Microscopic UrinalysisMicroscopic Urinalysis

Vicki S. Freeman, Ph.DVicki S. Freeman, Ph.D

Clinical Laboratory MethodsClinical Laboratory Methods

General ConsiderationsGeneral Considerations

• The results of the microscopic should The results of the microscopic should correlate with physical and chemical test correlate with physical and chemical test results.results.

• Contamination is common; especially in Contamination is common; especially in voided specimens when no effort is made to voided specimens when no effort is made to obtain a “clean catch” specimen.obtain a “clean catch” specimen.

• Results more reliable with concentrated, but Results more reliable with concentrated, but fresh specimen, as cellular elements tend to fresh specimen, as cellular elements tend to lyse in dilute, hypotonic urine or alkaline lyse in dilute, hypotonic urine or alkaline urine.urine.

More General ConsiderationsMore General Considerations

• Urine should be examined within one hour Urine should be examined within one hour of collection. If not, specimen should be of collection. If not, specimen should be refrigerated.refrigerated.

• Normal values vary considerably due to Normal values vary considerably due to variation in concentration of the specimen variation in concentration of the specimen and different methods used to concentrate and different methods used to concentrate the sediment by centrifugation (volume, the sediment by centrifugation (volume, speed, etc.)speed, etc.)

Sediment Preparation & ProcedureSediment Preparation & Procedure

1 Centrifuge 10 ml of well-mixed urine Centrifuge 10 ml of well-mixed urine specimen (1500-2000 rpm) for 5 minutes.specimen (1500-2000 rpm) for 5 minutes.

2 Suction or pour off all but 1.0 ml of urineSuction or pour off all but 1.0 ml of urine

3 Resuspend sediment and place Resuspend sediment and place approximately .05 ml on a glass slide - add approximately .05 ml on a glass slide - add coverslip on topcoverslip on top

Sediment Preparation & ProcedureSediment Preparation & Procedure

• Examine under low power with dimmed Examine under low power with dimmed light or with phase contrast microscopy to light or with phase contrast microscopy to estimate urine sediment (casts and crystals). estimate urine sediment (casts and crystals). Report numbers per low power field (LPF). Report numbers per low power field (LPF).

• Examine under high power objective to Examine under high power objective to estimate #s of RBCs, WBCs, and renal estimate #s of RBCs, WBCs, and renal tubular epithelial cells (RTE); report per tubular epithelial cells (RTE); report per high power field (HPF).high power field (HPF).

Typical Urine Report Typical Urine Report on Normal Maleon Normal Male

• Specific GravitySpecific Gravity 1.0201.020• pH rangepH range 4.8 - 7.54.8 - 7.5• ProteinProtein NegativeNegative• GlucoseGlucose NegativeNegative• KetonesKetones NegativeNegative• UrobilinogenUrobilinogen 0.8 EUs0.8 EUs• BilirubinBilirubin NegativeNegative• Occult BloodOccult Blood NegativeNegative• WBC EsteraseWBC Esterase NegativeNegative• NitriteNitrite NegativeNegative

•

• ColorColor StrawStraw

• AppearanceAppearance ClearClear

• MicroscopicMicroscopic• 0-8 WBC/HPF0-8 WBC/HPF

• 0-2 RBC/HPF0-2 RBC/HPF

• 0-1 Hyaline Cast0-1 Hyaline Cast

• Few BacteriaFew Bacteria

• 0-1 RTE/HPF0-1 RTE/HPF

• Few Ca oxalate crystalsFew Ca oxalate crystals

Epithelial CellsEpithelial Cells

• Squamous epithelial cellsSquamous epithelial cells

• Transitional epithelial cellsTransitional epithelial cells

• Renal tubular epithelial cellsRenal tubular epithelial cells

• Oval Fat BodiesOval Fat Bodies

• Clue CellsClue Cells

Squamous Epithelial CellsSquamous Epithelial Cells

• 30-50 microns30-50 microns

• large, flat cells with small nucleilarge, flat cells with small nuclei

• Appear flat with abundant cytoplasmAppear flat with abundant cytoplasm

• Originate from the superficial lining of the Originate from the superficial lining of the urethra and vaginaurethra and vagina

• Common contaminant; seen in female Common contaminant; seen in female voided specimenvoided specimen

Transitional epithelial cellsTransitional epithelial cells

• 20 -30 microns20 -30 microns

• Polyhedral shaped but swell in urine to Polyhedral shaped but swell in urine to spheroidal shapespheroidal shape

• Have round or pear-shaped contours with Have round or pear-shaped contours with small central nucleus (may be bi-nucleated)small central nucleus (may be bi-nucleated)

• Originate from transitional epithelial lining Originate from transitional epithelial lining of the renal pelvis, ureter, urinary bladder of the renal pelvis, ureter, urinary bladder and proximal urethraand proximal urethra

• A few are seen in normal urine; large A few are seen in normal urine; large clumps suggest possible carcinoma.clumps suggest possible carcinoma.

Renal tubular epithelial cells (RTE)Renal tubular epithelial cells (RTE)

• 14 - 60 microns from proximal and distal 14 - 60 microns from proximal and distal convoluted tubulesconvoluted tubules

• Single, oblong or egg-shaped cells with Single, oblong or egg-shaped cells with coarsely granular eosinophilic cytoplasmcoarsely granular eosinophilic cytoplasm

• Nuclei may be multiple but are small with Nuclei may be multiple but are small with dense chromatindense chromatin

• Seen in cases of acute tubular necrosis and Seen in cases of acute tubular necrosis and drug or heavy metal toxicitydrug or heavy metal toxicity

Oval Fat BodiesOval Fat Bodies

• Renal tubular cells that have absorbed lipids.Renal tubular cells that have absorbed lipids.

• Are highly refractile and produce a Are highly refractile and produce a characteristic Maltese cross appearance with characteristic Maltese cross appearance with polarized light.polarized light.

• Extremely significant finding. Seen in lipid Extremely significant finding. Seen in lipid nephrosis and terminal kidney disease.nephrosis and terminal kidney disease.

Clue Cells

• Squamous epithelial cells covered with Squamous epithelial cells covered with coccobacilli, Gardnerella vaginaliscoccobacilli, Gardnerella vaginalis

Blood CellsBlood Cells

• Red Blood CellsRed Blood Cells

• White Blood CellsWhite Blood Cells

Red Blood CellsRed Blood Cells• Normal size 6-8 microns, biconcave discsNormal size 6-8 microns, biconcave discs• Swollen in hypotonic, crenated in hypertonic urineSwollen in hypotonic, crenated in hypertonic urine• Empty RBC membranes may be seen from lyzed Empty RBC membranes may be seen from lyzed

cells in alkaline urinecells in alkaline urine

• Confusing artifacts Confusing artifacts • oil droplets, yeast, uratesoil droplets, yeast, urates

Red Blood CellsRed Blood Cells

• NormalNormal– MaleMale 1-2 RBC/HPF1-2 RBC/HPF– FemaleFemale 3-12 RBC/HPF3-12 RBC/HPF

• Increased RBC seen in Increased RBC seen in – Renal disease such as glomerulonephritis, lupus Renal disease such as glomerulonephritis, lupus

nephritis, kidney stones, tumors and traumanephritis, kidney stones, tumors and trauma– Lower urinary tract disease such acute and Lower urinary tract disease such acute and

chronic infection, tumors and strictureschronic infection, tumors and strictures– Extrarenal disease such as acute appendicitis.Extrarenal disease such as acute appendicitis.

White Blood CellsWhite Blood Cells

• 10-12 microns, swell to 15 microns in 10-12 microns, swell to 15 microns in alkaline or hypotonic urine, nuclei more alkaline or hypotonic urine, nuclei more distinct in acid urinedistinct in acid urine

• Mainly neutrophils and have a granular Mainly neutrophils and have a granular cytoplasm and lobed nucleuscytoplasm and lobed nucleus

White Blood CellsWhite Blood Cells

• Normal 0-8 WBC/HPFNormal 0-8 WBC/HPF

• Increased inIncreased in– pyelonephritis, cystitis, urethritis, prostatitispyelonephritis, cystitis, urethritis, prostatitis

• ““Glitter cell” term used to describe large Glitter cell” term used to describe large WBC seen in hypotonic urine that have WBC seen in hypotonic urine that have Browian movement of granules in Browian movement of granules in cytoplasmcytoplasm

• Clumps of WBCs considered very Clumps of WBCs considered very significant in indicating an infectionsignificant in indicating an infection

Other Urinary ElementsOther Urinary Elements• BacteriaBacteria

• Yeast Yeast – confused with red cellsconfused with red cells– look for budding, look for budding,

doubly refractive walldoubly refractive wall

• TrichomonasTrichomonas– confused with white cellsconfused with white cells– look for undulating look for undulating

membrane movementmembrane movement

• SpermSperm

Urine CastsUrine Casts• Hyaline CastsHyaline Casts

• Red Blood Cell CastsRed Blood Cell Casts

• Hemoglobin or Blood Cell CastsHemoglobin or Blood Cell Casts

• White Blood Cell CastsWhite Blood Cell Casts

• Renal tubular epithelial cell castsRenal tubular epithelial cell casts

• Granular castsGranular casts

• Waxy castsWaxy casts

• Fatty castsFatty casts

Urinary CastsUrinary Casts• Cylindrical structure which consists of Cylindrical structure which consists of

– jelled protein (Tamm-Horsfall mucoprotein)jelled protein (Tamm-Horsfall mucoprotein)– clumping of the protein or conglutination of material clumping of the protein or conglutination of material

within the lumen of the renal tubuleswithin the lumen of the renal tubules– Albumin or globulins may be mixed with the Albumin or globulins may be mixed with the

mucoproteinmucoprotein

• Conditions that increase urine cast formationConditions that increase urine cast formation– Increased concentration of the urine]Increased concentration of the urine]– Increased acidity of the urineIncreased acidity of the urine– High protein concentration in the urineHigh protein concentration in the urine– Stasis or obstruction of the nephrons by cells or debrisStasis or obstruction of the nephrons by cells or debris

Hyaline CastsHyaline Casts

• Formed in the lumen of the distal Formed in the lumen of the distal convoluted tubules or collecting ducts and convoluted tubules or collecting ducts and serve as the matrix of all castsserve as the matrix of all casts

• Pale, smooth and usually cylindrical, Pale, smooth and usually cylindrical, homogeneous gel-like forms of low homogeneous gel-like forms of low refractive index. Mainly Tamm-Horsfall refractive index. Mainly Tamm-Horsfall mucoproteinmucoprotein

• Narrower casts form in the convoluted Narrower casts form in the convoluted tubules while broader casts form in the tubules while broader casts form in the collecting ducts.collecting ducts.

Red Blood Cell CastsRed Blood Cell Casts

• These casts are always pathologicThese casts are always pathologic

• Diagnostic of glomerular disease or damageDiagnostic of glomerular disease or damage

• Classically found in acute glomerulonephritisClassically found in acute glomerulonephritis

• RBC outline must be sharply defined in at RBC outline must be sharply defined in at least part of the castleast part of the cast

Hemoglobin CastHemoglobin Cast

• An RBC cast in which the red cells have An RBC cast in which the red cells have ruptured and disintegrateruptured and disintegrate

• Cast appears reddish-brown due to acid Cast appears reddish-brown due to acid hematin formationhematin formation

• Diagnostic of glomerular disease or damage Diagnostic of glomerular disease or damage such as acute glomerulonephritissuch as acute glomerulonephritis

WBC CastWBC Cast

• Hyaline cast with WBCs embedded in Hyaline cast with WBCs embedded in matrixmatrix

• Indicate inflammation/infection in kidneyIndicate inflammation/infection in kidney

• Seen in acute pyelonephritis and other Seen in acute pyelonephritis and other nephritis conditionsnephritis conditions

Renal Tubular Epithelial CastRenal Tubular Epithelial Cast

• Hyaline cast with renal tubular epithelial Hyaline cast with renal tubular epithelial cells embedded in the hyaline matrixcells embedded in the hyaline matrix

• Form as result of stasis and necrosis of the Form as result of stasis and necrosis of the tubulestubules

• Seen in severe chronic renal disease, Seen in severe chronic renal disease, exposure to nephrotoxic agents or viruses exposure to nephrotoxic agents or viruses and rejection in kidney transplantsand rejection in kidney transplants

Granular CastsGranular Casts

• Results of the degeneration of cellular Results of the degeneration of cellular components of casts or direct aggregation components of casts or direct aggregation of serum proteins into a matrix of Tamm-of serum proteins into a matrix of Tamm-Horsfall mucoproteinHorsfall mucoprotein

• Usually indicates significant renal diseaseUsually indicates significant renal disease

• Thought to be the result of breakdown of Thought to be the result of breakdown of cellular casts with the progression of cellular casts with the progression of cellular to coarsely to finely granular to cellular to coarsely to finely granular to waxy.waxy.

Waxy CastsWaxy Casts

• Smooth, homogeneous, highly refractive Smooth, homogeneous, highly refractive appearance. Typically have blunt, broken appearance. Typically have blunt, broken ends and cracked or serrated edgesends and cracked or serrated edges

• Seen in patients with severe chronic renal Seen in patients with severe chronic renal failure, malignant hypertension, diabetic failure, malignant hypertension, diabetic nephropathynephropathy

• May also be seen in acute renal disease and May also be seen in acute renal disease and renal allograft rejectionrenal allograft rejection

Fatty CastsFatty Casts

• Casts that have incorporated either free fat Casts that have incorporated either free fat droplets or oval fat bodies.droplets or oval fat bodies.

• In the fat is cholesterol, the droplets will be In the fat is cholesterol, the droplets will be demonstrate a “Maltese cross” appearance demonstrate a “Maltese cross” appearance under polarized light.under polarized light.

• Droplets which consist of triglycerides or Droplets which consist of triglycerides or neutral fat will not polarize but will stain neutral fat will not polarize but will stain with Sudan III or Oil Red O stains for fat.with Sudan III or Oil Red O stains for fat.

Crystals seen in Acidic UrineCrystals seen in Acidic Urine• Calcium oxalateCalcium oxalate

– envelope, dumbbell or ring formsenvelope, dumbbell or ring forms– colorless, do not polarizecolorless, do not polarize– Common cause of kidney stonesCommon cause of kidney stones

• Uric acidUric acid– rhombic plates, rosettes, wedges, needlesrhombic plates, rosettes, wedges, needles– polarize to multicoloredpolarize to multicolored– found in goutfound in gout

• Amorphous uratesAmorphous urates– clumps of brownish-yellow granulesclumps of brownish-yellow granules

Crystals seen in Alkaline UrineCrystals seen in Alkaline Urine

• Triple phosphateTriple phosphate– Coffin lid crystalsCoffin lid crystals– colorless prismscolorless prisms

• Ammonium biurateAmmonium biurate– thorn-apple crystalsthorn-apple crystals– yellow-brown, spicule covered spheresyellow-brown, spicule covered spheres

Crystals with Pathogenic Crystals with Pathogenic SignificanceSignificance

• CystineCystine– colorless hexagonal plates colorless hexagonal plates

– do not polarizedo not polarize

– associated with inborn errors of metabolismassociated with inborn errors of metabolism

• CholesterolCholesterol– rectangular plate with a notched rectangular plate with a notched

corner or edgecorner or edge

– polarize as multicolored platespolarize as multicolored plates

– seen in nephritis and nephrosis conditionsseen in nephritis and nephrosis conditions

Gladys Glomerulus (35 yr old)• ColorColor PalePale• AppearanceAppearance ClearClear• Specific GravitySpecific Gravity

1.0351.035• GlucoseGlucose NegNeg• BilirubinBilirubin NegNeg• KetoneKetone NegNeg• BloodBlood NegNeg• pHpH 5.05.0• ProteinProtein 1+1+• UrobilinogenUrobilinogen 0.20.2• NitriteNitrite NegNeg• LeukocytesLeukocytes TraceTrace

• Epi cellsEpi cells ManyMany• CastsCasts 2-5 Hyaline2-5 Hyaline• RBCs/HPF RBCs/HPF 0-20-2• WBCs/HPFWBCs/HPF 10-2510-25• CrystalsCrystals 2-5 Triple 2-5 Triple

PO4PO4

Many Amorp UratesMany Amorp Urates• BacteriaBacteria Few (10-50)Few (10-50)

Tammy Tubule (25 yr old)• ColorColor YellowYellow

• AppearanceAppearanceCloudyCloudy

• Specific GravitySpecific Gravity 1.0031.003

• GlucoseGlucose NegNeg

• BilirubinBilirubin NegNeg

• KetoneKetone NegNeg

• BloodBlood NegNeg

• pHpH 8.58.5

• ProteinProtein 4+4+

• UrobilinogenUrobilinogen 0.20.2

• NitriteNitrite NegNeg

• LeukocytesLeukocytes TraceTrace

• Epi cellsEpi cells 5-205-20• CastsCasts NoneNone• RBCs/HPF RBCs/HPF 50-10050-100• WBCs/HPFWBCs/HPF 0-20-2• CrystalsCrystals

Many Amorp UratesMany Amorp Urates• BacteriaBacteria NoneNone

Bowman S. Capsule (2 yr old)• ColorColor YellowYellow

• AppearanceAppearance HazyHazy

• Specific GravitySpecific Gravity 1.0111.011

• GlucoseGlucose NegNeg

• BilirubinBilirubin NegNeg

• KetoneKetone NegNeg

• BloodBlood NegNeg

• pHpH 5.05.0

• ProteinProtein NegNeg

• UrobilinogenUrobilinogen 0.20.2

• NitriteNitrite PosPos

• LeukocytesLeukocytes ModMod

• Epi cellsEpi cells FewFew• CastsCasts 2-5 Hyaline2-5 Hyaline

5-10 Fine gran5-10 Fine gran

>10 Coarse >10 Coarse grangran

• RBCs/HPF RBCs/HPF 2-52-5• WBCs/HPFWBCs/HPF NoneNone• CrystalsCrystals

Few Amorp UratesFew Amorp Urates• BacteriaBacteria NoneNone

Ned Nephron (23 yr old)• ColorColor AmberAmber

• AppearanceAppearance ClearClear

• Specific GravitySpecific Gravity 1.0061.006

• GlucoseGlucose NegNeg

• BilirubinBilirubin NegNeg

• KetoneKetone SmallSmall

• BloodBlood ModMod

• pHpH 6.06.0

• ProteinProtein NegNeg

• UrobilinogenUrobilinogen 1.01.0

• NitriteNitrite PositivePositive

• LeukocytesLeukocytes ModMod

• Epi cellsEpi cells >100>100• CastsCasts NoneNone• RBCs/HPF RBCs/HPF NoneNone• WBCs/HPFWBCs/HPF 25-5025-50• CrystalsCrystals NoneNone• BacteriaBacteria Mod (50-Mod (50-

200)200)• OtherOther Budding yeastBudding yeast

Renal DiseasesRenal Diseases

• NephrosisNephrosis

• CystitisCystitis

• Acute pyelonephrititsAcute pyelonephritits

• Acute glomerulonephritisAcute glomerulonephritis

Sediment ProcedureSediment Procedure Centrifuge 10 ml of well-mixed urine specimen (1500 - Centrifuge 10 ml of well-mixed urine specimen (1500 -

2000 rpm) for 5 minutes2000 rpm) for 5 minutes Pour off all but 1.0 ml of the urinePour off all but 1.0 ml of the urine Resuspend sediment and place approximately 0.05 ml on a Resuspend sediment and place approximately 0.05 ml on a

glass slide and add coverslipglass slide and add coverslip Examine under low power with dimmed light to estimate Examine under low power with dimmed light to estimate

urine sediment (casts and crystals). Report numbers per urine sediment (casts and crystals). Report numbers per low power field (lpf)low power field (lpf)

Examine under high power to estimate #s of RBCs, WBCs Examine under high power to estimate #s of RBCs, WBCs and renal tubular epithelial cells; report per high power and renal tubular epithelial cells; report per high power field (hpf).field (hpf).