Microscopic Analysis of Urine

Microscopic Urine Analysis Third part of routine urinalysis Used to detect/ identify/ quantify insoluble materials present in urine (sediments, crystals, cells) Most time consuming part of routine urinalysis LEAST STANDARDIZED among all analyses conducted in urine sample. Also known as chemical seiving. Urine Sediment Preparation and Examination Standardization started in 1926 by Thomas Addis (Addis Count) uses hemocytometer 12 hour urine sample Used to monitor the course of diagnosed cases of RENAL DISEASE. Commercialized methods have been developed throughout the years. e.g. KOVA CenSlide Urisystem R/S Workstation Count-10 Quick-Prep CenSlide Doesnt require manual charging of urine sample on slides Closed-system procedure to eliminate exposure Especially designed tube to facilitate urine microscopy R/S Workstation Glass flow cell through which urine sediment is pumped, examined and discarded from the system. Specimen Preparation Freshly collected then thoroughly mixed, midstream clean-catch sample 10-15 mL (standard amount of urine ) Centriguation time: 5 minutes at 400 RCF Decantation (rapid pouring of centrifuged urine sample) Charging the sediment on the slide (20uL/ 0.02 mL) Examination under the microscope Reporting the microscopic exam semiquantitatively (rare, few, moderate, many) Checking the Correlation Correlation between physical and chemical exam plus the microscopic exam must be observed to ensure accuracy and reliability of reports. Sediment Examination Techniques STAINS Increases the overall visibility of sediments being analysed. Imparts identifying characteristics to cellular structures: nuclei, cytoplasm, and inclusions. Sediment/ Cellular Stain 1. Sternheimer-Malbin Stain Supravital stain (stains living cells) Made up of crystal violet and safranin O Absorbed well by WBC, EC, Cast Provides clearer delineation of cells Provides contrasting color of the nucleus and cytoplasm 2. 0.5% Solution of Toluidine Blue Metachromatic stain 3. 4.

Enhances nuclear detail of cells Differentiates WBCs from RTE cells Useful for cell identification of other body fluids

2% Acetic Acid Lyses RBCs Enhances nuclear detail WBCs. Hansel Stain Composed of methylene blue and eosin Y Preferred stain for urinary eosinophls Dried smear of centrifuged urine is used Wrights Stain Primarily used for peripheral blood smear but can also be use as a substitute for urinary staining (particularly eosinophil) Not as specific as Hansel stain for eosinophil Prussian Blue Stain Stains hemosiderin granules as seen in hemoglobinuria Hemosiderin granules stain BLUE

5.

6.

Lipid Stains 1. Oil red O, Sudan III & Sudan IV For the identification of lipid containing cells and free fat bodies. Triglycerides and neutral fats stains well with a ORANGE-RED color Bacterial Stain 1. Grams Stain Differentiates Gram positive from Gram negative bacterial infection For bacterial cast identification 2. Ziehl Neelsen Stain Differentiates acid fast organisms from non acid fast organisms. For urinary tubercular infection

Cytodiagnostic Testing 1. Papanicolaous Stain Used for preparing fixed slides for urine sediment identification and cytology. For the detection of malignancies in lower urinary tract. Detects and monitors renal diseases Provides more definitive information about: transplant rejection, inflammatory conditions, pathologic casts First morning urine sample or suprapubic aspirate. MICROSCOPE Bright-field Microscopy Most common type of microscopy done in the lab Object appears dark against light background When using this method, decreased light level must be employed. (Adjust the rheostat level) Phase-Contrast Microscopy

Polarizing Microscopy Confirms the identification of fat droplets, oval fat bodies and fatty casts that produce MALTESE CROSS APPEARANCE Interference Contrast Microscopy Provides a three dimensional image by showing very fine structural detail ADVANTAGE: object appears dark against light background without any halo associated with phase contrast microscopy Dark-Field Microscopy Enhances visualization of specimens that cannot be seen easily with bright field microscopy For identification of spirochetes (Treponema, Borrelia, etc) Fluorescence Microscopy Used to detect bacteria and viruses within cells and tissues through immunofluorescence. FLUORESCENCE: property by which an atom absorbs light of a particular wavelength and emit light of a longer wavelength, fluorescence lifetime Urine Sediments Red Blood Cells Smooth, non-nucleated, biconcave discs 7um in diamter MOST DIFFICULT to recognize by students because of variation in sizes (anisocytes), characteristic structures and close resemblance to YEAST, BUBBLES, OIL DROPLETS and other microscopic sediments. HYPERSTHENURIC SAMPLE: RBCs shrink= CRENATED= loss of water HYPOSTHENURIC SAMPLE: RBCs swell, lyse, relase Hgb= GHOST CELLS DYSMORPHIC RBC Aids in determining the site of renal bleeding Associated with glomerular bleeding ACANTHOCYTES CLINICAL SIGNIFICANCE Glomerular bleeding Lower urinary tract bleeding Trauma Acute infection/ inflammation Coagulation disorder MANNER OF REPORTING: / hpf White Blood Cells Larger than RBCs 12 mm in diameter Stains well with Sternheimer-Malbin Stain NEUTROPHIL- predominating urinary WBC At HYPOTONIC ENVIRONMENT = SWELL BROWNIAN MOVEMENT = GLITTER CELLS Eosinophil Stains well with Hansel Stain Seen in: Drug- induced interstitial nephritis (allergy) UTI (parasitic in origin) Renal transplant rejection (anaphylactic reaction) NOT NORMALLY SEEN IN URINE= (at least 1%) CLINICALLY SIGNIFICANT!

Mononuclear cells Not identified in wet mount preparation of urine LYMPHOCYTES resembles RBCs increased in early stages of renal rejection MONOCYTES, MACROPHAGE, HISTIOCYTES large cells with vacuoles The primary concern of mononuclear cells is to differentiate them from RTE CLINICAL SIGNIFICANCE Pyuria (increased WBC in urine) Pyelonephritis (ascending UTI; involve the kidneys) Bacteuria (increased bacterial multiplication) Cystitis (inflammation of the bladder) Epithelial Cells Normally seen in urine except in large amounts or abnormal forms 3 Types of EC: 1. Squamous EC 2. Transitional EC (urothelial cells) 3. Renal Tubular Cells Squamous Epithelial Cells Largest cells found in urine sediment Originates from the linings of vagina and female urethra, and the lower portion of the male urethra. CLUE CELLS infected epithelial cells Seen in bacterial vaginosis Gardnerella vaginalis Transitional Epithelial Cells Originates from the lining of renal pelvis, calyces and bladder Smaller than epithelial cells Spherical, polyhedral or caudate in shape due to the ability to absorb water. Pathologically seen in Malignancy and Viral Infection SYNCTIA increased numbers of transitional cells seen in clumps. Seen in catheterization collection Considered as NORMAL Renal Tubular Epithelial cells Slightly larger than WBC May be flat, cuboidal, columnar Must be identified under HPO THE MOST CLINICALLY SIGNIFICANT URINARY EPITHELIAL CELL (e.g. oval fat bodies) CLINICAL SIGNIFICANCE Malignancy Exposure to heavy metals Tubular damage Pyelonephritis Kidney transplant rejection Oval Fat Bodies RTE cells that absorb lipid lipid containing RTEC Lipiduria presence of lipid/ fat in urine Most frequently associated with nephrotic syndrome Bacteria

 Yeast

Not normally present in urine especially if specimen is taken under normal condition. Most indicative of urinary tract infection Most common cause of UTI are the Enterobacters

Small, refractile oval structures, usually mistakes as RBC Candida albicans is the most common cause of yeast UTI. CLINICAL SIGNIFICANCE Diabetes Immunocompromised patients Vaginal Moniliasis Parasites Trichomonas vaginalis The most frequent parasite encountered in urine samples Resmbles WBC, transitional cells or RTE; hard to identify when not moving. Can be seen in wet mount preparation Schistosoma haematobium Parasite of the urinary bladder that can be excreted in urine. Enterobius vermicularis Most common ova contaminant in urine Spermatozoa Oval, slightly tapered heads with long, flagella- like tails. Occasionally found in both male and female following sexual intercourse Not usually reported in the laboratory due to possible legal consequences Mucus Thread-like structure that is made of protein materials TAMM-HORSFALL PROTEIN Produced at the distal convoluted tubule by RTE excretion Major component of mucus and casts. Casts The only element found in the urinary sediments that are unique to the kidney Made up of TAMM-HORSFALL PROTEIN Cannot be detected by reagent strips, therefore, increased or positive CHON strip is frequently associated with renal disease.

Type: RBC Description: RBCs in cast matrix. Yellowish to orange color or orange-red color Significance: Acute glomerulonephritis. Strenous exercise Comments: Pinpoints source of bleeding in kidney. Most fragile of casts. Often in fragments. Type: Blood Description: Contain hemoglobin. Yellowish to orange color. Significance: Same as RBC cast Comments: From disintegration of RBC casts Type: WBC Description: Leukocytes incorporated into cast matrix. Irregular in shape. Significance: Pyelonephritis Comments: Pinpoints kidneys as the site of infection Type: Epithelial cell Description: Renal tubular epithelial cells incorporated into cast matrix Significance: Renal tubular damage Comments: Transitional and squamous epithelial cell casts do not exist. These cells are found distal to renal tubules and collecting ducts where casts are formed. Type: Waxy Description: Homogenous, opaque, notched edges, broken ends Significance: Urinary stasis Comments: From degeneration of cellular and granular casts. Unfavorable sign. Type: Fatty Description: Casts containing lipid droplets Significance: Nephrotic syndrome Comments: Maltese crosses with polarized light. Stain with Sudan and oil red O. Type: Broad Description: Wide. May be cellular, granular, or waxy. Significance: Advanced renal disease Comments: Formed in dilated distal tubules and collecting ducts. Renal failure casts.

Crystals in Acidic/Neutral Urine Crystal: Amorphous urates Description: Irregular granules Significance: None Comments: From pink precipitate in bottom of tube. May obscure significant sediment. Dissolved by warming to 60C. Crystal: Uric acid Description: Very pleomorphic. Four-sided, sixsided, star-shaped, rosettes, spears, plates. Colorless, red brown, or yellow. Significance: Usually normal Comments: Birefringent. Polarized light. Crystal: Calcium oxalate Description: Octahedral (eight-sided) envelope form is most common. Also dumbbell and ovoid forms. Significance: Normal Comments: Occasionally found in slightly alkaline urine. Monohydrate form may be mistaken for RBCs. Most common constituent of renal calculi.

CYLINDURIA Presence of casts in urine Type: Hyaline Description: Homogenous with parallel sides and rounded ends Significance: 0-2/low-power field (LPF) are normal. Increased with stress. Fever, trauma, exercise, renal disease Comments: Most common type. Least significant. Contain Tamm-Horsfall protein only . May be overlooked if light is too bright Type: Granular Description: Same as hyaline, but contains granule Significance: 0-1/LPF is normal. Increased with stress, exercise, glomerulonephritis, Pyelonephritis. Comments: May originate from disintegration of cellular casts.

Crystal: Leucine Description: Yellow, oily-looking spheres with radial and concentric striations Significance: Severe liver disease Comments: Often accompanied by tyrosine Crystal: Tyrosine Description: Fine yellow needles in sheaves or rosettes Significance: Severe liver disease Comments: Often accompanied by leucine Crystal: Cystine Description: Hexagonal (six-sided) Significance: Cystinuria Comments: Must be differentiated from uric acid. Does not polarize light. Crystal: Cholesterol Description: Flate plate with notched out corner. Star-step. Significance: Nephrotic syndrome Comments: Birefringent Crystal: Bilirubin Description: Yellowish-brown needles, plates, and granules Significance: Liver disease Comments: Reagent strip or Ictotest should be positive for bilirubin.

Crystals Found in Alkaline Urine Crystal: Amorphous phosphates Description: Irregular granules Significance: None Comments: Form white precipitate in bottom of tube. Dissolve with 2 acetic acid. Crystal: Triple phosphate Description: Coffin-lidcrystal Significance: None Crystal: Ammonium biurate Description: Yellow-brown thorn apples and spheres Significance: None Comments: Seen in old specimens Crystal: Calcium phosphate Description: Needles, rosettes, pointing finger Significance: None Comments: Only needle form seen in alkaline urine Crystal: Calcium carbonate Description: Colorless dumbbells Significance: None