Microscopic Examination Of Urine PDF

MICR O S CO P IC ANALY SIS O F U RIN E A ND R EILLE R. GATA , R M T B Y: L AR S ADDIS COUNT First procedure to standardize the quantitation of formed elements, used a hemocytometer NORMAL VALUE OF ADDIS COUNT RBCs = 0 to 500,000 cells /uL WBCs and Epi...

MICR O S CO P IC ANALY SIS O F U RIN E A ND R EILLE R. GATA , R M T B Y: L AR S ADDIS COUNT First procedure to standardize the quantitation of formed elements, used a hemocytometer NORMAL VALUE OF ADDIS COUNT RBCs = 0 to 500,000 cells /uL WBCs and Epithelial cells =0 to 1,800,000 cells /uL Hyaline casts = 0 to 5000 cells/uL  SPECIMEN PREPARATION URINE 10 -15 ML ↓ CENTRIFUGE AT 400 RCF FOR 5 MINUTES ↓ DECANT ↓ GET THE SEDIMENT (0.5-1.0ML) ↓ PLACE THE SEDIMENT ON THE MICROSCOPIC SLIDE (20 UL OR 0.02ML) ↓ COVERED BY GLASS COVER SLIP (22X22MM) ↓ OBSERVE UNDER THE MICROSCOPE (BRIGHT FIELD –REDUCED LIGHTNING) To correct for differences in the diameter of centrifuge heads, RCF rather than revolutions per minute (RPM) is used. Formula: RCF(g) = 1.118 x 10^-5 x radius (cm) x RPM^2  SEDIMENT STAINS STAIN ACTION FUNCTION Sternheimer-Malbin(a supravital stain Delineates structure and contrasting Identifies WBCs, epithelial cells, and consisting of Crystal violet and colors of the nucleus and cytoplasm casts safranin) 0.5%Toluidine blue (a metachromatic Enhances nuclear detail Differentiates WBCs and renal tubular supravital stain) epithelial (RTE) cells 2% acetic acid Lyses RBCs and enhances nuclei of Distinguishes RBCs from WBCs, yeast, WBCs oil droplets, and crystals Lipid Stains: Oil Red O and Sudan III Stain triglycerides and neutral fats Identifies free fat droplets and lipid orange-red containing cells and casts Gram stain Differentiates gram-positive and gram Identifies bacterial casts negative bacteria Hansel stain Methylene blue + EosinY Stains Identifies urinary eosinophils eosinophilic granules Prussian blue stain Stains structures containing iron Identifies yellow-brown granules of hemosiderin in cells and casts Sedi and KOVA stain Modified Sternheimer Malbin The dye Hyaline cast appears as pink Motile is absorbed well by WBCs, epithelial bacteria are unstained Non-Motile cells, and casts, providing clearer bacteria stains purple T.vaginalis stains delineation of structure and contrasting Light blue-green colors of the nucleus and cytoplasm NOTE 1. In Oil Red O and Sudan III, cholesterol and cholesterol esters do not stain and must be confirmed by polarizing Microscopy 2. Wright’s stain or Giemsa stain also distinguishes urinary eosinophils, but Hansel stain is preferred. COMMERCIAL SYSTEMS FOR URINE SEDIMENT PREPARATION UriSystem The UriSystem tube is designed such that after centrifugation, it can be decanted with a quick smooth motion and consistently retains 0.4 mL of urine for sediment resuspension. KOVA System The KOVA System uses a specially designed pipette that snuggly fits the diameter and shape of the tube to retain 1 mL of urine during decanting. Count-10 System The Count-10 System offers several options to retain 0.8 mL for sediment resuspension SEDI M ENT CO NS T I T UEN T S FOUN D I N U RI NE  I. RED BLOOD CELLS Appear as smooth, non-nucleated, biconcave disk measuring approximately 7 um in diameter Most difficult to recognize The observation of microscopic hematuria can be critical to the early diagnosis of glomerular disorders and malignancy of the urinary tract and to confirm the presence of renal calculi The presence of not only RBCs but also hyaline, granular, and RBC casts may be seen following strenuous exercise If the specimen is not fresh when it is examined, erythrocytes may appear as faint, colorless circles or “shadow cells,” because the hemoglobin may dissolve out They may become crenated in hypertonic urine and appear as small, rough cells with crinkled edges In Concentrated /Hypersthenuric urine: Crenated cells /ECHINOCYTES / Irregularly shaped In Diluted / Hyposthenuric urine: Ghost cells / Swollen RBC Dysmorphic or Distorted RBC – vary in sizes, mainly they are acanthocytes, it is associated with glomerular bleeding  Because their hemoglobin has been lost, ghost cells are difficult to see using brightfield microscopy; however, they are readily visible with phase-contrast or interference contrast microscopy When viewed from the side, RBCs have an hourglass shape; when viewed from above, they appear as disks with a central pallor Hypotonic and Alkaline urine promotes formation of ghost cells in urine Normal RBC in normal urine is 0–2 cells/hpf ; more than 3 cells/hpf is considered abnormal Source of identification error: Yeast cell, oil droplets, air bubbles Look-alike crystal: Monohydrate calcium oxalate crystals NOTE Wright’s stain can be used to further analyze the dysmorphic RBCs. Analysis shows the cells to be hypochromic and better delineates the presence of cellular blebs and protrusions  II. WHITE BLOOD CELLS WBCs are larger than RBCs, measuring average of about 12 um in diameter Pyuria or leukocytoruia- Term used to denote increase urinary WBCs and is associated with bacterial infection (UTI), Interstitial nephritis, and SLE Neutrophil is the predominant WBC found in urine Neutrophils lyse rapidly in dilute alkaline urine and begin to lose nuclear detail. In Hypotonic urine, white blood cell swells and become spherical balls that lyse as rapidly as 50% in 2 to 3 hours at room temperature Hypotonic Urine: Glitter Cells – WBC with sparkling appearance due to Brownian movement of the granules. When stained with Sternheimer-Malbin stain, these large cells stain light blue as opposed to the violet color usually seen with neutrophils  In hypertonic urine, leukocytes become smaller as water is lost osmotically from the cells, but they do not crenate. Another degenerative change in WBC is the development of numerous finger-like or wormlike projections protruding from their surfaces. These long filaments, termed myelin forms, result from the breakdown of the cell membrane Eosinophil - The presence of urinary eosinophils is primarily associated with drug- induced interstitial nephritis; however, small numbers of eosinophils may be seen with urinary tract infection (UTI) and renal transplant rejection. Eosinophils are not normally seen in the urine; therefore, the finding of more than 1% eosinophils is considered significant Lymphocytes predominate in urine from patients experiencing renal transplant rejection. Normal WBC in urine = 0-5 WBC/hpf for male, and 0-8 WBC/hpf for female  III. EPITHELIAL CELLS A. Squamous Epithelial cell Originates from the linings of the vagina and female urethra and the lower portion of the male urethra. Squamous cells are the largest cells found in the urine sediment. They contain abundant, irregular cytoplasm and a prominent nucleus about the size of an RBC. They may appear as flagstone-shaped with distinct cell borders The point of reference in microscopic analysis They may occasionally appear folded, possibly resembling a cast, and will begin to disintegrate in urine that is not fresh. Increased amounts are more frequently seen in females. Clue cells: pathologic squamous epithelial cell covered with the Gardnerella vaginalis coccobacillus To be considered a clue cell, the bacteria should cover most of the cell surface and extend beyond the edges of the cell. This gives the cell a granular, irregular appearance.  B. Transitional Epithelial (Urothelial) cells/ Bladder epithelial cells Transitional epithelial cells originate from the lining of the renal pelvis, calyces, ureters, and bladder, and from the upper portion of the male urethra. Transitional epithelial cells are smaller than squamous cells and appear in several forms, including spherical, polyhedral, and caudate. The differences are caused by the ability to absorb large amounts of water. They are two to four times as large as white cells. They may be round, pear-shaped, or may have taillike projections. Occasionally, these cells may contain two nuclei. Spherical forms of transitional epithelial cells are sometimes difficult to distinguish from RTE cells. The presence of a centrally located rather than eccentrically placed nucleus, and supravital staining, can aid in the differentiation. Increased numbers of transitional cells seen singly, in pairs, or in clumps (syncytia)are present following invasive urologic procedures such as catheterization and are of no clinical significance. An increase in transitional cells exhibiting abnormal morphology such as vacuoles and irregular nuclei may be indicative of malignancy or viral infection.  C. Renal Tubular Epithelial Cells (RTE cells) Renal tubular epithelial (RTE) cells vary in size and shape depending on the area of the renal tubules from which they originate. The cells from the proximal convoluted tubule (PCT) are larger than other RTE cells. They tend to have a rectangular shape and are referred to as columnar or convoluted cells. Cells from the distal convoluted tubule (DCT) are smaller than those from the PCT and are round or oval. Collecting duct RTE cells are cuboidal and are never round. Along with the eccentrically placed nucleus, the presence of at least one straight edge differentiates them from spherical and polyhedral transitional cells. Cells from the collecting duct that appear in groups of three or more are called renal fragments. They are frequently seen as large sheets of cells. Renal fragments are an indication of severe tubular injury with basement membrane disruption. RTE cells often resemble casts and they should be closely examined for the presence of a nucleus, as a nucleus would not be present in a cast. Tubular Injury: presence of more than 2 RTE/HPF RTE cells are the most clinically significant of the epithelial cells and the presence of increased amounts is indicative of necrosis of the renal tubules They are the precursor of oval fat bodies  LOCATION OF RTE CELL APPEARANCE PCT (Proximal convoluted tubules) Rectangular DCT (Distal convoluted tubules) Round or Oval Collecting duct Cuboidal and can be seen as large sheets of cells NOTE Conditions producing tubular necrosis include exposure to heavy metals, drug-induced toxicity, hemoglobin and myoglobin toxicity, viral infections (hepatitis B), pyelonephritis, allergic reactions, malignant infiltrations, salicylate poisoning, and acute allogenic transplant rejection Bubble cells – RTE cells containing large, nonlipid-filled vacuoles that is mainly associated with Acute tubular necrosis. They appear to represent injured cells in which the endoplasmic reticulum has dilated prior to cell death IV. OVAL FAT BODIES These are lipid-containing RTE cells They are highly refractile RTE cells They are usually seen in conjunction with free-floating fat droplets When monocytes or macrophages have ingested lipoproteins and fat, these globular inclusions are distinctly refractile. Called oval fat bodies, these cells are impossible to distinguish from renal tubular cells that can also absorb fats Identification of oval fat bodies is confirmed by staining the sediment with Sudan III or Oil Red O fat stains and examining the sediment using polarized microscopy. Examination of the sediment using polarized light results in the appearance of characteristic Maltese cross formations They are present in disorders such as: Nephrotic syndrome, DM, Severe tubular necrosis, and in trauma cases that cause release of bone marrow fat from the long bones NOTE In lipid-storage diseases, large fat-laden histiocytes may also be present in urine. They can be differentiated from oval fat bodies by their large size. -Strasinger V. BACTERIA They appear as small spherical and rod-shaped structures Bacteria are not normally present in urine To be considered significant for UTI, bacteria should be accompanied by WBCs. They are motile and is useful to differentiate from similar appearance, amorphous urates and phosphates The actual bacteria producing an UTI cannot be identified with the microscopic examination. VI. YEAST Yeast cells appear in the urine as small, refractile oval structures that may or may not contain a bud. In severe infections, they may appear as branched, mycelial forms Yeast cells, primarily Candida albicans, are seen in the urine of diabetic, immunocompromised patients and women with vaginal moniliasis. A true yeast infection should be accompanied by the presence of WBCs. FAVORABLE URINE CONDITION: ACIDIC urine and with glucose VII. PARASITES Trichomonas vaginalis – most frequent parasite encountered in urine Schistosoma haematobium – bladder parasite, associated with bladder tumors Enterobius vermicularis- most common contaminant ova Cyst of Giardia lamblia- observed in urine sediment as the result of fecal contamination of infected individuals When not moving, Trichomonas is more difficult to identify and may resemble a WBC, transitional, or RTE cell. Use of phase microscopy may enhance visualization of the flagella or undulating membrane. VIII. SPERMATOZOA Spermatozoa are easily identified in the urine sediment by their oval, slightly tapered heads and long, flagellalike tails Urine is toxic to spermatozoa; therefore they rarely exhibit the motility observed when examining a semen specimen. They are rarely of clinical significance except in cases of male infertility or retrograde ejaculation in which sperm is expelled into the bladder instead of the urethra. Laboratory protocols vary with regard to reporting or not reporting the presence of spermatozoa in a urine specimen IX. MUCUS Mucus is a protein material produced by the glands and epithelial cells of the lower genitourinary tract and the RTE cells. Mucus appears microscopically as thread-like structures with a low refractive index Uromodulin / Tamm-Horsfall protein is the major constituent or matrix of the mucus Mucus is more frequently present in female urine specimens. It has no clinical significance when present in either female or male urine. Increase in numbers are found in cases of UTI. X. HEMOSIDERIN GRANULES Hemosiderin granules are found in the urine sediment 2 to 3 days after a severe hemolytic episode (e.g., transfusion reaction, paroxysmal nocturnal hemoglobinuria). Hemosiderin granules may be found free floating or within macrophages, casts, or tubular epithelial cells. The Prussian blue reaction, also known as the Rous test, is used to identify hemosiderin in the urine sediment and in tissues. The urine sediment is suspended in a freshly prepared solution of potassium ferricyanide– HCl and is allowed to stand at room temperature for 10 minutes. After centrifugation and discarding of the supernatant, the sediment is reexamined for the presence of coarse blue granules URINARY CAST URINARY CAST CYLINDRURIA– presence of urinary cast Casts are the only elements found in the urinary sediment that are unique to the kidney. The major constituent/mould /template/ matrix of cast is UROMODULIN which is secreted by the RTE Cells. The protein (uromodulin) gels more readily under conditions of urine-flow stasis, acidity, and the presence of sodium and calcium. Uromodulin protein is found in both normal and abnormal urine Casts are Formed in DCT, and Collecting duct Examination of casts should be performed along the edges of the cover slip. Cylindroids – formed at the ALH and DCT with tapered end or have a tail at the other tail. They have the same significance as casts (hyaline cast). Cylindroids are product of incomplete cast formation,or cast disintegration.  CAST FORMATION From least significant to the most significant Hyaline Cast→ Cellular cast→ Coarse granular cast → Fine granular cast → Waxy cast → Broad Cast STEP BY STEP ANALYSIS OF THE FORMATION OF TAMM-HORSFALL PROTEIN MATRIX 1. Aggregation of Tamm-Horsfall protein into individual protein fibrils attached to the RTE cells 2. Interweaving of protein fibrils to form a loose fibrillar network (urinary constituents may become enmeshed in the network at this time) 3. Further protein fibril interweaving to form a solid structure 4. Possible attachment of urinary constituents to the solid matrix 5. Detachment of protein fibrils from the epithelial cells 6. Excretion of the cast NOTE Hypotonic and alkaline urine promotes the disintegration of casts in the urine sediment. Acid pH, increased solute concentration, urine stasis, and increased plasma proteins (particularly albumin) enhance cast formation If the conventional glass-slide method is being used, casts have a tendency to locate near the edges of the cover slip; therefore, low-power scanning of the cover-slip perimeter is recommended 1. HYALINE CAST Most frequently seen cast which consists almost entirely of uromodulin Pro – cast Hyaline casts appear colorless in unstained sediments and have a low refractive index similar to that of urine; thus, they can easily be overlooked if specimens are not examined under subdued light The morphology of hyaline casts is varied, consisting of normal parallel sides and rounded ends, cylindroid forms, and wrinkled or convoluted shapes that indicate aging of the cast matrix The accompanying dehydration of the protein fibrils and internal tension may account for the wrinkled and convoluted appearance of older hyaline casts Physiologic increase: 1. Strenuous exercise 2. Dehydration 3. Heat exposure 4. Emotional stress Pathologic increase: 1. Acute glomerulonephritis 2. Pyelonephritis 3. Chronic renal disease 4. Congestive heart failure Sternheimer-Malbin stain and KOVA stain: Pink color Normal value: 0-2 /low power field 2. RBC CAST Seen during bleeding in the nephron, especially associated with glomerulonephritis RBC casts are easily detected under low power by their orange-red color. They are more fragile than other casts and may exist as fragments or have a more irregular shape as the result of tightly packed cells adhering to the protein matrix They have also been observed in healthy individuals following participation in strenuous contact sports As an RBC cast ages, cell lysis begins and the cast develops a more homogenous appearance, but retains the characteristic orange-red color from the released hemoglobin. These casts may be distinguished as blood casts, indicating greater stasis of urine flow 3. WBC CAST The appearance of WBC casts in the urine signifies infection or inflammation within the nephron. They are most frequently associated with pyelonephritis and are a primary marker for distinguishing pyelonephritis (upper UTI) from Cystitis (lower UTI) They are also present in non-bacterial inflammations such as acute interstitial nephritis and may accompany RBC casts in glomerulonephritis WBC casts are visible under low-power magnification but must be positively identified using high power Staining and the use of phase microscopy can be helpful to enhance the nuclear detail needed for differentiating WBC cast from Epithelial cast 4. BACTERIAL CAST Bacterial casts containing bacilli both within and bound to the protein matrix are seen in pyelonephritis. Their presence should be considered when WBC casts and many free WBCs and bacteria are seen in the sediment Confirmation of bacterial casts is best made by performing a Gram stain on the dried or cytocentrifuged sediment. 5. FATTY CAST Fatty casts are seen in conjunction with oval fat bodies and free fat droplets in disorders causing lipiduria. They are most frequently associated with the nephrotic syndrome, but are also seen in toxic tubular necrosis, diabetes mellitus, and crush injuries Confirmation of fatty casts is performed using polarized microscopy and Sudan III or Oil Red O fat stains. 6. GRANULAR CAST Coarsely and finely granular casts are frequently seen in the urinary sediment and may be of pathologic or nonpathologic significance. The origin of the granules in non-pathologic conditions appears to be from the lysosomes excreted by RTE cells during -normal metabolism. Granular casts are easily visualized under low-power microscopy. However, final identification should be performed using high power to determine the presence of a cast matrix. Pathologic increase: glomerulonephritis and pyelonephritis Physiologic increase: strenuous exercise 7. WAXY CAST Waxy casts are representative of extreme urine stasis, indicating chronic renal failure. They are brittle, and highly refractive compared to hyaline cast They often appear fragmented with jagged ends and have notches in their sides Ground glass appearance, homogenous matrix, with cracks or fissures from margins or along the length of the cast With supravital stains, waxy casts stain a homogenous, dark pink Waxy casts are more easily visualized than hyaline casts because of their higher refractive index 8. BROAD CAST Often referred to as renal failure casts Broad casts may result from tubular distension or, in the case of extreme urine stasis, from formation in the collecting ducts The presence of broad casts indicates destruction (widening) of the tubular walls. Also, when the flow of urine to the larger collecting ducts becomes severely compromised, casts form in this area and appear broad. All types of casts may occur in the broad form Bile-stained broad, waxy casts are seen as the result of the tubular necrosis caused by viral hepatitis Two most commonly seen broad cast: granular and waxy 9. GRANULAR DIRTY BROWN CAST Granular, dirty, brown casts representing hemoglobin degradation products such as methemoglobin may also be present They are associated with the acute tubular necrosis often caused by the toxic effects of massive hemoglobinuria that can lead to renal failure. These dirty, brown casts must be present in conjunction with other pathologic findings such as RTE cells and a positive reagent strip test for blood SOURCES OF ERROR Cast Sources of error Hyaline Cast Mucus threads, fibers, hair, increased lighting RBC Cast RBC clumps WBC Cast WBC clumps Bacterial Cast Granular casts Epithelial / RTE Cast WBC cast Granular Cast Artifacts such as Clumps of small crystals, Fecal debris Columnar RTE cells Waxy Cast Cotton threads or diaper fibers, Fibers and fecal material Fatty Cast Fecal debris Broad Cast Fecal material, fibers CLASSIFICATION OF URINARY CASTS (BRUNZEL, 3RD ED.) Homogenous Hyaline and waxy cast Pigmented Bilirubin, Myoglobin, and hemoglobin Size Broad cast Inclusions Cellular inclusions: Red blood cells, Leukocytes, Renal tubular epithelial cells, Mixed cells, Bacteria Others: Granular, Fat globules—cholesterol, triglycerides, Hemosiderin granules, Crystals