrigeration: Precipitation of amorphous urates/phosphates, other non-pathologic crystals o Cold specimens (from ice bag/storage) must be set at room temp before examination o Warming @ 37°C: may dissolved some crystals Specimen Volume o Standard amount of urine: usually 10 and 15 mL o 12 mL is frequently used o Note to the report: if the urine volume is not adequate ▪ Note if pediatric as well Centrifugation: 5 minutes at 400 RCF o Centrifugation may vary on the SOP of the lab Sediment Preparation o Volumes of 0.5 mL and 1 mL are frequently used (uniform amount of urine and sediment) Volume of Sediment Examined o Conventional Glass Slide Method: recommended volume is 20 uL (0.02 mL) covered by a 22 x 22 mm glass cover slip o Others do not use coverslip due to tendency of casts may reside on the edges/periphery of the coverslip Examining the Sediment o LPO: detects casts, ascertain general composition of sediment o HPO: identification of urinary sediments o Conventional Glass Slide Method: casts have a tendency to locate near the edges of the coverslip (Low power scanning of the coverslip perimeter is recommended) ▪ Begin with a dim light, do not max out brightness o Point of Reference: Epithelial cells Reporting the Microscopic Examination o Average number per LPF: casts o Average number per 10 HPF: RBCs and WBCs o Semi-quantitative (Rare, Few, Moderate, Many / 1+, 2+, 3+, 4+): epithelial cells, crystals, & other sediments • • Sediment Stains o Sternheimer-Malbin Stain ▪ Most frequently used stain in urinalysis ▪ Components: Crystal violet & Safranin O ▪ Commercially available: Sedi Stain, KOVA Stain o Lipid Stains, Oil Red O, Sudan III & Polarizing Microscopy ▪ Used to confirm the presence of triglycerides, neutral fats, and cholesterol o Gram stain ▪ Confirmation & identification of bacterial casts, which can be confused from granular casts ▪ Components: Crystal violet (10 stain), Iodine (Mordant), Alcohol (decolorizer), Safranin (counterstain) ▪ Gram (+) bacteria: purple/violet ▪ Gram (-) bacteria: red o Hansel Stain ▪ Preferred stain for urinary eosinophils (in cases of drug-induced allergic reaction producing inflammation of the renal interstitium) ▪ Components: Methylene blue and Eosin Y o Prussian Blue Stain ▪ Stain for iron (Hemosiderin Granules: blue) Microscopy o The type of microscopy used depends on the specimen type, the refractive index of the object, and the ability to image unstained living cells o Bright-field microscopy - objects appear dark against a light background, is most frequently used in the clinical laboratory ▪ Most commonly used microscopy o Dark-field microscopy - objects appear against a dark background, aids in the identification of Treponema pallidum o Phase-Contrast microscopy - enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads, and Trichomonas ▪ Must be observed in a dim light o Polarizing microscopy - aids in identification of cholesterol in oval fat bodies, fatty casts, and crystals which polarize under this microscopy o Fluorescence microscopy - allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye including labeled antigens and antibodies ▪ Computer-aided to improve visuallization o Interference contrast - produces a three-dimensional microscopy image and layer-by-layer imaging of a specimen Cytodiagnostic Urine Testing o Frequently performed independently of routine urinalysis for detection of malignancies of the lower urinary tract o Preparation of permanent slides using cytocentrifugation followed by staining with papanicolau stain o Papanicolau stain - provides an additional method for detecting and monitoring renal disease 1 ▪ URINARY SEDIMENTS • • Red Blood Cell o Appearance: smooth, non-nucleated biconcave disc (7mm) o Hypersthenuric (Concentrated): crenated or irregularly shaped (shrinkage due to loss of water) o Hyposthenuric (Diluted): ghost cells (empty cells) ▪ Swelling due to absorption of water which lead to lysis & release of cellular components (hemoglobin) ▪ Only cellular membrane is left & observable o Dysmorphic RBC (cellular protrusions, fragmented): associated with Glomerular Bleeding o Normal: 0-2 cells/10 hpf ▪ Check whether presence physiologic or pathologic • Physiologic - menstrual contamination o Clinical Significance: ▪ Associated with damage to the glomerular membrane or vascular injury within the genitourinary tract • Damaged membranes allow passage of RBCs in the urine ▪ The number of cells present is indicative of the extent of the injury o Macroscopic/Gross Hematuria ▪ Urine appears cloudy with a red to brown color ▪ Frequently associated with advanced glomerular damage but is also seen with damage to the vascular integrity of the urinary tract caused by trauma, acute infection or inflammation, and coagulation disorders o Microscopic Hematuria ▪ Urine appears normal but more RBCs are present under microscope ▪ Can be critical to the early diagnosis of glomerular disorders and malignancy of the urinary tract and to confirm the presence of renal calculi (kidney stones) White Blood Cell o Pyuria: increase in urinary WBC o Neutrophil: predominant ▪ Granulated with multi-lobed ▪ Appear as glitter cells in Hypotonic • Neutrophil tends to absorb water in hypotonic environment and swell • Granules exhibit Brownian movement causing sparkling appearance o Eosinophil: associated w/ drug-induced interstitial nephritis ▪ Small number of eosinophils is seen in UTI, renal transplant rejection o Mononuclear cells (lymphocytes, monocytes, macrophages, histiocytes) ▪ Lymphocytes - resemble RBC; smallest of WBC ▪ Monocytes, macrophages, histiocytes appear as vacuolated large cells which may or may not contain inclusions • Vacuole - holes in cytoplasm o Clinical Significance: ▪ Pyuria - increase in urinary WBCs which indicates the presence of an infection or inflammation in the genitourinary system ▪ Lymphocytes - seen in increased numbers in early stages of renal transplant rejections • • Primary concern in identification of WBC in urine: differentiation of mononuclear cells and disintegrating neutrophils from round renal tubular epithelial (RTE) cells (usually larger than WBCs with an eccentrically located nucleus) ▪ Supravital staining/addition of acetic acid - used to enhance nuclear detail Epithelial Cell o Squamous EC: Normal, Sloughing Off of old cells ▪ Derived from the linings of genitourinary tract; ▪ Largest cells found in urine sediment ▪ Point of reference; first structure to be observed ▪ Represent normal cellular sloughing and have no pathologic significance o Transitional EC: aka urothelial cell ▪ Smaller than Squamous EC and appear in several forms, including spherical, polyhedral, and caudate ▪ These differences are caused by the ability of transitional epithelial cells to absorb large amounts of water ▪ Increased in numbers is due to invasive urologic procedures like catheterization ▪ Syncytia - transitional EC in clumps ▪ Vacuolated and irregular nucleus transitional EC - assoc. with malignancy or viral infection ▪ Originate from the lining of the renal pelvis, calyces, ureters, and bladder, and from the upper portion of the male urethra • Can change its appearance based on the amount of water it can absorb o Renal Tubular Epithelial (RTE) Cell: PCT, DCT, CD – renal fragments (if they appear in groups of 3) ▪ Size and shape depending on the area of the renal tubules from which they originate • PCT - proximal convoluted tubule o Larger than any RTE cells o Rectangular in shape; referred to as columnar or convoluted cells; resembles a cast • DCT - distal convoluted tubule o Smaller, round, or oval o Can be mistaken for WBC or spherical transitional EC • CD - collecting duct o Cuboidal and are never round o If they appear in groups of three or more, they are called as renal fragments ▪ Oval Fat Bodies: lipid-containing RTE cells ▪ Bubble Cells: RTE cells containing non-lipid filled vacuoles (acute tubular necrosis) o Clue Cells ▪ Squamous EC covered with Gardnerella coccobacillus (Bacterial vaginosis) Bacteria o Not normally present in urine as urinary bladder is sterile o Presence of bacteria may indicate infection, specimen contamination o Significant UTI: bacteria should be accompanied by WBC o Cocci: Spherical-shaped o Bacilli: Rod-shaped o Enterobacteriaceae: gram-negative rods, most frequently associated with UTI 2 • • • • • • • Yeast o Two forms: ▪ Budding yeast - may resemble RBC • Addition of KOH lyses RBC and leaves yeast ▪ Mycelial form o Small Refractile oval structures (may or may not contain bud) o Candida albicans: DM, Vaginal Moniliasis, Immunocompromised Individual Parasites o Trichomonas vaginalis - most frequent parasite encountered in the urine. ▪ Sexually transmitted ▪ Often associated with vaginal inflammation ▪ Infection of male urethra and prostate due to T. vaginalis are asymptomatic ▪ Disintegrates during prolonged standing leading to resemblance with WBC o Schistosoma haematobium - bladder parasite (ova) ▪ Prominent terminal spine ▪ Often associated with bladder cancer in other countries o Enterobius vermicularis - most common fecal contaminant ▪ Its egg resides in the perianal region causing ▪ D-shaped ova Spermatozoa o Routine UA: do not report o Significant: male infertility, retrograde ejaculation (sperm is expelled into bladder instead of urethra) o Urine is toxic to spermatozoa ▪ Rarely exhibit motility when present in urine o Can be present in women following sexual intercourse o Present in men after masturbation or nocturnal emission Mucus o Uromodulin - major constituent o Thread-like structures (Very Low Refractive Index) ▪ Fiber-like o Frequently seen in females ▪ Commonly seen with unhygienic collection Casts o Only elements found in urine ▪ Unique to the kidney o Cylinduria - presence of urinary cast o The width of the cast depends on the size of the tubule in which it is formed Crystals o Appearance: true geometrically structure or amorphous materials Artifacts o Starch - highly refractile sphere with dimpled center o Oil Droplets o Air Bubbles o Pollen Grains - appear as spheres with a cell wall and occasional concentric circles o Fibers/Hair - may resemble casts however fibers polarized while casts do not o Fecal Contamination - appear as plant and meat fibers or as brown amorphous material URINARY CASTS • • • • • • • • Hyaline Casts o Most frequently seen o Normal: 0-2 / LPF ▪ Forms: normal parallel sides, rounded ends, cylindroid form o Has low refractive index (low microscope light) o Normally Increased in: strenuous exercise, dehydration, heat exposure, emotional stress o Appear as colorless in unstained urine o Appear pink in Sternheimer-Malbin Stain o Pathologically Increased in: acute glomerulonephritis, pyelonephritis, chronic renal disease, congestive heart failure (CHF) RBC Casts o Can be observed in health individuals following strenuous contact sport o Blood Cast - greater stasis of urine ▪ Under LPO, appear as orange-red due to hgb ▪ Indication: bleeding within the nephron o Glomerular damage (glomerulonephritis) is associated with proteinuria and dysmorphic erythrocytes WBC Casts o Usually composed of neutrophil & may appear as granular o Non-Bacterial Inflammation: acute interstitial nephritis ▪ Only WBC cast is seen ▪ Accompanying RBC cast may be seen o Bacterial Inflammation: pyelonephritis ▪ Bacteria + WBC o Signifies infection or inflammation within the nephron o Associated with pyelonephritis and are a primary marker for distinguishing pyelonephritis (upper UTI) from cystitis (lower UTI) Bacterial Casts o Bacterial Inflammation: pyelonephritis o Bacterial casts containing bacilli both within and bounf to the protein matrix are seen in pyelonephritis o Pure bacterial cast or mixed with WBCs o May resemble granular cast o Confirmation: Gram stain Epithelial Cell Casts o Significant: Cast containing RTE Cells o Indication: Advance tubular obstruction producing urinary stasis along with disruption of tubular linings Fatty Casts o Lipiduria: Fatty Casts + Oval Fat Bodies + Free Fat Droplets o Frequently Associated with: nephrotic syndrome o Maybe seen in toxic tubular necrosis, DM, crush injuries Mixed Cellular Casts o Casts containing multiple cell types o Most frequent encountered include RBC and WBC casts in glomerulonephritis and WBC and RTE cell casts, or WBC and bacterial casts in pyelonephritis Granular Casts o Result of cellular disintegration o May contain cells that may or may not be recognized o In longer periods, it disintegrates and become resemblance of waxy cast o Increased cellular metabolism occurring during periods of strenuous exercise accounts for the transient increase of granular casts that accompany the increased hyaline casts 3 • • Waxy Casts o Appearance: fragmented with jagged ends and have notches on sides o Indication: extreme urine stasis (chronic renal failure) o Appear homogenous dark pink in supravital stain Broad Casts o Renal failure casts o Indication: destruction “widening” of the tubular walls o o ABNORMAL URINARY CRYSTALS • • NORMAL URINARY CRYSTALS • • • • • • • • Uric Acid o Urine pH: Acid o Appearance: rhombic, four-sided flat plates (whetstones), wedges, rosettes ▪ Yellow-brown but may be colorless if are 4- or 6sided o Highly Birefringent: distinguishing feature from cystine crystals o Significantly Increased in: Gout, Lesch-Nyhan Syndrome Amorphous Urates o Urine pH: acid o Appearance: yellow-brown granules o Refrigerated: pink sediment Amorphous Phosphates o Urine pH: alkaline o Appearance: similar to amorphous urates (distinguished by urine pH) o Refrigeration: white sediment Calcium Oxalate (CaOx) o Urine pH: acid/neutral/alkaline o Dihydrate (most common form): colorless, octahedral envelope or as two pyramids joined at their base o Monohydrate: oval or dumbbell shaped associated with ethylene glycol “antifreeze” poisoning o Majority of renal calculi is composed of “CaOx” Calcium Phosphate o Urine pH: alkaline o Appearance: colorless, flat rectangular plates, thin prisms in rosette form o Confused with: sulfonamide crystals (when urine pH is in neutral range) o Distinguished by addition of dilute acetic acid: calcium phosphate (dissolves); sulfonamide (doesn’t dissolve) Triple Phosphate o Urine pH: alkaline o A.K.A.: “Ammonium Magnesium Phosphate” o Appearance: prism shape, resembles “coffin-lid” Ammonium Biurate o Urine pH: acid o Appearance: brown color; “thorny apples” - spicule covered spheres o Dissolves at 60 degC and convert into uric acid crystals when glacial acetic acid is added o Most often encountered in: old specimen ▪ Associated with presence of ammonia produced by urea-splitting bacteria Calcium Carbonate o Urine pH: alkaline Appearance: small, colorless, dumbbell of spherical shapes Resemble amorphous material – distinguished by addition of acetic acid (formation of gas) • • • • • • • Most often found in acidic urine or rarely in neutral urine Cystine o Appearance: colorless, hexagonal plates (thick or thin) o Distinguished from distinguished from uric acid crystals (HIGHLY BIREFRINGENT) o Confirmation: Cyanide-Nitroprusside Test o Cystinuria - Metabolic disorder that prevents reabsorption of cysteine by renal tubules ▪ Has tendency to form renal calculi at an early age Cholesterol o Appearance: rectangular plates with notch on one or more corners o Can be seen in: Nephrotic syndrome in conjunction with fatty casts and oval fat bodies Radiographic Dye o Appearance: colorless, flat plates similar to cholesterol crystals o Specific Gravity of urine is marked elevated Sulfonamides o Appearance: colorless to yellow-brown, needles, rhombics, whetstones, “sheaves of wheat” and rosettes o Distinguished from calcium phosphate o Does not dissolved upon addition of Dilute Acetic Acid o Inadequate patient hydration is the primary cause of sulfonamide crystallization o Correlation must be aided by medical history to confirm and identify sulfonamide Ampicillin o Appearance: colorless needles (tend to form bundles following refrigeration) o Indication: precipitation of antibiotics following massive dosage of this penicillin compound without adequate hydration Leucine o Appearance: yellow-brown spheres (concentric circles with radial striations) ▪ May resemble pollen grains o Should be accompanied by: tyrosine crystals o Liver Disorder Tyrosine o Appearance: fine, colorless to yellow needles (in clumps or rosettes) o Seen in conjunction with: leucine crystals & (+) chemical test for bilirubin o Liver Disorder Bilirubin o Appearance: yellow, clump needles or granules o (+) Bilirubin in Urine o Liver Disorder 4