MODULE 4

MICROSCOPIC EXAMINATION OF
URINE
Part of the complete urine analysis scheme is the evaluation of the physical, chemical and microscopic
characteristics of urine. From the previous module, concepts and principles on the physical and chemical
examination of urine were discussed and elaborated. This module is intended to describe how microscopic
elements are examined, identified and quantified. Different microscopic techniques, along with methods of
sediment preparation, will also be explained.
At the end of this module, the students will be able to:
• describe the microscopic composition of urine
• understand the microscopic examination of urine; and
• correlate abnormal microscopic test results with pathologic conditions.

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 UNIT 1: SEDIMENT PREPARATION AND
EXAMINATION
At the end of this unit, the student should be able to:
• discuss the importance of standardizing the microscopic examination of urine.
• describe the microscopic and staining techniques used to enhance visualization of the formed
elements in urinary sediment.

ENGAGE: RECALL
DEFINITION OF TERMS

Cytocentrifugation. This is a specialized centrifuge procedure used to produce a monolayer of the cellular
constituents in various body fluids, such as urine, on a microscopic slide.

Rous Test/Prussian blue staining. A chemical reaction used to identify the presence of iron in body fluids.

Uromodulin/Tamm-Horsfall protein. A glycoprotein that is secreted by the renal tubular cells of the thick
ascending loop of Henle and distal convoluted tubules.

Urinary sediment. This is the cellular pellet remaining after urine centrifugation. A normal urinary sediment
should be free of crystals, contain less cells and very low concentration of proteins.

Birefringent/Double Refractile. This refers to the ability of a substance to refract light in two directions.

Brightfield microscopy. This is a type of microscopy that produces a magnified image that appears dark against
a bright or white background.

Polarized microscopy. Type of microscopy that illuminates the specimen with polarized light (light waves in
which the vibrations occur in a single plane). This is used to identify and classify birefringent substances.

Field of view. The circular field observed through a microscope.

Kohler illumination. Type of microscopic illumination in which a lamp condenser located above the light
source focuses the image of the light source onto the front focal plane of the condenser.

Parfocal. Term describing objective lenses that remain in focus when the user switches from one objective to
another of a different magnification.

Addis Count. A traditional method developed by Addis in 1926 to standardize the quantitation of formed
elements in the urine. It uses a hemocytometer to count the number of RBC, WBC, casts and epithelial cells
present in a 12-hour specimen.

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 EXPLORE: UNLOCKING CONCEPTS
The microscopic examination is a vital part of the routine urinalysis. Its purpose is to detect and to
identify insoluble materials present in the urine for the evaluation of renal and urinary tract disorders as well as
other systemic diseases. The blood, kidney, lower genitourinary tract and external contamination all contribute
formed elements to the urine. These include RBC, WBC, epithelial cells, casts, bacteria, yeasts, parasites,
mucus, spermatozoa, crystals and artifacts. Because some of these components are of no clinical significance
and others are considered normal unless they are present in increased amounts, examination of the urinary
sediment must include both identification and quantitation of the elements present.
To ensure the accuracy and precision of the urine microscopic examination, protocols on the
standardization of sediment preparation should be implemented. Hence, the laboratory should adopt a regulated
speed, time, and amount for the centrifugation of the urine specimens.
1. Specimen Preparation
The ideal specimen for routine urinalysis is a first morning specimen (midstream clean catch).
Specimens should be examined while fresh or adequately preserved. Formed elements such as RBC,
WBC, and hyaline casts disintegrate rapidly in dilute, alkaline urine. Refrigeration may cause
precipitation of amorphous urates and phosphates and other non-pathologic crystals that can obscure
other elements in the urine sediment. Warming the specimen to 37 degrees Celsius prior to
centrifugation may dissolve some of these crystals.

2. Specimen Volume
The volume of urine recommended for routine urinalysis is 30-50 ml. However, for sediment
preparation, 12 ml or volumes ranging from 10 to 15 mL is used. This volume from a well-mixed
specimen will contain a representative sampling of urine formed elements. If obtaining a 12 ml
specimen is not possible, as for pediatric patients, the volume of urine can be reduced to 6 ml. In some
laboratories, when less than 3 mL of urine is available for testing, the urine is examined
microscopically, without concentration of the sediment.

If the volume is below 12 ml, this should be noted on the report form to allow necessary corrections.
For example, if 6 ml of urine is centrifuge, the results are multiplied by 2.

3. Centrifugation
A well-mixed urine is poured into a centrifuge tube, covered and centrifuged at 400 to 450 g for 5
minutes. This centrifugation speed allows for optimal sediment concentration without damaging fragile
formed elements such as cellular casts.

It is very important that the centrifuge brake is not used because this will cause the sediment to
resuspend, resulting in erroneously decreased numbers of formed elements in the concentrated
sediment. To prevent biohazard aerosols, all specimens must also be centrifuged in capped tubes.

4. Sediment concentration
Most commercial systems produce sediment concentration ranging from a 12: 1 to a 30: 1. However,
in manual techniques, a 24:1 or 12:1 concentration is desirable. This is achieved by using a 12 ml of
urine, leaving a sediment volume of 0.5 to 1 ml.

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 The volume of urine centrifuged divided by the sediment volume equals the concentration factor. The
sediment concentration factor relates to the probability of detecting elements present in low quantities
and is used when quantifying the number of elements present per milliliter. This means that a
concentration factor of 24 indicates a higher probability of detecting elements present in low amounts
as compared to a concentration factor of 12.

12÷0.5= 24
Hence, 24:1 concentration
The supernatant urine is removed by decanting or using a disposable pipette until 0.5 to 1 mL of urine
is retained. However, to maintain a uniform sediment concentration factor, urine should be aspirated
off rather than poured off, unless otherwise specified by the method in use. A pipette is used to gently
suspend the sediment by gently pipetting it through and through. Resuspension may also be done by
gently flicking the bottom of the tube. Thorough resuspension is essential to provide equal distribution
of elements in the microscopic examination field. Too vigorous agitation of the sediment can cause
fragile and brittle formed elements, such as red blood cell casts and waxy casts, to break into pieces.

5. Volume of sediment examined

When using the conventional glass slide method, the recommended volume is 20 ul covered by a 22x22
mm glass cover slip. The 20 ul is a representative sample for detecting formed elements. Allowing the
specimen to flow outside of the coverslip may result in the loss of heavier elements such as casts.

6. Examination of sediment
A standardized slide should be used for the microscopic examination of urine sediment to ensure that
the same volume of sediment is presented for viewing each time. Commercial standardized slides made
of molded plastic and have a built-in coverslip are available for use (Figure 1). With a disposable
transfer pipette, urine sediment is presented to a chamber, which fills by capillary action. This technique
facilitates uniform distribution of the formed elements
throughout the viewing area of the slide.
A minimum of 10 fields under both low and high-power
objectives is examined. The slide is first examined under low
power to detects casts and to ascertain the general composition of
the sediment. The magnification is switched to high power field
to delineate the structures that are seen 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.

Figure 1. Standardized Slide

When the sediment is examined unstained, many sediment constituents have a refractive index similar
to urine. Therefore, it is essential that sediments be examined under reduced light when using bright
field microscopy to provide adequate contrast. This is obtained by partially closing the iris diaphragm
and then adjusting the condenser downward until optimum contrast is achieved. If there is too much
light, some of the structures will be missed. For example, hyaline casts, they have a very low refractive
index and will be overlooked if the light is too bright or if there is not enough contrast.

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 It is also important that the fine adjustment should be continuously adjusted to enable the viewer to see
the depth of the object as well as other structures that may be on a different focal plane. Focusing with
the fine adjustment also aids in obtaining a complete representation of the sediment constituents.
The sediment should be examined as soon as possible after collection, but it may be refrigerated for a
few hours if the examination cannot be performed immediately.
7. Format of reporting
In a manual microscopic examination, the amount of some components is qualitatively assessed per
field of view in descriptive/semiquantitative or numeric terms. Table 1 lists commonly used terms and
typical descriptions. Other sediment components are also enumerated as a range of formed elements
present (e.g., 0 to 2, 2 to 5, 5 to 10). Note that these formed elements may be reported per low power
field or high-power field.
Table 1. Qualitative Terms and Descriptions for Fields of Views

Term Numeric Term Qualitative Terms and Descriptions

Rare 1+ Present, but hard to find
Few 1+ One (or more) present in almost every field of view
Moderate 2+ Easy to find; number present per field of view varies but it should be
more than few and less than many
Many 3+ Prominent; large number present in all field of views
Packed 4+ Field of view is crowded with the element
Note: The term ‘Packed’ is not routinely used in the laboratory for describing quantities of formed
elements.

NOTE!
The ‘Range’ of formed elements observed in 10 fields is enumerated by considering the
most consistent number seen per field. For example, if the number of hyaline casts in 10
different fields is 0, 2,2,0,0,2, 2, 3, 1, and 2, then the report would be 0-2 hyaline cast/low
power field.

The terminology and methods of reporting may differ slightly among laboratories but must be
consistent within a particular laboratory system. Routinely, casts and parasites are reported as the
average number per low-power field (in range) following examination of 10 fields. Red blood cells,
white blood cells, transitional epithelial cells and renal epithelial cells are reported as the average
number per 10 high-power fields (in range). Squamous epithelial cells, crystals, mucus threads and
amorphous materials are reported in semiquantitative/descriptive terms per low power field while
bacteria and yeasts per high power field. Note that although a component may be reported using low-
power magnification, high-power magnification may be needed to specifically identify or categorize it.
In addition, the absence of the formed element is reported as NEGATIVE.

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 Table 2. Sample reference values of different formed elements in urine

Component Number
RBC 0-3/hpf
WBC 0-8/hpf
Hyaline Cast 0-2/lpf
Fine granular cast 0-2/hpf
Transitional Epithelial cell 0-2/hpf (few)
Renal Tubular Epithelial cell 0-2/hpf (few)

Laboratories must also determine the particular reference values for the formed elements (see Table 2).
Take note, however, that these reference values are affected by the sediment concentration factor in
use. For example, with a concentration factor of 30, the reference value for WBC is zero to eight per
hpf, as opposed to the conventional value of zero to five per hpf used with a concentration factor of 12.
In some laboratories, the average number of elements per lpf or hpf is converted to the number per
milliliter. Here is a guide on how to do it.
1. The area of the field of view for the low- and high-power fields must be determined first using the
formula: Area = πr 2

For example:
Diameter of high-power field view: 0.35 mm
Radius of hpf view: 0.175 mm (This is obtained by dividing the diameter by 2).
Area of hpf view= 3. 1415 x (0.175 mm)2
= 0.096 mm2
2. Calculate the maximum number of lpf or hpf in the viewing area by dividing the area of the
coverslip used for viewing with that of the area per field of view.
Number of field views possible= Coverslip area ÷ area of field of view
For example:
Number of hpf views possible= (22 mm x 22 mm) ÷ 0.096 mm2
= 484 mm2 ÷ 0.096 mm2
= 5040 fields of view possible using hpf
NOTE: The 22 mm x 22 mm is the dimension of the ordinary microscopic slide used in the laboratory.
Other standardized microscopic slides have their own area.
3. Calculate the field conversion factor or the number of hpf/lpf per mL of urine testing using the
concentration factor and the volume of sediment examined.
Field conversion factor= Number of view fields possible ÷ (volume of sediment viewed per ml x
concentration factor)
NOTE: The volume of sediment viewed per ml refers to the volume of sediment that is placed onto the
slide for microscopic viewing.

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 For example:
Field conversion factor= 5040 ÷ (0.02 x 24)
= 5040 ÷ 0.48
= 10, 500 hpf/ml of urine
NOTE: The concentration factor is obtained assuming that the volume of urine used is 12 ml with 0.5
ml as the final volume of sediment formed.
4. Calculate the number of formed elements per ml of urine by multiplying the field conversion factor
to the average number of formed elements per field.
For example:
4 WBC/hpf x 10, 500 hpf/ml = 42, 000 WBC per ml of urine

SUMMARY OF SEDIMENT PREPARATION AND EXAMINATION

12 ml of urine is placed into a graduated centrifuge tube

Urine is centrifuged for 5 minutes at 400-450 RCF

Decant the sample (180 degrees full inversion with 1 to

2 seconds hang time)
* Apirating the sample is better to maintain a uniform
concentraiton factor. If the volume is less than 0.5 ml
after decantation or aspiration add urine sample up to
the desired volume

Thoroughly re-suspend the sediments by gentle agitation.

Using an automatic pipette, transfer 20 ul of urine

sediment onto a clean slide and cover it with a coverslip.

Observe a minimum of 10 fields under both low (10x) and

high (40x) power.

EXPLAIN: LET’S GO INTO DETAILS

The normal urine sediment may contain a variety of formed elements. These include red blood
cells, white blood cells, epithelial cells, casts and a variety of crystalline and amorphous materials. These formed
components can originate from throughout the urinary tract, from the glomerulus to the urethra, or can result
from contamination (such as menstrual blood, spermatozoa, fibers, starch granules). Opportunistic
microorganisms such as bacteria, yeast, and trichomonads may also be encountered in urine sediment.

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 Not all of these formed elements indicate an abnormal or pathologic process. Normally, a few red blood
cells, white blood cells, epithelial cells, and hyaline casts are observed in the urine sediment from normal,
healthy individuals. The presence of large numbers of “abnormal” components is diagnostically significant.
Note, however, that because changes occur in unpreserved urine, factors such as the type of urine collection
and how the specimen has been stored must be considered during microscopic examination.
As discussed previously, sediment preparation methods determine the actual concentration of the
sediment which reflects the number of elements that may be present in a microscopic field. To put this in better
perspective, the sediment constituents will be discussed individually in this section and in other units of this
module.

RED BLOOD CELLS(RBCs)

Red blood cells are anucleated, biconcave, refractile cells that measures 8
um in diameter and 3 um in depth. When viewed from the side, they have
an hourglass shape and when viewed from above, they appear as disks with
a central pallor.
When present in hypertonic urine, RBCs become smaller as intracellular
water is lost from the cell by osmosis, which causes them to become
crenated. As they crenate, erythrocytes lose their biconcave shape and
Figure 1. Red blood cells become spheres covered with spicules or crenations (see Figure 2). Because
of these membrane changes, these crenated cells appear rough
microscopically compared with normal erythrocytes.
In hypotonic urine, erythrocytes swell and release their hemoglobin to
become “ghost” cells, which are cells with intact cell membranes but no
hemoglobin. These empty cells, outlined by their membranes, appear as
colorless, empty circles (see Figure 3 and 4). Ghost cells are difficult to see
using brightfield microscopy; however, they are readily visible with phase
contrast or interference contrast microscopy. Alkaline urine promotes red
Figure 2. Crenated red blood blood cell lysis and disintegration, resulting in ghost cells and erythrocyte
cells remnants.

Figure 3. Ghost cells Figure 4. Ghost cells

Dysmorphic or distorted erythrocytes can also be observed (see Figure 5A and 5B). Occasionally, these
cells are present with normal erythrocytes in the urine of healthy individuals. However, increased numbers of

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acanthocytes are associated with glomerular disorders and sickle cells with sickle cell disease. It has been
demonstrated also after strenuous exercise, indicating a glomerular origin.
The number and appearance of the dysmorphic cells must be considered, because abnormal urine
concentration affects RBC appearance, and small numbers of dysmorphic cells are found with non-glomerular
hematuria.

Figure 5A. Dysmorphic RBC Figure 5B. Acanthocytes

All RBCs in urine originate from the vascular system. The presence of RBCs in the urine is associated
with damage to the glomerular membrane or vascular injury within the genitourinary tract. The number of cells
present is indicative of the extent of the damage or injury; hence, these urines also have significant proteinuria.
Increased numbers of RBCs along with red blood cell casts indicate renal bleeding, either glomerular or tubular,
such as in cases of glomerulonephritis, pyelonephritis, cystitis and presence of calculi.
When an increased number of RBCs is present without casts or proteinuria, the bleed is occurring below
the kidney or may be caused by contamination (e.g., menstrual blood, hemorrhoid). Non-renal disorders such
as hypertension, smoking, appendicitis, tumors, trauma, and drugs (anticoagulant drugs and drugs that induce
a toxic reaction such as sulfonamides) may also be attributed to the occurrence of hematuria.
Macroscopically, the urine sediment is characteristically red in color. If specimens have a positive
chemical test for blood but the microscopic examination reveals no RBCs, this can be explained by the fact that
RBCs readily lyse and disintegrate in hypotonic or alkaline urine wherein such lysis can also occur within the
urinary tract before urine collection. It is important to note
NICE TO KNOW! that other substances, such as myoglobin, microbial
peroxidases, and strong oxidizing agents, can cause a
Hemoglobin at a concentration greater positive blood chemical test and these reactions are
that 10 mg/dl can be detected by the considered “false-positive” reactions because RBCs or blood
protein reagent strip. is not present.
In specimens in which RBCs are present microscopically but the chemical screen for blood is negative,
ascorbic acid interference should be suspected. If ascorbic acid is ruled out, it is possible that the formed
elements observed are not RBCs but a “look-alike” component such as yeast, oil droplets, bubbles or
monohydrate calcium oxalate crystals. In these cases, their identity should be confirmed by an alternative
technique such as staining or using polarizing microscopy.

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 A Sternheimer-Malbin stain characteristically colors RBCs, whereas neither yeast nor calcium oxalate
crystals stain. Polarizing microscopy can also identify calcium oxalate crystals. The addition of 2% acetic acid
lyses RBCs but does not eliminate yeast or calcium oxalate crystals. Yeast varies in size, tends to be spherical
or ovoid rather than biconcave, and often exhibits budding. Small bubbles or droplets of oil contaminating the
urine sediment can be distinguished from RBCs by their variation in size, uniformity in appearance, and high
refractility. Bubbles and fat droplets (see Figure 6A and 6B) are larger, highly refractile and occur in different
sizes. Even white blood cells can be difficult to distinguish from crenated RBCs in a hypertonic urine specimen.
In the latter case, using acetic acid or toluidine blue stain can be advantageous because these solutions make it
easier to see the nuclei of white blood cells.

Figure 6A. Oil Droplets Figure 6B. Bubbles.

WHITE BLOOD CELLS (WBCs)

The distribution of white blood cells in the urine essentially mirrors that of peripheral blood. The five
types of cells that can be present are neutrophils, lymphocytes, eosinophils, basophils, and monocytes
(macrophages). Because neutrophils predominate in the peripheral blood, they are the white blood cell most
often observed in urine. However, with some renal conditions, other leukocytes predominate in the urine.
Normally, NEUTROPHILS are much easier to identify than RBCs
because they contain granules and multilobed/segmented nuclei.
They are larger than RBC, with a diameter that can range from 10-
20 um depending on the tonicity of urine, and can be similar in size
to the small epithelial cells that line the collecting ducts of nephrons.
In unstained preparation, neutrophils have a grayish hue and appear
grainy. Neutrophils lyse rapidly in dilute alkaline urine. Hypotonic
urine causes white blood cells to swell and become spherical balls
that lyse as rapidly as 50% in 2 to 3 hours at room temperature. In
Figure 7. White blood cells these large swollen cells, Brownian movement of the refractile
cytoplasmic granules is often evident, giving the descriptive name
“glitter cells” (see Figure 8). Brownian movement refers to the uncontrolled and fast movement of granules
within the cytoplasm as a result of collision forces.

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 As neutrophils age and begin to disintegrate, their lobed nuclei
fuse, and they can resemble a mononuclear cell (see Figure 9).
These changes can make neutrophils difficult to distinguish
from renal tubular collecting duct cells. In addition to fusion
of lobed nuclei, further evidence of cellular disintegration is
seen in the formation of blebs (see Figure 10). These vacuoles
develop within the cell periphery or on their outer membranes
and they appear to be empty or may contain a few small
granules. As these changes continue, the blebs or vacuoles can
detach and become free floating in the urine. They may also
Figure 8. Glitter cells develop and remain within the cell, pushing the cytoplasm to
one side and giving rise to large pale areas intracellularly.

Figure 10. Bleb formation Figure 9. WBCs with fused lobes

Another degenerative change is the development of numerous finger-like or wormlike projections

protruding from their surfaces (see Figure 11). These long filaments, termed myelin forms, result from the
breakdown of the cell membrane. As these cells die, additional
vacuolization, rupturing, or pseudopod formation may be
observed.
In hypertonic urine, neutrophils and other leukocytes become
smaller as water is lost osmotically from the cells, but they do not
crenate.

Figure 11. Filament formation

EOSINOPHILS have a diameter that ranges from 12-17 um and are easily identified by the preferred
stain which is Hansel stain (see Figure 12); though Wright stain can also be used. Eosinophils have bilobed
nuclei and contain acidophilic cytoplasmic granules. Eosinophils are not normally seen in the urine; therefore,
the finding of more than 1% eosinophils is considered significant.

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 MONONUCLEAR CELLS such as lymphocytes (see Figure
13), monocytes (see Figure 14), macrophages (Figure 15), and
histiocytes may be present in small numbers and are usually not
identified in the wet preparation urine microscopic analysis.
Because lymphocytes are the smallest WBCs (6-9 um), they may
resemble RBCs. They are characterized by a single, round to
slightly oval nucleus and scant clear cytoplasm that usually
extends out from one side of the cell.
Monocytes, macrophages, and histiocytes are large cells and
may appear vacuolated or contain inclusions. Macrophages are
derived from monocytes; when they reside in interstitial tissues,
Figure 12. Eosinophils they are often called histiocytes. Although macrophages average
30 to 40 µm in diameter, they can be as small as 10 µm or as
large as 100 µm in diameter. When they are small, their oval
nuclei and azurophilic granules make them difficult to
distinguish from neutrophils. When they are large, they usually
have irregular, kidney-shaped nuclei and abundant cytoplasm.
Macrophages and monocytes are actively phagocytic cells that
are capable of phagocytizing bacteria, viruses, antigen-antibody
complexes, RBCs, and organic and inorganic substances such as
fats and hemosiderin. The primary functions of these cells are to
defend against microorganisms, to remove dead or dying cells
and cellular debris, and to interact immunologically with
lymphoid cells.
Figure 13. Lymphocytes

Figure 15A. Monocytes Figure 15B. Monocytes

An increase in urinary WBCs is termed leukocyturia and indicates the presence of an infection or
inflammation in the genitourinary system. In contrast, pyuria refers to the increased pus excretion in urine,
where pus usually contains white blood cells. Leukocytes are capable of ameboid migration through the tissues
to sites of infection or inflammation. Bacterial infections, including pyelonephritis, cystitis, prostatitis, and
urethritis are frequent causes of leukocyturia. However, it is also present in nonbacterial disorders such as
glomerulonephritis, lupus erythematosus, interstitial nephritis, chlamydia, mycoplasmosis, tuberculosis,
presence of tumors in the urinary tract, trichomonads and mycoses. The latter two organisms, trichomonads and

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mycoses, often appear in urine from women as contaminants from vaginal secretions. Although they can infect
the urinary tract, infection is rare. In contrast, when these organisms are present in the urine from a male, a
urinary tract infection is implied.
Specifically, lymphocytes also predominate in renal transplant rejection. In the same way, the presence
of urinary eosinophils is primarily associated with drug-induced interstitial nephritis; however, small numbers
of eosinophils may be seen with chronic urinary tract infection (UTI) and renal transplant rejection. Overall,
eosinophiluria is a good predictor of acute interstitial nephritis (AIN) associated with drug hypersensitivity,
particularly hypersensitivity to penicillin and its derivatives. Untreated AIN can lead to permanent renal
damage. In cases of acute allograft rejection, the presence of large numbers of eosinophils in a kidney biopsy
specimen is considered a poor prognostic indicator.
When WBCs are present in the urine in increased numbers, the urine may appear cloudy, with
characteristics gray-white sediment button. Depending on the extent of infection, the urine may have a strong,
foul odor. The specific gravity is increased due to the density of white blood cells and pH is also increased due
to bacterial activity that allows conversion of urea to ammonia.
The leukocyte esterase (LE) test detects the presence of WBCs in urine, hence, in most cases,
leukocyturia results to a positive LE test. If bacteria are present, nitrite test will also be positive since nitrite test
detects presence of bacteriuria. However, since leukocytes readily lyse in urine, discrepancies can occur
between the number of cells seen microscopically and the leukocyte esterase (LE) screening test.
A positive LE test, despite few or no white blood cells present microscopically, can occur because of
WBC lysis and disintegration. Also, different populations of WBCs have varying quantities of cytoplasmic
granules and therefore differing amounts of leukocyte esterase. In
fact, lymphocytes have no leukocyte esterase. When increased
numbers of WBCs are present in urine, but the LE test is negative,
the microscopist must ensure that the cells are granulocytic
leukocytes, and that the reagent strips are functioning properly. This
may also possibly indicate that “look-a- like” elements such as RTE
may be present (see Figure 16).
Although the LE screening test usually detects 10 to 25 white blood
cells per microliter, the amount of esterase present may be
Figure 16. RTE insufficient to produce a positive response. Note that owing to
hydration, hypotonic urine could cause the leukocyte esterase to be
diluted such that it is below the detection limit of the LE reaction.
The primary concern in the identification of WBCs is the differentiation from renal tubular epithelial
(RTE) cells. RTE cells are usually larger than WBCs and more polyhedral in shape, with an eccentrically located
denser nuclei. WBCs in the process of ameboid motion may be difficult to distinguish from epithelial cells
because of their irregular shape. Monocytes and macrophages are identified more easily by using supravital
stains on the urine sediment or by making a cytocentrifuged preparation followed by Wright’s or Papanicolaou’s
stain. The addition of acetic acid can be used to enhance nuclear detail.

EPITHELIAL CELLS
It is not unusual to find epithelial cells in the urine, because they are derived from the linings of the
genitourinary system. Presence of epithelial cells result from normal turnover of old cells and epithelial damage

14
and sloughing caused by inflammatory processes/renal disease. Three types of epithelial cells are seen in urine
which includes squamous, transitional (urothelial), and renal tubular.
Squamous epithelial cells are the largest (40-60 um) and most
common cells found in the urine sediment (see Figure 17). They
contain abundant, irregular cytoplasm with granulations referred as
keratohyalin granules (increases as the cell ages) and a prominent
centrally located nucleus about the size of an RBC (8-14 um). They
are often the first structures observed when the sediment is examined
under low-power magnification appearing as thin, flagstone-shaped
elements with distinct cell border; and can be anucleated or
multinucleated.
Usually at least a few squamous epithelial cells are present in the
sediment and can serve as a good reference for focusing of the
microscope. Squamous epithelial cells can assume different
Figure 17A. Squamous Epithelial cell
conformations because their edges can fold over (possibly resembling
a cast) or curl while they are suspended in urine.
Difficulty identifying squamous cells is rare. However, they may
begin to disintegrate in urine that is not fresh. In sediments containing
large amounts or clumps of squamous cells, it may be more difficult
to enumerate smaller pathologic elements, such as RBCs and WBCs,
and they should be carefully examined.
Squamous epithelial cells originate from the linings of the vagina and
entire female urethra and the lower/distal portion of the male urethra.
They represent normal cellular sloughing and have no pathologic
significance. In women, large numbers of squamous epithelial cells in
the urine sediment often indicate vaginal or perineal contamination.
Figure 17B. Folded squamous Similarly, in uncircumcised men, large numbers suggest specimen
Epithelial cell contamination.
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. Clue cells are indicative of vaginal infection by the bacterium Gardnerella vaginalis. They appear
as squamous epithelial cells covered with the Gardnerella coccobacillus (see Figure 18). 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. Routine testing for clue cells is performed by examining a vaginal wet
preparation for the presence of the characteristic cells.

Figure 18. Squamous Epithelial

cells embedded with bacilli

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 Transitional epithelial cells are smaller than squamous cells and appear in several forms, including
spherical, polyhedral, and caudate (see Figure 19). These differences are caused by the ability of transitional
epithelial cells to absorb large amounts of water, them becoming spherical in form and much larger than the
polyhedral and caudate cells. All forms have distinct, centrally located nuclei.
Transitional (urothelial) epithelial cells vary considerably in size (see Figure 20). This size variation
relates primarily to the three principal layers of transitional epithelium in the bladder.
a. Superficial cells
This is the most prevalent transitional epithelial cell. It is round or pear-shaped (30-40 um),
with a dense oval to round centrally located nucleus and abundant cytoplasm. The nucleus is about the
size of a red or white blood cell, and the peripheral borders of the nucleus and cell membrane are
distinctly outlined.

b. Intermediate cells
It is smaller than superficial cells and appears round with a diameter of 20-30 um.

c. Basal Cells
It is a columnar form of transitional 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 except the distal portion. They are usually present in small
numbers in normal urine (0-2/hpf), representing normal cellular sloughing. Increased numbers of transitional
cells seen singly, in pairs, or in clumps (syncytia) are present following invasive urologic procedures such as
catheterization or other types of instrument procedures (chemotherapy and radiation) and are of no clinical
significance. However, in some cases, it may imply urinary tract infection.
At times, cluster or sheets of transitional epithelium are observed. An increase in transitional cells
exhibiting abnormal morphology such as vacuoles and irregular nuclei may be indicative of malignancy (such
as transitional cell carcinoma and neoplasia in the genitourinary tract) or viral infection. In such cases, whenever
epithelial cells with abnormal characteristics are observed, such as unusual size, shape, inclusions, or nuclear
chromatin pattern, additional cytologic studies are necessary.
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.
Urine specimens with increased transitional epithelial cells will appear turbid and is positive for blood
and protein is malignancy is considered.

16
 Figure 19. Transitional Epithelial Figure 20. Types of Transitional
cell Epithelial cell

Renal tubular epithelial cells vary in size and shape depending on the area of the renal tubules from
which they originate. These would include convoluted tubular cells and collecting duct cells.

Convoluted Tubular cells

Proximal Convoluted tubular cells. These cells are larger (20-60 um) than other
RTE cells. They tend to be oblong or cigar-shaped with a grainy cytoplasm- a
characteristic that makes them resemble granular casts with indistinct cell
membrane. They have a nucleus with a dense chromatin pattern that is usually
eccentric, and they can be multinucleated. When PCT cell had absorbed fat
globules, it could easily be mistaken for a granular or fatty cast (difference is that
casts do not have nucleus).

Distal Convoluted tubular cells. Cells from the distal convoluted tubule are
smaller (14-25 um) than those from the PCT and are round or oval. It has a small,
dense, round nucleus that is usually eccentric. The cytoplasm is granular, much like
that of the PCT cells. They can be mistaken for WBCs and spherical transitional
epithelial cells. Observation of the eccentrically placed round nucleus aids in
differentiating them from spherical transitional cells. Differentiation between
proximal and distal convoluted tubular cells is not necessary, and these cells are
collectively reported as “convoluted” renal tubular cells.
Collecting Duct Cells
In contrast to proximal and distal convoluted tubular cells, collecting duct cells can be observed
as fragments of undisrupted tubular epithelium (see Figure 21). To be identified as a fragment, at least three
cells must be sloughed together with a bordering edge intact.

17
 Figure 21. Collecting duct cell

Small Duct cells. These are polygonal or cuboidal in shape with a size of 12-20
um. They have a single, large, moderately dense nucleus that occupies about 60-
70% of the cytoplasm. These cells are rarely round or spherical. Therefore, one
distinguishing characteristics in a flat corner/edge on the membrane of the cell.
However, one must consider that over time, when these cells in urine absorb water,
they become swollen and the flat edge might not be observable. Also, these cells
may be mistaken as macrophages or monocytes if they assume a round or spherical
shape.
Large Duct cells. The small duct cells become wider as they approach the renal
calyces, and their epithelial cells become larger and more columnar. They measure
about 6-10 um, with a nucleus occupying about 90 % of the cytoplasm (6-8 um)
and is eccentrically located.

RTE cells are the most clinically significant of the epithelial cells. The presence of increased amounts
(usually more than 2 per high power field) is indicative of necrosis of the renal tubules, with the possibility of
affecting overall renal function.
Proximal and distal convoluted tubular cells are found in the urine as a result of acute ischemic or toxic
renal tubular disease (e.g., acute tubular necrosis) from heavy metals or drug (aminoglycosides) toxicity.
Similarly, collecting duct fragments are also found following trauma, shock, or sepsis, and indicate ischemic
necrosis and severe tubular injury with basement membrane disruption. Specifically, large duct cells are often
observed in cases of pyelonephritis, acute tubular necrosis, kidney transplant rejection, malignant infiltration,
and salicylate poisoning.
Because RTE cells are often present as a result of tissue destruction, the nucleus is not easily visible in
unstained sediment. Cytocentrifugation followed by Papanicolaou’s staining of the urine sediment can be used
to specifically identify these cells.
RTE participates in reabsorption. It can absorb bilirubin present in the filtrate as the result of liver
damage and appear deep yellow. Hemoglobin present in the filtrate is also absorbed by the RTE cells and
converted to hemosiderin. Therefore, following episodes of hemoglobinuria (transfusion reactions and

18
paroxysmal nocturnal hemoglobinuria), the RTE cells may contain the characteristic yellow-brown hemosiderin
granules.
Urine specimens with increased RTE would appear turbid and color varies depending on the etiology.
Chemical tests for blood, protein, LE and nitrite become positive.

OVAL FAT BODIES

RTE cells absorb lipids that are present in the glomerular filtrate. They appear to be highly refractile,
with small number of glistening granules, and the nucleus may be more difficult to observe. These lipid-
containing RTE cells are called oval fat bodies (see Figure 22). They are usually seen in conjunction with free-
floating fat droplets. Note however that when monocytes or macrophages have ingested lipoproteins and fat,
these globular inclusions are distinctly refractile and are also called oval fat bodies.
Oval fat bodies indicate glomerular dysfunction and renal tubular cell death. It is also observed in cases
of lipiduria caused by nephrotic syndrome, sever tubular necrosis, diabetes mellitus and trauma that cause
release of bone marrow fat from long bones.
In cases of acute tubular necrosis, RTE cells containing large, nonlipid-filled vacuoles may be seen
along with normal renal tubular cells and oval fat bodies. Referred to as “bubble cells,” they appear to represent
injured cells in which the endoplasmic reticulum has dilated prior to cell death.
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. The droplets are composed of triglycerides,
neutral fats, and cholesterol. Fat stains stain triglycerides and neutral fats, producing orange-red. Examination
of the sediment using polarized light results in the appearance of characteristic Maltese cross formations in
droplets containing cholesterol (see Figure 23). Sediments negative for fat after staining should still be checked
using polarized light in case only cholesterol is present. Likewise, staining should be performed on sediments
negative under polarized light.

Figure 22. Oval fat bodies Figure 23. Cholesterol with

maltese cross appearance

19
 ELABORATE: CONNECTING CONCEPTS
Many factors can influence the appearance of the urinary sediment, including cells and casts in
various stages of development and degeneration, distortion of cells and crystals by the chemical content of
the specimen, the presence of inclusions in cells and casts, and contamination by artifacts. Identification can
then be enhanced through the use of sediment stains and different types of microscopy
Staining increases the overall visibility of sediment elements being examined using bright-field
microscopy by changing their refractive index. It allows easy identification of cellular structures, such as the
nuclei, cytoplasm, and inclusions.
Sternheimer-Malbin stain. This is a supravital stain, consisting of crystal violet and safranin, most
frequently used in urinalysis. Epithelial cells, WBCs, RBCs, casts inclusions and low refractile
elements (hyaline casts, mucus threads) are stained (pink to purple), providing clearer delineation of
structure and contrasting colors of the nucleus and cytoplasm.
0.5% solution of toluidine blue. A metachromatic stain that provides enhancement of the nuclear
details of a cell. It can be useful in the differentiation between WBCs and renal tubular epithelial cells
and is also used in the examination of cells from other body fluids. Nuclear detail is also enhanced by
the addition of 2% acetic acid to the sediment.
Oil Red O and Sudan III. With the use of polarizing microscopy, it can be used to confirm the presence
of fats. Triglycerides and neutral fats stain orange-red whereas cholesterol does not stain but is capable
of polarization.
Gram stain. This is used primarily in the microbiology section for the differentiation between gram-
positive (blue) and gram-negative (red) bacteria. Its role in routine urinalysis is limited to the
identification of bacterial casts, which can easily be confused with granular casts.
Prussian blue stain. To facilitate the visualization of hemosiderin (appears 2-3 days after intravascular
hemolysis), free floating or in epithelial cells and casts, the Prussian blue reaction is used.

Aside from the use of stains, visualization and identification of urine sediment components can also be
enhanced by employing appropriate microscopy.
Bright-field microscopy. This technique is most frequently used in the laboratory. It allows
examination of formed elements by making it appear dark against a light background. Use of bright-
field microscopy for the examination of urine sediment can present problems when the amount of light
reaching the specimen is not properly controlled. Sediment constituents with a low refractive index will
be overlooked when subjected to light of high intensity. Therefore, sediments must be examined using
decreased light controlled by adjusting the light source.
Phase-Contrast microscopy. To facilitate the visualization of low-refractility components such as
hyaline casts, mucous threads, and bacteria, the use of phase microscopy is very helpful.
Polarizing microscopy. This is used in urinalysis to confirm the identification of fat droplets, oval fat
bodies, and fatty casts that produce a characteristic Maltese cross pattern. Birefringent uric acid crystals
can be distinguished from cystine crystals, monohydrate calcium oxalate crystals from nonpolarizing
RBCs, and calcium phosphate crystals differentiated from nonpolarizing bacteria by their polarizing
characteristics. Substances that polarize light includes monohydrate calcium oxalate crystals, fibers

20
 (clothing, diapers), plastic fragments, amorphous crystals (urates: strongly; phosphates: very weakly),
cholesterol globules and starch granule; while substances that do not polarize light includes RBCs,
casts, mucus, bacteria and cellular debris (membrane phospholipids).
Interference-contrast microscopy. This provides a three-dimensional image of an element, either of
low or high refractive index, thus enhancing the cellular details.
Dark-Field Microscopy. Dark-field microscopy is a technique used in the clinical laboratory to
enhance visualization of specimens that cannot be seen easily viewed with a bright-field microscope.
It is often used for unstained specimens, and, in particular, to identify the spirochete Treponema
pallidum.
Fluorescence microscopy. This is used to detect bacteria and viruses within cells and tissues through
a technique called immunofluorescence.

Although it is not a part of the routine examination of the urine sediment, the preparation of permanent
slides using cytocentrifugation followed by staining with Papanicolaou stain provides an additional method for
detecting and monitoring renal disease. Cytodiagnostic urine testing is frequently performed independently of
routine urinalysis for detection of malignancies of the lower urinary tract. A voided first morning specimen is
recommended for testing, which is performed by the cytology laboratory.
Cytodiagnostic urine testing also provides more definitive information about renal tubular changes
associated with transplant rejection; viral, fungal, and parasitic infections; cellular inclusions (pathologic casts),
and inflammatory conditions.

21
 EVALUATE: ACTIVITY ALERT
This is a graded activity worth 10 points.
To assess your understanding on the concepts presented, answer the given questions below.

MULTIPLE CHOICE (10 POINTS). Select the BEST answer for each of the questions.

1. Which focusing knob should you use with 10x and 40x objectives?
A. Fine focusing knob
B. Respectively coarse and fine focusing knobs
C. Both fine focusing knob and the iris diaphragm
D. Coarse focusing knob
2. Which of the following is a sensitive early indicator of renal disease?
A. Intact red blood cells
B. Dysmorphic red blood cells
C. Ghost cells
D. Bubble cells

3-10. Match the elements with the following statements:

A. Eosinophils
B. Glitter cells
C. Lymphocytes
D. Neutrophils
E. Oval fat bodies
F. RTE
G. Squamous epithelial cell
H. Transitional epithelial cell

3. Indicate active kidney disease or tubular injury

4. May be seen with infection and after urethral catheterization
5. Early indicator of renal transplant rejection
6. Associated with drug induced interstitial nephritis
7. Associated with nephrotic syndrome
8. Presence usually indicates vaginal contamination
9. Swollen neutrophils associated with hypotonic urine
10. Most common leukocyte indicating inflammation in the urogenital tract

22
 UNIT 2: URINARY CASTS
ENGAGE: RECALL
Unique to the kidney, urinary casts are formed in the distal and collecting tubules with a core matrix
of uromodulin or formerly known as Tamm-Horsfall protein. This glycoprotein is secreted by the renal tubular
cells of the thick ascending limb of the loop of Henle and by the distal convoluted tubules. Other proteins such
as albumin and immunoglobulins are also incorporated into the cast matrix. Any urinary component, whether
chemical or a formed element, can be found incorporated into a cast. Eventually, the formed cast detaches from
the tubular epithelial cells and is flushed through the remaining portions of the nephron with the lumen fluid.

EXPLORE: UNLOCKING CONCEPTS

Urinary casts are formed in the lumen of the tubules of the kidney. Their shape and size can vary
greatly depending on the diameter and shape of the tubule in which they form. They are cylindrical in shape
and do not have dark edges. They have nearly parallel sides and rounded or blunted ends, with a thick middle
portion.
The narrower the tubular lumen, the narrower is the resulting cast. As an example, the ascending loop
of Henle and DCT produce casts with tapered end. Similarly, broad casts, which can be from two to six times
wider than ordinary casts, are formed either in pathologically dilated or atrophied tubules or in collecting
tubules.
Casts can be short and stubby, long and thin, or any combination of these. They may be straight, curved,
or convoluted as well. A cast becomes convoluted when after formation and release from a tubule, it encounters
a tubular obstruction, such as another cast being formed. The first (narrower) cast becomes compressed to form
a cast that appears convoluted (see Figure 1).

Figure1. Convoluted cast

Casts are always renal in origin, and they are important indicators of intrinsic renal disease. Disorders
in which cast may be present include glomerular damage, tubular damage, renal inflammation, and renal
infection.
Factors involved in Cast Formation
Factors that are involved in cast formation include urinary stasis (marked decrease in urine flow),
increased acidity, high solute concentration (sodium and calcium), and the presence of abnormal ionic or protein

1
constituents. Under normal conditions, Tamm-Horsfall protein is excreted at a relatively constant rate and the
rate of excretion appears to increase under conditions of stress and exercise. In an acidic environment, gelation
of protein and the precipitation of solutes are enhanced. Because acidification and concentration of urine occur
in the distal and collecting tubules, these tubules are the sites of most cast formation.
Urinary stasis can occur because of obstruction from disease processes or congenital abnormalities.
This stasis promotes the accumulation and concentration of ultrafiltrate components. As the cast forms, urinary
flow within the tubule decreases as the lumen becomes blocked. The internal tension formed may account for
the wrinkled and convoluted appearance of older casts.
In conditions that cause increased quantities of plasma proteins (e.g., albumin, globulins, hemoglobin,
myoglobin) in the lumen ultrafiltrate, cast formation is enhanced greatly. These proteins become incorporated
into the uromodulin protein matrix, along with any cells and cellular or granular debris that happen to be present.
The appearance of a cast is also influenced by the materials present in the filtrate at the time of its
formation and the length of time it remains in the tubule. Any elements present in the tubular filtrate, including
cells, bacteria, granules, pigments, and crystals, may become embedded in or attached to the cast matrix.
Casts will dissolve in alkaline urine and in neutral urine having a specific gravity of 1.003 or less. The
presence of casts in the urine referred to as cylindruria is frequently accompanied by proteinuria. However,
casts can be seen in the absence of protein. Because casts can be retained in the tubule for varying lengths of
time, the substances enmeshed in their matrices can disintegrate. In addition, the cast matrix itself can undergo
changes that become apparent microscopically, for example, transition from a granular to a waxy cast.

Cylindroids
Sometimes casts are well formed at one end but are tapered or have a tail at the other end (see Figure
2). These casts, often called cylindroids, result from incomplete cast formation, formation of a cast in a tubule
where the lumen width differs (naturally or from disease), or cast disintegration. Because they have the same
clinical significance as completely formed casts, cylindroids are not enumerated separately.

Figure 2. Cylindroids

2
 EXPLAIN: LET US GO INTO DETAILS

CLASSIFICATION OF CASTS
Classification of casts is made on the basis of their appearance and the cellular components that they
contain. The different types of casts are hyaline, red cell, white cell, epithelial cell, granular (coarse and fine),
waxy, and fatty. At times, it may be difficult to distinguish the various casts because of degeneration, or because
the cast may contain a variety of structures (mixed casts). Casts are reported according to type and the number
that is present per low-power field. Ranges reported are usually none seen, 0–2, 2–5, 5–10, 10–20/LPF.
Casts with homogenous matrix composition
Hyaline casts. Hyaline casts are the most commonly observed casts in the urine sediment (see Figure
3). It is composed primarily of a homogeneous uromodulin protein matrix that gives it a low refractive index
that is similar to that of urine. These casts appear colorless in unstained urine sediment, consisting of normal
parallel sides and rounded ends, cylindroid forms, and wrinkled or convoluted shapes that indicate aging of the
cast matrix (see Figure 4). The accompanying dehydration of the protein fibrils and internal tension may account
for the wrinkled and convoluted appearance of older hyaline cast.
When phase-contrast or interference contrast microscopy is used, their fibrillar protein matrix is more
apparent and often includes some fine granulation (see Figure 5). Stained with Sternheimer-Malbin, these casts
appear pink with a more defined edge.
In healthy individuals, two or fewer hyaline casts per low-power field is considered normal. Increased
numbers of hyaline casts can be found following extreme physiologic conditions such as strenuous exercise
(>10/lpf), dehydration, fever, or emotional stress. They may also be found along with other casts in cases of
pyelonephritis, glomerulonephritis, chronic renal disease and congestive heart failure.
Occasionally, a hyaline cast may have a single epithelial or blood cell within its matrix. These casts, as
well as cylindroids, are enumerated as hyaline casts and have no diagnostic significance.
Even if matrix of hyaline cast is made up of protein, this is not detected by the reagent strip test for
protein. They are often mistakenly identified with mucus thread and fibers.

Figure 3. Hyaline casts Figure 4. Degenerating hyaline

casts

3
 Figure 5. Fibrillar matrix of
hyaline casts

Waxy Cast. Waxy casts are named as such because of their waxy appearance. These casts appear
homogeneous, with their edges well defined, and often have sharp, blunt, or uneven ends. Cracks or fissures
from their lateral margins or along their axes are often present also (see Figure 6).
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. As a result, they often appear fragmented with jagged
ends and have notches in their sides (see Figure 7). With supravital
stains, waxy casts stain a homogenous, dark pink and have a
diffuse, ground glass appearance (see Figure 8).
Waxy casts indicate prolonged stasis and tubular obstruction.
These are found most frequently in patients with chronic renal
failure; hence they are frequently referred to as renal failure casts.
Figure 6. Waxy cast They are also encountered in patients with acute renal disease (e.g.,
acute glomerulonephritis, nephrotic syndrome), tubular
inflammation, malignant hypertension, renal amyloidosis, diabetic nephropathy and during renal allograft
rejection.
Presence of waxy casts in urine is often accompanied by hematuria (may be negative), proteinuria,
leukocyturia and the presence of other casts such as granular and cellular casts. They are often mistakenly
identified with fiber and fecal materials.

Figure 7. Waxy cast with jagged sides

Figure 8. Waxy cast with a supravital stain

4
Cellular Inclusion Casts
RBC cast. In unstained urine sediments, erythrocytes within the cast matrix cause them to be
characteristically yellow. In SternheimerMalbin–stained sediments, intact RBCs may appear colorless or
lavender in a pink homogeneous matrix.
The microscopic appearance of red blood cell casts varies such that some casts are packed with RBCs
while others may present principally as a hyaline cast with several clearly defined RBCs embedded within its
matrix (see Figure 9).
Occasionally, an RBC cast may be observed in the urine of a healthy
individual. This finding usually is noted after strenuous exercise
(referred to as athletic pseudonephritis), particularly after participation
in contact sports such as football, basketball, or boxing. As with other
urine findings associated with this condition, the urine sediment returns
to normal within 24 to 48 hours.
Red blood cell casts are diagnostic of intrinsic renal disease. The RBCs
are most often of glomerular origin (e.g., passage across glomerular
filtration barriers as in glomerulonephritis) but can result from tubular
damage (e.g., blood leakage into the tubules, as with acute interstitial
Figure 9. RBC cast
nephritis).
Red blood cell casts are associated with the acute tubular necrosis
often caused by the toxic effects of massive hemoglobinuria that can
lead to renal failure. These casts must be present in conjunction with
other pathologic findings such as RTE cells and a positive reagent
strip test for blood.
In the presence of massive hemoglobinuria or myoglobinuria and
with sufficient urine stasis, homogenous orange-red or red-brown
casts may be observed which are referred to as blood casts or muddy
brown casts (see Figure 10). These red to golden-brown granular
casts contain no distinct RBCs in their matrix because cells have
Figure 10. Muddy brown cast lysed and undergone degeneration. This process may also occur in
vitro when the urine specimen is old and improperly stored.
RBC casts are fragile, and overly vigorous resuspension of the urine sediment can result in breakage of
the casts into pieces. Microscopically, chunks of casts would be present and may be difficult to identify.
Phase-contrast and interference contrast microscopy aid in identification of red blood cell casts by
enhancing the detail of cells trapped within the cast matrix. Because free-floating RBCs are also present, one
must ensure that cells are actually within the cast matrix and are not simply superimposed on its surface.

WBC casts. These highly refractile casts consist of leukocytes embedded in a hyaline cast matrix (see
Figure 11). Because of the refractility of the cells within them, leukocyte casts are readily apparent and
identifiable with brightfield microscopy.
The presence of WBC casts indicates inflammation of the upper urinary tract, particularly of the
glomerulus or the tubules. In most glomerular infections (e.g., glomerulonephritis, pyelonephritis), red blood

5
cell casts will also be present and in greater numbers than white blood cell casts, along with bacteriuria,
proteinuria and hematuria. It is also seen in cases of non-bacterial inflammations such as acute interstitial
nephritis.
Most of the time, these casts are mistaken as RTE cell casts. The presence of increased numbers of
white blood cells, free-floating or in clumps, would suggest strongly that the it is a WBC cast.
RTE cell cast. These colorless, highly refractile casts contain individual renal tubular cells or fragments
of the tubular lining removed intact from the tubule. Some are stained will bilirubin (in cases of hepatitis) and
thus appear yellow.
The presence of degenerating tubular cell casts in urine sediment indicates intrinsic renal tubular
disease, along with severe urinary stasis. Proteinuria, hematuria and often granular casts accompany renal
tubular cell casts. Similar to RTE cells, they are associated with heavy metal and chemical or drug-induced
toxicity, viral infections, and allograft rejection. They also accompany WBC casts in cases of pyelonephritis.

Figure 11. WBC cast Figure 12. RTE cell cast

Mixed Cellular cast. This cast is composed of a combination of multiple cell types such as RBCs, WBC
and RTE cells. Most frequently encountered include combination of RBC and WBC in glomerulonephritis and
WBC and RTE cell or WBC and bacteria in pyelonephritis (see Figure 12)
Identification of these casts is difficult and thus requires
staining and phase microscopy. When mixed casts are present,
there should also be homogenous casts of at least one of the cell
types, which will be the primary diagnostic marker. For
example, in glomerulonephritis, the predominant casts will be
RBC, and in pyelonephritis, the predominant casts will be
WBC.
Mixed cellular casts often are reported as such with a
description of the cells involved. For example, cellular cast—
leukocytes and renal tubular cells. When a cast of two matrix
types (half granular and half waxy) is encountered, the cast is
identified using the term that has the greatest clinical
Figure 13. Cellular Cast-WBC and RBC significance. In this example, the cast should be enumerated and
reported as a waxy cast.

6
 Bacterial cast. Bacterial cast contain bacilli both within and bound
to the protein matrix (see Figure 14). They usually include
leukocytes and thus often reported as leukocyte casts. These casts are
often observed in cases of pyelonephritis.
Identification of bacterial casts can be difficult, because casts packed
with bacteria can resemble granular casts. 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.
Contrast interference microscopy allows even better visualization of
Figure 14. Bacterial cast bacteria within casts. Correlating with a positive protein, LE, nitrite
and blood reagent strip test results facilitates identification.

Cast with Inclusions

Granular casts. These are highly refractile casts composed of small, fine granules (fine granular cast)
or large, coarse granules (coarse granular cast) with a uromodulin protein matrix. Fine granules may appear
gray or pale yellow in color while coarse granules are darker in color and these casts often appear black because
of the density of the granules.
The granules in finely granular casts are by-products of lysosome and protein metabolism that are
excreted by renal tubular epithelial cells. Initially, the granules are large and coarse that results from
degeneration of cellular casts; but when urine stasis is prolonged, these granules break down to fine granules.
Further degeneration would then result to formation of waxy casts.
Granular casts almost always indicate significant renal disease. However, fine granular casts may be
present in the urine for a short time following strenuous exercise.
As mentioned earlier, columnar RTE and clumps of small crystals may also resemble granular casts,
and thus must be differentiated properly.

Figure 15. Fine granular cast Figure 16. Coarse granular cast

7
 Fatty casts. Fatty casts contain highly refractile free fat globules, oval
fat bodies, or both, and their matrix can be hyaline or granular (see
Figure 17). In unstained urine sediment examined by brightfield
microscopy, lipid globules may appear light yellow or darker. If fat
stains such as Sudan III or Oil Red O are used, triglyceride (neutral
fat) globules within casts stain characteristically orange or red,
whereas cholesterol and cholesterol esters do not.
Fatty casts are accompanied by significant proteinuria and most
frequently associated with the nephrotic syndrome, but are also seen
in toxic tubular necrosis, lipid nephrosis, chronic glomerulonephritis,
Figure 17. Fatty cast Kimmelstiel-Wilson syndrome (diabetic nephropathy), lupus, crush
injuries and degenerative tubular diseases.

Figure 18 shows a fatty cast loaded with fat. With prolong

storage and cooling, cholesterol crystal (arrow) has started to
form from the fat (cholesterol). This can be often mistaken as a
fecal debris.

Figure 18. Fatty cast

Casts with non-cellular Inclusions

Hemosiderin granules, sulfonamide crystals and calcium oxalate crystals have been found in casts.
Because crystals can aggregate along mucous threads, it is important that the hyaline matrix is observed and
that it actually encases the crystals. The presence of crystal casts indicates crystal precipitation within the
tubules, which can damage tubular epithelium as well as cause tubular obstruction. As a result, varying amounts
of hematuria usually accompany crystalline casts in the urine sediment.
Casts with Pigments
Pigments such as hemoglobin, myoglobin, or bilirubin (bile) can be incorporated in a hyaline matrix
and produce characteristic colors. Hemoglobin and myoglobin casts appear yellow to brown and are
accompanied by hematuria while bilirubin casts appear yellow- or golden-brown. Similarly, highly pigmented
drugs, such as phenazopyridine, can also characteristically color sediment elements. In contrast, urobilin
pigment that can impart an orange-brown color to urine does not color the formed elements of the sediment.

8
 Figure 19. Bilirubin stained RTE cast Figure 20. Hemosiderin granules in cast

Broad Cast
Broad casts indicate cast formation in dilated tubules or in the large collecting ducts. Because several
nephrons empty into a single collecting duct, cast formation here indicates significant urinary stasis due to
obstruction or disease. 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. However,
considering the accompanying urinary stasis, the most commonly seen broad casts are granular and waxy.
In chronic renal diseases in which nephrons have sustained previous damage, broad hyaline casts may
be encountered. These casts form as a result of continued proteinuria and other factors that enhance their
formation.

Figure 21. Broad cast

9
 ELABORATE: CONNECTING CONCEPTS
Several formed elements in urine sediment can be confused with casts. Mucous threads can be
misidentified as hyaline casts (see Figure 22). Although mucous threads have a similar low refractive
index, they are ribbon-like, and their ends are not rounded but are serrated. They are irregular, whereas hyaline
casts are more formed.
Various fibers, such as cotton threads or diaper fibers, can resemble waxy casts. Fibers tend to be flatter
in the middle and thicker at their margins, whereas casts are cylindrical and thicker in the center. In addition,
fibers are more refractile than casts. Under polarizing microscopy, fibers polarize light, whereas casts do not
Finally, fibers may contaminate the urine at any time, whereas casts, particularly waxy casts, must be
accompanied by proteinuria.
Other entities, such as squamous epithelial cells folded into a tubular shape or scratches on the coverslip
surface, may be misidentified as casts. Crystals such as amorphous urates and phosphates can aggregate together
or along a mucous thread to simulate a cast. With polarizing microscopy, their birefringence identifies them as
crystalline entities, and the lack of a distinct matrix differentiates them from a true cast.

Figure 21. Cloth fibers

Figure 24. Mucus threads

Figure 23. Hair fiber

10
 EVALUATE: ACTIVITY ALERT
This is a graded activity worth 10 points.
To assess your understanding on the concepts presented, answer the given questions below.
MATCHING TYPE (5 POINTS). Match the following type of casts with the BEST disease/condition
associated.

A. Epithelial cell cast

B. Fatty cast
C. Fine granular cast
D. RBC cast
E. Waxy cast

1. Acute glomerulonephritis
2. Strenuous exercise
3. Chronic renal failure
4. Diabetic nephropathy
5. Nephrotoxic poisoning

TRUE OR FALSE (5 POINTS). Write TRUE if the statement is correct and FALSE if incorrect.

1. Red blood cell degeneration results in the formation of coarse granular cast.
2. Casts easily degenerate in a hypertonic, alkaline urine.
3. WBC casts appear pink in Sternheimer-Malbin stain.
4. Hyaline cast is the most common form of cylindroid encountered.
5. Casts are primarily formed in the Loop of Henle.

11
 UNIT 3: URINARY CRYSTALS AND
MISCELLANEOUS ELEMENTS
ENGAGE: RECALL
Crystals are usually not found in freshly voided urine. When the urine is supersaturated with a
particular crystalline compound, or when the solubility properties of that compound are altered, the result is
crystal formation. This may occur when urine samples are allowed to stand for a long period of time. In some
cases, this precipitation occurs in the kidney or urinary tract and can result in the formation of urinary calculi
(stones).
Microscopic evaluation of urine is important for detection of crystals because no chemical test detects
the presence of crystals. Many of the crystals that are found in the urine have little clinical significance, except
in cases of metabolic disorders, calculus formation, and the regulation of medication. Some crystals indicate a
pathologic process; therefore, it is important that they are correctly identified and reported. The most important
crystals that may be present are cystine, tyrosine, leucine, cholesterol, and sulfa.
Crystals can be identified by their appearance, birefringence (crystals that can refract light into two
directions), pH dependency, and solubility characteristics. Take note that almost all crystals are birefringent
except for bilirubin, tyrosine and hemosiderin crystals.

EXPLORE: UNLOCKING CONCEPTS

Crystals result from the precipitation of urine solutes. They are not normally present in freshly voided
urine but form as urine cools to room or refrigerator temperature. When crystals are present in freshly voided
urine, they indicate formation in vivo and are always clinically significant. Regardless of the crystal type, crystal
formation within the nephrons can cause significant tubular damage. Several factors affect the formation of
crystals.
Concentration of the solute in the urine. As the glomerular ultrafiltrate passes through the tubules,
solutes within the lumen fluid are concentrated. As the concentration of urinary solutes increases, their
ability to remain in solution decreases, resulting in crystal formation. This can result in precipitation of
the solute into its characteristic crystalline form.
Urine pH. Because solutes differ in their solubility, urine pH provides a means of identifying and
differentiating them. For example, inorganic salts such as oxalate, phosphate, calcium, ammonium, and
magnesium are less soluble in neutral or alkaline urine. As a result, when the urine pH becomes neutral
or alkaline, these solutes can precipitate out in their crystalline form. In contrast, organic solutes such
as uric acid, bilirubin, and cystine are less soluble in acidic conditions and can form crystals in acidic
urine.
Flow of urine through the tubules. Flow reduction allows time for maximum concentration of solutes
in the ultrafiltrate. This flow reduction allows time for maximum concentration of solutes in the
ultrafiltrate.
Temperature. Solutes precipitate more readily at low temperatures. For example, amorphous urates
frequently form in refrigerated specimens and may dissolve if the specimen is warmed. Therefore, the
majority of crystal formation takes place in specimens that have remained at room temperature or been

1
 refrigerated prior to testing. Crystals are extremely abundant in refrigerated specimens and often present
problems because they obscure clinically significant sediment constituents.

EXPLAIN: LET US GO INTO DETAILS

NORMAL URINE CRYSTALS

Urine crystals occur in acidic or alkaline urine. Phosphates represent the majority of the crystals seen
in alkaline urine and include amorphous phosphate, triple phosphate, and calcium phosphate. Other normal
crystals associated are calcium carbonate and ammonium biurate. On the other hand, those crystals which are
frequently found in acidic urine are uric acid, calcium oxalate, and amorphous urates. Crystals which occur less
frequently include calcium sulfate, sodium urates, hippuric acid, cystine, leucine, tyrosine, cholesterol, and
sulfa.
Crystals in Acidic Urine
Amorphous Urates. Urate salts such as sodium, potassium, magnesium and calcium can precipitate in
amorphous or non-crystalline forms. Microscopically, these precipitates appear as small, colorless to yellow-
brown granules much like sand, with strong birefringence (see Figure 1). Sometimes, they may appear in clumps
resembling granular casts.
Amorphous urates are frequently encountered in specimens that have been refrigerated and produce a
very characteristic pink sediment. Accumulation of the pigment, uroerythrin, on the surface of the granules is
the cause of the pink color. Amorphous urates are found in
acidic urine with a pH greater than 5.5, whereas uric acid
crystals can appear when the pH is lower. Thus, if concentrated
acetic acid is added and time allowed, amorphous urates will
convert to uric acid crystals.
Amorphous urates have no clinical significance and are
distinguished from amorphous phosphates on the basis of urine
pH, their macroscopic appearance, and their solubility
characteristics. Note that amorphous urates can also occur in a
neutral urine.
Figure 1. Amorphous urates
Acid Urates. These are sodium, potassium, and ammonium
salts of uric acid that appear as small, yellow-brown balls or
spheres with strong birefringence (see Figure 2). They appear as
larger granules and may have spicules similar to the ammonium
biurate crystals seen in alkaline urine. Because of their small,
spherical shape and color, they may be misidentified as leucine
crystals
Acid urate crystals can be present when the urine pH is neutral
to slightly acidic but frequently are not observed in fresh urine.
Similar to other urate crystals, acid urates dissolve at 60° C and
Figure 2. Acid urates

2
can be converted to uric acid crystals by the addition of glacial acetic acid. They have no clinical significance
and are reported as “urate crystals.”
Monosodium urates. These are distinct form of a uric acid salt which appear as colorless to light-yellow
slender, pencil-like prisms, with moderate birefringence (see Figure 3). They may be present singly or in small
clusters, and their ends are not pointed. Monosodium urate crystals can be present when the urine pH is acid
and dissolve at 60° C. They have no clinical significance and usually are
reported as “urate crystals.” However, they are seen in synovial fluid
during episodes of gout, but do appear in the urine.
Calcium Oxalate. Two forms of calcium oxalate exists. The most common
is the dihydrate form which is octahedral or pyramidal in shape - two
pyramids joined at their bases (see Figure 4). When viewed from one end,
they appear as squares scribed with lines that intersect in the center, hence
they are sometimes called envelope crystals. Moreso, when focusing on
other orientation, the viewer will see the “X” of the crystal popping out of
the field. The less coomon form is the calcium oxalate monohydrate
crystals (see Figure 5) which are small and ovoid or dumbbell shaped. It
can resemble RBCs and may require differentiation by polarizing
Figure 3. Monosodium urates microscopy to demonstrate the strong birefringence of these crystals.
Calcium oxalate crystals are colorless and can vary significantly in size. Usually the crystals are small
and require high-power magnification for identification. On occasion, the crystals may be large enough to be
identified under low-power magnification. Calcium oxalate crystals may cluster together and can stick to
mucous threads. When this occurs, they may be mistaken for crystal casts.
Calcium and oxalate are solutes normally found in the urine of healthy individuals. Approximately 50%
of the oxalate typically present in urine is derived from ascorbic acid (vitamin C) or from oxalic acid. Foodstuffs
high in oxalic acid or ascorbic acid include vegetables (rhubarb, tomatoes, asparagus, spinach) and citrus fruits.
In addition, beverages that are high in oxalic acid include cocoa, tea, coffee, and chocolate. As urine forms in
the renal tubules, oxalate ions associate with calcium ions to become calcium oxalate. When conditions are
optimal, calcium oxalate can precipitate in a crystalline form. Increased numbers of calcium oxalate crystals
are often observed following ingestion of the oxalate precursor ethylene glycol (antifreeze) and during severe
chronic renal disease, diabetes mellitus and liver disease.
Calcium oxalate crystals are the most frequently observed crystals in human urine, in part because they
can form in urine of any pH.

Figure 4. Dihydrate form Figure 5. Monohydrate form

3
 Uric acid. Uric acid crystals are seen in a variety of shapes, including rhombic or diamond, four-sided
flat plates (whetstones/cubes), barrel, wedges, bands and may cluster together to form rosettes (see Figure 6).
They may also have a six-sided shape, similar to cystine crystals. They usually appear yellow to golden brown
and the intensity of their color varies directly with the thickness of the crystal. As a result, they may be colorless
when they are thin or when urine is low in uroerythrin. Similarly, with the use of polarizing microscopy, uric
acid crystals exhibit strong birefringence and produce a variety of interference colors.
Uric acid crystals can be present only if the urine pH is less than 5.7. At a pH greater than 5.7, uric acid
is in its ionized form as urate and forms urate salts. Generally, uric acid crystals are 17 times less soluble than
urate salt crystals. If urine with uric acid crystals is adjusted to an alkaline pH, the crystals readily dissolve.
Uric acid is a normal urine solute that originates from the catabolism of purine nucleosides (adenosine
and guanosine from RNA and DNA). Hence, uric acid crystals can appear in urine from healthy individuals.
Increased amounts of urinary uric acid can be present following the administration of cytotoxic drugs (e.g.,
chemotherapeutic agents) and with gout and patients with Lesch-Nyhan Syndrome.

Figure 6. Rhombic form Figure 6. Rosette form

Figure 6. Barrel form

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Crystals in Alkaline Urine
Amorphous phosphates. These are non-crystalline form of phosphates that resembles fine, colorless
grains of sand in the sediment (see Figure 7). These strongly birefringent granular particles, which are also
found in neutral urine, have no definite shape and they are usually microscopically indistinguishable from
amorphous urates.
Amorphous phosphates are differentiated from
amorphous urates on the basis of urine pH, their solubility
characteristics, and, to a lesser degree, their macroscopic
appearance. Large quantities of amorphous phosphates cause a
urine specimen to appear cloudy. The precipitate/sediment button
is white or gray which is in contrast to the pink-orange color of
amorphous urates. Unlike urates, amorphous phosphates are
soluble in acid and do not dissolve when heated to approximately
60° C.
Similar to amorphous urates, amorphous phosphates have
Figure 7. Amorphous phosphates no clinical significance and can make the microscopic
examination difficult when a large quantity is present. Because
refrigeration enhances their deposition, specimens maintained at room temperature and analyzed within 2 hours
of collection minimize amorphous phosphate formation.
Triple phosphate. This is referred to as triple phosphate since this is composed of ammonium,
magnesium and phosphate materials. This is a colorless crystal with moderate birefringence that appear in
several different forms, both in alkaline and neutral urine. The most common forms are three- to six-sided
prisms with oblique terminal surfaces, described as “coffin lids” (see Figure 8). With prolonged storage, these
crystals can dissolve, taking on a feathery form that resembles a fern leaf.
Ammonium magnesium phosphate is a normal urine solute, hence triple phosphate crystals can be
present in urine from healthy individuals. Triple phosphate crystals have little clinical significance but have
been associated with UTIs and have been implicated in the formation of renal calculi.

Figure 8. Coffin-lid appearance Figure 8. Fern leaf appearance

5
 Calcium phosphate. This is present in any urine pH (alkaline, neutral and slightly acidic) as dibasic
(calcium monohydrogen phosphate) and monobasic calcium phosphate (calcium biphosphate). Dibasic calcium
phosphate crystals, sometimes called stellar phosphates, appear as colorless, thin, wedgelike prisms arranged
in small groupings or in a rosette pattern (see Figure 9). Each prism has one tapered or pointed end, with the
other end squared off. Others may appear as long needles arranged in bundles or sheaves (see Figure 9).
This dibasic form can be confused with sulfonamide crystals when the urine pH is in the neutral range.
To differentiate, this dissolves in dilute acetic acid while sulfonamides do not.
Monobasic calcium phosphate crystals appear microscopically as irregular, granular sheets (see Figure
10) or flat plates that can be large and may be noticed floating on the top of a urine specimen. These colorless
crystalline sheets can resemble large degenerating squamous epithelial cells. They are weakly birefringent with
polarizing microscopy.
Both types of calcium phosphate have no clinical significance, though calcium phosphate is a common
constituent of renal calculi.

Figure 9. Needle-like appearance Figure 9. Wedge-like appearance

Figure 10. Monobasic form

Figure 11. Magnesium phosphate

Magnesium phosphate. Magnesium phosphate crystals are large, colorless crystals that appear as
elongated rectangular or rhomboid plates. These flattened prisms can be notched and their edges can be irregular
or eroded (see Figure 11). They are weakly birefringent under polarizing microscopy. Although magnesium
phosphate crystals form from normal urine solutes in neutral, slightly acidic or alkaline urine, they are rarely
seen.
Ammonium biurate. These are yellow-brown spheres with striations/projections or spicules on the
surface giving these crystals a “thorny apple” appearance (see Figure 12). Ammonium biurate is a normal urine

6
solute that can also form in a neutral urine. These crystals occur most frequently in urine specimens that have
undergone prolonged storage. However, when they form precipitate in fresh urine specimens (such as when
following iatrogenically induced alkalinization and inadequate hydration of the patient), they are clinically
significant because in vivo precipitation can cause renal tubular damage.
Ammonium biurate crystals are strongly birefringent and dissolve in acetic acid or on heating to
approximately 60° C. Similar to other urate salts, ammonium biurate crystals can be converted to uric acid
crystals with the addition of concentrated hydrochloric or acetic acid. An important note is that ammonium
biurate crystals can resemble some forms of sulfonamide crystals. One differentiates between them on the basis
of urine pH, a sulfonamide confirmatory test, and the solubility characteristics of the crystals.
Calcium Carbonate. These are tiny, colorless granular crystals that
assume dumbbell, spherical or tetrad shapes and is slightly larger than
amorphous material (See Figure 13). They may occur in clumps that
resemble amorphous material, but they can be distinguished by the
formation of gas after the addition of acetic acid. They are also strongly
birefringent, which differentiates them from bacteria.
Present primarily in alkaline urine, calcium carbonate crystals are not
found frequently in the urine sediment. Although they have no clinical
significance, one can identify calcium carbonate crystals positively
through the production of carbon dioxide gas (effervescence) with the
addition of acetic acid to the sediment.

Figure 12. Ammonium biurate

Figure 13. Calcium carbonate

ABNORMAL URINE CRYSTALS

Abnormal urine crystals are found in acidic urine or rarely in neutral urine. Most abnormal crystals
have very characteristic shapes. However, their identity should be confirmed by chemical tests or by any
medication of the patient. Iatrogenic (drugs and medications) crystals can be caused by a variety of compounds,
particularly when they are administered in high concentrations. They may be of clinical significance when they
precipitate in the renal tubules.

7
Crystals of Metabolic Origin
Bilirubin. Bilirubin crystals usually appear as yellow-brown small clusters of fine needles (20 to 30
µm in diameter), and sometimes in granules and plates (see Figure
14) and only form in an acidic urine. They dissolve when alkali or
strong acids are added. They are classified as abnormal crystals
because bilirubinuria indicates a metabolic disease process.
However, because these crystals form in the urine after excretion
and cooling (refrigeration), they are not frequently observed and
they are not usually reported.
Bilirubin crystals are confirmed by correlation with the chemical
examination, that is, the crystals can be present only if the chemical
screen for bilirubin is positive.

Figure 14. Bilirubin Crystals

Cystine. These are clear, colorless, hexagonal plates with uneven sides and are weak to moderately
birefringent (see Figure 15). They are often refractile, laminated or layered and tend to clump. They are present
primarily in acidic urine but can be found in neutral and alkaline urine.
Cystine crystals are found in the urine of persons who inherit a
metabolic disorder that prevents reabsorption of cystine by the renal
tubules (cystinuria). Persons with cystinuria have a tendency to
form renal calculi, particularly at an early age.
Cystine crystals can be present in urine with a pH of less than 8.3.
They dissolve in alkali and hydrochloric acid (pH <2). In vivo, the
solubility of cystine rises exponentially with urine pH such that it
is almost four times more soluble at pH 8 than at pH 5. Cystine
crystals are insoluble in acetic acid, alcohol, acetone, ether, and
Figure 15. Cystine Crystals boiling water and are soluble in hydrochloric acid.
Disintegrating forms of cystine may be seen in the presence of ammonia. They may be difficult to
differentiate from colorless uric acid crystals. Uric acid crystals are very birefringent under polarized
microscopy, whereas only thick cystine crystals have polarizing capability. Cystine can be detected chemically
with the sodium cyanide–sodium nitroprusside test.
Tyrosine. Tyrosine crystals in acidic uric appear as fine, delicate needles that are colorless or yellow
(see Figure 16). They frequently aggregate to form clusters or sheaves but also may appear singly or in small
groups. The needle clusters often appear to be black, especially in the center, but they may take on a yellow
color in the presence of bilirubin. Tyrosine crystals are soluble in ammonium hydroxide and hydrochloric acid
but insoluble in acetic acid. In addition, these crystals are rarely seen because they require refrigeration to force
them out of solution.
Tyrosine crystals are often present in the urine of patients with tyrosinosis and oasthouse urine disease.
Although rare, these crystals have been observed in the urine of patients with severe liver disease.
Leucine. Leucine crystals are highly refractile, strongly birefringent, yellow to brown spheres and can
resemble fat globules when observed in an acidic urine (see Figure 17). They have concentric circles or radial
striations on their surface.

8
 Just like tyrosine, these crystals are rarely seen because they require refrigeration or addition of alcohol
to force them out from the sample. Of the two, tyrosine is found more often in urine because leucine is more
soluble. These amino acid crystals are abnormal and are present
in the urine of patients with cystinuria, an overflow
aminoaciduria and a rare inherited metabolic disorder. Although
uncommon, these crystals have been observed in the urine of
patients with severe liver disease. These abnormal crystals are
confirmed, preferably by chromatographic methods, before they
are reported.

Figure 16. Tyrosine Crystals

Cholesterol. These are clear, flat, rectangular plates
with notched corners (see Figure 18) present in acidic urine and
can be in neutral urine and, because of their organic
composition, are soluble in chloroform and ether. They are
characterized by weak birefringence.
Rarely observed in urine sediment, cholesterol crystals
indicate large amounts of urine cholesterol and ideal Figure 17. Leucine Crystals
conditions that have promoted supersaturation and precipitation. Other evidence of lipids, such as free-floating
fat droplets, fatty casts, or oval fat bodies and large amounts of protein, accompany these crystals.
Cholesterol crystals can be seen with the nephrotic syndrome and in conditions resulting in chyluria:
the rupture of lymphatic vessels into the renal tubules as a result of tumors, filariasis, and chronic renal diseases.
Intravenous radiopaque contrast media (x-ray dye) such as
meglumine diatrizoate and diatrizoate sodium form flat and
layered rectangular crystals that are morphologically similar to
and need to be differentiated from cholesterol crystals. For
example, radiopaque contrast media produces an abnormally
high urine specific gravity and they are not associated with
proteinuria or lipiduria. In contrast, cholesterol crystals are seen
in urine with a normal specific gravity and must be
accompanied by proteinuria and lipiduria.

Figure 18. Cholesterol Crystals

9
Crystals of Iatrogenic Origin
Iatrogenic crystals are crystals induced in patients as a result of a particular treatment (e.g.,
medications). Generally, most medications and their metabolites are eliminated from the body by the kidneys.
As urine forms within the nephrons, high concentrations of these agents can cause their precipitation out of
solution. Proper identification and reporting of drug crystals are important because if the crystals are forming
in vivo, such as in the renal tubules, they can cause kidney damage. Two common drugs (antibiotics),
sulfonamides and ampicillin, are known for their ability to form crystals in urine.
Hippuric acid. Hippuric acid crystals are yellow–brown or colorless elongated prisms or plates (see
Figure 19). They may be so thin as to resemble needles, and they often cluster together. Hippuric acid crystals
are more soluble in water and ether than are uric acid crystals. They are rarely seen in the urine and have
practically no clinical significance.
Ampicillin. These are long, colorless, strongly birefringent thin prisms or needle-like crystals (see
Figure 20) that may aggregate into small groupings or, with refrigeration, into large clusters. In an acidic urine,
ampicillin crystals indicate large doses of ampicillin and are rarely observed with adequate hydration.
Indinavir. These are slender, feather-like crystals, with moderate to strong birefringence, that aggregate
into wing-like bundles, which can also associate into a rosette-like or cross form (see Figure 21). These can be
present in an acid urine but are more often observed in neutral and alkaline urines.
Sulfamethoxazole/Sulfonamides. These are highly refractile and strongly birefringent crystals that
appear as colorless to brown rosettes, spheres, whetstones or rhombic with irregular radial striations (see Figure
22 Sometimes, they appear as bundles of needles that resemble sheaves of wheat. The constriction of the bundle
may be located centrally or extremely eccentric, resulting in a fan formation.
Inadequate patient hydration was and still is the primary cause of sulfonamide crystallization. The
appearance of sulfonamide crystals in fresh urine can suggest the possibility of UTI or tubular damage if crystals
are forming in the nephron.
Radiographic contrast media crystals. Crystals of radiographic contrast media following retrograde
administration appear as colorless, long, rectangular needles that occur singly or clustered in sheaves (see Figure
23). When administered intravenously, they appear as flat, elongated rectangular plates with strong
birefringence. In contrast with cholesterol crystals, these types of crystals occur in large number in one field of
view.

Figure 19.. Hippuric acid Figure 20. Ampicillin

10
 Figure 21. Indinavir Figure 22. Sulfonamides

Figure 23. Radiographic

Contrast media

ELABORATE: CONNECTING CONCEPTS

MISCELLANEOUS ELEMENTS
Other structures which may be present in the urine include bacteria, yeast, spermatozoa, mucus, and
fats. Chemical analysis does not detect most of these types of sediment. Microscopic evaluation of urinary
sediment is important if these structures are to be detected.
Mucus threads
This is a fibrillar protein (some contain uromodulin) that comes from the renal tubules and vaginal
epithelium (women). It has a very low refractive index and thus better visualized when phase contrast or
interference contrast microscopy is used. They are readily identified
by their delicate, wavy, ribbon-like strands and irregular or serrated
ends. They can be present also as distinct strands or as a clumped
mass.
Mucus threads are present in normal urine in small numbers and has
no clinical significance. However, they may be very abundant in the
Figure 24. Mucus threads presence of inflammation or irritation of the urinary tract. Some of

11
the wider threads may be confused with cylindroids or hyaline casts. The cylindrical composition of casts and
their rounded ends aid in their differentiation from mucus.
Fats
These are found in urine as free-floating fat globules, within oval fat bodies, or within a cast matrix.
They are highly refractile and appear colorless to yellow-green or even brownish. These fats include
triglycerides and cholesterol.
Lipids are excreted in urine in cases of diabetes mellitus and when
there is damage to the glomerular filtration barrier like in nephrotic
syndrome. In preeclampsia, fat is often present and can persist for
several weeks after delivery. Extreme physical exercise (e.g.,
marathon) and crush injuries also result to lipiduria.
Oils and fats from lubricants, ointments, creams, and lotions can
contaminate urine. It is also important to note that urine from men
with prostatitis may contain oval-fat bodies and free-floating fat
globules. Regardless of the source, these contaminating lipids are
Figure 25. Fats often indicated by the lack of proteinuria, fatty casts, and oval fat
bodies.
Hemosiderin
Hemosiderin is a form of iron that results from ferritin
denaturation. These appear as coarse yellow-brown granules and are
difficult to distinguish from amorphous crystalline material in the
sediment. These are usually found 2 to 3 days after a severe hemolytic
episode such as in transfusion reactions and paroxysmal nocturnal
hemoglobinuria.
Hemosiderin granules may be found free floating or within macrophages,
casts, or tubular epithelial cells. The Prussian blue reaction, also known
Figure 26. Hemosiderin as the Rous test, is used to identify hemosiderin in the urine sediment and
in tissues.
Sperm
Sperm cells or spermatozoa may be present in urine sediment
from males and females. The presence of motile sperm indicates recent
intercourse or ejaculation. In urine from women, sperm are usually
considered a vaginal contaminant. It is important to report the presence of
sperm in urine from females because it could potentially identify sexual
abuse in underage and other vulnerable females. In urine from men, sperm
can be present owing to nocturnal emissions, from normal or retrograde
ejaculation.

Figure 27. Sperm cells

12
Microorganisms
Normally, the urinary tract is sterile such that no microorganisms are present. Consequently, the
presence of bacteria, yeast, trichomonads, or parasites in urine indicates an infection or that contamination
occurred during the collection process.
Bacteria. The most commonly encountered bacteria in urine are bacilli but
coccoid forms can also be present. In wet preparations, their motility often
distinguishes bacteria from amorphous substances that may be present.
Because the skin, vagina, and gastrointestinal tract normally contain
bacteria, the presence of bacteria in urine often reflects contamination from
these sources. However, it may also imply UTI or because of a fistula
between the urinary tract and the bowel.
Contaminating bacteria multiply rapidly in improperly stored urine.
Therefore, the presence of bacteria has clinical significance only if the urine
specimen has been properly collected and stored. When significant bacteriuria
Figure 28. Bacteria is present without leukocytes, the specimen collection and handling should be
investigated.
Yeast cells. Yeasts are ovoid, colorless cells, about 10-12 um, that are more refractile than erythrocytes
and have a characteristic budding form and pseudohyphae (in severe infections). They do not dissolve in acid
and usually do not stain with supravital stains.
In women, yeast in the urine sediment most often indicates
contamination of the urine with vaginal secretions. Similarly, because it
can be found in the air and on skin, their presence in urine could indicate
contamination from these sources.
Urinary tract infections resulting from yeasts are possible. Certain
situations such as pregnancy, use of oral contraceptives, vaginal
moniliasis, kidney disease, immunocompromised patients and diabetes
mellitus may also promote the development of vaginal yeast infection.
The most commonly encountered yeast in urine sediment is Candida
Figure 29. Yeast cells albicans and rarely found is Candida glabrata, the latter not involved in
pseudohyphae formation.
During the microscopic examination, only the presence of yeast can be determined. A true yeast
infection should be accompanied by the presence of WBCs. The identification of the species requires fungal
culture.
Parasites. Several parasites can be observed in the urine sediment.
These include the eggs of Enterobius vermicularis, cysts of Giardia
lamblia and eggs of the blood fluke Schistosoma haematobium; the
former two being associated with fecal contamination.
Trichomonas vaginalis is the most common cause of parasitic
Figure 30. T. vaginalis gynecologic infection in female patients. They appear as turnip-
shaped flagellates with unicellular bodies averaging 15 µm in length.
Transmitted sexually, trichomonads most frequently represent an

13
infection of the vagina and/or urethra, and their presence in the urine often indicates contamination with vaginal
secretions. In male patients, trichomonad infections of the urethra are usually asymptomatic.
Urine to be analyzed should be freshly collected as old specimen may no longer demonstrate their
motility.
Contaminants
Starch granules. Starch granule contamination may occur when cornstarch is the powder used in
powdered gloves. The granules are highly refractile spheres, usually with a dimpled center. They resemble fat
droplets when polarized, producing a Maltese cross formation. Starch granules may also occasionally be
confused with RBCs.
Fibers (Hair, Cloth, Diaper). Fibers are short to long, flat structures often mistaken for casts. To
differentiate, they usually have dark edges while casts do not have dark edges. Second, most of the fibers are
flat while casts are cylindrical.

Diaper fiber Cloth fiber

Glass chards Talcum powder

14
Pollen debris with concentric Fecal contaminant
circle

Starch Granules

15
 EVALUATE: ACTIVITY ALERT
This is a graded activity worth 10 points.
To assess your understanding on the concepts presented, answer the given questions below.
TRUE OR FALSE (10 POINTS). Write TRUE if the statement is correct and FALSE if incorrect.

1. Cystine is an abnormal crystal that can be associated with stone formation.

2. Bilirubin crystals are seen in an acidic urine.
3. All crystals seen in urine exhibit birefringence.
4. The use of polarizing microscopy is not particularly helpful in the identification of urine crystals.
5. With bright microscopy, starch granules demonstrate a maltese cross appearance.
6. Calcium oxalate may appear in any urine pH.
7. The presence of bilirubin crystals does not affect urine clarity and specific gravity.
8. The rhombic form of uric acid is often mistaken as cystine.
9. Amorphous urates are differentiated with phosphates in terms of solubility, pH and microscopic
appearance.
10. Thorny apple appearing crystals are commonly observed in a freshly voided, alkaline urine.

16