Urinalysis and other Body Fluids - Microscopic Urine Analysis

ANALYSIS OF URINE AND OTHER BODY FLUIDS CLINICAL AND LABORATORY STANDARDS INSTITUTE (CLSI) MICROSCOPIC ANALYSIS OF URINE OUTLINE • Introduction • Microscopic Screening of Urine • Macroscopic Screening o Clinical and Laboratory Standards Institute o Specimen Preparation o Specimen Volume o Centrifugation o Sediment Preparation o Volume of Sediment Examined o Commercial Systems o Examining the Sediment o Reporting the Microscopic Examination o Correlating Results • Sediment Examination Techniques o Sediment Stains o Cytodiagnostic Urine Testing o Microscopy • Urine Sediment Constituents o Red Blood Cells o White Blood Cells o Epithelial Cells o Bacteria o Yeast o Parasites o Spermatozoa o Mucus o Casts o Urinary Crystals o Urinary Sediment Artifacts INTRODUCTION • • Urine microscopic analysis has been part of the urinalysis examination since the introduction of the Addis count in 1926. Although considered to be a highly accurate method for evaluating kidney function, the Addis count is no longer routinely performed largely due to the introduction of additional chemical testing for urine. Urine microscopic analysis still plays a large role in the examination of urine, however. During microscopic examination, urine is evaluated for the presence of formed elements such as red blood cells, white blood cells, epithelial cells, casts, crystals. The presence of increased amounts of these formed elements or abnormal cells correlate with diseases of the urinary tract. • • • SPECIMEN PREPARATION • • • • • Microscopic examination of the urinary sediment Identification of insoluble substances (formed elements) o Red blood cells (RBCs) o White blood cells (WBCs) o Epithelial cells o Casts o Bacteria o Yeast o Parasites o Mucus o Spermatozoa o Crystals o Artifacts (resemble other formed elements) Least standardized, most time consuming Examine when fresh or preserved o RBCs, WBCs, casts disintegrate in dilute, alkaline urine o Casts - first morning specimen Refrigeration precipitates crystals o Can obscure other elements Less contamination (epithelial cells) from a midstream clean-catch specimen Thoroughly mix specimen before decanting to the centrifuge tube SPECIMEN VOLUME • • • • Centrifuge 10-15 mL urine (reagent strips fit into 12 mL) Quantities <12 mL should be documented Too little volume = fewer formed elements Some laboratories correct for volume CENTRIFUGATION • • • • • • Standardize speed and time of centrifugation 5 min at relative centrifugal force (RCF) of 400 is ideal RCF corrects for variations in the diameter of centrifuge heads; revolutions per minute does not RCF = 1.118 × 10−5 × radius in centimeters × RPM2 Do not brake the centrifuge Cap all specimens SEDIMENT PREPARATION • MICROSCOPIC EXAMINATION OF URINE • • Requested by the physician Laboratory-specified population Any abnormal physical or chemical result • Sediment Standardization o Preparation of sediment o Volume of sediment examined - 0.5 to 1.0 mL o Methods of visualization - LPO, HPO o Reporting of results o Commercial systems: KOVA ▪ Calibrated centrifuge tubes, special slides to control volume, decanting pipettes, grids for better quantitation Postcentrifuge Sediment o 0.5 to 1.0 mL after decantation o Concentration factor: volume of urine centrifuged/ sediment volume ▪ Probability of detecting low quantities of formed elements o Aspirate rather than pour off urine (pipettes available for this) o Mix sediment gently, not vigorously (may damage) MACROSCOPIC SCREENING VOLUME OF SEDIMENT EXAMINED • • • Microscopic is performed based on physical and chemical results Color, clarity, blood, protein, nitrite, leukocyte esterase, and possibly glucose Special populations: pregnant women; pediatric, geriatric, diabetic, immunocompromised, and renal patients • • • Be consistent Commercial systems control this Glass slide method o 20 μL o 22 × 22 glass cover slip o Do not overflow cover slip ▪ Heavier elements (casts) flow outside COMMERCIAL SYSTEMS • • • • • • SEDIMENT EXAMINATION TECHNIQUES Chambers capable of containing a standardized o Chamber volume o Size of the viewing area o Approximate number of low-power and high-power viewing areas Based on the area of the field of view using a standard microscope CLSI recommends these systems together with standardization of all phases of the methodology Capped, calibrated centrifuge tubes Decanting pipettes to control sediment volume Slides that o Control the amount of sediment examined o Produce a consistent monolayer of sediment for examination o Provide calibrated grids for more consistent quantitation • SEDIMENT STAINS • • • EXAMINATION OF SEDIMENT • • • • • • Be consistent Minimum 10 low (10×) and 10 high (40×) fields Low power: casts, general composition o Scan edges for casts with glass slide method High power: identification of type Initial focusing: low power, reduced light o Focus on epithelial cell, not artifacts that are in a different plane Use fine adjustment continuously for best view REPORTING THE MICROSCOPIC EXAMINATION • • • • Consistent within laboratory Casts: average per lpf RBCs, WBCs: average per hpf Epithelial cells, crystals, etc., in semiquantitative terms o Few, moderate, many o 1+, 2+, 3+, 4+ o Follwed by /lpf or /hpf • • • • • CORRELATING RESULTS Microscopic Elements RBCs WBCs Low refractive index elements are often difficult to see under bright-field microscopy Sternheimer-Malbin stain: crystal violet /Safranin O o Increases refractive index o Stains nuclei, cytoplasm, inclusions o Sedi-Stain, KOVA stain, etc. o Identifies WBCs, epithelial cells, and casts 0.5% solution of toluidine blue enhancement of nuclear detail o Differentiates WBCs & renal tubular epithelial (RTE) cells Acetic acid will enhance WBC nuclei o RBCs are lysed by this o Distinguishes RBCs from WBCs, yeast, oil droplets, and crystals Lipid stains o Oil Red O and Sudan III for triglycerides and neutral fats; cholesterol polarizes o Do not stain cholesterol Gram stain o Identification of bacterial casts o Differentiates gram-positive & gram-negative bacteria Hansel stain o Urinary eosinophils o Methylene blue and eosin Y: better than Wright stain Prussian blue stain o Hemosiderin granules seen with hemoglobinuria o Stains structures containing iron o Identifies yellow-brown granules of hemosiderin in cells and cast CYTODIAGNOSTIC URINE TESTING Physical Turbidity Red color Turbidity Epithelial cells Turbidity Casts Bacteria Turbidity Crystals Sediment appearance o Cells and casts in various stages of development and degeneration o Distortion of cells and crystals by the chemical content of the specimen o The presence of inclusions in cells and casts o Contamination by artifacts Turbidity Color Chemical + Blood + Protein + Protein + Nitrite + LE + Protein ↑ pH + Nitrite + Leukocytes pH + Bilirubin Exceptions Number Hemolysis Number Lysis • • • Number Number Number and type Number and type Cytodiagnostic urine testing is frequently performed to detect and monitor renal disease/malignancies Preparation of permanent slides using cytocentrifugation Papanicolaou stain o Transplant rejection o Viral, fungal, and parasitic infections o Cellular inclusions o Pathologic casts o Inflammatory conditions MICROSCOPY • Bright field - most common in urinalysis o Reduced light is essential o Magnification is 10× (LPF) and 40× (HPF) o Par focal means minimal adjustment when changing objectives (use fine adjustment) o Lower light using the rheostat o Condenser can be raised up and down o Do not use the aperture diaphragm • • • • • • Others include phase contrast, polarizing, dark field, fluorescence, and interference contrast Phase-contrast microscopy o Increases refractive index o For pathologic microscopy o Enhances visualization of elements with low refractive indices, such as hyaline casts, mixed cellular casts, mucous threads, and Trichomonas Polarizing microscopy o Crystals and lipids o Ability to split light into two beams o Crystals are multicolored o Cholesterol produces Maltese cross formations Interference-contrast microscopy o Three-dimensional images Dark-field microscopy o Aids in identification of Treponema pallidum Fluorescence microscopy o Allows visualization of naturally fluorescent microorganisms or those stained by a fluorescent dye including labeled antigens and antibodies • WHITE BLOOD CELLS • • • • • 12 µm Neutrophil is predominant Identify under high power Nucleated cells Glitter cells o Hypotonic urine o Brownian movement (multidirectional) o Swell; granules sparkle o Pale blue if stained o Nonpathologic • Eosinophils o Drug-induced interstitial nephritis o Renal transplant rejection Hansel stain o Percent per 100 to 500 cells o >1% significant o Concentrate sediment, centrifuge, or cytocentrifuge URINE SEDIMENT CONSTITUENTS • • • • Clinical Significance o Normal value: 0–3 to 5/hpf o Damage to glomerular membrane or vascular injury to the genitourinary tract o Number of cells = extent of damage o Macroscopic versus microscopic hematuria ▪ Macro - Cloudy, red urine, advanced disease, trauma, acute infection, coagulation disorders ▪ Micro - Clear urine, early glomerular disease, malignancy, strenuous exercise, renal calculi confirmation (renal stone) Small amounts of constituents can be normal or pathogenic based on the clinical picture Many urines have just a rare epithelial cell Some constituents are easily distorted o Concentrations, pH, and presence of metabolites Normals are not clearly defined RED BOOD CELLS • • • • • Identification difficulties o Yeast: look for buds (C. albicans), similar to RBCs, can be mistaken for RBC o Oil droplets: refractility o Air bubbles: refractility and possibly in a different plane o Starch: refractile, polarizes o Reagent strip correlation Smooth, nonnucleated, biconcave disks ~7 µm Crenated in hypersthenuric urine Ghost cells in hyposthenuric urine Identify using high power • • • Dysmorphic RBCs (ghost cell) o Glomerular bleeding o Strenuous exercise o Acanthocytic, blebs o Fragmented, hypochromic o Aid in diagnosis Mononuclear cells o Lymphocytes, monocytes, macrophages, histiocytes are rare o Differentiate from renal tubular epithelial (RTE) cells ▪ Staining o Lymphocytes may resemble RBCs; seen in early transplant rejection o May need to refer to cytodiagnostic testing • Clinical Significance o Normal = <5 per hpf, more in females o May enter through glomerulus or trauma but also by amoeboid migration o Increased WBCs = pyuria o Infections: cystitis, pyelonephritis, prostatitis, urethritis o Glomerulonephritis, lupus erythematosus, interstitial nephritis, tumors o Report presence of bacteria EPITHELIAL CELLS • • • Three types o Squamous o Transitional (urothelial) o Renal Tubular Epithelial (RTE) Classification o Squamous: vagina, male and female urethra o First structures observed o Transitional: bladder, renal pelvis, calyces, ureters, upper male urethra o RTE: renal tubules Squamous Epithelial Cells o Largest cell in urine o Good for focusing microscope o Rare, few, moderate, many o lpf or hpf per laboratory o Normal sloughing o Contamination if not midstream clean-catch • Transitional Epithelial (Urothelial) Cells o Three forms ▪ Spherical: absorb water in bladder and become large and round ▪ Caudate: appear to have a tail ▪ Polyhedral: multiple sides o Differentiate from RTE ▪ Centrally located nucleus o Syncytia = clumps ▪ Catheterization ▪ Malignancy Polyhedral Spherical Caudate • Renal Tubular Epithelial (RTE) Cells o Size and shape vary with renal tubular area o Columnar = proximal convoluted tubule (PCT) o Round, oval = distal convoluted tubule (DCT) o Cuboidal = collecting duct o Three or more cuboidal cells = renal fragment o Clinical Significance ▪ RTE cells are the most clinically significant urine epithelial cells; indicate tubular necrosis; fragments indicate severe destruction • Heavy metals, drug toxicity, hemoglobin, myoglobin, viral infections, pyelonephritis, transplant rejection, salicylate poisoning ▪ Single cuboidal cells = salicylate poisoning ▪ Absorb: bilirubin, hemoglobin, lipids ▪ Hemosiderin stains with Prussian blue o o Clue Cells ▪ Squamous cell with pathologic significance ▪ Gardnerella vaginalis: vaginal infection, can be considered as STD ▪ Coccobacillus sp. covers most of the cell and extends over the edges ▪ Seen in urine but more common in vaginal wet preparation Oval Fat Bodies ▪ RTE cells that have absorbed lipid in the filtrate ▪ Also free-floating refractile droplets ▪ Maltese cross formation with polarized light ▪ If negative check with Sudan III or oil red O stain ▪ Stain polarizing negative structures ▪ Cholesterol polarizes ▪ Triglycerides and neutral fats stain ▪ Lipiduria: nephrotic syndrome, acute tubular necrosis, diabetes, crush syndromes BACTERIA • • • • • Urine is usually sterile, contaminated on the way out; contaminants multiply fast WBCs should accompany bacteria in UTI Report few, moderate, many per hpf Rods and cocci may be seen o Rods most common (Escherichia coli) Nitrite helps to confirm rods, not cocci (gram negative bacilli) YEAST • • • • • • • Single, refractile, budding structures Mycelial forms may be present Report: few, moderate, many Diabetic urine: ↑ glucose and acid ideal for yeast growth Immunocompromised, vaginal moniliasis Nitrite negative, WBCs present Confuse with RBCs PARASITES • • • • Most common: Trichomonas vaginalis o Pear-shaped flagellate o Swims across field rapidly Report few, moderate, many If not moving, may resemble WBC, transitional, or RTE cells Also Schistosoma haematobium and Enterobius vermicularis (Enterobiasis in vagina, cervix) MUCUS • • • • • Protein from RTE, glands, squamous cells Threadlike, low refractive index Confuse with casts o Irregular, composed of uromodulin protein Female specimens, no clinical significance Usually found in male specimens CASTS • • • • • • • • • Elements unique to the kidney Formed in DCT and collecting duct Parallel sides, rounded ends, inclusions Detect under low power, ID high power Scan edges of glass cover slip Low light is essential Report number per lpf Many pathologic and nonpathologic causes Usually observed in first morning urine specimen COMPOSITION AND FORMATION • • • • • • • • Uromodulin protein secreted by RTE of DCT and collecting duct Consistent excretion normally o ↑ stress and exercise Formation of protein fibrils into matrix o Urine stasis, acid pH, Na, and Ca Uromodulin protein not detected by reagent strips ↑ protein is from renal disease Formation o Aggregated uromodulin fibrils attached to RTEs o Interweaving to loose network, traps elements o More interweaving to form solid matrix o Attachment of elements to matrix o Detachment of fibrils from RTEs o Excretion of cast Cylindroids o Tapered ends, one or both o Same significance as cast Sequence Hyaline → Cellular → Coarsely Granular → Finely Granular → Waxy HYALINE CASTS UL-T. vaginalis, LL-S. haematobium, UR&LR-E. vermicularis SPERMATOZOA • • • • • • • Oval, tapered heads and long tail Urine is toxic to sperm, so they are immobile Rarely significant, infertility: sperm expelled into bladder instead of urethra May cause positive protein Reporting varies with laboratories Lack of clinical significance, legal consequences • • • • • • Low refractive index Colorless when unstained Uromodulin protein Use low light or phase Normal parallel sides or convoluted, wrinkled, cylindroid, occasional adhering cell or granule Clinical Significance o Most frequently seen o 0 to 2 is normal o Nonpathologic: stress, exercise, fever, heat exposure, dehydration o Pathologic: glomerulonephritis, pyelonephritis, chronic renal disease, congestive heart failure RBC CASTS • • • • • • Orange-red color Embedded and adhering cells May be fragmented Confirm seeing free RBCs & positive reagent strip for blood Look for cast matrix to avoid mistaking a RBC clump for a cast Clinical Significance o Bleeding within the nephron, casts are more specific than free RBCs in urine o Glomerular damage or nephron capillary damage o Glomerular damage: dysmorphic RBCs and elevated protein o May be seen following strenuous exercise o Cells begin to disintegrate with more stasis of urine flow o Hemoglobin and myoglobin damage tubules o Hemoglobin degraded to methemoglobin = dirty brown casts o Look for RTE cells to confirm tubular necrosis WBC CASTS • • • • • • • Mostly neutrophils and lobed nucleus and granules are seen Staining helps differentiate from RTE cells May be tightly packed; look for cast matrix to distinguish from WBC clump (clump = no matrix) WBC casts are seen with infection and inflammation of the tubules Pyelonephritis: WBC casts, bacteria Acute interstitial nephtitis: WBC casts, no bacteria May accompany RBC casts • Clinical Significance o Tubular damage, heavy metals, viral infections, drug toxicity, graft rejection, pyelonephritis o Cells may appear bilirubin stained o Look for matrix to distinguish fragments FATTY CASTS • • • • Seen with oval fat bodies (OFBs) and fat droplets Highly refractile, OFBs may attach to matrix Polarized microscopy and lipid stains Nephrotic syndrome, diabetes, crush trauma, tubular necrosis MIXED CELLULAR CASTS • • • • RBC and WBC casts in glomerulonephritis WBC and RTE cell casts, or WBC and bacterial casts in pyelonephritis Identification difficult Staining or phase microscopy aids in the identification GRANULAR CASTS • • • • • Coarse and finely granular Granule origin o RTE lysosomes, excreted in normal metabolism, more after exercise and activity o Disintegration of cellular casts and free cells Detect with low power, ID w/ high power Granules disintegrate to form waxy casts Differentiate granular casts from clumps of debris and crystals; look for matrix WAXY CASTS BACTERIAL CASTS • • • • • • • May be pure bacteria or mixed with WBCs Resemble granular casts Look for free WBCs and bacteria Confirm with Gram stain Seen in pyelonephritis Mixed cellular casts o Glomerular nephritis: RBCs and WBCs Look for predominant type of cell • • • • • • Brittle, highly refractile Often fragmented with jagged ends and notches Well visualized with stain Degenerated hyaline and granular casts Extreme urine stasis Renal failure BROAD CASTS EPITHELIAL (RTE) CELLS • • • • Formed in DCT = small, round cells Fibrils forming cast pull cells from damaged tubules Majority of cells are on the cast matrix Differentiate from WBCs: stain to show single nucleus • • • • • • Renal failure casts Destruction and widening of the CTs Formation in the upper collecting duct All types of casts may be broad Most common are granular and waxy Bilirubin stained from viral hepatitis URINARY CRYSTALS • • • • • • • • Most are not clinically significant but are reported True geometrically formed structures or as amorphous material Must differentiate from the few abnormal crystals indicating liver disease, inborn errors of metabolism, and damage to tubules Iatrogenic: caused by medications or treatments Report: rare few, moderate, many Crystal Formation o Precipitation of urine solutes: salts, organic compounds, and medications o Formation based on temperature, solute concentration, and pH o Many crystals in refrigerated specimens o High specific gravity needed in fresh specimens General Identification Techniques o Most have characteristic shapes and colors o Most valuable ID is urine pH o Classification: normal acid, normal alkaline o All abnormal crystals are found in acid urine o Polarized microscopy characteristics are valuable in ID Solubility Characteristics o Temperature and pH contribute to formation and solubility o Amorphous urates form in refrigerated acid urine; will dissolve with heat o Amorphous phosphates form in refrigerated alkaline urine; will dissolve in acetic acid; so will RBCs • NORMAL CRYSTALS IN ALKALINE URINE • • • NORMAL CRYSTALS IN ACID URINE • • • Amorphous urates o Yellow-brown granules microscopically o Urine sediment has pink color due to the pigment uroerythrin attaching on surface of granules o Often in clumps; may resemble casts o pH usually greater than 5.5 Uric Acid Crystals o Rhombic, whetstones, wedges, rosettes o Yellow-brown color o May resemble cystine crystals but always polarize o ↑ purines, nucleic acids o Chemotherapy for leukemia, gout Calcium Oxalate Crystals o Acid and neutral pH o Dihydrate is envelope or two pyramid–shaped ▪ Most common o Monohydrate is oval or dumbbell shaped ▪ Antifreeze poisoning o Calcium oxalate is a major component of renal calculi Amorphous Phosphates o May appear similar to amorphous urates o Differentiate ▪ Alkaline pH and heavy white precipitate after refrigeration Triple phosphate o Colorless, prism, or coffin-lid shaped o Highly alkaline urine and urinary tract infections (UTIs) o Polarize o No clinical significance Calcium Phosphate and Carbonate o Phosphate ▪ Flat rectangles and thin prisms in rosettes ▪ No clinical significance o Carbonate ▪ Small, dumbbell, and spherical shapes ▪ Gas produced with addition of acetic acid ▪ No clinical significance Ammonium Biurate Crystals o Yellow-brown, spicule-covered spheres; “thorny apples” o Only urates in alkaline urine o Old specimens and with urea-splitting bacteria o No clinical significance on adults, significant to babies ABNORMAL CRYSTALS • • Cystine crystals o Hexagonal, thin and thick plates o Similar to uric acid o UA polarizes but only thick cystine crystals polarize o Seen in cystinuria: inability to reabsorb cystine o Confirm: cyanide nitroprusside Cholesterol Crystals o Refrigerated specimens o Rectangular plates with characteristic notched corners o Highly birefringent o Nephrotic syndrome accompanying fatty casts and OFBs (oval fat bodies) • • • • Radiographic Dye Crystals o Radiographic dye ▪ Similar to cholesterol crystals, polarize ▪ Patient history ▪ Very high SG with refractometer Liver Disease Crystals o Tyrosine crystals ▪ Fine yellow needles in clumps or rosettes ▪ Seen with leucine crystals ▪ Inherited amino acid disorders o Leucine crystals ▪ Yellow-brown spheres with concentric circles and radial striations o Bilirubin crystals ▪ Clumped needles or granules ▪ Characteristic yellow color ▪ Viral hepatitis with tubular damage ▪ Positive reagent strip for bilirubin Sulfonamide Crystals o Possibility of tubular damage if crystals are forming in the nephron o Shapes most frequently encountered include needles, rhombics, whetstones, sheaves of wheat, and rosettes with colors ranging from colorless to yellow-brown Ampicillin Crystals o Ampicillin crystals appear as colorless needles that tend to form bundles following refrigeration URINARY SEDIMENT ARTIFACTS • • Material fibers, meat and vegetable fibers, and hair Starch, oil droplets, air bubbles, pollen grains, vegetable fiber, hair, diaper fiber Starch pollen grain, fiber, vegetable fiber