AUBF Microscopy

• Purpose

  Detect and identify insoluble materials present in the urine
• Formed elements in urine

  • RBCs
  • WBCs
  • Epithelial cells
  • Casts
  • Bacteria
  • Yeast
  • Parasites
  • Mucus
  • Spermatozoa
  • Crystals
  • Artifacts
• Microscopic examination of urine

  • Least standardized and most time-consuming part of the routine urinalysis
• Macroscopic screening tests

  • Color
  • Blood
  • Clarity
  • Hematuria vs hemoglobinuria vs myoglobinuria
  • Confirm pathologic or non-pathologic cause of turbidity
  • Blood
  • RBCs, RBC casts
  • Protein
  • Casts, Cells
  • Nitrite
  • Bacteria, WBCs
  • Leukocyte Esterase
  • WBCs, WBC casts, Bacteria
  • Glucose
  • Yeast
• Macroscopic screening correlation

  Specimens should be examined while fresh or adequately preserved
• Formed elements

  Disintegrate rapidly, particularly in dilute alkaline urine
• Refrigeration


  Causes precipitation of amorphous urates and phosphates and other non-pathologic crystals
• Midstream clean-catch specimen


  Minimizes external contamination of the sediment
• Specimen preparation

  Care must be taken to thoroughly mix the specimen prior to decanting a portion into a centrifuge tube
• Specimen volume


  10 and 15 mL -- provides an adequate volume to obtain a representative sample of the elements present in the specimen
• Sediment preparation

  • A uniform amount of urine and sediment should remain in the tube after decantation
  • Volumes of 0.5 and 1.0 mL are frequently used
  • To maintain a uniform sediment concentration factor, urine should be aspirated off rather than poured off
  • Sediment must be resuspended by gentle agitation
  • Vigorous agitation should be avoided, as it disrupt some cellular elements
• Volume of sediment examined

  • When using the conventional glass-slide method, the recommended volume is 20 uL (0.02 mL) covered by a 22 x 22 mm glass cover slip
  • Do not allow the specimen to flow outside of the cover slip -- result in the loss of heavier elements such as casts
• Examination of the sediment

  • Observe in a minimum of 10 fields under LPO and HPO
  • Slide is first examined under LPO to detect casts and to ascertain the general composition of the sediment
  • If conventional glass-slide method is being used, casts have a tendency to locate near the edges of the cover slip; therefore, LPO scanning of the cover-slip perimeter is recommended
• Reporting the microscopic examination

  • Casts are reported as the average number per low-power field (lpf) following examination of 10 fields
  • RBCs and WBCs, as the average number per 10 high-power fields (hpf)
  • Epithelial cells, crystals, and other elements are frequently reported in semi-quantitative terms: rare, few, moderate, many, 1+, 2+, 3+, 4+
• Sediment stain characteristics

  • Sternheimer-Malbin (Crystal Violet & Safranin) - Delineates structure and contrasting colors of the nucleus and cytoplasm, Identifies WBCs, epithelial cells, and casts
  • Toluidine Blue - Enhances nuclear detail, Differentiates WBCs and renal tubular epithelial cells
  • 2% Acetic Acid - Lyses RBCs and enhances nuclei of WBCs, Distinguishes RBCs from WBCs, yeast, oil droplets, and crystals
  • Lipid Stains (Oil Red O, Sudan III) - Stains triglycerides and neutral fats orange-red
  • Gram Stain - Differentiates gram-positive and gram-negative bacteria, Identifies bacterial casts
  • Hansel Stain (Methylene blue & Eosin Y) - Methylene blue and eosin Y stain eosinophilic granules, Identifies urinary eosinophils
  • Prussian Blue Stain - Stains structures containing iron, Identifies yellow-brown granules of hemosiderin in cells and casts
• Red blood cells (RBCs)


  • Appear as smooth, non-nucleated, biconcave disk
  • Measure approximately 7 mm in diameter
  • Identified using HPO (average number seen in 10 hpfs)
  • In concentrated (hypersthenuric) urine, cells shrink due to loss of water and appear crenated or irregularly shaped
  • In dilute (hyposthenuric) urine, cells absorb water, swell, and lyse rapidly, releasing their hemoglobin and leaving only the cell membrane – GHOST CELLS (not examined under reduced light)
• RBCs in urine

  • RBCs are the most difficult for students to recognize: RBCs' lack of characteristic structures, Variations in size, Close resemblance to other sediment constituents
  • Frequently confused with yeast cells, oil droplets, and air bubbles - Yeast cells usually exhibit budding, Oil droplets and air bubbles are highly refractile when the fine adjustment is focused up and down
  • To confirm RBC vs. Yeast cells: Add Acetic acid – dissolves RBC, not the Yeast cells, Stain with Eosin – RBC is red, Yeast is colorless
  • Studies have focused on the morphology of urinary RBCs as an aid in determining the site of renal bleeding
  • Dysmorphic RBC found in: Abnormal urine concentration, Glomerular bleeding, Non glomerular hematuria (in small amount), Strenuous exercise (in small amount)
  • RBC with RBC cast – Renal Disease, RBC without cast and protein – Damaged distal to the kidney
  • Presence of RBCs in the urine is associated with: Glomerular membrane damage or vascular injury within the genitourinary tract, UTI, Extra Renal Diseases, Toxic conditions, Physiologic conditions
  • The number of cells present is indicative of the extent of the damage or injury
  • Macroscopic Hematuria - cloudy with a red to brown color. Microscopic analysis may be reported in terms of greater than 100/hpf.
• White blood cells (WBCs) in urine

  • Larger than RBCs, measuring an average of about 12 mm in diameter
  • Normal: less 5 WBC/hpf
• Types of WBCs in urine

  • Neutrophil
  • Eosinophil
  • Mononuclear cells (Lymphocytes, Monocytes, Macrophage, Histiocytes)
• Neutrophils


  • The predominant WBC in urine
  • Exhibit Brownian movement (false motility) – because of the presence of granules with sparkling appearance
  • Stain: Sternheimer Malbin – for glitter cell
  • Associated with drug-induced interstitial nephritis, Small amount urinary tract infection (UTI) and renal transplant rejection
• Eosinophils


  • Not normally seen in the urine; therefore, the finding of more than 1% eosinophils is considered significant
  • Stain: Hansel Stain
• Mononuclear cells

  • Lymphocytes, Monocytes, Macrophage, Histiocytes
  • Found in small amount
  • Lymphocytes – mistaken as RBC because of its small size
  • Increased amount in early renal transplant rejection
• Pyuria


  • Increased urinary WBC
  • Due to: Infection in the genitourinary system, Bacterial Infections (Pyelonephritis, Cystitis, Prostatitis, Urethritis), Non Bacterial Infections (Glomerulonephritis, LE, Interstitial nephritis), Tumors
• Types of epithelial cells in urine

  • Squamous
  • Transitional / Urothelial / Caudate
  • Renal tubular
• Squamous epithelial cells

  • Derived from the linings of the vagina and female urethra and the lower portion of the male urethra
  • Increased amounts are more frequently seen in urine from female patients
  • Specimens collected using the midstream clean-catch technique contain less squamous cell contamination
  • A variation of the squamous epithelial cell is the clue cell, which does have pathologic significance - indicative of vaginal infection by Gardnerella vaginalis
• Transitional (urothelial) epithelial cells

  • Originate from the lining of the renal pelvis, calyces, ureters, bladder, and upper portion of male urethra
  • Increased numbers of transitional cells seen singly, in pairs, or in clumps (syncytia) are present following invasive urologic procedures such as catheterization (no clinical significance) or malignancy or viral infection
• Renal tubular epithelial (RTE) cells

  • Vary in size and shape depending on the area of the renal tubules from which they originate – cuboidal, columnar with eccentrically located nucleus
  • More than 2 RTE cells/hpf = Tubular injury
  • Oval fat bodies – lipid containing RTE cells, seen in lipiduria
  • RTE from proximal convoluted tubule (PCT): larger than other RTE cells, rectangular shape, referred as columnar or convoluted cells
  • RTE from the distal convoluted tubule (DCT): smaller than those from the PCT, round or oval
• Casts


  • Only elements found in urinary sediment that are unique to the kidney
  • Formed within the lumens of the DCT and collecting ducts, providing a microscopic view of conditions within the nephron
  • Detection of casts is performed using LPO. When glass coverslip method is used, LPO scanning should be performed along the edges of the coverslip.
  • Once detected, casts must be further identified as to composition using HPO. They are reported as the average number per 10 lpf.
• Formation of casts

  • Uromodulin – main component of casts matrix
  • Matrix – Tamm Horsfall Protein (glycoprotein secreted by RTE cells in DCT and collecting ducts)
  • HYALINE CAST (prototype of all casts) à CELLULAR CAST à dissolve à COARSE/FINE GRANULAR CAST àWAXY CAST (final degenerative form of all types of casts)
  • CYLINDURIA – presence of cast in urine
• Hyaline casts


  • Most frequently seen cast
  • Consists almost entirely of Tamm-Horsfall protein
  • Presence of 0-2 hyaline casts /lpf is considered normal
  • Non-pathological causes of increase: strenuous exercise, dehydration, heat exposure, and emotional stress
  • Pathological causes of increase: acute glomerulonephritis, pyelonephritis, chronic renal disease, and congestive heart failure.
• RBC casts


  • Indicates bleeding from an area within the GUT
  • Presence of RBC casts is much more specific, showing bleeding within the nephron
  • Primarily associated with damage to the glomerulus (glomerulonephritis) that allows passage of the cells through the glomerular membrane
  • RBC casts associated with glomerular damage are usually associated with proteinuria and dysmorphic erythrocytes
  • Observed in healthy individuals following participation in strenuous contact sports
  • Easily detected under LPO by their orange-red color
  • Reported as the number of RBC casts per lpf
• WBC casts


  • Signifies infection/inflammation within the nephron
  • Most frequently associated with pyelonephritis and a marker for distinguishing upper UTI from lower UTIs
  • Present in nonbacterial inflammations such as acute interstitial nephritis and may accompany RBC casts in glomerulonephritis
  • WBC casts are composed of neutrophils; they appear granular, and, unless disintegration has occurred, multi
• Hyaline Casts


  • Indicates bleeding from an area within the GUT
  • Presence of RBC casts is much more specific, showing bleeding within the nephron
  • Primarily associated with damage to the glomerulus (glomerulonephritis) that allows passage of the cells through the glomerular membrane
  • RBC casts associated with glomerular damage are usually associated with proteinuria and dysmorphic erythrocytes
  • Observed in healthy individuals following participation in strenuous contact sports
  • Easily detected under LPO by their orange-red color
  • Reported as the number of RBC casts per lpf
• RBC Casts


  • Signifies infection/inflammation within the nephron
  • Most frequently associated with pyelonephritis and a marker for distinguishing upper UTI from lower UTIs
  • Present in nonbacterial inflammations such as acute interstitial nephritis and may accompany RBC casts in glomerulonephritis
  • WBC casts are composed of neutrophils; they appear granular, and, unless disintegration has occurred, multilobed nuclei will be present
  • Bacteria are present in cases of pyelonephritis, but are not present with acute interstitial nephritis
  • WBC casts - with matrix as compared to WBC in clumps
• Epithelial Cell Casts


  • Represent the presence of advanced tubular destruction, producing urinary stasis along with disruption of the tubular linings
  • Associated with heavy metal and chemical or drug-induced toxicity, viral infections, and allograft rejection
  • also accompany WBC casts in cases of pyelonephritis
  • Formed in the DCT, the cells visible on the cast matrix are the smaller, round, and oval cells
  • Fragments of epithelial tissue may also be attached to the cast matrix
  • Bilirubin-stained RTE cells are seen in cases of hepatitis
• Fatty Casts


  • Seen in conjunction with oval fat bodies and free fat droplets in disorders causing lipiduria
  • 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
  • Cholesterol demonstrates characteristic Maltese cross formations under polarized light, and triglycerides and neutral fats stain orange with fat stains
  • Fats do not stain with Sternheimer-Malbin stains
• Granular Casts


  • Coarsely and finely granular casts are frequently seen in the urinary sediment
  • May be of pathologic or non-pathologic significance
  • Granules in non-pathologic conditions - from lysosomes excreted by RTE cells during normal metabolism
  • Increased cellular metabolism occurring during strenuous exercise accounts for transient increase of granular casts that accompany increased hyaline casts
  • Granules in disease states – represent disintegration of cellular casts and tubule cells or protein aggregates filtered by the glomerulus
  • When granular casts remain in the tubules for extended periods, the granules further disintegrate, and the cast matrix develops a waxy appearance
• Waxy Casts


  • Representative of extreme urine stasis, indicating chronic renal failure
  • Usually seen in conjunction with other types of casts associated with the condition that caused the renal failure
  • The brittle, highly refractive cast matrix from which these casts derive their name is believed to be caused by degeneration of the hyaline cast matrix and any cellular elements or granules contained in the matrix
  • They often appear fragmented with jagged ends and have notches in their sides
• Broad/ Renal Failure Casts


  • Represent extreme urine stasis
  • Indicates destruction (widening) of the tubular walls
  • Also, when flow of urine to the larger collecting ducts becomes severely compromised, casts form in this area and appear broad
  • All types of cast may occur in broad form
  • Most commonly – granular and waxy casts
• Bacterial Casts


  • Contain bacilli both within and bound to the protein matrix – seen in pyelonephritis
  • May be pure bacterial casts or mixed with WBCs
  • Identification of bacterial casts can be difficult, because packed casts packed with bacteria can resemble granular casts
  • 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