Urine Analysis: Part 6 – Urine Microscopic Examination, and Interpretations

Sample

1. Freshly voided urine is the best sample.
2. If delayed, then refrigerate the urine.
3. The best volume to the centrifuge is 10 to 12 mL.

Procedure

1. Centrifuge the Urine, and clear supernatant is removed and leaves the uniform volume of sediment, usually 1 mL.
2. The recommended time for centrifugation is 5 minutes.
3. The standard centrifugation speed recommended is 400 g.

Normal sediment

• Normally there is no sediment or very little.
• Following table shows the normal contents of the sediments:

Normal contents of the urine	Findings in the normal sediments
White blood cells	Variable in males and females. Maybe 0 to 5 /HPF
Red blood cells	Variable and maybe 0 to 2 /HPF
Epithelial cells	Variable in male and females but more in females  1 to 5 /HPF
Bacteria	Negative
Hyaline cast	0 to 2 /HPF
Abnormal crystals	Not found

Microscopic contents

Red blood cells

1. The presence of RBC in the urine is called Hematuria.
2. RBCs are seen as biconcave, non-nucleated disc measuring 7 µm in diameter.
   

   Urine normal RBCs x 400

3. The presence of RBC is an indicator of renal disease.
4. These are reported in a routine as the average number seen in 10 HPF (x400).
5. RBCs swell and lyse rapidly in the diluted urine, releasing their hemoglobin and leaving only the cell membrane; these empty cells are called ghost cells. These can only be seen in the reduced light. Otherwise, these are missed.
6. RBCs are confused with:
   1. Yeast.
   2. Oil droplets.
   3. Air bubbles.
   4. How to differentiate RBCs:
      1. Oil droplets and air bubbles are highly refractile.
      2. These are seen on a different level than the other sediments.
      3. RBCs are smaller than the WBCs.
      4. Add acetic acid to the sediment, lyse the RBCs, and leave behind yeast, oil droplets, and air bubbles.
      5. Or do the supravital staining.
   5. Dysmorphic RBCs:
      1. These are the RBCs that vary in size, have cellular protrusions, or maybe fragmented.
      2. Wright’s stain can also help to differentiate these RBCs as hypochromic and prominent cellular blebs and protrusion.
         

         Urine Dysmorphic RBCs

      3. These RBCs are associated with glomerular bleeding.
      4. These are also seen after the strenuous exercise indicating glomerular RBCs.
   6. Macroscopic hematuria shows cloudy urine with red to brown color.
      1. This is seen in:
         1. Trauma.
         2. Acute infection.
         3. Inflammation.
         4. Coagulation disorders.
   7. Microscopic hematuria is seen in:
      1. Glomerular diseases.
      2. Malignancy of the urinary tract.
      3. Renal calculi.
   8. The possibility of menstrual contamination should be considered in females.
7. Hematuria may be caused by:
   1. Renal disease
   2. Infections
   3. Renal stones
   4. Renal tumors
   5. Bleeding disorder.
   6. Anticoagulant therapy.
      

      Urine hematuria

White Blood Cells (WBC), Neutrophils

1. WBCs are bigger than the RBCs measuring an average of 12 µm in diameter. In the urine sediment to recognize the WBCs is easy as a comparison to RBCs.
   1. Neutrophils contain multilobate nuclei and granules in the cytoplasm.
   2. WBCs can be identified in the high power and reported as the average number seen in  10 HPF.
      

      Urine WBCs in the sediment

2. Neutrophils lyse rapidly in the dilute alkaline urine and begin to lose nuclear details.
3. An increased number of WBCs in the urine is called Pyuria.
4. Pyuria may be caused by :
   1. Bacterial infection of the urinary tract
   2. Acute pyelonephritis shows casts along with WBCs, protein, and bacteria.
   3. Lower urinary tract infection also shows WBCs but a small amount of protein.

5. Increased neutrophils are seen in:
   1. 1. These are seen in all renal inflammatory diseases.
      2. Glomerulonephritis.
      3. Cystitis and urethritis.
      4. Chronic pyelonephritis.
      5. Prostatitis.
      6. Pyogenic infection.
      7. Acute appendicitis.
      8. Acute pancreatitis.
      9. Tuberculosis.
      10. Urinary bladder tumors.
   2. Nonbacterial increased WBCs are seen in:
      1. SLE.
      2. Interstitial nephritis.
      3. Glomerulonephritis.
      4. Tumors.
   3. Normal:
      1. Normally few Neutrophils are seen.
      2. Usually 4 to 5 /HPF.
      3. >30 cells /HPF is considered an infection.
      4. WBCs clumps are a sign of infection and must be reported.

Eosinophils

1. Eosinophils are normally not seen in the urine sediment. Few may be seen along with neutrophils.
2. On wet preparation are difficult to recognize except for the presence of 2 or 3 lobes.
3. These are detected by the supravital stain.
4. Can use wright’s stain or special stain for eosinophils like Methylene-blue and eosin-Y in methanol.
5. Eosinophils may be seen in :
   1. Drug-induced interstitial nephritis (hypersensitivity), e.g, penicillin and its derivatives.
   2. Acute genitourinary diseases like tubulointerstitial disease.
   3. These are also seen in renal transplantation rejection.
   4. Procedure to see eosinophils:
      1. Centrifuge the urine; concentrated sediments is needed to stain it.
      2. Or prepare the smear with a cytospin centrifuge.
      3. The best stains are Hansel’s stain, but you can use Wright’s stain.
      4. >1% eosinophils are a significant number.
         

         Urine eosinophils with prominent granules

 Lymphocytes 

1. Normal urine may contain few small lymphocytes, and these are not recognized.
2. Wright’s stain can recognize them.
3. An increased number of lymphocytes is seen in:
   1. The first few weeks of renal transplantation are a sign of rejection.
   2. Many lymphocytes and plasma cells are the sign of renal transplantation rejection, acute renal allograft rejection.
      

      Urine lymphocytes and plasma cells in renal rejection

Monocytes, Histiocytes, and Macrophages

1. These cells are difficult to recognize in the wet preparation. Normally there are monocytes, macrophagic cells, and histiocytes.
2. Lymphocytes are smaller than WBCs and may resemble RBCs.
3. Monocytes, histiocytes, and macrophages are large cells and are vacuolated with inclusions.
4. To recognize these cells needs special stain (Wright’s and Papanicolaou stain).

Epithelial cells

1. Mainly these cells are shed from the urethra and urinary bladder.
2. Few renal epithelial cells are seen normally in the sediment.
3. Epithelial cells are the following types :
   1. Squamous epithelial cells
   2. Transitional epithelial cells
   3. Renal epithelial cells.

Squamous epithelial cells

1. These cells line the female urethra, trigone, and male urethra.
2. These cells also line the vagina, and in the female, these are the source of contamination.
3. Origin: These squamous epithelial cells take origin from the vagina, female urethra, and lower portion of the male urethra.
   1. These represent normal sloughing and have no pathologic significance.
   2. Midstream clean catch will have less number of these cells.
4. These cells have little significance but helpful to focus on the microscope.
5. Morphology:
   1. These cells are large, containing abundant irregular cytoplasm and a prominent nucleus, which may be around the size of RBC.
   2. The squamous epithelial cells are reported as:
      1. Rare
      2. Few.
      3. Moderate.
      4. Many.
         

         Urine squamous epithelial cells

6. Clue cells are a special type of squamous cell and take origin from the vagina. These are covered by coccobacilli  (Gardnerella vaginalis) and indicate bacterial vaginitis.
   1. A small number of the clue cells may be present in the urine sediment.
      

      Urine Clue cell

Transitional Epithelial cells

1. These are stratified epithelial lining of the urinary tract system starting from the kidney’s pelvis to the base of the urinary bladder in the female and proximal part of the male urethra.
2. Morphology:
   1. These are smaller than the squamous epithelial cells.
   2. These have various shapes like spherical, polyhedral, and caudate form.
   3. All forms have a central distinct nucleus.
   4. These cells take origin from the lining of the renal pelvis, calyces, ureter, urinary bladder, and upper part of the urethra.
3. Reporting of the Transitional cells:
   1. Normal urine may contain few transitional cells.
   2. Rare.
   3. Few.
   4. Moderate.
   5. Many.
4. Renal cells exfoliate more in :
   1. Inflammatory process and infection.
   2. Malignancies.
   3. After radiation and these become large in size and have multiple nuclei.
   4. After catheterization or washing of urinary bladder.
5. These cells need special stains.
6. An increase in the number of transitional epithelial cells with abnormalities like vacuoles in the cytoplasm and irregular nuclei may suggest malignancy or viral infection; such cases need cytological examination.

Renal  Tubular Epithelial cells

1. These cells line the proximal to distal convoluted tubules, small and large collecting ducts.
   1. Proximal convoluted tubular cells are rectangular, coarsely granular cytoplasm.
   2. Collecting duct cells are cuboidal to columnar, finely granular,  and not larger than WBcs.
2. Normally a few cells may be found in the urine sediment.
3. An increased number is significant, indicating renal disease.
4. The function of cells:
   1. The main function of these cells is reabsorption.
   2. In hemoglobinuria, these cells contain yellow-brown hemosiderin pigments.
   3. In the case of bilirubin, the color of these cells is deep yellow.
5. The critical number is more than 15 cells / 10 HPF. When >2 cells are found, then refer to cytologic examination.
6. Significance:
   1. When present in groups, it indicates severe renal damage.
   2. Acute tubular necrosis.
      1. It is seen in heavy metal poisoning.
      2. Drug-induced toxicity.
      3. Hemoglobin and myoglobin toxicity.
      4. Viral infections like HBV.
      5. Pyelonephritis.
   3. Viral infections.
   4. Allergic reactions.
   5. Acute allogenic Rejection phenomenon.
   6. Malignant infiltration.
7. These are not easily identified in the centrifuged material. It needs special stains.

Fat Globules and Fat Bodies 

1. Tubular cells absorb the fat, which is present in the glomerular filtrate.
   1. These cells containing the fats are refractile, and the nucleus difficult to see. These fat-containing renal tubular cells are called oval fat bodies.
   2. These oval fat bodies can be seen by the special stain Sudan black III or oil red O fat stain. These are seen in the polarized microscope.
   3. These are seen along with fat globules or droplets.
      1. These fat droplets consist of triglycerides, neutral fat, and cholesterol.
      2. Fat-stain will stain triglycerides and neutral fat and give orange-red color.
         

         Urine Oval fat bodies cell

2. The presence of fat in the urine is called lipiduria. This is usually seen in the case of damage to glomeruli in nephrotic syndrome.
3. Special stain Sudan 111 or oil red O is needed.
4. Lipiduria is seen in :
   1. Advanced diabetes mellitus.
   2. Severe tubular necrosis.
   3. Nephrotic syndrome.
   4. Lupus disease.
   5. In mercury poisoning.
   6. Ethylene glycol poisoning.
   7. In trauma where the bone marrow fats leak from the long bone.
   8. Lipid storage disease where there are large fat-laden histiocytes. These are differentiated from the oval fat bodies by their large size.

Spermatozoa

1. The spermatozoa are oval, slightly tapered heads and long flagellate-like tails.
2. Urine is toxic to sperms, so they are nonmotile in the urine.
   

   Spermatozoa structure

3. There should be a lab protocol when to report the spermatozoa in the urine, particularly female urine. There may be legal consequences.
4. These may be seen both in males and females following intercourse (coitus).
5. The presence of spermatozoa in the urine is significant in infertility cases and sexual abuse.
6. In male may be seen after a nocturnal emission or ejaculation.
7. Also seen in the diseases of the genital tract.
8. Their presence in the urine is of significance in the case of retrograde ejaculation, where the sperms go into the urinary bladder instead of the urethra.

Bacteria 

1. Normally a few bacteria are present in the urine unless you collect the urine in strict sterile conditions.
   1. There are bacteria due to contamination from the vaginal secretions, urethra, genitalia, or the container.
   2. These bacteria multiply if the urine is left at room temperature and give nitrite test positive and may result in pH 8, which indicates an unacceptable specimen.
2. If urine is kept at room temperature in the lab, then bacteria can multiply.
3. When bacteria are seen in the urine, they should be reported per high power as:
   1. Few.
   2. Moderate.
   3. Many.
4. When reporting bacteria, then you should see the WBCs in the urine.

Type of bacteria causing infection	% of urinary tract infections
E.Coli	72%
Klebsiella	16%
Proteus	5%
Staphylococcus	5%
Pseudomonas	1%
Streptococcus faecalis	1%

Yeast

1. The most common yeast is candida in the urine.
2. This may be vaginal contamination due to vaginal infection.
3.  These are yeast primarily candida seen in:
   1. The patients with diabetes. Because sugar is used as a source of energy for the growth of yeast.
   2. These are also seen in immunocompromised patients.
   3. Female with vaginal moniliasis.
   4. The infection is suspected when the presence of WBCs accompanies these.
4. Structure:
   1. These may be seen as a small, refractile oval structure that may be laking buds.
   2. These are strongly gram-positive.
   3. Wright’s stain differentiates yeast from the RBCs.
      

      Urine yeast showing mycelia

Trichomonas (parasites)

1. This protozoan is the common cause of vaginal infection called Trichomonas vaginitis caused by Trichomonas vaginalis.
2. Trichomonas is a sexually transmitted disease and causes inflammation in the female.
3. While the infection of the male urethra and prostate is asymptomatic.
4. Structure:
   1. Trichomonas is a pear-shaped flagellate with an undulating membrane.
   2. It is easily identified in a wet preparation and the urine sediment by its rapid darting movement under the microscope.
   3. When these are not moving, it is challenging to identify because it may resemble WBCs or transitional cells.
   4. The trichomonas is reported /HPF:
      1. Rare.
      2. Few.
      3. Moderate.
      4. Many.
5. These protozoa reside in the prostate and vagina.
6. Wet preparation from the vagina or urethra by direct swab preparation can diagnose it.

Casts

1. Formation of the cast:
   1. These casts are precipitated proteins in the tubules. It forms in the lumen of distal convoluted tubules and collecting ducts.
   2. Their shape is representative of the tubular lumen with parallel sides and mostly rounded ends.
   3. The shape and content of the cast depend upon:
      1. The material present in the filtrate at the time of formation.
      2. Length of the time it remains in the tubules.
      3. Any other components present in the filtrate at the time of formation, like bacteria, granules, pigments, and crystals, may become embedded in or attached to the cast matrix.
   4. The casts dissolve easily in the dilute alkaline urine.
2. Composition of the cast:
   1. The major component is the Tamm-Horsfall protein. This protein is produced from the renal tubular epithelium and upper collecting ducts.
   2. Other proteins are albumin and immunoglobulins.
   3. These proteins gels under the following conditions:
      1. Urine flow stasis.
      2. The acidity of the urine.
      3. In the presence of Na⁺and Ca⁺⁺.
      4. Protein glycosylation is also important.
      5. The width of the cast depends on where the cast is formed.
3. These casts lead to blockage of the urinary outflow in the tubules.
4. Reporting:
   1. These are screened under low power and usually seen at the edges of the coverslip.
   2. It also needs low light because the cast refractive index is low.
   3. After identifying under the low light, go to high-power magnification and report cast number / 10 HPF.
5. There are the following types of casts seen in the urine :
   1. Hyaline cast
   2. Cellular cast
   3. Waxy cast
   4. Granular cast
   5. Pigmented cast (maybe hemoglobin, myoglobin, bilirubin, and drugs casts)
   6. Fatty cast
   7. Inclusion cast

1. Hyaline casts

1. Hyaline casts are the most commons casts.
2. This cast mainly consists of Tamm-Horsfall protein. These are colorless in unstained sediments, and the refractive index is like urine.
   1. These can be easily seen in phase microscopy.
   2. Or stain with Sternheimer-Malbin, which produces the pink color of these casts.
3. These are of the least importance.
4. Normal:
   1. There may be <2 / PHF seen in the normal person (0 to 2 /HPF are normal).
5. These may be seen without any significant proteinuria.
6. These are seen in acidic urine and absent in the alkaline urine.
7. Normally Hyaline casts are seen in :
   1. After severe exercise.
   2. Dehydration.
   3. Emotional stress.
   4. Heat exposure.
8. Pathologically hyaline casts are seen in:
   1. Acute glomerulonephritis.
   2. Chronic renal disease.
   3. Pyelonephritis.
   4. Congestive heart failure.
   5. > 20 / PHF is seen in moderate or severe renal disease.
      

      Hyaline cast

2. Granular casts

1. Structure:
2. Granulat cast appears homogenous, coarsely granular, colorless, and very dense.
   1. These casts consist of a breakdown of cells, plasma protein aggregates (fibrinogen, immune complexes, and globulin in the Tamm-Horsfall matrix).
3. Normal:
   1. Few may be seen in a normal person or after vigorous exercise.
   2. Occasional 0 to 2 /HPF.
4. Granular casts are seen in:
   1. Acute tubular necrosis.
   2. Pyelonephritis.
   3. Advanced glomerulonephritis.
   4. Malignant nephrosclerosis.
   5. The increased number indicates severe renal disease.
5. Presence of cells in the cast:
   1. Red cells in the cast indicate a glomerular injury.
   2. The presence of epithelial cells indicates renal tubular injury.
   3. The presence of WBC indicates infection or interstitial injury.
      

      Granular cast

3. Waxy casts

1. These are not seen normally in the urine.
2. Structure and formation
   1. Waxy casts formed from the degeneration of the granular cast.
   2. These casts are larger than the other casts and are waxy and granular in appearance.
   3. These are broader than other casts and called broadcast or broad renal failure casts.
   4. These are like the hyaline cast, but these are more refractile and sharper outlines.
   5. These are also called renal failure casts.
3. Their presence indicates severe pathology.
4. Waxy casts are seen in:
   1. Chronic renal disease.
   2. Severe renal failure.
   3. Renal amyloidosis.
   4. These are rare in acute renal disease.
   5. Malignant hypertension.
   6. Renal amyloidosis.
   7. Diabetic nephropathy.
   8. Renal allograft rejection.
      

      Urine Waxy casts

4. Fatty casts

1. Formation:
   1. Fat casts are formed from the fat droplets’ attachment and degenerating oval fat bodies embedded in the protein matrix.
   2. Fatty casts are highly refractile and contain yellow-brown fat droplets or oval fat bodies.
2. These also indicate serious renal disease.
3. These are seen in:
   1. Nephrotic syndrome.
   2. Chronic glomerulonephritis.
   3. Severe diabetes mellitus.
   4. Toxic nephrosis due to mercury poisoning and ethylene glycol.
   5. SLE.
   6. Toxic renal poisoning.

5. Red Blood Cell cast

1. Fresh RBCs cast retain their brown pigments and consists of RBCs in tubular cast matrix.
2. These casts indicate intraparenchymal bleeding.
3. These are the hallmark of glomerulonephritis.
   

   Urine RBC casts

6. White Blood Cell cast

1. There are WBCs in a protein matrix.
   1. These take origin from kidney tubules.
      

      Urine WBC casts

2. WBC casts are seen in:
   1. These are characteristics of pyelonephritis.
   2. Interstitial nephritis.
   3. Tubulointerstitial infection.
3. These are used full to distinguish from the lower urinary tract infection.
4. Normal:
   1. Not seen normally.

7. Tubular Cell Cast

1. There are few tubular cells in a hyaline matrix of dense aggregates of shed tubular cells.
2. These are seen in concentrated urine.
3. The tubular cell sloughing occurs in acute tubular necrosis.

Table showing the type of cast and its association with various diseases:

Type of cast	Clinical disease association
Hyaline	After strenuous exercise
RBC	Glomerular disease
WBC	Tubular diseases
Granular	Tubulointerstitial diseases
Waxy	Severe chronic renal disease
Fatty	Diabetic nephropathy

Casts in the urine and their etiology:

Nature of the cast	Physical appearance	Possible reason
Hyaline cast	Structureless, colorless, and clear	Congestive heart failure Diabetic nephropathy Glomerulonephritis Chronic pyelonephritis Chronic renal failure After strenuous exercise
WBC cast	Cast shows WBCs	Lupus nephritis Acute pyelonephritis Interstitial nephritis
RBCs cast	RBCs are seen and maybe pigmented.	Subacute bacterial endocarditis Goodpasture’s syndrome Renal infarct Acute glomerulonephritis Lupus nephritis
Granular cast	Coarse or fine granules with or without cells fragments	Pyelonephritis Nephrotic syndrome Glomerulonephritis Transplant rejection Lead toxicity
Waxy cast	Strrutureless, broad, refractile, and sharply outlined.	Transplant rejection Renal failure Tubular atrophy severe
Epithelial cells cast	Difficult to differentiate from the WBCs cast	Transplant rejection Tubular necrosis Heavy metal toxicity Salicylates toxicity CMV infection

Mucous threads

1. Mucus is a protein produced by the glands and the lower genitourinary tract’s epithelial cells, and the renal tubular cells.
   1. The main components of the mucus are the Tamm Horsfall protein.
2. These may be mistaken for hyaline casts.
3. These are long threads, undefined edges, and pointed or irregular ends.
   1. Mucus has a low refractive index.
   2. Mucus threads are reported as:
      1. Rare.
      2. Few.
      3. Moderate.
      4. Many or abundant.
4. It has no clinical significance in males as well as females.
   

   Summary of urine analysis

   Please see more details in urine analysis part 3.

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