Study of Pattern of Urinary Sediments in Renal Diseases

STUDY OF PATTERN OF URINARY SEDIMENTS IN

RENAL DISEASES

DISSERTATION

SUBMITTED TO THE TAMILNADU DR.M.G.R. MEDICAL UNIVERSITY

CHENNAI

In partial fulfillment of the requirements for the degree of

M.D. (PATHOLOGY)

BRANCH – III

DEPARTMENT OF PATHOLOGY

TIRUNELVELI MEDICAL COLLEGE HOSPITAL

TIRUNELVELI – 627011

CERTIFICATE

This is to certify that the dissertation titled “STUDY OF PATTERN OF

URINARY SEDIMENTS IN RENAL DISEASES”, is a bonafide work done by

Dr.MOHAN MURUGESAN, Post Graduate Student, Department of Pathology,

Tirunelveli Medical College, Tirunelveli – 627011, in partial fulfillment of the

university rules and regulations for the award of MD DEGREE in PATHOLOGY

BRANCH-III, under my guidance and supervision, during the academic period

from 2014 – 2017.

Prof. SITHY ATHIYA MUNAVARAH. M.D.,

Dean,

CERTIFICATE

I hereby certify that this dissertation entitled “STUDY OF PATTERN OF

URINARY SEDIMENTS IN RENAL DISEASES” is a record of work done by

Dr.MOHAN MURUGESAN, in the Department of Pathology, Tirunelveli

Medical College, Tirunelveli, during his postgraduate degree course period from

2014- 2017. This work has not formed the basis for previous award of any

degree.

Prof. K. SHANTARAMAN. M.D.,

Department of pathology, Tirunelveli Medical College, Tirunelveli- 627011.

Prof. K. SHANTARAMAN. M.D.,

DECLARATION

I solemnly declare that the dissertation titled “STUDY OF PATTERN OF

URINARY SEDIMENTS IN RENAL DISEASES” was done by me at

Tirunelveli Medical College, Tirunelveli – 627011, during the period 2014 - 2017

under the guidance and supervision of Prof. K.SHANTARAMAN.M.D., to be

submitted to The Tamil Nadu Dr. M.G.R. Medical University towards the partial

fulfillment of requirements for the award of MD DEGREE in PATHOLOGY

BRANCH-III.

Place : Tirunelveli

Date :

Dr.MOHAN MURUGESAN, Post Graduate Student, Department of Pathology, Tirunelveli Medical College,

ACKNOWLEDGEMENT

I thank Professor Dr. SITHY ATHIYA MUNAVARAH. M.D., Dean,

Tirunelveli Medical College, for having permitted me to conduct the study and

use the hospital resources in the study.

I express my heartfelt gratitude to Professor Dr. SHANTARAMAN.K.

M.D., Professor and Head, Department of Pathology, for his inspiration, advice

and guidance in making this work complete.

I am extremely thankful to Professors Dr. VALLIMANALAN. S. M.D.,

Dr. SWAMINATHAN . K. M.D., Dr. SURESH DURAI. J. M.D., Dr. ARASI

RAJESH.M.D., DR.VASUKI.M.D., Additional Professors, Department of

Pathology, for guiding me academically and professionally during the period of

study. I also thank the Assistant Professors, for their encouragement and support.

I sincerely thank Professor Dr. RAMASUBRAMANIAN. V. M.D., D.M.,

and faculties of the Department of Nephrology for providing me the patients for

my study and guiding me during the period of study.

I also thank all the lab technicians especially Mr. Balamurugan and

Mrs. Premalatha for their valued assistance and my fellow postgraduates for their

cooperation which enormously helped me in the study. I am also indebted to thank

all the patients and their caring relatives for without their humble cooperation, this

ABBREVIATIONS

GFR GLOMERULAR FILTERATION RATE

SLE SYSTEMIC LUPUS NEPHRITIS

RBCs RED BLOOD CORPUSCLES

WBCs WHITE BLOOD CORPUSCLES

HDL HIGH DENSITY LIPOPROTEIN

RTECs RENAL TUBULAR EPITHELIAL CELLS

THG TAMM HORSFALL GLYCOPROTEIN

MCD MINIMAL CHANGE DISEASE

FSGS FOCAL SEGMENTAL GLOMERULOSCLEROSIS

IgAN IgA NEPHROPATHY

HPF HIGH POWER FIELD

MPGN MEMBRANOPROLIFERATIVE

GLOMERULONEPHRITIS

LN LUPUS NEPHRITIS

DN DIABETIC NEPHROPATHY

AIN ACUTE INTERSTITIAL NEPHRITIS

NSAIDS NON STEROIDAL ANTI INFLAMMATORY DRUGS

CIN CHRONIC INTERSTITIAL NEPHRITIS

AN ANALGESIC NEPHROPATHY

ATN ACUTE TUBULAR NECROSIS

IMH ISOLATED MICROSCOPIC HAEMATURIA

AKI ACUTE KIDNEY INJURY

AKIN ACUTE KIDNEY INJURY NETWORK

LPF LOW POWER FIELD

S.D. STANDARD DEVIATION

CONTENTS

1. INTRODUCTION 1

2. AIMS AND OBJECTIVES 3

3. REVIEW OF LITERATURE 4

4. MATERIALS AND METHODS 49

5. RESULTS AND OBSERVATION 55

6. DISCUSSION 72

7. SUMMARY AND CONCLUSION 80

8. BIBLIOGRAPHY

1

INTRODUCTION

Urinary microscopy is an integral part of the clinical evaluation of patients

with kidney disorders and frequently is used to differentiate a number of clinical

conditions (eg, nephrotic syndrome, nephritic syndrome). The findings also can

guide medical interventions and improve patient management. The examination of

the urinary sediment, coupled with the assessment of proteinuria, allows the

identification of different urinary profiles, which can be caused by various

clinical conditions. These urinary profiles are: the nephrotic pattern; the nephritic

pattern; the nephrotic and nephritic pattern; the sediment pattern with many renal

tubular epithelial cells; the sediment pattern with increased numbers of

erythrocytes and the leukocyturia. Hyaline cylindruria with a few erythrocytes

and leukocytes may be seen in normal subjects. The concentration of creatinine in

serum is the most widely used and commonly accepted measure of renal function

in clinical medicine. The clinical utility of the serum creatinine concentration

centers on its relation to the glomerular filtration rate (GFR). In renal patients, the

serum creatinine level may remain normal with abnormal urinalysis as in case of

non proliferative glomerulonephritis; increased in level with abnormal urinalysis

as seen in diseases like proliferative glomerulonephritis, acute kidney injury with

acute tubular necrosis; increased level with normal urinalysis as seen in cases of

hypertensive nephrosclerosis and ischemic renal diseases.

Urinary sediment scoring system can be used to assess the role of urine

2

tubular epithelial cell casts constitute two parameters needed for calculating a

score.

Renal diseases are seldom diagnosed in the early stages because the

biochemical parameters are not elevated until late.Investigations for detection of

early stages of renal disease are of immense importance. Urinalysis yields a lot of

valuable information regarding the functioning of the kidney, when properly

performed, hence is called “liquid renal biopsy”.Properly conducted urine

examination can indicate subtle changes in renal function. Urine examination is

simple and cost effective. It has added values in the diagnostic workup of renal

diseases. Several systemic diseases like diabetic mellitus, systemic lupus nephritis

(S.L.E) causes significant damage to kidneys where regular monitoring is required

for detection of renal injury. In these conditions, which necessitates repeated

3

AIMS AND OBJECTIVES

1. To describe the various patterns of urinary sediments in patients with renal

diseases.

2. To correlate these urine sediment patterns described with type and severity

of renal damage namely the histopathological patterns in renal biopsy,

4

REVIEW OF LITERATURE

The earliest microscopic examination of urine was done by Nicolaus

Fabricius de Peiresc [1] in 1630, to study the nature of the urinary stones. Pierre

Rayer and Eugene Napoleon Vigla introduced urine microscopy into clinical

practice[1].Their study was comprehensive and of paramount importance. They

explained about the handling of urine sample, microscope, and the importance of

correlating microscopic examination with biochemical parameters. They

described in detail about the crystals, and squamous epithelial cells, mucus, pus

cells, erythrocytes, lipids, sperm and yeasts. They mentioned about thin and light

lamellae which was probably casts and also described the urinary findings in

normal subjects and many pathological conditions. They stated that apparently

normal urine can contain erythrocytes or leukocytes which can be identified only

with the microscope, thus presenting the concepts of microscopic hematuria and

pyuria. In acute nephritis plenty of blood corpuscles, mucus globules, squamous

epithelial cells, and fibrin strands. In patients with nephrotic syndrome, thin

lamellae of amorphous material probably casts, mucus globules, lipids, blood

corpuscles, uric acid crystals, scanty phosphates. In acute and chronic

pyelonephritis, pus globules, blood corpuscles, necrotic cellular debris, uric acid

crystals or crystals of triple phosphate. Finally, the association between lipiduria

and albuminuria. All these observations were done during a period in which

microscopy as a diagnostic tool was at its initial stages. In 1837, Gabriel Gustav

5

tubules[1]. In 1842 Johann Franz Simon, described them in urine[1]. Henry Bence

Jones, discovered light chain proteinuria[1]. He described urinary casts and

explained the association between casts, Bright's disease and albuminuria.

Thomas Addis in 1920, performed microscopic examination of urine in patients of

glomerulonephritis serially over the years and detected that an abundant sediment

containing large numbers of erythrocytes (RBCs), leucocytes(WBCs), epithelial

cells and casts could become more and more scanty over time[1]. This specified

the transformation of an active disease into a chronic process. For quantification

of urinary sediments Addis introduced collection of timed urine samples and the

use of counting chambers, which was termed as "Addis count"[1]. Based on the

results Bright's disease was classified into three types, that is, haemorrhagic,

degenerative, and arteriosclerotic. Addis also defined broad renal failure casts in

renal failure patients in a period in which biochemical tests were not readily

available as they are today. In 1960s urinary sediments were studied using new

microscopic techniques. The final evidence to previous in vitro studies that

Tamm-Horsfall glycoprotein is the matrix of casts was provided by McQueen

using immunofluorescent-labelled antibodies [1]. In early 1950s phase contrast

microscopy was discovered and recommended for urinalysis in 1968 by Robert

Kark[1].In early 1970s transmission electron microscopy was used to identify

amyloid fibrils in urine samples of patients with renal amyloidosis, and then in

acute tubular necrosis. Few years later scanning electron microscope was utilised

6

The examination of the urinary sediment, which is an integral part of urinalysis, is

an irreplaceable tool for the diagnosis and monitoring of the diseases of the

kidneys and the urinary tract [2,3]. However, reliable results can be obtained only

by using the correct methodology.

Collection of urine :

International guidelines suggest the procedures for urine collection [4,5]. For

routine examination any fresh specimen of urine is adequate. It is best to collect

an early morning specimen, which is voided when the patient first arises from a

night’s sleep, as it is the most concentrated single specimen, and it has the lowest

pH, which tends to preserve the formed elements well. The second urine of the

morning, is usually concentrated and acidic, and without the lysis of the elements

which can occur in overnight urine due to the prolonged permanence in the

bladder[6]. Highly alkaline urine favours the lysis of leukocytes[7], casts [8] and

precipitation of phosphates which can mask other urinary elements. In order to

minimize contamination, hands and external genitalia must be cleaned with water,

for females, spreading of the labia of vagina, for males the retraction of the

foreskin of the glans. The midstream technique is the recommended procedure in

which the first portion of the urine is discarded, since it may be contaminated with

cellular elements and bacteria from the external urinary tract and genital area [5].

Macroscopic examination:

Macroscopic inspection reveal the presence of turbidity and of abnormal

changes of the urine colour. Turbidity is caused by large numbers of squamous

7

urates[9] in majority of cases. However, pathological samples are often perfectly

clear. Therefore, the absence of turbidity in itself is not a reliable criterion to

judge a urine sample. Abnormal urine colour can be due to a large number of

causes (Tables1 and 2)[10]. In some conditions such as gross haematuria,

bilirubinuria, or chyluria , the examination of the urinary sediment allows us to

identify the cause of abnormal urine colour. It is best to examine the samples

within 3 hours of collection, to avoid bacterial overgrowth, dissolution of casts

and cells, and contamination from the environment. This is because in some

instances leukocytes can lyse in less than 1 hour especially in samples with a high

pH ( > 7.0) and/or a low specific gravity ( < 1.010). This can be prevented by

refrigeration of the samples at + 2 °C to + 8 °C[4,5]. However at these temperatures

phosphates and urates may precipitate with a masking of important sediments. A

possible alternative is the use of preservatives such as formaldehyde[11],

glutaraldehyde[12] and cellfix[13].

Table 1 : The main causes of abnormal urine colour.

ENDOGENOUS CAUSES EXOGENOUS CAUSES

Haemoglobin Vegetable derived substances

Myoglobin Alimentary pigments

Bilirubin , biliverdin Drugs

8

Table 2 : Differentiation of the causes of changes in the colour of the urine.

COLOR CAUSES

Reddish brown Increased urobilinogen or porphyrins

Bright red Large amount of fresh blood

Pink Small amount of blood

Smoky brown Blood pigments

Brownish yellow or green Bile pigments

Milky white Chyluria

International guidelines recommend standardized procedures for

centrifugation and the subsequent steps of urine preparation and examination.

Centrifugation is used to concentrate the formed elements of the urine, but if it is

not performed properly, it may introduce relevant biases, mainly due to partial

recovery of the elements present in the urine. Gadeholt [14] showed that the best

recovery of erythrocytes and leukocytes was obtained at 2,500 r.p.m. (~ 1,120 g),

whilst a lower recovery was found at 1,000 and 3,000 r.p.m, using a centrifuge

with a radius of 16 cm and a centrifugation time of 5 min. Therefore, the yield is

strongly influenced by the speed of centrifugation. Other factors are the duration

of centrifugation and the volume of urine centrifuged. Hence the same

combination of speed and duration of centrifugation, as well as the same volume

of urine is to be used for obtaining reproducible results[15].The speed of

centrifugation is most often expressed as rotations per minute (rpm) or as “relative

centrifugal force” (RCF) or “g”. To calculate this, one must know the radius of the

centrifuge used. The RCF is obtained by the following formula [4]:

9

where: r = the radius in cm from the center of the spindle to the bottom of the

tube;

N = rotations per minute.

Centrifugation is important, for instance, for erythrocytic casts which, in

patients with isolated microscopic haematuria, we find in low numbers even in

centrifuged samples. After centrifugation, the supernatant is discarded. While in

many laboratories this is still done by pouring off but international guidelines

recommend removing a standardized volume of urine. This helps to standardize

the procedure and increases its reproducibility. To resuspend the pellet, the test

tubes were agitated gently by hand. A standardized volume of the resuspended

urine should be transferred to the slide.

Microscopic examination:

The urinary sediment is to be examined after obtaining the results for urine

protein level mainly. The knowledge of the dipstick results are also important

because it helps to direct the examination of the urinary sediment. At high specific

gravity, erythrocytes and leukocytes become smaller and at a specific gravity of ≤

1.010, erythrocytes and leukocytes swell and can undergo considerable lysis [16].

In addition, cytoplasmic Brownian movements and loss of nuclear segmentation

of leukocytes occurs [17]. In fact, a negative dipstick for haemoglobin, leukocyte

esterase, nitrites, or albumin has a high probability of being associated with

negative microscopy, while a dipstick positive for one or more such analytes

directs microscopy investigation towards the search of erythrocytes, leukocytes,

10

haemoglobin or leukocyte esterase but with no or only few erythrocytes or

leukocytes at microscopy, suggests cell lysis, which may be due to low urinary

specific gravity and/or high pH. In such a case, microscopy without dipstick

would give a false negative result. On the contrary, a case with negative dipstick

for haemoglobin or leukocyte esterase but with microscopy positive for

erythrocytes or leukocytes may be due to the presence in the urine of ascorbic acid

(which reduces the sensitivity of the pad for haemoglobin) or of cephalotine

(which reduces the sensitivity of the pad for leukocyte esterase) [2,3].

Once the slide is put under the microscope, it is to be examined without

delay to avoid changes due to the heat caused by the light beam of the microscope

or the drying up of the sample. The slide is to be examined at low magnification.

This is essential for an overview of the sample, and to analyse the distribution of

the elements. Casts tend to collect at the edges. Then, proceed to higher

magnification to identify the elements properly. Since these usually lie on

different planes, frequent adjustments of focus are necessary. For every case,

examine at least 20 random low and high microscopic fields. The following

microscopic particles were examined:

- erythrocytes (glomerular or non glomerular )

- leukocytes

- renal tubular epithelial cells

- transitional epithelial cells

- squamous epithelial cells

11

- casts with their subtypes

- other elements

The microscope for the examination of the urinary sediment must be of

good quality and equipped with at least a low magnification (e.g., x 100) and a

high magnification (x 400). Bright field microscopy has traditionally been used

for the analysis of urinary sediment, and is still widely used today [18]. However,

with this type of microscopy, all the elements of the urinary sediment are poorly

differentiated from the background with some exception for lipids, crystals, and

waxy casts. Therefore, particles with low refractive index such as hyaline casts

and erythrocytes with low haemoglobin content can easily be missed. In addition,

cellular details are poorly distinguishable. Some improvement may be obtained by

downward adjustment of the condenser, by closing the diaphragm of the

condenser. The urine can contain different types of cells, some of which derive

from the circulation, while others derive from the epithelia of the urinary system.

Other cells, such as podocytes [19] , basophilic leukocytes, platelets or monocytes

have also been described in urine. However, they were identified with

sophisticated techniques.

Erythrocytes :

Erythrocytes are a frequent finding in urine. Erythrocyte diameter,

refractivity index and morphology can vary under various conditions. The

diameter of erythrocytes ranges from 4.0 to about 10 μm, and influenced by

changes in specific gravity (or osmolality), increasing as specific gravity

12

about ≤ 1.010, the erythrocytes tend to undergo lysis, a fact which can cause false

negative results and discrepancies between microscopy and dipstick for

haemoglobin. The refractivity index of erythrocytes varies according to their

haemoglobin content. When this is very low, the erythrocyte is hardly discernible,

especially with bright field microscopy. In such a case, a thin cell membrane is the

only identifiable structure (the so-called “ghost cell”).

The morphology of urinary erythrocytes ranges from perfectly round cells

to particles with very changed shape. Fairley and Birch in 1982 [24] confirmed that

altered erythrocytes were typical of patients with Bright’s disease (i.e.,

glomerulonephritis).These investigators were the first to report in modern times

that in haematuria of glomerular origin, erythrocytes have an abnormal shape (the

so-called “dysmorphic erythrocytes”) , while in haematuria of non-glomerular

origin, erythrocytes have a normal appearance, similar to that of erythrocytes seen

in peripheral blood smears (the so-called “isomorphic erythrocytes”). Pollock

C.et.al confirmed the utility of examining urinary erythrocyte morphology [20].

The evaluation of urinary erythrocytes morphology is associated with certain

limitation [21] with lack of univocal criteria to define a haematuria as glomerular or

non-glomerular. In fact, the discriminating cut off was as low as 10% [22]. Koene

R.A.P.et.al.[23] defined a haematuria as glomerular when more than two

erythrocyte subtypes were found in the same sample. Köhler H.et.al. [25] defined it

when ≥ 5% “acanthocytes” were seen, which are doughnut-shaped dysmorphic

erythrocytes with one or more vesicle-like protrusions, which can be identified

13

The wide spectrum of appearances that erythrocytes may have in the urine,

in the category of both dysmorphic and isomorphic erythrocytes may lead to low

inter-observer reproducibility [26].

A non glomerular haematuria can be found in patients with a glomerular

disease due to gross haematuria [27], renal insufficiency, increased diuresis after

furosemide administration or necrotizing glomerulonephritis [28]. Haematuria is

considered as glomerular when we find ≥ 40% dysmorphic erythrocytes and /or ≥

5% acanthocytes [25] and/or ≥ 1 erythrocytic casts/50 low power fields. With this

approach, Fogazzi G.B.et al. have been able to find a close correlation with the

presence of glomerular changes at renal biopsy were present[29].

The cause of glomerular erythrocyte dysmorphism is not entirely known.

In vitro experiments have shown that neither osmolality nor pH changes of

solutions in which erythrocytes are suspended are sufficient to cause dysmorphic

morphology [30]. On the contrary, this can be produced if osmolality or pH

changes are coupled with the passage of erythrocytes through membranes with

pores having a diameter of 3.0 μm. In addition, erythrocytes develop dysmorphic

features if they are serially incubated with different solutions corresponding to

those of the different tubular segments, and finally are also incubated with a

solution containing a haemolytic substance, derived from red cell lysate [31]. These

data led to the hypothesis that in vivo erythrocytes become dysmorphic as a

consequence of a dual injury [32]. The first injury is thought to result from the

passage through “gaps” in the glomerular basement membrane, while the second

14

pH/osmolality changes or unidentified substances interfere with the ability of the

cells to regain their original shape. Rarely, urinary erythrocytes may have

morphological changes due to causes unrelated to glomerular diseases. Urinary

erythrocytes may have morphological changes due to causes unrelated to

glomerular diseases like in cases of haematuria caused by sickle cell disease,

whose urine can show sickle erythrocytes [33, 34], and in patients with urological

haematuria and concomitant iron deficiency anaemia, whose urine can contain

anisocytes and poikilocytes [35].

Leukocytes:

Neutrophils are the leukocytes most frequently found in the urinary

sediment. Typically they appear as round granular cells, granules representing

cytoplasmic organelles. Their diameter ranges from about 7.0 to 15.0 μm.

However, substantial differences in diameter and morphology may be caused by

differences in urine specific gravity or osmolality. In diluted urine, the cell is

larger and in case of concentrated urine, the identification of the lobulated nucleus

may be difficult. Occasionally, for unknown reasons, neutrophils may show blebs

protruding from the cell body or may have an elongated shape. On microscopic

examination of the sample, neutrophils undergo degeneration and transform into

larger cells with irregular shape and a thin transparent granular cytoplasm, hardly

distinguishable from the background. Neutrophils may also appear in clumps,

which is seen especially in urinary tract infection. Bacterial urinary tract infection

is the most frequent cause of neutrophiluria. They are seen in a wide spectrum of

15

interstitial nephritis, polycystic kidney disease, or urologic disorders. In women,

neutrophils may be found in the urine because of contamination by genital

secretions with presence of large amounts of squamous epithelial cells with or

without bacteria, Candida, or Trichomonas vaginalis.

Eosinophils too may be present in the urine. They can definitely be

identified only by using special stains like May-Grünwald-Giemsa, Wright’s or

Hansel’s stains. Eosinophiluria can be found in a wide spectrum of diseases

including acute interstitial nephritis caused by methicillin[37] urinary tract

infection, prostatitis, extracapillary glomerulonephritis, Henoch- Schonlein

purpura nephritis, acute allograft rejection or urinary schistosomiasis.

Eosinophiluria is now considered as an unspecific finding of much less diagnostic

importance [38].

Lymphocytes can be identified with certainty only with specific or general

stain preparations, such as Papanicolou’s stain. Lymphocyturia is considered as

an early and sensitive marker of acute cellular rejection in renal allograft

recipients [39,40,41]. It is also common in chyluria, a condition which is

characterized by “milky” urine [42].

Renal tubular epithelial cells:

The different segments of the renal tubules are lined by different types of

epithelial cells. Their size ranges from about 9.0 to 25.0 μm. Most of the renal

tubular epithelial cells (RTECs) in the urinary sediment are derived from the

proximal segments. These are round to oval or rectangular, have a large central or

16

abundant organelles, and a mean diameter of about 14.0 μm .Other RTECs from

distal tubules, are polygonal with a central nucleus and are smaller, and those

deriving from the collecting ducts have a columnar shape with a nucleus in the

basal position containing prominent nucleoli. At times, RTECs show degenerative

changes or appear in aggregates which indicates a particularly severe tubular

damage. RTECs are usually accompanied by elements indicative of parenchymal

renal disease such as casts, dysmorphic erythrocytes or lipids. RTECs are found in

disorders that primarily involve the tubules, such as acute tubular necrosis [43, 44],

acute interstitial nephritis [45] or acute rejection of a renal allograft. However, they

may also be seen in the urine of patients with glomerular diseases, as a

consequence of the tubular damage caused by inflammation and/or proteinuria.

Transitional epithelial cells:

Transitional epithelial cells derive from the urothelium, which lines the

urinary tract from the calyces to the bladder in women, and to the proximal

urethra in men. Transitional cells of the urothelium may have various shapes, but

they are mostly of ovoid or club-like appearance, having a central or peripheral

nucleus with one or two nucleoli, and a thin cytoplasm. Their longitudinal

diameter ranges from about 10.7 to 38.0 μm .Ovoid cells may at times be difficult

to distinguish from round or ovoid renal tubular cells. However, ovoid deep

transitional cells have a thinner cytoplasmic rim than renal tubular cells, and are

not associated with other particles suggestive of renal damage such as casts or

dysmorphic erythrocytes. Transitional cells are seen in large quantities (i.e. one or

17

layers of the urothelium such as urolithiasis, bladder carcinoma, or

hydronephrosis [46].

Squamous epithelial cells:

Squamous epithelial cells found in the urine mostly derive from the

superficial layers of vaginal epithelium. They are the largest cells in the urinary

sediment, their diameter ranging from about 17.0 to 118.0 μm. These cells are

quadrangular to polygonal in shape, and have a broad cytoplasm containing few

granules and a small central nucleus. Frequently, squamous cells are folded or are

aggregated in clumps.

Occasionally, bacteria are attached to their cell membrane , reflecting

colonization by bacteria. This process is thought to be an indispensable step

preceding urinary tract infection. If urine contains large numbers of squamous

cells, free nuclei of these cells are often seen, which represent remnant debris after

cell degeneration. Squamous cells are constantly shed from the urethra and

vagina, and small numbers are almost invariably present in the urinary sediment

of females. If urine is not collected properly (without spreading the labia and

without discarding the first portion of the voided urine), or if there is a vaginal

discharge, the squamous cells can be so abundant that proper analysis of the

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Lipids:

In the urine sediment lipids appear as free lipid droplets, oval fat bodies,

fatty casts or cholesterol crystals. Free lipid droplets, isolated or in aggregates,

appear as translucent round particles of very variable size, with a bright yellow

colour. The oval fat bodies are macrophages [47] or renal tubular epithelial cells

with lipid droplets[48] . Fatty casts are cylinders which contain lipid droplets in

their matrix. The amount of lipids can vary from a few and isolated droplets to

tightly packed droplets, which mask the matrix of the cast. Cholesterol crystals are

thin, colourless and transparent plates with well-defined edges, which can be

isolated or in aggregates. Lipid droplets can usually be identified without

difficulty. However, larger fat globules may be confused with isomorphic

erythrocytes, yeasts or round calcium oxalate crystals. Lipids can also be

identified by stains such as Oil-Red O or Sudan III. Lipiduria can be found in

several renal diseases, but especially in nephrotic syndrome . In this condition,

lipiduria is due to free cholesterol, cholesterol esters, triglycerides, free fatty acids

and phospholipids, the main lipoprotein being represented by HDL[49]. However,

they may also be found in patients with non-nephrotic proteinuria, in some

patients with non- glomerular diseases [50] or in patients with polycystic kidney

disease and low grade proteinuria [51]. In the latter condition, the lipid droplets are

thought to derive from renal cysts containing degraded blood. In patients with

glomerular diseases, lipids enter the urine because of abnormal glomerular

19

tubular cells and transported for hydrolysis into lysosomes. Then, they re-enter the

tubular urine by active expulsion , or as a result of cellular breakdown.

Casts:

Casts are cylindrical elements of variable diameter and length which form

in the distal tubules and collecting ducts of the kidneys. They can also form in the

branching collecting ducts, as demonstrated by the occasional finding of branched

casts. The matrix of casts is made of Tamm-Horsfall glycoprotein (THG) which is

synthesized and secreted by the cells of the thick ascending limb of Henle’s loop.

THG contains 616 amino acids and carbohydrates, which account for

approximately 30% of its molecular weight. THG is the major protein of the

normal urine but its biologic role is still unclear [52]. Under several physiological

and pathological conditions, fibrils of THG tend to aggregate and to interweave

within the tubular lumen, forming a cylindrical structure. The formation of this is

favoured by low intratubular pH, high osmolality and high sodium concentration,

or by interaction with myoglobin, haemoglobin, Bence-Jones protein and other

substances. Initially, the forming cast remains anchored to the tubular cells by fine

fibrils, but subsequently it is washed away by the tubular urine flow and finally

reaches the bladder as a cast[53].

Casts may be hyaline, if they consist of THG only, or complex if they also

contain other elements. In fact, whichever particles are passing through the

tubular lumen during the formation of the cast (e.g., cells, lipids, granules, crystals

or microorganisms)they can be trapped in its matrix. This explains the large

20

significance .The final morphology of casts also depends on the diameter of

tubules in which they were formed. When the tubules are dilated, as in tubular

atrophy or renal obstruction, large casts are seen in the urine, a finding which is

therefore indicative of renal failure.

Since casts are formed in the renal tubules, all particles they contain derive

from the kidneys. Unfortunately, several types of casts of diagnostic importance

are often not recognized in community or hospital based laboratories [54, 55].

Table 3: Classification of casts.

TYPE SUBTYPE

Hyaline

Granular Finely, coarse

Waxy

Cellular Erythrocytic, leucocytic , epithelial casts

Fatty

Pigmented Haemoglobinic, myoglobinic, bilirubinic

Hyaline casts:

Hyaline casts contain only THG, which confers a low refractive index.

Consequently, these casts may be overlooked if only bright field microscopy is

used. Variable amounts of hyaline casts can be found in the normal subject. They

can also be seen in subjects without renal disease after physical exercise, during

episodes of fever or dehydration, or during acute congestive heart failure.

Moreover, Imhof et al. [56] found transient abundant hyaline cylindruria after a

21

However, hyaline casts are also found in renal diseases, mostly in combination

with other types of casts [36] .

Granular casts:

Typical granular casts have their surface covered by granules which vary in

number and size. Granules can be fine or coarse, and clear, dark or pigmented.

Granules of casts are lysosomes containing reabsorbed ultrafiltered proteins

which, due to active expulsion from the tubular cell or tubular cell damage, fall

into the tubular lumen, where they are trapped in the matrix of the forming cast.

However, since granular casts are also found in renal diseases without proteinuria

such as acute tubular necrosis [57], it is accepted that granules might also derive

from cellular degeneration. Coarse granules are formed by degenerated cells such

as leukocytes or renal tubular epithelial cells [53]. The above mechanisms explain

why granular casts are usually not seen in the urine of normal subjects and why

their finding strongly indicates the presence of a renal disease. Granular casts,

together with renal tubular epithelial cell casts, are a distinguishing finding in

patients with acute tubular necrosis . However, they are also frequent in patients

with glomerulonephritis [36].

Waxy casts:

Waxy casts derive their name from their appearance, which is reminiscent

of melted wax. They have a high refractive index, dark colour, broad diameter and

hard, frequently indented and cracked edges. Occasionally, their surface is not

smooth but somewhat irregular. It has been claimed that waxy casts may derive

22

frequent finding in patients with renal failure, both acute and chronic. In few

patients with glomerular diseases without significant differences between

proliferative and non- proliferative histologic types [36].

Cellular casts:

Casts may contain different types of cells, namely erythrocytes, leukocytes

or renal tubular epithelial cells (RTEC). Therefore, cell-containing casts are

classified as erythrocytic, leukocytic and RTEC casts. The erythrocytes within the

cast may be so tightly packed that the matrix of the cast can hardly be seen and

individual erythrocytes can hardly be discernible. Alternatively, only a few

erythrocytes may be trapped in the hyaline matrix. The erythrocytes within the

casts can have a normal or reduced haemoglobin content (so-called “ghost”

erythrocytes), and can be either isomorphic or, more rarely, dysmorphic.

Erythrocytic casts are a marker of glomerular bleeding and should be examined in

all patients with isolated microscopic haematuria of unknown origin. In patients

with overt glomerulonephritis, erythrocytic casts are found in 85% of cases [23],

which depends on the types of glomerular disease investigated and on the

methodology used to search them. The prevalence being significantly higher in

proliferative disorders than in non proliferative ones. Together with dysmorphic

erythrocytes, erythrocytic casts are a distinguishing feature of the nephritic

sediment. Very rarely they may be found in patients with haematuria caused by

acute interstitial nephritis. The degradation of erythrocytes within the casts leads

to the formation of so-called haemoglobin casts, whose clinical significance is the

23

Leukocytic casts can contain variable amounts of leukocytes, from few to

so many that the matrix of the cast is completely masked .Leukocytes may be well

preserved or degenerated, in which case they are hardly distinguishable from renal

tubular epithelial cells. Leukocytic casts are found in patients with active

proliferative lupus glomerulonephritis [58], other glomerular diseases [36] or acute

interstitial nephritis.

Renal tubular epithelial cells (RTEC) casts, which are also known as

epithelial casts, can contain variable amounts of RTECs, from few to many .These

cells are identical to RTECs seen outside casts, which have a well evident nucleus

and a granular cytoplasm. However, when the cells are degenerated, these

distinguishing details are lost and differentiation between leukocytic casts and

RTEC casts can be impossible.

RTEC casts are found in all conditions associated with severe tubular

damage such as acute tubular necrosis [54] and acute interstitial nephritis of

whatever cause. However, these casts are a very frequent finding also in patients

with glomerular diseases.[36]

Fatty casts:

Fatty casts can contain lipid droplets (isolated or in clumps), oval fat

bodies or cholesterol crystals. The lipid droplets within the casts may be few,

small and scattered or so abundant and packed that they completely mask the

matrix of the cast. Cholesterol plates may at times protrude from the edges of the

24

sediment. Therefore, they are typical of patients with nephrotic range proteinuria

but also seen in patients without nephrotic range proteinuria [36].

Pigmented casts:

Pigmented casts includes haemoglobin, myoglobin and bilirubin casts.

Haemoglobin casts derive from erythrocytes which have undergone degeneration.

Haemoglobin casts have a typical brownish to reddish-brown colour and a

granular appearance.

Their identification is facilitated by careful focusing, which may reveal the

remnants of erythrocyte membranes. In typical cases, haemoglobin casts are

associated with erythrocytic casts and erythrocytes, and indicate renal bleeding.

Myoglobin casts have a brown to reddish-brown colour similar to that of

haemoglobin casts. The surface can be either smooth or granular, but careful

focusing does not show any remnants of erythrocytes. The knowledge of the

clinical context is indispensable to distinguish myoglobin casts from haemoglobin

casts. Myoglobin casts are seen in the urine of patients with acute renal failure

associated with rhabdomyolysis, which occurs in crush syndrome [59]. Casts of any

type i.e., hyaline, granular, waxy or cellular, may be stained by the typical yellow

colour of bilirubin. Bilirubin casts are observed in the urine of patients with

25

Other elements :

Pseudocasts:

Pseudocasts are particles which morphologically resemble casts without

being formed in the renal tubules. Many particles in the urine can resemble casts.

Among these crystals (especially when in clusters or aggregates), cells, mucus and

most frequently, contaminants such as cloth or synthetic fibres. Compared to

casts, pseudocasts may show harder edge, more irregular contours, more variable

size , unusual colours, differing from the colour due to haemoglobin, myoglobin

or bilirubin.

Mucus :

Mucus is a substance derived from the secretion of the accessory glands.

Usually, mucus appears as ribbon-like threads with irregular contours and fibrillar

structure. The fibrils tend to be larger and more loosely textured than fibrils seen

in hyaline casts. Less frequently, mucus threads aggregate to form large masses or

networks of fine fibrils. Occasionally, threads of mucus resemble hyaline casts

(pseudocasts). Cells may be trapped in mucus which leads to a grossly

inhomogeneous distribution across the slide .

Crystals:

Crystals are a frequent finding. There are many types of urinary crystals ,

such as common crystals like uric acid, amorphous urates and amorphous

phosphates, calcium oxalate, calcium phosphate, triple phosphate. Pathological

26

crystals due to drugs and other crystals like hippuric acid, calcium carbonate,

ammonium biurate.

Micro-organisms :

Several organisms can be identified in urinary sediments. Rods may be

isolated, in pairs or in long chains. The same is true for cocci . Rods and cocci are

easily identifiable, but sometimes cocci may be confused with amorphous urates

or phosphates but can be differentiated based on movement typical for cocci.

Bacteria may adhere to squamous epithelial cells, or clump into masses of

variable size. Urinary infection can reasonably be suspected if bacteria are present

in freshly voided midstream urine, particularly if numerous leukocytes are also

present. In women, bacteria and leukocytes in the urine can be due to

contamination from vaginal secretions, as a consequence, for instance, of

vaginitis. This situation is usually associated with massive amounts of squamous

epithelial cells with or without Candida and/or Trichomonas vaginalis. In urinary

tract infections with involvement of the kidney, besides leukocytes and bacteria,

leukocytic casts and even bacterial casts can be found. Yeasts are unicellular

organisms which reproduce by budding. Candida albicans is the most frequent

yeasts found in the urine. Candida appears as pale-green cells with smooth and

well-defined walls. The nucleus is at times visible, and the cytoplasm is

homogenous without apparent organelles. Round Candida cells may resemble

erythrocytes and some types of monohydrated calcium oxalate crystals, but

Candida are often nucleated and, especially, show budding. After letting the urine

27

clumps, can be seen. The most frequent cause of Candida in the urine sediment is

contamination by vaginal discharge in women with vaginitis. In this condition,

Candida is usually associated with massive amounts of squamous epithelial cells,

bacteria, and leukocytes .Candida, however, can also cause a true urinary

infection, especially in patients with diabetes mellitus, structural abnormalities of

the urinary tract, in-dwelling catheters, prolonged antibiotic treatment or

immunosuppression. Under these conditions, Candida in the urine may reflect

invasive candidiasis, which may cause urethritis, cystitis or renal infection. In case

of renal involvement, candidal casts can be found in the urine.

Patterns of urinary sediments:

Normal individual:

Many studies have been carried out on the urine sediment of normal

subjects. All studies showed that erythrocytes, leukocytes, renal tubular epithelial

cells and casts could be present. Casts ranged from zero to variable numbers[60].

Casts were almost invariably of the hyaline type, even though granular casts or

even epithelial casts could be found. Birch et al. studied the erythrocyturia of 376

healthy adult subjects [61]. Using samples centrifuged at 750 g for 5 min, a

Fuchs-Rosenthal counting chamber and a phase contrast microscope, it was found that

males gave a median count of 2,500 erythrocytes/mL (range 250-13,000/mL) and

a modal count of 2,000 cells/mL. Females gave a median count of 4,000

erythrocytes/ mL (range 250-16,000) and a modal count of 3,000/mL. Counts did

not appear to be age dependent. Since the 95% percentile was 8,000

28

8,000 erythrocytes/mL. Pollock et al. studied the excretion of erythrocytes of 27

healthy volunteers [20]. After centrifugation at 2,000 rotations per minute for 4

minutes, the cells were counted in a Fuchs-Rosenthal chamber using phase

contrast microscopy. The excretion rate of erythrocytes was < 1,000/mL (95%

confidence). Loh et al. studied the excretion of erythrocytes and leukocytes of 419

children [62]. Using uncentrifuged urine, a Neubauer counting chamber and phase

contrast microscopy, it was found that 95% of children excreted < 14 × 10 6

erythrocytes/L (i.e. < 14,000/mL) and < 4 × 106 leukocytes/L (i.e. < 4,000/mL).

Erythrocyturia was significantly higher in children aged 2-5 years, and leukocyte

excretion was significantly higher in females than in males (2.5 × 106/L versus 1.2

× 106/L). Interestingly, in the studies of Birch et al. [61] and of Loh et al. [62], the

morphology of erythrocytes was also evaluated. This was found to be consistently

dysmorphic, i.e. of glomerular origin. These results were confirmed partially by

Fasset et al., who studied 50healthy adult subjects and found that most subjects

had red cells similar to those seen in patients with glomerulonephritis, but many

also had some non- glomerular red cells [63]. Thus, these studies described give

very different figures in the excretion of erythrocytes and leukocytes in the urine

of the normal subject, and explains the different definitions of pathologic

microscopic haematuria which can be found in the literature [64, 65].The difference

in results may be partially explained by the methods used to collect, prepare and

analyse the urine samples were not the same in the different studies, and there

were large differences in the number of subjects studied. The difference in the

29

to the lack of consistent results, and may explain why several laboratories do not

provide normal values for urinary erythrocytes and leukocytes. Without these

figures, it is impossible to define microscopic haematuria or pathological

leukocyturia correctly, which is done by a careful selection of the subjects to be

studied and by using a standardized method for urine collection, handling and

analysis.

Minimal change disease and focal segmental glomerulosclerosis:

The urine findings in MCD and FSGS are those of the nephrotic syndrome

namely, marked proteinuria associated with variable numbers of renal tubular

epithelial cells (RTECs), marked cylindruria (hyaline, hyaline-granular, granular

and RTEC casts), and lipiduria. In MCD, lipiduria is less frequent than in other

glomerular diseases and microscopic haematuria is absent or mild. In primary

FSGS, microscopic haematuria is more frequent and less mild than in MCD. Any

deviation from the above patterns is suspicious. For instance, the sudden

appearance of RTECs and RTEC casts associated with a rapid decline of renal

function may point to superimposition of acute tubular necrosis, which in

nephrotic patients is often the consequence of hypovolaemia. The sudden

appearance of a severe microscopic haematuria and leukocyturia, accompanied by

a rise in serum creatinine, may suggest the presence of superimposed acute

interstitial nephritis, which occasionally is the result of diuretic treatment. The

sudden appearance of gross haematuria, particularly in the presence of unilateral

30

Membranous nephropathy:

Proteinuria in the nephrotic range is usually associated with microscopic

haematuria, heavy cylindruria, and lipiduria. The sudden appearance of gross

haematuria, with or without unilateral enlargement of the kidney, is suggestive of

renal vein thrombosis, which in membranous nephropathy is more frequent than

in any other glomerular disease. Rapid transformation into a full-blown nephritic

sediment, accompanied by a rise in serum creatinine, is suggestive of a possible,

although rare, superimposition of extra capillary proliferation [66].

IgA Nephropathy:

Proteinuria in nephrotic range with a mild to very severe haematuria , a

mild to moderate leukocyturia and few renal tubular epithelial cells . Erythrocytic

casts and fatty casts are the most frequent, being followed by renal tubular

epithelial cell casts and leukocytic casts, which are rare. Ibels et al. [67], who

studied 174 patients with IgA Nephropathy, and found increased red cells and

increased white cells in 94% and 46% of samples, respectively. Interestingly, they

also found that the total number of casts and the number of hyaline-granular casts

at presentation correlated significantly with the worsening of serum creatinine at

follow-up. Microscopic haematuria (defined as > 5% RBCs/HPF) was a marker of

IgAN also in the study by Nakayama et al.[68], who found it in 92% of 364

patients. These authors also observed that there was a significant correlation

between the total number of hyaline, granular, erythrocytic, leukocytic and fatty

casts and of oval fat bodies in the urine and the severity of the histological lesions.

31

isolated microscopic haematuria. Interestingly, when the morphology of urinary

red blood cells was evaluated in this disease, a mixed haematuria, containing

isomorphic and dysmorphic erythrocytes in the same proportion was found by

some investigators [24], while others found mainly dysmorphic erythrocytes or ≥

5% acanthocytes [25].

Membranoproliferative glomerulonephritis:

A wide spectrum of urinary changes is possible in MPGN of whatever

type. These include isolated dysmorphic microscopic haematuria, microscopic

haematuria and proteinuria, nephrotic syndrome and macroscopic haematuria.

Thus, the finding of a nephritic or of a nephritic and nephrotic sediment is not

uncommon in patients with MPGN. Interestingly, in a large study on the

prognostic factors of primary MPGN [69], it was found that the presence of

granular casts in the urine at baseline correlated significantly with the logarithm of

serum creatinine, the degree of proteinuria and albuminaemia, and with acute

tubular damage, mesangial sclerosis, and glomerular crescents or necrosis at renal

biopsy. In addition, it was found that patients with urinary granular casts had a

significantly higher probability of progression to end stage renal failure at 3 years

than the patients without granular casts.

Acute post-streptococcal glomerulonephritis:

In the acute phase, the urinary findings correspond to those of a full-blown

nephritic syndrome .However, as in lupus nephritis, diffuse and active glomerular

changes may occasionally be associated with normal or only mildly altered

32

disappear by the end of the first year, but in some isolated microscopic haematuria

may persist for years.

Extra capillary glomerulonephritis:

In the active phase, extra capillary glomerulonephritis is typically

associated with rapidly progressive renal failure, mild to moderate proteinuria and

the most severe haematuria which can be observed in patients with

glomerulonephritis. When the glomerular lesions heal with appropriate therapy,

erythrocytes and erythrocytic casts usually decrease to complete disappearance.

Thus, in extracapillary glomerulonephritis, the examination of the urinary

sediment is a valuable tool for the evaluation of the activity or inactivity of the

disease, with relapses frequently being heralded by the appearance of an active

sediment and a rise in the number of urinary erythrocytes. One must be aware that

these findings can be observed in whatever type of extra capillary

glomerulonephritis, including Goodpasture’s syndrome. Interestingly, the latter

condition can occasionally occur with only mild microscopic haematuria with or

without mild proteinuria, in spite of a clear cut linear deposition of IgG in the

kidney [71].

Lupus nephritis:

The finding of persistent proteinuria > 0.5 g/24 hours (or > 3+ if

quantification is not available) or of cellular casts including red blood cell,

haemoglobin, tubular, granular, or mixed in the urine sediment, are among the

criteria of the American College of Rheumatology for the diagnosis of systemic

33

patients with known or suspected lupus should undergo urinalysis at regular

intervals[73].The examination of the urinary sediment with the assessment of

proteinuria and of serum creatinine is mandatory for the identification of renal

flares and the guidance of therapeutic intervention [74]. Urinary sediment

examination is also useful for recognizing the severity of the renal disease. As a

general rule, the milder the renal lesions the fewer the urinary changes, and vice

versa [75], though there are exceptions however. Thus, in active class III and class

IV lupus nephritis, discrete to severe proteinuria is almost invariably present,

while the sediment reflects the inflammatory injury of the glomerulus.

Consequently, it comprises a frequent and moderate to severe erythrocyturia, mild

to moderate leukocyturia, a few RTECs, and abundant and pleomorphic

cylindruria, including erythrocytic casts in the vast majority of patients. However,

rare cases of active lupus nephritis with minor urinary changes and inactive

sediment can occur.

When the disease is controlled by therapy, the former elements usually

decrease or disappear. Reappearance of erythrocyturia and, especially, of cellular

casts, quite often indicates imminent relapse. In patients with the LN class V,

proteinuria is usually, but not always, marked. In contrast with class III and IV

LN, microscopic haematuria, leukocyturia, erythrocytic casts and leukocytic casts

may be either absent or mild. A change in the amount of proteinuria and/or the

character of the urinary sediment may indicate a change in the type of glomerular

lesions with transformation into another class [75]. In a lupus patient with a

34

absence of inflammatory changes of the sediment correctly, since this finding

usually indicates that progressive renal failure is due to non-immune mechanisms

which will not respond to immunosuppression.

Schönlein-henoch purpura nephritis:

A minority of patients have only dysmorphic microscopic haematuria,

which can be transient and of short duration. About 50% of patients have

persistent microscopic haematuria and proteinuria. In patients with nephrotic

syndrome, there is usually mild haematuria associated with abundant cylindruria

and fatty particles. In patients with nephritic syndrome and extracapillary

proliferation at renal biopsy, the sediment shows severe erythrocyturia and

cylindruria including erythrocyte/haemoglobin casts. About 20% of patients,

however, present with both nephrotic and nephritic sediment. In some patients,

urinary changes recur or worsen during, or shortly after, skin rash recurrences [76].

Diabetic nephropathy:

Initially, only microalbuminuria is found, the detection of which

necessitates the use of specific methods such as immunochemical assays utilizing

anti-albumin antibodies, high performance liquid chromatography, or appropriate

dipsticks. In advanced stages, non-selective proteinuria develops .Urinary

sediment is usually defined as unremarkable in DN, apart from some occasional

erythrocytes. However, a number of studies have shown that microscopic

haematuria is not uncommon in diabetic nephropathy, being found in 15% to 35%

of patients with biopsy-proven DN associated with type 2 diabetes mellitus [77,78].

35

found in 62% of patients with clinically diagnosed DN, a prevalence which

increased to 82% when three consecutive samples from the same patients were

analyzed [79]. Interestingly, ≥ 5% acantocyturia, a marker of glomerular bleeding,

was found in only 4% of diabetic patients in contrast with 75% of patients with

glomerulonephritis. This finding makes the origin of microscopic haematuria in

DN largely unclear. Besides microscopic haematuria, it should be remembered

that the appearance of an active urine sediment with many erythrocytes,

leukocytes and pleomorphic cylindruria in a diabetic patient should always be

considered as a possible sign of superimposed proliferative and active

glomerulonephritis such as IgAN, acute post-infectious glomerulonephritis, or

extracapillary glomerulonephritis [80]. Leukocyturia associated with bacteriuria is

suggestive of urinary tract infection which, in diabetes, is frequent and may be

associated with pneumaturia i.e., the passage of gas into the urine due to bacteria

such as Escherichia coli and Enterobacter aerogenes [81]. Another complication of

urinary tract infection in diabetics is septic papillary necrosis, which can present

with flank pain, gross haematuria and papillary fragments in the urine. Candida is

also frequently found in the urine of diabetic patients.

Nephropathies due to plasma cell dyscrasias:

In patients with myeloma cast nephropathy, proteinuria is mainly due to the

excretion of monoclonal light chains (the so-called Bence Jones proteinuria),

which are identified by immunefixation. It is important to remember that light

chains are not detected by dipsticks commonly used for urinalysis. In fact, these

36

light chains. On microscopy, myeloma cells appears with oval to round eccentric

nuclei showing peripheral clumping of nuclear chromatin, prominent nucleoli and

a high nucleus/cytoplasm ratio. Myeloma casts i.e., casts surrounded by

multinucleated cells, may also be seen [82].Patients with amyloidosis or light chain

deposition disease usually have proteinuria in the nephrotic range, which is

mainly of the glomerular type. Thus, the urinary sediment usually contains

abundant cylindruria (hyaline, hyaline-granular, granular, and RTEC cell casts)

and lipiduria. In amyloidosis, microscopic haematuria is usually absent. In both

amyloidosis and light chain deposition disease, the appearance of a nephritic

sediment may herald the superimposition of extra capillary proliferation.

Acute interstitial nephritis:

In AIN caused by antibiotics especially beta lactam drugs the proteinuria is

usually mild. Leukocyturia and haematuria are found in all patients in the

full-blown phase. Eosinophiluria seems to be always present. In AIN due to

non-steroidal anti-inflammatory drugs ( NSAIDS), due to the concomitant glomerular

disease, proteinuria is a constant finding, and frequently it is in the nephrotic

range with leukocyturia, haematuria or casts (hyaline, granular, waxy, containing

leukocytes but not erythrocytes) on sediment examination. In AIN associated with

bacterial infections, proteinuria is frequent but is usually < 1.5g/24 hours.

Microscopic haematuria is almost invariably seen but leukocyturia is rare.Of the

idiopathic forms, AIN associated with uveitis is the best characterized variety.

Proteinuria of < 1 g/24 hours is almost invariably present, which is caused by

37

both, but it may even be normal. In only a few patients has eosinophiluria been

found.

Two aspects of urinary sediment in AIN deserve a separate comment.

Eosinophiluria is usually considered as a marker of AIN. Old studies support this

view as well as several case reports in which AIN was caused by a wide spectrum

of drugs including ciprofloxacin, omeprazole, vancomycin, fluindione, or

linezolid. However ,one should consider that AIN encompasses a heterogeneous

group of diseases, and that eosinophiluria has a different prevalence in the

different forms. In fact, it seems to be more frequent in AIN associated with

antibiotics than in other types. In addition, with the use of Hansels’s stain, which

is more specific and sensitive than traditional Wright’s stain, eosinophiluria has

been found in a wide spectrum of disorders, including several types of

glomerulonephritis, prostatitis, chronic pyelonephritis, renal cholesterol

embolism, urinary schistosomiasis, etc. It is commonly thought that erythrocytic

casts are so rare in AIN that their presence should suggest the diagnosis of a

glomerular disease [83, 84]. Interestingly, erythrocytic cylindruria was associated

with many erythrocytic casts within the tubules at renal biopsy. In addition,

erythtrocytic casts have been found in 4/12 patients (33%) with AIN by Köhler et

al. [25]. Thus, the possibility that erythrocytic casts can be found in the urine of

38

Chronic interstitial nephritis:

In spite of the heterogeneous nature of CRIN, the urinary changes are

rather uniform. They are represented by the excretion of proteins of low molecular

weight such as β2- microglobulin, concentration and/or acidification defects, and

loss of glucose, bicarbonate, uric acid, phosphate and amino acids. The urinary

sediment may be normal or only mildly changed, containing sparse leukocytes

and a few hyaline or hyaline-granular casts, while microscopic haematuria is

uncommon. Analgesic nephropathy (AN) is the best studied form of CRIN. It is

characterized by early urinary concentration and acidification defects, with

proteinuria which is usually < 1 g/24 h, microscopic haematuria, as well as

leukocyturia. A sudden appearance or a worsening of haematuria and

leukocyturia, or even the appearance of gross haematuria in association with

lumbar colicky pain may indicate renal papillary necrosis. This event can also be

seen in diabetic patients usually in conjunction with a urinary tract infection,

sickle cell disease, renal tuberculosis, and urinary tract obstruction. When

papillary necrosis is suspected, fragments of the necrotic papilla should be looked

for in the urine, especially by the use of a filter paper or a gauze .A worsening of

haematuria with or without atypical urothelial cells may also be due to

uroepithelial cancer, whose incidence is increased in AN. Therefore, in patients

with AN the serial examination of urinary sediment is useful to reveal possible

39

Acute tubular necrosis:

In ATN, urine sediment shows variable numbers of RTECs, both necrotic

and viable, at times even fragments of the tubular epithelium, and abundant

cylindruria (granular casts and RTEC casts). In addition, depending on the cause

of the tubular damage, other particles can be seen. These other particles are

important in identifying the cause of the acute kidney injury. Thus the presence of

brownish pigmented casts in urine which does not contain erythrocytes suggests

ATN from myoglobinuria or haemoglobinuria. The presence of a massive

crystalluria can suggest ethylene glycol poisoning (which causes atypical

spindle-like monohydrate calcium oxalate crystals) [85], acute uric acid nephropathy

(which causes uric acid crystals), or ATN due to intra-renal precipitation of a

drugs in which the crystalluria can be either morphologically atypical (which may

be caused by sulfadiazine, amoxycillin, acyclovir, indinavir, felbamate, etc.) or

due to calcium oxalate (which may be caused by vitamin C, naftidrofuryl oxalate,

or orlistat).

The presence of severe haematuria associated with erythrocytic casts

strongly suggests a primary or secondary proliferative glomerulonephritis in an

active phase [86]. In addition to all this, in recent times, several urinary biomarkers

have been proposed for the early diagnosis of ATN and are currently under

investigation. These include interleukin 18, kidney injury molecule 1, and tubular

enzyme such as the intestinal form of alkaline phosphatase, N-acetyl-β

40

Interpretation of sediment findings:

The nephrotic sediment is observed in patients with nephrotic syndrome.

This is a condition characterized by proteinuria of > 3.5 g/24 hour associated with

hypoalbuminaemia, hypercholesterolaemia, and variable oedema. In most

instances, nephrotic syndrome is caused by glomerulopathies which at renal

biopsy have little or no evidence of glomerular inflammation (e.g., absence of

intra- or extra-capillary cell proliferation, necrotizing lesions, or insudation with

polymorphs and/or mononuclear cells).Any glomerular disease may cause a

nephrotic syndrome, but the most common are minimal change disease (MCD),

focal segmental glomerulosclerosis (FSGS), idiopathic membranous nephropathy,

diabetic nephropathy, membranous lupus nephritis, amyloidosis and light chain

deposition disease. Lipiduria and marked cylindruria, especially fatty casts and

renal tubular epithelial cell (RTEC) casts, are the hallmarks of the nephrotic

sediment. Microscopic haematuria is variable according to the type of

glomerulopathy. Usually, it is absent or mild in minimal change disease, while it

is more frequent in focal segmental glomerulosclerosis and idiopathic

membranous nephropathy, and it is variable in diabetic nephropathy. In these

conditions, leukocyturia is uncommon and, if present, mild .Although lipiduria is

the distinguishing feature of the nephrotic sediment, this is not invariably present .

Lipids enter the urine because of impaired glomerular basement membrane

permeability, their passage through the glomerular barrier being also influenced

by the selectivity of proteinuria (the higher the selectivity the lower the lipiduria).