Overview and Objectives
Main Topic
Subtopic 1: Physical Examination of Urine
  Activities
1.1, 1.2, 1.3
  Subtopic 1 Summary
Subtopic 2: Chemical Testing
  Activities
2.1, 2.2, 2.3
  Subtopic 2 Summary
Subtopic 3: Microscopic Examination of Urine Sediment
  Activities
3.1, 3.2
  Subtopic 3 Summary
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CELLS OF THE URINARY SEDIMENT


Contents of This Section

(All links are to subsections within this file.)

Introduction
Leukocytes
  About Leukocytes
  Neutrophils
  Pus
  Eosinophils
  Lymphocytes
  Monocytes
  Macrophages
Erythrocytes
Epithelial Cells
  About Epithelial Cells
  Squamous Cells
  Renal Tubular Cells
    About Tubular Cells
    Proximal Renal Tubular Cells
    Collecting Duct Renal Tubular Cells
    Necrotic Renal Tubular Cells
    Renal Epithelial Fragments
    Renal Cells and Fragments from the Terminal Section
  Transitional Cells (Urothelials)
    About Transitional Epithelium
    Transitional Cells
    Transitional Eipthelial Fragments
Oval Fat Bodies
  Definition
  Origin of the Fat Droplets
  Clinical Value


Introduction

To identify urinary sediment cells is a difficult task. Some cells have characteristic features, therefore easily identifiable. On the other hand, some cells, even with sophisticated stains, remain a challenge. The diversity of cells that one can meet is notable. Cells found in urine can belong to the reticuloendothelial system (leucocyte, macrophage) or to the epithelial system. Urinary cells can originate from the kidney or from the lower urinary tract, from the superficial lining or from a deeper source. In an adult male, cells can also originate from the prostate and the urethra.

Urine is not a favorable media for maintaining cell structures. Most cells undergo rapid changes (degeneration, rupture, vacuolization, granulation of the cytoplasm) that profoundly affect the visual aspect of the cell. These changes are more pronounced if the cell comes from the higher urinary tract because aggression due to osmotic variation adds to the energy lack and other causes of morphological change. The best preserved cells are usually bladder cells, while a well preserved proximal renal tubular cell is exceptional. Some pathologies, like inflammation, metaplasia, neoplasia, are known to affect the cellular aspect.

All experienced urinary sediment microscopists have seen some cases of cells, so special, that identification was a best guess based on the sediments context. This situation, while being frustrating, should not be considered as a failure. Identification of all the figures seen in the urinary sediment is quite impossible; so,one must focus his effort on element of clinical value.

Leukocytes

About Leukocytes

  

Leukocytes or white blood cells designate all the hemoglobin free blood cells. These cells belong to the reticuloendothelial system. Based on their nuclear aspect, white blood cells can be divided into two categories: mononuclear cells and polynuclear cells. Lymphocytes and monocytes are the principal mononuclear cells and polynuclear are subdivided into neutrophilic, eosinophilic and basophilic cells. In the urinary sediment, the term leukocyte is usually interpreted as polynuclear, mostly neutrophils. The reason for this situation is that the neutrophils are, by far, the most abundant leukocytes in urine. In a normal specimen, up to 6 or 7 neutrophils / high-power field can be observed. High neutrophil counts are usually related to an inflammation process.

Mononuclear leukocytes are occasionally seen. High mononuclear counts are usually related to a high blood count pathology, infiltrating the urinary space.

Under bright field microscopy and without staining, identification of the different types of white blood cells is almost impossible. The term leukocytes is indicated for the routine microscopy. The subclasses of leukocytes can be identified by staining with the Wright or PAP stain. Efficiency of these staining procedures is highly dependent of the conservation state of the cells. Staining with these represents a workload, not justified for the routine specimen.


Neutrophils

A polynuclear neutrophil (neutro) is a plurilobular nucleus bearing cell with a slightly granulated cytoplasm. Neutros have two types of granulations (lysozome) named azurophilic and specific. Neutros lysozomes havediverse enzyme activities, some being specific like the peroxydase. Neutrophils also possess a heterogenic group of hydrolase gathered under the term esterase. One of these esterases, the Naphtyl AS-D Chloroacetate esterase, seems to be specific for the polynuclear lineage. This activity is shared with the mastocytes and some macrophages (some think that this activity is due to phagocyted neutros). Since mastocytes are not seen in urine, and that the macrophage size and aspect is quite different from leukocytes, this activity is used in a specific staining procedure for leukocytes.

Because of a pycnotic nucleus or of an unfavorable refractive index of the urine, the plurilobulated nucleus are not always obvious. Acidification of the sediment, with one drop of 2% acetic acid enhances the contrast of the preparation.

In the urinary sediment, there are two types of neutrophils. The first type is the usual named "Old" by Stamey. When numerous, these cells are related to inflammation.

The second type, named "Fresh" by Stamey, and "Pale cells" by Sternheimer, is bigger in size and resistant to some stains. If the urine density is lower than 1,019 this cell will demonstrate a brownian movement of its granules wich will give a glittering cytoplasm. These cells are then called glitter cells. For a time, these cells were thought to be specific to pyelonephritis. Since these were found in other conditions the accepted interpretation is relating them to an active inflammation process of the urinary tract.


Pus

One characteristic of the activated neutrophil is it's adherent capacity. This characteristic is essential for the migration of the cell. Because of this, neutrophils can easily aggregate. In some cases, it is important not to confuse cell aggregation and pus. Pus is formed of degenerated neutrophils (pyocytes) and cellular debris compacted into a mass where cell identity is lost. This discrimination is not commonly used with the urinary sediment, so that many aggregates are reported as pus. The term pus should be restrained to real pus.


Eosinophils

This cell is different from the other polynuclear cells because of the affinity of its granules for the acid stains like eosin. To be able to distinguish this cell, it is absolutely necessary to stain. In a comparative study, by Nolan and Kelleher, the former has shown that the Hansel stain was superior to the Wright stain for eosinophiluria. The presence of urinary eosinophils is a useful indicator of acute allergic interstitial nephritis. Eosinophiluria is also seen in conditions other than nephritis.

Causes of Eosinophiluria
Acute allergic interstitial nephritis
Rapidly progressive glomerulonephritis
Acute glomerulonephritis
IgA nephropathy, Henoch-Schönlein purpura
Prostatitis
Schistosomiasis
Chronic pyelonephritis
Graft rejection (acute phase)


Lymphocytes

Lymphocytes can occasionally be seen in a normal sediment. High counts have been reported in cases of acute allergic interstitial nephritis, rapidly evolutive glomerulonephritis, and graft rejection.

With bright field microscopy, the cell cannot be differentiated from the usual leukocytes. With the PAP stain, the cell is small, as a round nucleus with little cytoplasm.


Monocytes

In a study using monoclonal antibodies, a high number of urinary monocytes has been reported in cases of acute allergic interstitial nephritis and of rapidly evolutive glomerulonephritis. In necrosis, these were rare or absent.

Like the lymphocytes, monocytes cannot be identified with bright field microscopy. With the PAP stain, the cell is larger than a neutrophil and has a characteristic bean shaped nucleus.


Macrophages

The macrophage is, after the fibroblast, the most abundant cell in connective tissue. The activated macrophage is difficult to describe because it has a very variable aspect. This cell often presents itself with so many cytoplasmic inclusions that the cell's structures are completely masked. Inclusions are of several types, but the droplet is the most frequent. A classic easy to identify macrophage, is the giant cell that contains one or two smaller phagocyted cells in its cytoplasm. But this form is exceptional; the majority of the macrophages are of average size with a lot of inclusions.

The macrophages are frequent in acute inflammations. The macrophage loaded with fat droplets is frequently found in many body fluids. These are usually related to a chronic inflammation process. In urine, when these droplets form a maltese cross when viewed between crossed polarized filters, the macrophages are then called oval fat bodies.


Erythrocytes (Hematuria)

Hematuria is defined as a high urine red blood cell count sustained over three specimens taken on different days. In a normal urine, less than 1,5 million erythrocytes are found in a 24-hour specimen. This normal value represents a count of less than 5 RBC/hpf. Hematuria is normally associated with a urinary tract disease. Some cases of idiopathic hematuria have been reported in the literature. Red blood cells originating from an external source, like vaginal bleeding, is not a true hematuria.

Two types of hematuria can be seen in urine, lower urinary tract hematuria and dysmorphic hematuria.

Lower urinary tract hematuria—In the first type hematuria, the red cells have their typical shape and color. This hematuria is usually associated with a lower urinary tract disease.

Dysmorphic hematuria—The second type of hematuria is called dysmorphic or renal hematuria. This hematuria is characterized by: a great variation in the size of the cells (anisocytosis), many ghost cells, and by a high percentage of dysmorphocytosis (>20%). This hematuria is usualy related with a glomerular bleeding.

Dysmorphocytosis is characterized by bizarre shapes and projections of the cell membrane called blebs. Schramek has demonstrated that the dysmorphocytosis can be reproduced in vitro, by osmotic shocks in a hemolytic media. This situation compares well with the travel of a red blood cell from the glomerule to the bladder. In glomerulonephritis, the dysmorphic cells can represent up to 80% of the erythrocytes. A value of 14% was proposed by Pillsworth, as a cut-off value for the differentiation of renal from non-renal hematuria. At room temperature, the specimen is stable for up to 5 hours. It is not rare to see, in a sediment, what seems to be two populations of erythrocytes. This situation could correspond to a mixed hematuria.


Epithelial Cells

About Epithelial Cells

Epithelium is one of the four primary tissues (epithelium, connective, muscle, nervous) found in the human organism. This tissue is characterized by little or no intercellular matrix, the latter being responsible for its compacted aspect. Epithelium is subdivided into two types: the first is called a lining epithelium, the second a glandular epithelium. The lining epithelium has a sheet-like structure laid over a basement membrane. This membrane separates the epithelium from the underlaying connective tissue. The lining epithelium is further divided into two types: the first type is said simple, since it is formed of a single layer of cells; the second type is called stratified. This type has a multiple layers of cells, from 3 to 7 in the urinary tract.

The surface cells of a simple or stratified epithelium can be squamous, cuboidal or columnar. The squamous cells are flat, with an irregular border. The cuboidal cells have a polygonal aspect, with a width to height ratio of around one. Columnar cells have a tall aspect with the nucleus often at one end. The renal tubular cells found in the collector tubes are cuboidal while the proximal tubular cells are columnar.

The stratified epithelium can also be squamous, cuboidal, columnar or transitional. Transitional epithelium (urothelial epithelium) forms the lining of the urinary tract from the pelvis to the bladder. In the urothelial lining's stratification, the bottom cells are rather cylindrical while the intermediate cells have a variable aspect. The surface cells are rounded, with some having characteristic shapes such as kite cells, umbrella cells, and so on. This urothelium is seven layers thick in the bladder, 4 to 5 layers in the urethra, and 2 to 3 in the pelvis.

Exfoliation is the elimination of a cell or a group of cells (paired cells is frequent) through a secretion (urine). A normal exfoliation is the result of the renewal of the epithelium. An abundant exfoliation can be the result of a disease, increasing the cellular mortality rate or lowering the linkage of the cells with the basement membrane.

The aspect of cells found in urine, can be quite different from its original tissue-bonded aspect. Because of the lost tissue constraint, the cells adopt a more rounded shape. Because the dying cells cannot resist osmotic attack of urine, all kinds of degenerating transformations occur. These transformations will be more pronounced in cases where there is urinary stasis. The course is long for proximal tubular cells. Normally, well-preserved cells are from the lower urinary tract.

Positive identification of urinary cells is possible with the use of monoclonal antibodies. Segathosy has proposed the use of the URO series monoclonal antibodies to count the different renal cells.

For the routine urinalysis, the cell name used must be sufficiently large to include a great variety of cells (different shapes but of related origin). The names suggested are: renal tubular cells, urothelial or transitional cells, and squamous cells.


Squamous Cells

The squamous cells seen in the normal urine usually does not come from urinary tract. Only the last two third of the male urethra is lined with squamous cells. The urinary tract is lined with transitional, cubic, or cylindrical cells. A large number of squamous cells are mostly seen with woman's specimen and are probably the result of an accidental washing of the external genitalia.

Nevertheless, it is necessary to be vigilant with elderly patient's specimens. Patients in this group could have a squamous metaplasia of the bladder. The frequency of this anomaly is low, but is not negligible in the elderly population.

NB! Proliferative lesions of the lower urinary tract are sometimes of squamous cells type (squamous cell carcinoma).


Renal Tubular Cells (RTC)

About tubular cells—For the routine examination, the different renal tubular epithelial cells are regrouped under the term: renal cells. This term implies several types of cells. As it has been mentioned, it is impossible to know with confidence the origin of a tubular cell without using a sophisticated method, like staining specific markers with conjugated monoclonal antibodies.

Schumann has described some particular tubular cells based on usual microscopic characteristics. The cells described are: the convoluted proximal tubular cells (convulted RTC2), the collecting duct renal tubular cells (collecting duct RTC), the necrotic renal tubular cells (necrotic RTC) and finally the epithelial fragments.

In a wet preparation, the distinction between some tubular cells and the leukocytes is not always obvious. To be able to evaluate the number of tubular cells in a specimen with a marked leukocyturia, the naphtyl AS-D chloroacetate esterase is used.

Proximal renal tubular cellsThis cell is described by Schumann as a large size cell (20 to 60 m) with an abundant granular cytoplasm, and a blunted cell membrane. The nucleus is round and eccentric. In a wet preparation and under bright field microscopy, this cell can easily be confused with a small granular cast. The proximal tubular cells are known to have an elaborate brush border. This fragile structure is constructed from folds of the cellular membrane. In a toxic or ischemic state, the brush border is eliminated and is found in the urine in the form of granules. These granules can be free, but can also be embedded in a cast matrix (granular casts).

The exfoliated proximal tubular cell undergoes many changes, due to the travel in the osmotic changing environment, from the proximal tubule to the bladder . It is unlikely to find these cells intact.

Collecting duct renal tubular cellsThe collecting duct renal tubular cells are originally of a cubic shape; but once exfoliated, these adopt a rounded shape. These cells are slightly larger than leukocytes (10-14 mm) with a lightly granular cytoplasm. The nucleus is round and well defined (due to the thick nuclear membrane) and usually centric. The cytoplasm shows a perinuclear halo, when stained or under phase contrast.

The RTC casts are frequent with high exfoliation specimens. This fact can be of help for identification, since the similarities between the free cells and the cast cells should be evident.

These cells are the most frequently observed renal tubular cells. With the cytodiagnostic method, Schumann has reported a normal value of less than 20 cells per 10 fields at hpf. Values of over a 100 cells/10 x hpf is indicative of a renal parenchym disease. For the routine analysis, a value of less than 1 cell/hpf (a few cells) is a normal expected value.

Necrotic renal tubular cells—The necrotic renal tubular cell is an important element of Schumann cytodiagnostic method. This cell is described, with the PAP stain, as ghost cells, with the form and size of normal cells, and with poorly stained nuclei. The cytoplasm of these cells is highly granular.

Due to special conditions, the collecting duct renal tubular cells are unable to resist to the low osmolality of the urine. As water and salts get in the cells, the cytoplasm starts to disorganize. The cell swells, the cytoplasm takes a granular aspect and the nucleus shrinks and becomes pycnotic. At the end of the process, the cells are completely granular. The granules are quite similar to those found in granular casts (it is frequent to observe identical granules in cells and in casts).

In an acute tubular necrosis of ischemic origin, granules in renal tubular cells and in granular casts (dirty brown cast) bare the same dark red-brown pigmentation. The pigments are related to hemoglobin and methemoglobin.

Renal epithelial fragmentsFor Schumann, renal epithelial fragments have a high clinical value. The renal epithelial fragments are quite different from cell clusters. The epithelial fragment is a piece of tissue. It is not normal to have tissue fragments in urine.

The fragments are described as a structure of at least three tubular cells with an intercellular cohesion. The distinction between fragments and renal tubular cell casts is not always easy. This identification can be made on the basis of the presence, or not of a matrix. The renal epithelial fragments also have to be distinguished from the urothelial fragments. The identification is made upon cellular criteria.

Schumann has proposed criteria, characteristics to renal epithelial fragments.

Characteristics of Renal Epithelial Fragments
Found in Urine Sediment
Configuration characteristics of a renal epithelium
Encasement of casts
Cylindrical (sleeve-like) arrangement
Sheets with "honeycomb"arrangement
Spindle or elongated cells

Additional characteristics of a renal epithelium
Encasement of crystals
Intracytoplasmic lipid
Intracytoplasmic cast material

In histological cut sections, fragments are associated to ruptures of the basement membrane at the collecting duct level. The presence of epithelial fragments must always be considered as abnormal. Schumann has reported characteristic epithelial fragments in: acute tubular necrosis (5 cases), renal allograft reject (20 cases), papillary necrosis (2 cases) and renal infarction (12 cases). (original in Acta cytol 25:147-152, 1981)

Renal cells and fragments from the terminal sectionIn some cases, one can find, in a specimen, isolated cells fragments that originate from the terminal part of the large tubes (tubes of Bellini). These large tubes are lined with a cylindrical epithelia, and form the urinary canal of the papilla. Considering the proximity of the region, and due to the fact that at this level the urine is no longer modified, isolated cells and fragments can have a well preserved morphology. These cells have an average size of (2030 mm) and are round or clearly cylindrical; the nuclei are eccentric in the cylindrical cells. The presence of some rare cells of this type can be considered as a normal renewal.

These cells can represent an identification difficulty.

While being renal cells, in the routine microscopy, these are reported as an urothelial cells. These cells are often found in various urological problems like in obstruction, urolithiasis and others. In the presence of similar cells or fragments, without cast, this type of urothelial cells should be considered.


Transitional Cells (Urothelials)

About transitional epithelium—From the pelvis to the beginning of the urethra, the walls of the urinary tract is lined with a stratified transitional epithelia. In the bladder, the epithelia is formed of around seven layers of cells. The deepest cells are cylindrical while the intermediate layer cells are variable. The surface cells are typically transitional. These cells are also named urothelial cells.

Transitional cellsTransitional cells are normal elements of the urinary sediment. The cell's shape changes slightly according to the section of the lower urinary tract. The typical bladder cells are round with a round centered nucleus. In the cytological nomenclature, one speaks of balloon cells, umbrella cells, kite cells, and so on. All these designations correspond to transitional cells originating from different levels: bladder, uretere, pelvis.

The presence of transitional cells is more frequent in the elderly population. Occasionally, because of morphological changes, the identification of transitional cells is difficult. Although these changes are not always associated to a pathology, atypical cells must be watched for in this prevalent population. Holmquist has demonstrated that a simple urinary routine sediment can play an important role in the early detection of TCC (transitional cell cancer).

Transitional epithelial fragmentsContrarily to the isolated cells, the presence of transitional epithelial fragments is almost always associated to an abnormal situation. In the majority of cases, the cells have a normal aspect and form a thin sheet where it is easy to delimit each cell. These sheets are said to have a brick-wall-like aspect. The presence of this type of fragments can be the result of a urinary catheter or of another condition that provokes an erosion of the surface of the bladder's epithelium.

In some sustained irritating conditions, transitional cells can become reactive. In these conditions, the cells and the nucleus increase in size. The size of cells can be variable, but the ratio nucleus/cytoplasm is well preserved. This situation is quite different from the atypical fragments as seen in a TCC of high grade. The finding of atypical urothelial fragments is an important element in the detection of an otherwise silent TCC.

Characteristics of the Atypical
Urothelial Fragments (Holmquist)
Cells
Variable size (anisocytosis)
Anarchic cluster
Nuclear crowding
Necrosis, vacuolation
Mitosis, frequent bi and multinucleation
Leukocytes margination (inflammation)

Nucleus
Irregular nucleus, variable size et shape
Cariolysis
Clumped chromatin
Variable nuclear/cytoplasm ratio
Thick nuclear membrane

Nucleolus
Multiple nucleolus
Variable shape and size
Variable nucleolus/nucleus ratio (macronucleolus)


Oval Fat Bodies

Definition

Oval fat bodies are cells with birefringent fat droplets within their cytoplasm. Under low power magnification, oval fat bodies are often seen as large brown spots (sometimes almost black). This coloration is due to the yellowish brown pigmented fat making the droplets. These cells are usually seen in a context of heavy proteinuria.

The lipiduria manifests itself as lipid droplets:

  • Free as the birefringent fat droplets

  • Intracellular in the oval fat bodies

  • Imbedded within a cast matrix in the fatty cast


Origin of the Fat Droplets

Fat droplets can originate from a vacuolar fatty degeneration of intracytoplasmic membranes. This fatty degeneration phenomenon is frequent. Thus, cells kept for several days in a urine develop all kinds of vacuoles. In some cases, droplets are quite similar to those found within the oval fat bodies, except for the birefringence. The intracytoplasmic droplets can also originate from phagocyted material followed by an intensive lysozome digestion.

True oval fat bodies show a typical "maltese cross" interference pattern when viewed under polarized light. This interference is a crystalline structure property, and is due to the presence of esterified cholesterol in a liquid crystal state. Naturally, cholesterol-free droplets are not birefringent. It is possible to stain the fat droplets with a suitable fat stain like Sudan, Fat Red 7B, and others. To our view, staining is of little use, since the birefringent criteria is easy to observe and that staining has a tendency to stain degenerated cells that are erroneously interpreted as oval fat bodies.

Some fat droplets lose their birefringent characteristics. It is sometimes possible to correct the crystalline disorganization by gently heating the slide, followed by a rapid cooling.


Clinical Value

The nature of oval fat bodies is debated. For Schumann, the cell is an oval renal proximal tubular cell with a fat droplets filled cytoplasm. For Stamey, oval fat bodies are in fact macrophages also known as foam cells. The latter has proposed the use of the term oval fat macrophages. This apparent controversy is probably due to the fact that both possibilities exist.

Oval fat bodies, in a high proteinuria context, are associated with the nephrotic syndrome (nephrosis). The link between the nephrotic syndrome and lipiduria is not known. Lipiduria seems to be related to the proteinuria, and not to plasmatic lipids level. Some have suggested that the lipiduria could be a consequence of a specific apolipoprotein accessing, like the albumin, to the urinary space through the glomerule. Filtrated free fatty acid adsorbed to the urinary albumin could play a role in the intracytoplasmic accumulation of fat by the renal proximal tubular cell. (Oval fat bodies are often seen with the droplets filling one side of the cell)

Oval fat bodies are not specific to the nephrotic syndrome. These cells are sometime seen in specimens with a normal proteinuria. This situation can be explained by the presence of fatty macrophages often seen in chronic inflammation sites.

Stamey has reported the presence of oval fat macrophages in seminal fluid with patients having prostatitis.

Foam cells can be seen in many human fluids: bile, bronchial, and others.