Study of Pattern of Urinary Sediments in Renal Diseases

Home , Addis count, Bilirubinuria, Hyperuricosuria

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

APRIL-2017

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, Tirunelveli Medical College, Tirunelveli- 627011.

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., Professor and Head, Department of Pathology, Tirunelveli Medical College, Tirunelveli- 627011.

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, Tirunelveli – 627011.

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 study would not have been possible.

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

MH MICROSCOPIC HAEMATURIA

IMH ISOLATED MICROSCOPIC HAEMATURIA

AKI ACUTE KIDNEY INJURY

AKIN ACUTE KIDNEY INJURY NETWORK

LPF LOW POWER FIELD

S.D. STANDARD DEVIATION

WHO WORLD HEALTH ORGANIZATION

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

9. ANNEXURES

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 sediment in predicting the severity of renal injury. The granular casts and/or renal

1 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

monitoring, urine sediment examination is of paramount value.

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,

serum creatinine and urinary protein excretion levels.

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

Valentin, described tubular casts, which were first observed within the renal

4 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 to study the ultrastructure of urinary casts[1].

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

epithelial cells of vaginal origin, leukocytes, bacteria, amorphous phosphates or

6 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

Fat Chemicals

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]:

RCF = 1.118 × 10– 5 × r × N2

8 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, bacteria, or casts. For instance, a case with dipstick clearly positive for

9 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

- lipids

- 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

decreases and vice versa . It is important to remember that at a specific gravity of

11 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 easily than other dysmorphic erythrocytes.

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 insult is thought to occur during the passage along the tubular system, in which

13 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

non-infectious renal diseases including glomerulonephritis[36], acute and chronic

14 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

eccentric nucleus containing one or two nucleoli, a granular cytoplasm showing

15 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 more per high power field) in conditions characterized by damage to the deep

16 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

urinary sediment is difficult or impossible.

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

ultrafiltration. Within the tubules, they are partially reabsorbed by proximal

18 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 variety of casts, which differ in morphology, composition and diagnostic

19 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

single oral dose of furosemide (80-160 mg) or ethacrynic acid (50-100 mg).

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

from hyaline casts that have been altered by urine products [54]. Waxy cast are a

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

patients with glomerular diseases without significant differences between

proliferative and nonproliferative 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

same as that of erythrocytic casts.

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 cast. Fatty casts are usually associated with other fatty particles in the urine

23 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 jaundice associated with increased direct (conjugated) bilirubin.

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 crystals include cholesterol, cystine, leucine, tyrosine, 2,8-dihydroxyadenine and

25 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

stand , abundant pseudomycelia, i.e. chains of elongated unseparated Candida or

26 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 erythrocytes/mL, a pathological haematuria was described as the excretion of >

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

male to female ratio, and the different ages of the subjects studied also added up

28 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

renal swelling, is suggestive of renal vein thrombosis.

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.

Among glomerular diseases, IgAN is also one of the most frequent causes of

30 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

urine[70]. In patients who go into remission, proteinuria and haematuria usually

31 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

lupus [72]. Early diagnosis of renal disease is of paramount importance, and

32 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

progressive increase in serum creatinine, it is extremely important to interpret the

33 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].

More recently, microscopic haematuria, defined as ≥ 8 erythrocytes/μL, was

34 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 react with amino groups of proteins, which are numerous in albumin but absent in

35 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

tubular proteins. The urinary sediment may contain leukocytes, erythrocytes or

36 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

patients with AIN should be considered.

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 superimposed disorders.

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-

glucosaminidase, and alanine aminopeptidase .

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).

Within the tubules, lipids are partially reabsorbed by proximal tubular cells and

40 transported for hydrolysis into lysosomes .Then, they re-enter the tubular urine as

a result of cellular breakdown. In nephrotic syndrome, cylindruria has several causes, but the presence of high concentrations of ultrafiltered serum proteins in

the tubular urine is certainly an important causative factor. As nephrotic syndrome

reverses (either spontaneously or under treatment), urinary changes also clear up.

Occasionally, however, a nephrotic sediment may transform into an active

nephritic sediment. This may happen in lupus nephritis, in which transformation

from the histological class V into a proliferative class (III or IV) occurs in about

7% of patients. The sudden appearance of a nephritic sediment may also occur in

membranous nephropathy, diabetic nephropathy or amyloidosis due to the

superimposition of extracapillary proliferation.

The nephritic sediment is observed in patients with acute nephritic syndrome. This is defined as the sudden increase of serum creatinine associated with the appearance of haematuria, variable proteinuria, oliguria, and hypertension. Usually, acute nephritic syndrome is caused by glomerulopathies characterized by intracapillary proliferation with or without insudation of polymorphs or mononuclear cells, crescent formation (extracapillary proliferation), or glomerular fibrinoid necrosis. The most common causes of acute nephritic syndrome are IgA Nephropathy, membranoproliferative glomerulonephritis, acute post-infectious glomerulonephritis, extracapillary

/necrotizing glomerulonephritis, active class III and IV lupus nephritis, Henoch-

Schönlein purpura nephritis and cryoglobulinaemic glomerulonephritis. The hallmarks of the nephritic sediment are erythrocyturia, leukocyturia, shedding of

RTECs and erythrocytic cylindruria .Interestingly, the number of leukocytes in the urine significantly correlated with the severity of intracapillary proliferation, extracapillary proliferation and fibrinoid necrosis as well as the absence or presence of crescents at renal biopsy. The number of erythrocytes and RTECs, instead, correlated with fibrinoid necrosis only. The main glomerular lesion responsible for the appearance of a nephritic sediment is the formation of breaks in the glomerular basement membrane, which are caused by immunologically- mediated inflammatory mechanisms. These breaks, which have a diameter of 5-10

μm, have been well demonstrated by transmission and scanning electron microscopy in patients with proliferative or necrotizing glomerulonephritis[87,88,89], while they were not found in non-proliferative glomerular disorders [90]. Thus, through breaks in the glomerular basement

membrane, erythrocytes and polymorphs reach Bowman’s space and the tubular

system, and ultimately the urine. Other mechanisms involved in the pathogenesis

of a nephritic sediment are the formation of casts within the tubular lumen, with

entrapment into their matrix of erythrocytes and leukocytes deriving from

damaged glomeruli; and the co-existence of tubular damage, which is observed in

the most active forms of glomerulonephritis [86]. These last mechanisms explain

the presence in the urine of RTECs and of RTEC casts. As a general rule, there is

a positive correlation between the intrarenal changes and the severity of the urinary findings [36, 68]. Therefore, the persistence of a nephritic sediment usually

indicates the persistence of proliferative changes in the glomeruli, while the

clearing up of the urinary abnormalities, especially when confirmed by repeated

42 examinations, indicates a decrease in the activity of the renal disease, due either to healing or to the transformation into a chronic disease. The reappearance of a nephritic sediment, on the other hand, is usually associated with a relapse of the disease. This is seen especially in patients with lupus nephritis or pauci-immune renal vasculitis. However, there may be cases with active renal disease and mild

or no changes of the urinary sediment, as repeatedly demonstrated in both acute post-infectious glomerulonephritis and proliferative lupus nephritis. In the

nephritic sediment, haematuria is expected to be dysmorphic. In some instances, it

may be isomorphic in spite of the glomerular origin, which may be due to the co- existence of renal insufficiency, necrotizing glomerulonephritis [28] or the use of

Henle’s loop diuretics.

A urine profile with both nephrotic and nephritic features may be found in

virtually all proliferative glomerulonephritis.

Urinary sediment containing many renal tubular epithelial cells is found in

conditions associated with tubular damage. This can occur in a wide spectrum of

diseases not necessarily associated with renal function impairment. Consequently,

according to the nature of the causative disorder, different elements can be

associated with tubular epithelial cells. This gives rise to the following urinary

profiles, which are diagnostically important. RTECs cells with degenerative

aspects associated with RTEC casts and dark granular casts without any other

elements suggests the presence of acute tubular necrosis caused by hypotension or

hypovolaemia. RTECs with brownish pigmented casts suggest myoglobinuria

from rhabdomyolysis, or haemoglobinuria. RTECs with a large number of

43 erythrocytes, some leukocytes and erythrocytic casts suggest the presence of a proliferative glomerulonephritis in active phase [86]. RTECs with lipids, e.g., fatty droplets, oval fat bodies, fatty casts suggest tubular damage as can be observed in glomerular disorders associated with nephrotic syndrome. RTECs with erythrocytes, leukocytes, and leukocytic casts (but usually without erythrocytic casts) suggest acute interstitial nephritis. RTECs with crystals (e.g., uric acid, monohydrated calcium oxalate, 2,8 dihydroxyadenine crystals or crystals due to drugs) suggest acute tubular damage caused by intratubular precipitation of crystals[91, 92].

While it is accepted that there is some excretion of red blood cells (RBCs) in normal subjects, microscopic haematuria (MH) is defined as the finding of >1

RBC/high power field (x 400). Once haematuria due to a contamination from genital secretions (which very frequently occurs during menstruation), has been ruled out, it is important to know whether haematuria is associated or not with proteinuria. The finding of MH associated with proteinuria is strongly suggestive of a haematuria of glomerular origin, as can be found in a wide spectrum of glomerular diseases, which can be diagnosed with the help of other laboratory tests and by renal biopsy.MH without detectable proteinuria defines a condition known as isolated microscopic haematuria (IMH). This can be caused by a large number of urological disorders (especially cancer of the urinary excretory system) as well as nephrological diseases (especially thin basement membrane disease and

IgA Nephropathy), whose identification can be complex and time consuming. In

IMH, the examination of the urinary sediment is of great value. In fact, through

44 the analysis of RBC morphology it is possible to diagnose, at the very beginning

of the workup, towards a glomerular or a non-glomerular (urological) origin of the haematuria[93,94]. The reliability of this approach has been proven by different

investigators. Schramek et al. investigated 316 patients with IMH of unknown

origin [95]. On the basis of the urinary erythrocyte morphology, they were divided into two groups: 123 patients with NGH haematuria and 193 with GH, which was diagnosed when 100% of urinary RBCs were dysmorphic. A urological disorder was identified in 85% of patients with NGH, while for the other 15% of patients, no cause for MH was identified. None of the patients with GH was submitted to renal biopsy. However, 122 of them were followed up for a mean period of 42 ±

11 months, and it was found that GH persisted unchanged in 112, spontaneously reversed in 5, became associated with proteinuria in 3, and transformed in mixed haematuria in 2, as a consequence of the appearance of a cancer of the urinary tract. McGregor et al. investigated 75 adults with renal biopsy with IMH [96] .

Forty-two patients had a non NGH and 33 had a GH, which was diagnosed when

>15% of RBCs were dysmorphic. All patients were submitted to renal biopsy, and it was found that a glomerular disease was present in 31/42 patients with NGH

(74%) and in 31/33 patients with GH (94%). Thus, the analysis of RBC morphology could identify a haematuria due to a glomerular disease with 60% sensitivity and 85% specificity. The analysis of the morphology of urinary RBCs is useful for patients with IMH of unknown origin in order to address the diagnosis towards a glomerular or non-glomerular cause of the haematuria.

Bacteriuria and leukocyturia are typical of urinary tract infection. In females this urinary pattern is frequently caused by urine contamination from vaginal discharge. This is suggested by the finding of large numbers of squamous epithelial cells (from both the vagina and urethra). In some instances, there may also be Candida and/or Trichomonas vaginalis, whose presence further reinforces the genital origin of bacteria and leukocytes. One should also consider that a mild isolated bacteriuria in urine collected and handled under non-sterile conditions may be a normal finding. Bacteriuria may even become abundant if there is a delay in urine handling and analysis. The presence of an isolated leukocyturia, if one excludes the conditions described above, may be due to a large spectrum of diseases including urinary tract tuberculosis, renal or perirenal abscesses, acute urethral syndrome, analgesic nephropathy or other chronic interstitial nephritis, polycystic kidney disease, urolithiasis, etc.

Minor urinary abnormalities encompasses a spectrum of nondescript abnormalities, the interpretation of which is possible only through adequate clinical information and the integration with other diagnostic tests. Variable cylindruria with mild erythrocyturia and/or leukocyturia associated with mild proteinuria (< 1 g/24 h) may be caused by a wide spectrum of glomerular, interstitial or vascular renal diseases, such as IgA Nephropathy, idiopathic membranous nephropathy or focal segmental glomerulosclerosis without nephrotic syndrome, lupus nephritis (especially class II), early diabetic nephropathy, chronic interstitial nephritis, benign nephrosclerosis, polycystic kidney disease, Alport’s syndrome, etc. In clinical practice, however, the pattern

46 described above is more frequently caused by glomerular diseases when they are

either mild or in the healing phase or evolving towards chronicity. Hyaline

cylindruria with a few erythrocytes and leukocytes may even be seen in normal

subjects. The finding of mild uric acid or calcium oxalate crystalluria is relatively

common, especially in the hot season. In most instances, the precipitation of

crystals is due to transient supersaturation of urine caused by some food ingestion

or mild dehydration. Frequently crystalluria is also due to changes of urine

temperature and/or pH which occur upon samples being left to stand in the

laboratory. Less frequently, crystalluria reflects a permanent abnormality of

mineral metabolism (e.g., hypercalciuria, hyperuricosuria, hyperoxaluria),

suggested by the finding of the same type of crystalluria in repeated samples.

Urine sediment scoring system

Recently, 2 different groups have brought the value of urinary microscopy

back to the clinical arena. In a pilot study, Chawla et al [97] developed an AKI cast scoring index to standardize urine sediment analysis . Score precision was evaluated in 30 patients with a clinical syndrome compatible with ATN, with an interobserver index of 99.8% and coefficient of variation of 1.24%. Urine sediment was correlated further with outcomes in 18 patients with ATN. The investigators found that renal recovery was worse in patients with a higher cast scoring index and area under the receiver operating characteristic curve of the cast scoring index for prediction of nonrenal recovery was 0.79. In another study,

Perazella et al [98] proposed a different scoring system for differentiating ATN

from decreased kidney perfusion in AKI (pre-renal AKI). Using final AKI

47 diagnosis at discharge as the gold standard, urinary microscopy on the day of

nephrology consultation was highly predictive of ATN. The odds ratio for ATN incrementally increased with a higher score . In patients with an initial diagnosis of ATN, any granular casts or renal epithelial tubular cells (corresponding to a score of 2)resulted in a positive predictive value of 100% and negative predictive value of 44%. Lack of renal epithelial tubular cells or granular casts in patients with an initial diagnosis of decreased kidney perfusion (prerenal AKI) had sensitivity of 0.73 and specificity of 0.75 for the final diagnosis of ATN. More recently, a scoring point system of urinary microscopy findings was used to predict adverse outcomes [99].Correlation of urinary sediment score and AKI

Network (AKIN) stage at nephrology consultation was shown, and the score was

associated with higher risk of worsening AKI in a dose-dependent manner.

Although these studies are not novel, they highlight the importance of good

urinary microscopy examination in patients with AKI.

The Chawla et al [97] and Perazella et al [98,99] studies emphasize the

importance of a simple, inexpensive, and noninvasive method to help determine

the differential diagnosis and cause of AKI, grade its severity, and predict

outcomes. Bhagyalakshmi A et al. [100] further evaluated the role of urinary sediment scoring system in predicting the extent of renal injury. Both the urine sediment scores had high positive predictive values and specificity in predicting the severity of renal damage and the values were 86.88%, 79.5% and 96.55%,

89.4% respectively and was statistically significant for both the scoring systems.

MATERIALS AND METHODS

Location of Study

The present study is carried out in the Central Laboratory, Department of

Pathology at Tirunelveli Medical College and Hospital. Part of the urine samples

for analysis, were utilised.

Study Population

Patients with clinical features pertaining to renal disease, admitted in the

Department Nephrology, General Medicine and Paediatrics in our hospital were

taken as study population.

Inclusion criteria:

Patients with clinical indications for renal biopsy who possess the evidence

of glomerular injury, tubular damage and tubulo-interstitial lesions were included

in the study. Patients presenting with progressive chronic renal failure with known

etiology, who had their renal biopsy done previously were also included in the

study as separate group.

Exclusion criteria:

Patients with absolute contraindications for renal biopsy were excluded

from the study. Absolute contraindications include uncontrolled severe

hypertension, uncooperative for renal biopsy procedure, solitary kidney and

uncontrollable bleeding diathesis[101].

Study Design

This is a cross sectional diagnostic study for a period of 1.5 years from

February 2015 to August 2016.

Sampling Methods

In this study a convenient sample size of 100 patients were taken. Case series of 80 patients posted for renal biopsy procedure within the study period of

18 months were included in the study and 20 patients with chronic renal failure

who had their biopsy done previously were also added to the study.

Study Plan

Complete clinical history of the patients presenting with facial puffiness,

pedal oedema, ascites, decreased urine output and haematuria were obtained

including onset and duration of symptoms, history of co-morbid conditions like

diabetes, hypertension, coronary artery disease autoimmune diseases and chronic

renal disease. A complete clinical examination performed. The values of blood

parameters, urine biochemical analysis, imaging study reports and previous

biopsy reports (in chronic renal failure patients) were obtained. Samples for urine

microscopic examination were collected and sediment pattern were analysed. The

extent of renal injury graded using cast scoring index. Renal biopsy taken and the

urine sediment patterns described were correlated with histopathological

diagnosis. Patients were grouped together based on the histopathological

diagnosis and sediment score was correlated with serum creatinine levels and

urinary protein excretion levels.

Data Collection

To obtain a urine sample suitable for examination, patients were instructed

to clean the external genitalia with soap and water, uncover the gland for male or

spread the labia of the vagina for female and collect the early morning sample

after discarding the ﬁrst part of micturition. After collection, the urine samples for

urine microscopy were handled in our laboratory according to the following

standardized method, which was shown to provide reproducible inter sample

quantitative results.

10mL of urine was centrifuged at 1500 rpm for 5 min in a standard

centrifuge. Removal by suction of 9.5mL of supernatant performed and followed

by gentle manual agitation of the test tubes [98]. A pipette was used to apply a

20microL of urine sediment on a glass slide, and coverslip of size 22x22mm is

gently applied. There is no variation in types of glass slides or coverslips used

during the study. Samples were examined on the bright-field microscope within

2 hours post – voiding. When the slides are not actively under review, the slides

are were stored in a covered opaque plastic box at room temperature.

Urinary sediments were analysed for RTECs, RBCs, WBCs, and casts such

granular, hyaline, waxy, fatty, cellular (RTEC, RBC, WBC) casts. Digital photos

of random HPFs for cells and random LPFs for casts were recorded. The pattern

of urinary sediments were noted and quantified as mild, moderate, severe and very

severe (Table 4).

Table 4 : Quantification of urinary sediments. [102]

PARTICLE NORMAL MILD MODERATE SEVERE VERY SEVERE CASTS <1 every 1 every 1 every 1 every ≥ 1per field (100X) 8-10 fields 8-10 fields 4-7fields 2-3 fields

RBCS,WBCS ≤1 >1-10 11-30 31-50 >50 per field (400X) per field per field per field per field

RTECS absent 1 every 1 every 1 every ≥1per field (400X) 8-10 fields 4-7fields 2-3 fields

Severity of renal damage were determined using Lakhmir S. Chawla et al. [97] cast

scoring index [Table 5].

Table 5 : Cast scoring index.

SCORE DEFINITION INTERPRETATION 1 NONE No evidence of granular casts or epithelial cell casts 2 RARE Rare granular casts or epithelial casts; at least 1 granular cast or epithelial cell cast seen on the entire slide, but <10% LPFs 3 MODERATE Many granular casts or epithelial cell casts, but not seen on every LPF; casts seen on >10% but <90% of LPFs 4 SHEETS Sheets of muddy brown casts; granular casts or epithelial cell casts seen on >90% of LPFs

Renal biopsy done and specimen were sent in two containers, one of

formalin and the other Michel's fixative. In formalin, submitted in its entirety for

52 light microscopy and in Michel's fixative submitted in its entirety for

immunofluorescence. For light microscopic examination sections were stained for

special stains (PAS, silver and trichrome) and include renal cortex and medulla.

Glomeruli present were evaluated using immunofluorescence. The section are

stained for IgG, IgM, IgA, C3, C1q, Kappa & Lambda light chains. The biopsy

reports were documented. The urine sediment patterns described were correlated

with histopathological diagnosis. Patients with similar diagnosis on renal biopsy

were grouped together. For each group with specific diagnosis, graphs were

plotted correlating urine sediment score and serum creatinine levels, wherever

possible. Within the individual groups, patients who do not show linear

correlation on graphs were taken as subgroup and the added value of urine

sediment examination in those subgroup were evaluated. Correlation coefficient

and statistical significance, p value were determined for each group separately and

then finally overall statistical values were calculated and later correlated with

urine protein level. Further for the subgroup of patients statistical values were

determined and later corrected overall statistical significance calculated excluding

these subgroup.

Statistical Methods:

Correlation is measured by a statistic called the correlation coefficient,

which represents the strength of the putative linear association between the

variables in question. In our study correlation coefficient were determined using

Spearman’s rho test and the level of significance was determined using Kendall’s

Tau test.

Table 6: Correlation coeffecient and its interpretation.[103]

VALUE INTERPRETATION 0 – 0.19 no correlation 0.2 – 0.39 possible correlation 0.4 – 0.59 moderate correlation 0.6 – 0.79 high correlation 0.8 – 1.0 very high correlation

If p value< 0.05 then it is statistically significant, if p value > 0.05 then it is not significant [104].

RESULTS AND OBSERVATION

80 patients admitted for renal biopsy in Tirunelveli Medical College

Hospital from February 2015 to August 2016 were enrolled in this study including

20 patients of chronic renal failure in whom biopsy procedure was already done.

Male predominance was seen forming 57% of cases with the mean age 34.94 years.

Chart 1: Gender distribution

Female 43% Male 57%

Table 7 : Age distribution.

NO OF PATIENTS MEAN S.D

Total 100 34.44 15.98

Male 34.94 17.8 57

Female 33.76 13.36 43

Chart 2 : Mean age distribution.

Overall mean value of age distribution 35.2 34.94 35 34.8 34.6 34.4 34.2 34 33.76 33.8 [years] 33.6 33.4 33.2 33 mean age within groups groups age within mean Male Female

Out of 100patients, 41 patients (41%) presented with nephrotic sediment followed

by nephritic sediment with 33patients (33%) and 20 patients(20%) with sediment

pattern of chronic renal failure. (Table 8)

Table 8 : Sediment finding of patients with renal diseases

SEDIMENT PATTERN NO OF CASES nephrotic 41 nephritic 33 nephrotic-nephritic 03 acute tubular injury 01 others 02 chronic renal failure 20 total 100

Based on the histopathological diagnosis, patients were grouped into 12 individual groups (Table 9) with chronic renal failure patients as group 13. Post infectious glomerulonephritis was the most common diagnosis on renal biopsy with 19 patients (19%) tailed by membranous nephropathy with 17 patients (17%) and minimal change disease 12 patients (12%). In lupus nephritis out of 9 patients, based on pattern defined by WHO: 2 patients were categorised as class

IV, 4patients as class V and 3 patients with mixed lupus class IIIa/V and class

IVa/V (Table 9)

Table 9: Histopathological diagnosis in renal patients and their groups

GROUP HISTOPATHOLOGICAL DIAGNOSIS NO OF CASES NO 1 Endocapillary-proliferative glomerulonephritis 19 2 Membranous nephropathy 17 3 Minimal change disease 12 4 Lupus nephritis 9 5 Mesangioproliferative glomerulonephritis 6 6 Focal segmental glomerulosclerosis 6 7 IgA Nephropathy 5 8 Diabetic nephropathy 2 9 Crescentic glomerulonephritis 1 10 Acute tubular injury 1 11 Acute tubulo-interstitial nephritis 1 12 Chronic interstitial nephritis 1 Total 80

On histopathological examination patients with endocapillary proliferative glomerulonephritis showed endocapillary and mesangial proliferation in all glomeruli, with cellular crescents and patchy inflammatory cell infiltrates in the interstitium noted in few patients. Patients with membranous nephropathy showed spikes and pinhole lesions on the glomerular basement membranes. In patients with minimal change disease no prominent histopathological changes were noted.

In patients with lupus nephritis WHO class V lesion, spikes and pinhole lesions were seen on the glomerular basement membranes. In mixed lupus patients with class IIIa / V lesion, increase in mesangial matrix with segmental endocapillary proliferation in all viable glomeruli and focal spike formation as well as double contours were seen on the glomerular basement membranes. In class IVa/ V lesion, glomeruli showed an increase in cellularity and mesangial matrix with double contours as well as spike formation and pinhole lesions seen on the glomerular basement membranes. Wire loop lesions were identified and hyaline thrombi also seen in few glomeruli. In diffuse lupus with class IVc lesion increase in mesangial matrix and a mild increase in mesangial cellularity were seen with prominent basement membranes showing double contours. Segmental sclerosis was identified in few glomeruli. Lymphocytic infiltrate along with lymphoid aggregates were seen in the interstitium. Interstitial fibrosis and tubular atrophy noted.

In patients with mesangioproliferative glomerulonephritis mild increase in mesangial cellularity and mesangial matrix with open capillary loops having delicate contours were noted. Patient with membranoproliferative

58 glomerulonephritis showed enlarged glomeruli with diffuse and segmental endocapillary proliferation and double contours were seen on the glomerular

basement membranes. Early cellular crescents were also identified in few

glomeruli. Patients with focal segmental glomerulosclerosis showed glomeruli

with segmental sclerosis with adhesions to the Bowmans capsule. In patients with

IgA Nephropathy an increase in mesangial cellularity and mesangial matrix was

seen in all viable glomeruli with segmental sclerosis identified in few glomerulus

and interstitial fibrosis and tubular atrophy also noted. Few cases showed global

sclerosis of glomeruli with advanced disease.

In a patient with nodular glomerulosclerosis there was diffuse and nodular

increase in mesangial matrix and cellularity with few glomeruli showing global

sclerosis. Tubules were dilated and tubular epithelial cells were swollen with

cytoplasmic vacuoles. Arteries showed moderate medial hypertrophy. In a patient

with diabetic nephropathy few globally sclerotic glomeruli were seen with few

showing periglomerular fibrosis. There was diffuse and nodular increase in

mesangial matrix and cellularity. Kimmelsteil Wilson nodules were seen, which

was PAS and PASM positive and blue on trichrome. Glomerular and tubular

basement membranes appears prominent with interstitial fibrosis and tubular

atrophy noted. Lymphocytic infiltration was seen in the fibrotic areas. Both

afferent and efferent arteriolar hyalinosis seen. Artery showed fibrointimal

hyperplasia.

Patient with crescentic glomerulonephritis showed cellular crescents in

glomeruli with endocapillary proliferation along with a mild influx of neutrophils

59 in all glomeruli.RBC casts were seen in some of the tubules and tubular epithelial cell injury was noted.

In a patient with acute tubular injury, tubules were dilated with loss of

brush border and tubular epithelial cells were swollen with cytoplasmic vacuoles and forms apical blebs focally. In some of the tubules epithelial cells were sloughed off forming granular debris. Interstitial fibrosis and tubular atrophy were noted and lymphocytic infiltration was seen in fibrotic areas. Arteries show moderate medial hypertrophy with arteriolar hyalinosis. Patient with acute tubulointerstitial nephritis showed dilated tubules with loss of brush border and swollen tubular epithelial cells with cytoplasmic vacuoles. Tubulitis was also present. Interstitium was edematous and was infiltrated with lymphocytes.

Arteries showed medial hypertrophy.

On immunofluorescence most of the patients with endocapillary proliferative glomerulonephritis showed C3 (+3) coarse granular positivity over the capillary walls with few showing varied positivity for IgG, IgM, IgA and C1q.

Patients with membranous nephropathy showed IgG (+3) granular positivity over the capillary walls in predominance. In patients of minimal change disease all stains were negative. Patients with lupus nephritis showed predominantly IgG

(+3) and varied positivity for IgM, IgA, C3 and C1q over the capillary walls and focally over the mesangium.

A patient of mesangioproliferative glomerulonephritis showed IgG (+3),

IgM (+1), IgA (+3), C3 (+2) and C1q (+2) positivity over the mesangium. In

patients with focal segmental glomerulosclerosis all stains were negative. Patients

60 with IgA Nephropathy showed IgA (+3) positivity over the mesangium. Diabetic nephropathy patient showed IgG (+1) linear positivity over the glomerular and tubular basement membranes. In a patient with crescentic glomerulonephritis C3

(+3) coarse granular positivity over the capillary walls was seen. In other groups all stains were negative.

The mean age of presentation in patients with proliferative

glomerulonephritis was 35.89 years and in patients of membranous nephropathy it

was 39.58 years (Table 10).

Table 10 : Age distribution within groups.

GROUP MEAN S.D 1 35.89 17.24 2 39.58 15.92 3 24.16 15.05 4 28.44 8.39 5 19.83 7.35 6 28.33 11.69 7 29 5.87 8 37.5 3.53 9 11 - 10 29 - 11 30 - 12 30 - 13 46.7 17.87

The following pattern of urinary sediments were seen on microscopic

examination of cases with various renal diseases. Patients with endocapillary

proliferative glomerulonephritis showed moderate to severe amount of

61 dysmorphic RBCs and WBCs, mild amount of RTECs, mild to moderate amount

of RBC casts, mild WBC casts and mild to severe granular casts. In membranous

nephropathy mild to moderate amount of dysmorphic RBCs, mild to very severe

fatty casts and granular casts. Minimal change disease patients showed mild

amount of RTECs, moderate to very severe hyaline casts and granular casts with mild to moderate oval fat bodies and fatty casts.

Patients with class III and class IV lupus nephritis showed moderate to severe amount of dysmorphic RBCs, mild to moderate WBCs, mild RTECs with mild to moderate RBC casts and very severe granular casts. In class V cases only mild dysmorphic RBCs with moderate to severe amount of granular casts were seen. In mesangioproliferative glomerulonephritis mild to moderate amount of dysmorphic RBCs with mild amount of WBCs and RTECs, mild to severe amount of granular casts. Patients with focal segmental glomerulosclerosis showed mild to moderate amount of RTECs and RBCs, moderate to very severe hyaline casts, granular casts and fatty casts with few cases showing oval fat bodies and cholesterol crystals.

In IgA Nephropathy patients mild to moderate dysmorphic RBCs, WBCs,

RBC casts, fatty casts and granular casts were seen. Patients with diabetic nephropathy showed mild to moderate amount of dysmorphic RBCs with mild

WBCs and RBC casts and severe to very severe granular casts. In rapidly progressive glomerulonephritis very severe amount of dysmorphic RBCs, moderate RBC casts with very severe granular casts. Patient with acute tubular injury showed moderate amount of RTECs and RTEC casts with very severe

62 granular casts. In acute tubulointerstitial nephritis moderate WBCs and dysmorphic RBCs with mild RTECs and very severe amount of granular casts was seen. A patient with chronic interstitial nephritis showed mild amount of

WBCs, RTECs and granular casts. Cases of chronic renal failure showed moderate to severe granular casts with few cases showing mild amount of broad waxy casts. (Table 11)

Table 11 : The pattern of urinary sediments in various renal diseases

HISTOPATHOLOGICAL SEDIMENTARY PATTERN DIAGNOSIS Endocapillary proliferative dysmorphic RBCs, WBCs, RTECs, glomerulonephritits RBC casts, WBC casts, granular casts Membranous nephropathy dysmorphic RBCs, fatty casts, hyaline granular casts, granular casts Minimal change disease RTECs, oval fat bodies, hyaline casts, hyaline granular casts, granular casts, fatty casts Class III/IV Lupus nephritis dysmorphic RBCs, WBCs, RTECs, RBC casts, granular casts Class V Lupus nephritis dysmorphic RBCs, granular casts

Mesangioproliferative dysmorphic RBCs, WBCs, RTECs, glomerulonephritis granular casts Focal segmental glomerulosclerosis RTECs, dysmorphic RBCs, oval fat bodies, fatty casts, hyaline casts, hyaline granular casts, granular casts, cholesterol crystals IgA Nephropathy dysmorphic RBCs, WBCs, RBC casts, fatty casts, granular casts Diabetic nephropathy dysmorphic RBCs, granular casts

Crescentic glomerulonephritis dysmorphic RBCs, RBC casts, granular casts Acute tubular injury RTECs, RTEC casts, granular casts Acute tubulo-interstitial nephritis WBCs, dysmorphic RBCs, RTECs, granular casts Chronic interstitial nephritis WBCs, RTECs, granular casts Chronic renal failure granular casts, broad waxy casts

Urine sediment scoring showed majority of patients with grade 3 score:

49 /100 patients (49%) (Table12).

Table 12: Urine sediment scoring by Lakhmir S.Chawla.et.al.2008 [93]

SCORE NO OF CASES 1 0 2 23 3 49 4 28

Graphs were plotted for all 13 groups correlating urine sediment score with serum creatinine level.

Chart no 3 : Group 1 – Endocapillary proliferative glomerulonephritis

GROUP 1 4 2.7 2.9 3 2.4 2.5 2.1 2.2 1.8 1.8 2 1.5 1.5 1.5 1.2 1.3 1.3 0.8 0.8 0.8 0.8 0.8 1 (mg/dL) 0 2222222223333333333 SERUM CREATININE LEVEL URINE SEDIMENT SCORE

Out of 19 patients, 2 patients did not show linear correlation case no 10 and

11. In these patients sediment score has added value of predicting the extent of damage well before the increase in serum creatinine levels. On statistical analysis the correlation coefficient showed “ high correlation” with value in between 0.6 –

0.8 ( 0.672 ) and p value <0.05 ( 0.002 ) , which is statistically significant.

Chart No 4 : Group 2 – Membranous nephropathy

GROUP 2 3 2.6

2 1.4 1.5 1.5 1.1 1.2 0.8 0.8 0.9 0.9 111 0.8 0.8 0.8 1 0.7

(mg/dL) 0 22222222333344444

SERUM CREATININE LEVEL URINE SEDIMENT SCORE

Out of 17 patients, 4 patients did not show linear correlation case no 28,

32, 33 and 34. In these patients early detection of injury by sediment scoring has added value in early initiation of treatment. The course of the diseasecan be modified by early immunotherapy. On statistical analysis the correlation

65 coefficient showed “no correlation” with value in between 0 – 0.2 (0.07) and p value >0.05 (0.722), which is statistically not significant.

Chart no 5 : Group 3 - Minimal change disease

GROUP 3 1.5 1 0.8 0.8 0.8 0.8 0.8 0.8 0.9 0.9 1 0.6 0.7 0.7 0.5

(mg/dL) 0 333333344444

SERUM CREATININE LEVEL URINE SEDIMENT SCORE

All 12 patients, showed linear correlation between urine sediment score

and serum creatinine level. On statistical analysis the correlation coefficient

showed “high correlation” with value in between 0.6 – 0.8 ( 0.708 ) and p value

<0.05 ( 0.01) , which is statistically significant.

Chart no 6 : group 4 - Lupus nephritis

GROUP 4 10 9.2 8 6 2.9 4 2.2 1.7 1.4 2 0.9 1.2 0.6 0.8

(mg/dL) 0 333344444

SERUM CREATININE LEVEL URINE SEDIMENT SCORE

Out of 9 patients, 3 patients did not show linear correlation case no 53, 54

and 55. In these patients sediment score has added value of early diagnosis of the

“nephritic flares” which is of paramount importance in the guidance of therapeutic

intervention. On statistical analysis excluding these 3 cases the correlation

66 coefficient showed “no correlation” with value 0 and p value >0.05 (1.000), which is statistically not significant.

Chart no 7 : Group 5 - Mesangioproliferative glomerulonephritis

GROUP 5 2 1.7 1.4 1.5 1.5 1.5 1 0.8 0.8 0.5

(mg/dL) 0 223333

SERUM CREATININE LEVEL URINE SEDIMENT SCORE

All 6 patients, showed linear correlation between urine sediment score and serum creatinine level. On statistical analysis the correlation coefficient showed

“very high correlation” with value in between 0.8 – 1.0 ( 0.853 ) and p value

<0.05 ( 0.031 ) , which is statistically significant.

Chart no 8 : Group 6 - Focal segmental glomerulosclerosis

GROUP 6 10 8.4 8

(mg/dL) 2 1.3 1.4 2 1.2 0.8 0 SERUM CREATININE LEVEL 333444 URINE SEDIMENT SCORE

Out of 6 patients, case number 67 did not show linear correlation. In this

patient, the sediment score has added value of diagnosing the increased severity of

67 injury. The cause of injury may bedue to associated increased endocapillary cellularity, collapse of the glomerular tufts, the so-called tip lesion. On statistical analysis the correlation coefficient showed “possible correlation” with value in between 0.2 – 0.4 ( 0.293 ) and p value >0.05 ( 0.573 ) , which is statistically not significant.

Chart No 9 : Group 7 -IgA Nephropathy

GROUP 7 4 3 3 2.5 2 1.9 2 1.5 1 (mg/dL) 0 22233

SERUM CREATININE LEVEL URINE SEDIMENT SCORE

Out of 5 patients, case number 73 and 74 did not show linear correlation. In these cases sediment score has added value of prognostic significance. The increase in sediment score may be due to associated tubulointerstitial lesion in renal parenchyma which along with proteinuria > 1.0 g per day and elevated serum creatinine at presentation is predictive of poor prognosis. On statistical analysis, the correlation coefficient showed “ negatively very high correlation” with value in between minus 0.8 – minus 1.0 (- 0.866 ) and p value >0.05 (0.058), which is statistically near significant.

In group 8 with patients of diabetic nephropathy, the urine sediment score did not show linear correlation with serum creatinine level and has added value of early diagnosis of progression to end-stage renal failure in association with

68 nephrotic range proteinuria and the possibility of superimposed proliferative and active glomerulonephritis such as IgA Nephropathy, acute post-infectious glomerulonephritis, or extracapillary glomerulonephritis. Calculation of correlation values was not possible because of less number of patients within the group.

In case of group 9, with a single patient of rapidly progressive renal failure linear correlation was seen.

In group 10 with a single patient of acute tubular injury linear correlation

was seen between the sediment score and serum creatinine value.

Group 11 with a patient of acute tubulointerstitial nephritis showed linear

correlation.

In group 12 with a single patient of chronic interstitial nephritis, case

number 80, the urine sediment score did not show linear correlation even though

the serum creatinine level is highly elevated. Sediment score had added value of

predicting the typical indolent clinical course with slowly progressive renal

insufficiency.

Chart No10 : Group 13 – Chronic renal failure

GROUP 13 20 17.9 17.2 15.515.516.8 14 14.114.4 14.2 15 11.411.512.1 11.7 9.9 10.310.7 10 6.5 7.9 4.1 5.5 5 (mg/dL) 0 33333333333333444444

SERUM CREATININE LEVEL URINE SEDIMENT SCORE

In group 13 with chronic renal failure patients, all patients have progressed to end stage renal disease with moderate to severe grade of injury.Sediment scoring had added value of predicting progression of disease on follow up and guide in treatment planning. On statistical analysis the correlation coefficient showed “ moderate correlation” with value in between 0.4 – 0.6 ( 0.587 ) and p value <0.05 ( 0.007) , which is statistically significant.

Table 13 : Statistical values of each groups

SEDIMENT CORRELATION P GROUP MEAN S.D SCORE COEFFICIENT VALUE 1 Creatinine 1.61 0.69 0.672 0.002 2 Creatinine 1.1 0.46 0.07 0.722 3 Creatinine 0.8 0.1 0.708 0.01 4 Creatinine 2.32 2.67 0 1.000 5 Creatinine 1.28 0.38 0.853 0.031 6 Creatinine 2.51 2.9 0.293 0.573 7 Creatinine 2.18 0.58 -0.866 0.058 13 Creatinine 12.06 3.9 0.587 0.007

When overall values of urinary sediment score was correlated with serum

creatinine levels, the correlation coefficient showed value in between 0 – 0.2

(0.173 ) interpreted as “no correlation” and p value > 0.05 ( 0.085 ) , which is

statistically not significant.

When 24hours urine protein excretion levels were compared with severity

of renal damage using urine sediment score, the correlation coefficient showed

positive correlation with value in between 0.2 – 0.4 ( 0.307 ) interpreted as

“possible correlation” and p value < 0.05 (< 0.0001), which is statistically significant.

Table 14 : Overall statistical values

CORRELATION OVERALL 100 PATIENTS P VALUE COEFFICIENT Sediment score Creatinine 0.173 0.085

Sediment score 24 hours urine protein level 0.307 <0.0001

On statistical analysis for the subgroup of 15 patients, correlation coefficient showed negative correlation with value in between minus 0.6 – minus

0.8 ( - 0.684 ) which is interpreted as ‘high correlation’ with p value of 0.005

which is statistically significant.

Table 15 : Statistical values for subgroup cases

CORRELATION SUBGROUP 15 PATIENTS P VALUE COEFFICIENT Sediment score Creatinine -0.684 0.005

On further calculation excluding these subgroup of patients (15 cases) from

the overall study population the correlation coefficient showed in between 0.2-0.4

(0.354) interpreted as possible correlation with p value < 0.05 ( 0.001) which is

statistically significant.

Table 16 : Corrected statistical values for 85 cases excluding the subgroups

CORRELATION OVERALL 85 PATIENTS P VALUE COEFFICIENT

Sediment score Creatinine 0.354 0.001

Figure 1: Urine sediment: coarse granular cast ( 10X)

Figure 2: Urine sediment: muddy brown broadcast ( 10X)

Figure 3: Urine sediment: broad waxy granular cast (10X)

Figure 4: Urine sediment: yeast mycelium( 10X)

DISCUSSION

Kidneys have large functional reserve and most of the renal diseases manifest symptoms later after progression of injury thus making them difficult to manage. The need for diagnosing renal injury when it is minimal is hence required. Microscopic examination of urine is a simple test for the evaluation of extent of renal injury. In the present study, 100 patients presenting with clinical manifestations of renal injury were completely evaluated which includes collection of clinically relevant data, measuring serum biochemical parameters and conducting proper urine examination. Most of the patients presented later in the clinical course with history of duration of illness being non reliable and baseline serum creatinine level could not be evaluated to determine the percentage increase for staging of acute renal injury.

In the present study male to female ratio was 1.3:1, with male predominance and mean age of 34.94 years. The commonest sediment pattern was nephrotic sediment with 41% cases, 33% cases presented with nephritic sediment

and 20% cases presented with chronic renal failure pattern of urinary sediment. In

the study done by U. Das.et. al.[105] the ratio of male : female was 1.4:1 and the most common indications of renal biopsy in their study were nephrotic syndrome

(49%), tailed by chronic renal failure (13.6%).

In the present study, the most common diagnosis on renal biopsy was infection related glomerulonephritis (19%) followed by membranous nephropathy

(17%). The mean age of presentation of infection related glomerulonephritis was

35.89 years and in patients of membranous nephropathy was 39.58 years. In the study done by U. Das et al. [105] primary glomerulonephritis comprised 69.1% and

among the primary glomerulonephritis patients, the commonest was minimal

change disease (21.8%), tailed by focal segmental glomerulosclerosis (15.3%). In

the study done by Muhammed Mubarak et al. [106], the ratio of male to female was

1.6:1 with 61.9% males and 38.1% females. The most common diagnosis was

focal segmental glomerulosclerosis (38.5%), tailed by minimal change disease

(23.2%). In the study done by A. S. Gyory et al.[107], the most common

histopathological lesion was IgA Nephropathy.

In the present study, on microscopic examination based on RBC

morphology, the glomerular (GH) or a non-glomerular (NGH/urological) origin of

the haematuria was determined when there were ≥ 40% dysmorphic RBCs and/or

≥ 5% acanthocytes. G.B.Fogazzi.et.al. [36] investigated 16 patients (10 children and 6 adults) with IMH, which was considered of glomerular origin when there were ≥ 40% dysmorphic RBCs and/or ≥ 5% acanthocytes. After repeated urinary sediment examinations (2-8/patient for a total of 55), all patients were submitted to renal biopsy. A glomerular disease was found in 14/16 patients (87.5%).

On examination of the urinary sediment, coupled with the assessment of proteinuria, various clinical conditions presented with different urinary profiles.

Nephrotic sediment was observed in cases of membranous nephropathy, minimal change disease, focal segmental glomerulosclerosis, membranous lupus nephritis and diabetic nephropathy. Marked cylindruria, especially fatty casts was the hallmark of the nephrotic sediment. Microscopic haematuria was variable

75 according to the type of glomerulopathy. It was absent or mild in minimal change disease, while it was more frequent in focal segmental glomerulosclerosis and idiopathic membranous nephropathy, and variable in diabetic nephropathy. In these conditions, leukocyturia was uncommon. The nephritic sediment was observed in acute post-infectious glomerulonephritis, active class III and IV lupus nephritis, membranoproliferative glomerulonephritis, IgA Nephropathy and necrotizing glomerulonephritis. Erythrocyturia, leukocyturia, shedding of RTECs and erythrocytic cylindruria was the hallmark sediments.G.B.Fogazzi.et.al. [36] in their study comparing 52 patients of proliferative glomerular diseases with 48 patients of non proliferative glomerular disorders, confirmed that the cohort of patients with a proliferative disorder had significantly higher prevalence of microscopic haematuria (98%vs 66.7%), leukocyturia (73.1% vs18.8%),shedding of RTECS (82.7%vs64.6%), erythrocytic cylindruria (84.6%vs39.6%), and RTEC casts (94.2%vs79.2%). In our study a urine profile with both nephrotic and nephritic features was found in 3/9 patients with lupus nephritis. Apple GB et.al.

[75] described that the 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. RTECs cells associated with RTEC casts and dark granular casts without any other elements was seen in a case of acute tubular necrosis.

In the present study sediment scoring was done using Lakhmir S. Chawla et al. cast scoring index. Out of the 100 patients, 23patients presented with score 2

(mild injury), 49 patients with score 3 (moderate injury) and 28 patients with

76 score 4 (severe injury).The role of urine microscopy in the assessment of severity of renal damage was evaluated by correlating with serum creatinine level as reference for extent of damage.

In correlation graphs, few cases within the groups did not show linear correlation between the urine sediment score and the serum creatinine level. In these subgroup of cases, urine sediment scoring was predictive of the extent of damage well before the increase in serum creatinine level. In membranous nephropathy the clinical course is highly variable. 30-40% of patients progress to end-stage renal failure while a few may go into spontaneous remission. The course of the disease can be modified by early immunotherapy. In lupus nephritis early diagnosis of the nephritic flares is of paramount importance in the guidance of therapeutic intervention. In focal segmental glomerulosclerosis urine sediment scoring was predictive of the progression of disease with increase in severity of injury, well in advance. The cause of injury may be due to associated increased

endocapillary cellularity, collapse of the glomerular tufts, the so-called tip lesion.

In IgA Nephropathy urine sediment score was of prognostic significance. The

increase in sediment score may be due to associated tubulointerstitial lesion in

renal parenchyma which along with proteinuria and elevated serum creatinine at

presentation is predictive of poor prognosis. In diabetic nephropathy early

diagnosis of progression to end-stage renal failure and the possibility of

superimposed proliferative and active glomerulonephritis such as IgA

Nephropathy, acute post-infectious glomerulonephritis, or extracapillary

glomerulonephritis can be determined using sediment score. In chronic interstitial

77 nephritis urine sediment score was predictive of the typical indolent clinical course with slowly progressive renal insufficiency.

In patients of chronic renal failure with known etiology (previous renal biopsy) presenting with severe azotemia and contracted kidney size which are relative contraindications for renal biopsy [101] , the sediment score may be of value in follow up of patients, evaluating the progression of disease to end stage renal failure and planning of early intervention like renal replacement therapy.

The statistical analysis was significant with p value < 0.05 in group 1, 3, 5,

13 with final diagnosis of endocapillary proliferative glomerulonephritis, minimal change disease, mesangioproliferative glomerulonephritis, chronic renal failure respectively and near significant in group 7, IgA Nephropathy. They also showed positive correlation with possible to very high correlation values with exception of group 7 which showed negative correlation. This signifies that the increase in sediment score can be correlated with increase in serum creatinine level except in cases of IgA Nephropathy where the increase in sediment score can be correlated with low serum creatinine levels.Vikse.B.E.et.al.[69] 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 cases of membranoproliferative glomerulonephritis. In case of IgA

Nephropathy the negative correlation may be due to low number of patients

within this group (5 patients) and it contains subgroup (2 patients) where the renal

damage was detected earlier using sediment score before the increase in serum

78 creatinine level, which is to be further evaluated using more number of study

population. Ibels.et.al. [67], studied 174 patients with IgA Nephropathy, and 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. Nakayama.et.al.[68] 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.

Group numbers 2, 4 and6with final diagnosis of membranous nephropathy,

lupus nephritis and focal segmental glomerulosclerosis showed no and possible

correlation with serum creatinine level and the p values were > 0.05which was

statistically not significant. This may be due to the subgroup of patients in which

urine sediment scoring had added values of detecting the damage when it is

minimal with low or near normal serum creatinine levels. In the study by

Balow.J.Eet.al [73],they described early diagnosis of renal disease is of paramount importance, and patients with known or suspected lupus should undergo urinalysis at regular intervals. Illei.et.al.[74] explained that 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.

In these groups further evaluation is to be done with more numbers of patients

within each group to determine the statistical significance and correlation

coeffecient between urine sediment score and serum creatinine levels. In

79 remaining groups statistical evaluation was not possible because of low number of cases.

On calculation of overall statistical values, the correlation coefficient

showed positive correlation with value in between 0 – 0.2 ( 0.173 ) interpreted as

“no correlation” and p value > 0.05 ( 0.085 ), which was statistically not significant. This may be due to less number of cases within few groups for calculating statistical values and the presence of subgroups which interferes with the evaluation. Demosthenes B.Panagiotakos [104] in his study described that p-

value is influenced by sample size and showed if the initial sample size is doubled

the study’s results achieve significance. In the study done by Mark A. Perazella et

al. [98] urine sediment scoring was significantly related with increased risk of deteriorating acute kidney injury and the sensitivity, specificity were 76% and

86% respectively. In the study done by Fogazzi GB et al. [36] the sensitivity and

specificity of urine sediment analysis in predicting histopathological changes were

80.8% and 79.2% respectively. In the study by Bhagyalakshmi A et al. [100] , the specificity and positive predictive value of Lakhmir S. Chawla et al. urine sediment score in predicting the severity of renal injury were 79.5% and 86.88% respectively with P value of 0.00082 which was statistically significant. Hence further studies with increased study population are needed for overall statistical significance. In our study when 24 hours urine protein values were correlated with urine sediment score as a measure of severity of renal injury, they had p value <

0.05 (<0.0001), which is statistically significant and showed positive correlation with correlation coefficient between 0.2 – 0.4 ( 0.307 ) interpreted as “possible

80 correlation”. This signifies that urine protein level can be used as a measure of extent of damage in the evaluation of patients with renal diseases where with the increase in sediment score, the urine protein level also increases. In the study by

Bhagyalakshmi A.et.al [100], the positive predictive value of 24 hour urine protein

value in estimating the severity of renal damage was 76.36%.

In our study an attempt was made to statistically analysis the subgroup of

15 patients and on analysis the correlation coefficient showed negative correlation

with value in between minus 0.6 – minus 0.8 ( - 0.684 ) which is interpreted as

high correlation with p value of 0.005 which is statistically significant. This

signifies that in these patients the increase in urine sediment score is correlated

with low values of serum creatinine, which may be due to early detection of renal

damage by sediment scoring well before the increase in serum creatinine levels.

Then the overall corrected statistical values on excluding these subgroup of

patients which had added value of early detection of renal damage were calculated

and the correlation coefficient was in between 0.2-0.4 (0.354) interpreted as

possible correlation with p value < 0.05 ( 0.001) which is statistically significant.

This signifies that increase in urine sediment score can be correlated with increase

in serum creatinine level in all groups of renal diseases.

SUMMARY AND CONCLUSION

On microscopic examination of urinary sediments it is possible to differentiate glomerular and non- glomerular causes of haematuria. Differentiation between the proliferative and nonproliferative causes of glomerulonephritis can be made with certainty based on the pattern of urinary sediments and can be correlated with the type of renal damage on renal biopsy.

As such, the urinary sediment score serves as an excellent proxy for the severity of renal injury and has additive value as an early predictor of damage, than the change in serum creatinine at the time of admission. The urine sediment score was predictive of worsening of clinical outcome at the time of admission.

One can speculate that increased numbers of RTECs and granular casts (in a graded fashion) reflect cell death and apoptosis that would be associated with more severe renal tubular injury, and thus worse outcomes. Urine protein excretion levels was also significant in prediction of the severity of renal injury and correlated with urine sediment score.

This was a pilot study attempted to correlate the pattern of urine sediments with type of histopathological lesion and the sediment score with concentration of creatinine in serum and urine protein excretion levels, which are most widely used as a measure of renal function and commonly accepted as a predictor of extent of damage in clinical medicine. Further studies are needed to evaluate the role of urinary sediments in predicting the histopathological diagnosis and

82 severity of renal diseases using larger study population with renal biopsy as gold standard.

 Based on the combination of meticulous study on pattern of urinary

sediments with serum and urine biochemical parameters it may be possible

to predict the histopathological diagnosis.

 Urine sediment scoring was significant in assessing the extent of renal

damage in non- neoplastic renal diseases.

 It has added values of early prediction of renal damage well before the rise

in serum creatinine levels.

 The decision of further evaluation, early treatment and regular follow-up

can be made by taking urine microscopy findings into consideration.

 Urinary microscopy should remain as a standard practice and valuable tool

in the diagnosis and assessment of renal diseases along with the serum

creatinine and urine protein level as markers of renal damage.

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ANNEXURES

ANNEXURE I

PROFORMA

NAME

AGE

SEX

ADDRESS

OCCUPATION

DURATION AND DETAILS OF ILLNESS

SYMPTOMS

 Facial fluffiness

 Bilateral leg swelling

 Decreased urine output

 Burning micturition

 Red or muddy colored urine

 Abdominal distention

 Breathlessness

PAST HISTORY

 Diabetes

 Hypertension

 Coronary artery disease

 Chronic kidney disease

 Connective tissue diseases

DRUG HISTORY

PERSONAL HISTORY

 Smoking

 Alcoholism

GENERAL EXAMINATION

 Pallor

 Pedal edema

 Ascites

SYSTEMIC EXAMINATION

CVS

ABDOMEN

CNS

INVESTIGATIONS

 CBC

 Blood urea

 Serum creatinine

 Urine biochemical analysis

 24hrs urine protein level

 Imaging studies

DEPOSITS

DYSMORPHIC RBCS RBC CASTS

WBCS WBC CASTS

RTEC RTEC CASTS

HYALINE CASTS BROAD WAXY CASTS

GRANULAR CASTS OTHERS

CHAWLA CAST SCORING INDEX

 Score 1

 Score 2

 Score 3

 Score 4

PATTERN OF URINARY SEDIMENT

 Nephrotic sediment

 Nephritic sediment

 Nephrotic – nephritic sediment

 Acute tubular injury

 Chronic renal failure

 Others

LIQUID RENAL BIOPSY DIAGNOSIS

RENAL BIOPSY REPORT

ஆராய்ச்சி தகவல்தாள் திெநல்ேவ மத்வ கல்ாி அரசு ெபா மத்வமைனக்கு

வம் சிநீரக ேநாய்கள் ெகாண்ட ேநாயாளிகக்கு சிநீர் பகள்

ைற பற்றிய ஒ ஆராய்ச்சி நைடெபற் வகிற.

சிநீர் பகள் ைற அப்பைடயில் அதற்கான காரணங்கள்

என்ன என்ப பற்றி அறிந் ெகாள்வேத இந்த ஆராய்ச்சியின்

ேநாக்கமாகும்.

நீங்கம் இந்த ஆராய்ச்சியில் பங்ேகற்க நாங்கள் விம்கிேறாம்.

கைள அல்ல கத்க்கைள ெவளியிம் ேபாேதா அல்ல

ஆராய்ச்சியின் ேபாேதா தங்கள ெபயைரேயா அல்ல

அைடயாளங்கைளேயா ெவளியிடமாட்ேடாம் என்பைதம் ெதாிவித்க்

ெகாள்கிேறாம்.

இந்த ஆராய்ச்சியில் பங்ேகற்ப தங்கைடய விப்பத்தின்

ேபாில்தான் இக்கிற. ேமம் நீங்கள் எந்ேநரம் இந்த

ஆராய்ச்சியித் பின்வாங்கலாம் என்பைதம் ெதாிவித்க்

ெகாள்கிேறாம்.

இந்த சிறப்ப் பாிேசாதைனகளின் கைள ஆராய்ச்சியின் ேபா

அல்ல ஆராய்ச்சியின் வில் தங்கக்கு அறிவிப்ேபாம் என்பைதம்

ெதாிவித்க் ெகாள்கிேறாம்.

ஆராய்ச்சியாளர் ைகெயாப்பம் பங்ேகற்பாளர் ைகெயாப்பம்

ேததி:

ஆராய்ச்சி ஒப்தல் கதம்

ஆராய்ச்சி தைலப் : சிநீரக ேநாய்கள் சிநீர் பகள் ைற ஆய்

ெபயர் : ேததி :

வய : உள்ேநாயாளிஎண்:

பால் : ஆராய்ச்சிேசர்க்ைகஎண்:

இந்த ஆராய்ச்சியின் விவரங்கம் அதன் ேநாக்கங்கம்

ைமயாக எனக்கு ெதளிவாக விளக்கப்பட்ட. எனக்கு விளக்கப்பட்ட

விஷயங்கைள நான் ாிந் ெகாண் என சம்மதத்ைத ெதாிவிக்கிேறன்.

சிநீரக ேநாய்கள் மற்ம் பாதிப்கள் குறித் ஆராய்ச்சியாளர் கூற

வம் விளங்கப் ெபற்ேறன்.

இதற்குத் ேதைவயான உடற்பாிேசாதைனக்கும், இரத்தம் மற்ம்

சிநீர் பகுப்பாய் சம்பந்தப்பட்ட பாிேசாதைனகக்கும் மனமார

சம்மதிக்கிேறன்.

ைகெயாப்பம்

GROUPS URINARY SEDIMENTS

URINE DIPSTICK ANALYSIS CELLS CASTS

1 10 male 60020 0.8 22 pink 1.015 7 50 positive 50 4 3 3.6 severe severe mild moderate mild normal normal normal mild normal NEPHRITIC 2 proliferative glomerulonephritis Negative 1

2 32 female 18952 0.8 34 yellow 1.02 6 30 negative 20 1 3 0.8 moderate moderate normal mild normal normal normal normal mild normal NEPHRITIC 2 encocapillary proliferative glomerulonephritis C3(+3) 1

3 12 female 50882 0.8 28 yellow 1.015 7 30 positive 50 1 3 0.6 moderate severe mild mild mild normal normal normal mild normal NEPHRITIC 2 encocapillary proliferative glomerulonephritis C3(+2) 1

4 32 female 73207 0.8 36 yellow 1.015 6 30 negative 20 1 3 0.9 moderate moderate normal mild normal normal normal normal mild normal NEPHRITIC 2 encocapillary proliferative glomerulonephritis Negative 1

5 15 female 10747 1.2 26 yellow 1.015 6 30 negative 20 4 3 3.2 moderate moderate normal mild normal normal normal normal mild normal NEPHRITIC 2 encocapillary proliferative glomerulonephritis C3 (+3), IgG (+1),IgM (+1) 1

6 9 male 76045 1.3 24 yellow 1.02 7 30 positive 50 1 3 0.7 moderate severe mild mild mild normal normal normal mild normal NEPHRITIC 2 encocapillary proliferative glomerulonephritis C3 (+3), IgG (+2) 1

7 50 female 46980 1.5 40 yellow 1.015 6 30 negative 20 1 2 0.4 moderate moderate normal mild normal normal normal normal mild normal NEPHRITIC 2 encocapillary proliferative glomerulonephritis C3 (+3), IgG (+1) 1

8 47 male 77749 1.5 46 pink 1.015 6 50 negative 30 1 2 0.6 severe moderate mild moderate mild normal normal normal mild normal NEPHRITIC 2 diffuse proliferative glomerulonephritis with crescents C3 (+3) 1

9 55 female 7249 1.5 48 yellow 1.015 6 30 negative 20 1 2 0.5 moderate moderate normal mild normal normal normal normal mild normal NEPHRITIC 2 encocapillary proliferative glomerulonephritis C3 (+3), IgG (+2) 1

10 16 female 36795 0.8 30 pink 1.015 7 30 positive 30 1 3 1.2 moderate moderate normal mild normal normal normal normal moderate normal NEPHRITIC 3 endocapillary proliferative glomerulonephritis C3 (+3) 1

11 42 female 25428 1.3 47 pink 1.015 6 30 negative 20 1 2 1 moderate moderate normal moderate normal normal normal normal moderate normal NEPHRITIC 3 endocapillary proliferative glomerulonephritis C3(+2) 1

12 47 male 7423 1.8 38 pink 1.015 6 30 negative 20 1 3 1.4 moderate moderate normal moderate normal normal normal normal moderate normal NEPHRITIC 3 endocapillary proliferative glomerulonephritis IgG (+3), C3 (+3), IgM (+1),C1q (+1) 1

13 31 female 8697 1.8 40 yellow 1.02 6 30 negative 20 3 3 2.2 moderate moderate normal moderate normal normal normal normal moderate normal NEPHRITIC 3 encocapillary proliferative glomerulonephritis C3 (+3) 1 IgG (+3), IgM (+3), IgA (+1), C3 (+3),C1q 14 40 female 26945 2.1 52 yellow 1.02 6 30 negative 20 1 2 1.1 moderate moderate normal moderate normal normal normal normal moderate normal NEPHRITIC 3 encocapillary proliferative glomerulonephritis 1 (+2) 15 48 female 1629 2.2 46 yellow 1.015 6 30 negative 20 1 2 1.3 moderate moderate normal mild normal normal normal normal moderate normal NEPHRITIC 3 encocapillary proliferative glomerulonephritis C3 (+3),IgG (+3) 1 IgG (+3), IgM (+1), IgA (+2), C3 (+3), C1q 16 65 female 76036 2.4 54 pink 1.015 6 30 negative 20 3 3 2.7 moderate moderate normal mild normal normal normal normal moderate normal NEPHRITIC 3 encocapillary proliferative glomerulonephritis 1 (+1) 17 30 female 78323 2.5 48 yellow 1.015 6 30 negative 20 1 2 1.2 moderate moderate normal moderate normal normal normal normal moderate normal NEPHRITIC 3 encocapillary proliferative glomerulonephritis C3(+3) 1

18 41 female 30546 2.7 34 pink 1.015 6 50 negative 20 1 3 1.4 severe moderate mild moderate mild normal normal normal severe normal NEPHRITIC 3 focal proliferative glomerulonephritis IgG (+3), C3 (+3), C1q (+1) 1

19 60 female 39271 2.9 52 yellow 1.015 6 30 negative 20 1 2 1 moderate moderate normal moderate normal normal normal normal moderate normal NEPHRITIC 3 endocapillary proliferative glomerulonephritis C3 (+3) 1

20 42 female 34157 0.8 34 yellow 1.015 6 10 negative negative 3 4 3.4 mild normal normal normal normal normal moderate normal mild normal NEPHROTIC 2 membranous nephropathy IgG (+3),C3(+2) 2

21 35 female 71368 0.8 38 yellow 1.015 6 10 negative negative 1 3 1.1 mild normal normal normal normal normal moderate normal mild normal NEPHROTIC 2 membranous nephropathy Negative 2

22 50 male 27487 0.9 42 yellow 1.015 6 10 negative negative 1 3 1.2 mild normal normal normal normal normal mild normal mild normal NEPHROTIC 2 membranous nephropathy IgG (+3) 2

23 20 female 40071 0.9 32 yellow 1.015 6 10 negative negative 3 4 3.2 mild normal normal normal normal normal mild normal mild normal NEPHROTIC 2 membranous nephropathy IgG (+3), IgM (+1), C3 (+3),C1q (+1) 2

24 30 male 54332 1 34 yellow 1.015 6 10 negative negative 1 3 1.4 mild normal normal normal normal normal mild normal mild normal NEPHROTIC 2 membranous nephropathy Negative 2

25 23 male 41281 1 30 yellow 1.015 6 10 negative negative 1 3 1.2 mild normal normal normal normal normal moderate normal mild normal NEPHROTIC 2 membranous nephropathy Negative 2

26 43 male 18720 1 20 yellow 1.015 6 10 negative negative 1 3 1.2 mild normal normal normal normal normal moderate normal mild normal NEPHROTIC 2 membranous nephropathy IgG (+3), C3 (+1) 2

27 29 male 43684 1.1 24 yellow 1.015 6 30 negative negative 3 4 3.2 moderate normal normal normal normal normal moderate normal mild normal NEPHROTIC 2 membranous nephropathy IgG (+3),C3(+2) 2

28 74 male 32658 0.8 38 yellow 1.015 6 30 negative negative 3 4 3.6 moderate normal normal normal normal normal severe normal moderate normal NEPHROTIC 3 membranous nephropathy IgG (+3) 2

29 20 male 36071 1.2 32 yellow 1.015 6 30 negative negative 3 4 3.2 moderate normal normal normal normal normal severe normal severe normal NEPHROTIC 3 membranous nephropathy IgG (+3) 2

30 25 male 4589 1.4 36 yellow 1.015 6 30 negative negative 3 4 3.8 moderate normal normal normal normal normal severe normal moderate normal NEPHROTIC 3 membranous nephropathy IgG (+2) 2

31 38 male 31429 1.5 16 yellow 1.015 6 10 negative negative 1 2 1.8 mild normal normal normal normal normal moderate normal moderate normal NEPHROTIC 3 membranous nephropathy IgG (+3), C3 (+1) 2

32 28 female 2175 0.7 26 yellow 1.015 6 30 negative negative 2 3 3.6 moderate normal normal normal normal normal moderate normal very severe normal NEPHROTIC 4 membranous nephropathy IgG (+3) 2

33 51 male 27510 0.8 30 yellow 1.015 6 30 negative negative 2 3 3.4 moderate normal normal normal normal normal severe normal very severe normal NEPHROTIC 4 membranous nephropathy IgG (+3) and C3 (+2) 2

34 43 male 18962 0.8 24 yellow 1.015 6 30 negative negative 2 3 3.2 moderate normal normal normal normal normal very severe normal very severe normal NEPHROTIC 4 membranous nephropathy IgG (+3) and C3 (+3) 2

35 67 male 44056 1.5 36 yellow 1.015 6 30 negative negative 4 3 4.6 moderate normal normal normal normal normal severe normal very severe normal NEPHROTIC 4 membranous nephropathy Negative 2

36 55 male 43942 2.6 42 yellow 1.015 6 30 negative negative 3 4 4.2 moderate normal normal normal normal normal severe normal very severe normal NEPHROTIC 4 membranous nephropathy IgG (+2) 2

37 13 male 24106 0.6 19 yellow 1.02 6 negative negative negative 2 3 2.2 normal normal normal normal normal normal mild moderate moderate normal NEPHROTIC 3 minimal change disease Negative 3

38 52 female 31609 0.7 16 yellow 1.02 6 negative negative negative 2 3 2.4 normal normal normal normal normal normal moderate moderate severe normal NEPHROTIC 3 minimal change disease Negative 3

39 23 female 51533 0.7 34 yellow 1.015 6 negative negative negative 2 3 2.1 normal normal normal normal normal normal mild moderate severe normal NEPHROTIC 3 minimal change disease Negative 3

40 13 female 24307 0.8 22 yellow 1.015 6 negative negative negative 2 3 2.2 normal normal normal normal normal normal mild moderate severe normal NEPHROTIC 3 minimal change disease Negative 3

41 12 male 59473 0.8 24 yellow 1.015 6 negative negative negative 2 3 2.4 normal normal normal normal normal normal mild moderate severe normal NEPHROTIC 3 minimal change disease Negative 3

42 35 female 67432 0.8 36 yellow 1.015 6 negative negative negative 2 3 2.1 normal normal normal normal normal normal moderate moderate severe normal NEPHROTIC 3 minimal change disease Negative 3

43 16 male 32323 0.8 20 yellow 1.015 6 negative negative negative 1 2 2.4 normal normal normal normal normal normal mild moderate moderate normal NEPHROTIC 3 minimal change disease Negative 3

44 13 male 41592 0.8 18 yellow 1.015 6 negative negative negative 1 2 1.8 normal normal mild normal normal normal mild severe very severe normal NEPHORTIC 4 minimal change disease Negative 3

45 14 male 76554 0.8 26 yellow 1.02 6 negative negative negative 4 4 5.6 normal normal mild normal normal normal mild severe very severe normal NEPHROTIC 4 minimal change disease Negative 3

46 41 male 22375 0.9 29 yellow 1.02 6 negative negative negative 3 4 4.3 normal normal mild normal normal normal mild moderate very severe normal NEPHROTIC 4 minimal change disease Negative 3

47 12 male 26329 0.9 18 yellow 1.015 6 negative negative negative 4 4 4.8 normal normal mild normal normal normal mild moderate very severe normal NEPHROTIC 4 minimal change disease Negative 3

48 46 female 28466 1 24 yellow 1.01 6 negative negative negative 3 4 4.4 normal normal mild normal normal normal mild moderate very severe normal NEPHROTIC 4 minimal change disease Negative 3

49 37 female 72179 0.9 28 yellow 1.015 6 10 negative negative 1 3 1.8 mild normal normal normal normal normal normal normal moderate normal NEPHROTIC 3 membranous lupus nephritis class V IgG (+3), IgM (+1), C3 (+3),C1q (+2) 4

50 31 female 75503 1.2 32 yellow 1.015 6 10 negative negative 1 2 1.2 mild normal normal normal normal normal normal normal moderate normal NEPHROTIC 3 membranous lupus nephritis class V IgG (+3), IgM (+1), C3 (+3),C1q (+2) 4 URINARY SEDIMENTS

URINE DIPSTICK ANALYSIS CELLS CASTS

UREA 24 HRS UPL CASE AGE SEX IP NO Cr mg/dL COLOR S.G. pH RBCs/uL NITRITE WBCs/uL PROTEIN g/L UPL RBCs WBCs RTECs RBC WBC RTEC FATTY HYALINE GRANULAR BROAD WAXY PATTERN USS RENAL BIOPSY REPORT IMMUNOFLUORESCENCE GROUP mg/dL g/day IgG (+3), IgM (+3), IgA (+2), C3 (+3), C1q 51 21 female 62629 1.7 35 yellow 1.02 6 10 negative negative 1 2 1.6 mild normal normal normal normal normal normal normal severe normal NEPHROTIC 3 diffuse proliferative lupus with crescent transformation 4 (+2) IgG (+3), IgM (+3), IgA (+2), C3 (+3), C1q 52 27 female 76913 2.2 39 yellow 1.015 6 10 negative negative 1 2 1.4 mild normal normal normal normal normal normal normal moderate normal NEPHROTIC 3 diffuse and membranous lupus nephritis class V 4 (+3) NEPHRI- IgG (+3), IgM (+2), IgA (+3), C3 (+3), C1q 53 14 male 24962 0.6 19 yellow 1.015 6 30 negative 20 1 3 1.8 moderate mild normal mild normal normal normal normal very severe normal 4 focal proliferative and membranous lupus nephritis classIIIa/V 4 NEPHROTIC (+3) NEPHRI- IgG (+3), IgM (+1), IgA (+3), C3 (+2), C1q 54 40 female 37372 0.8 32 yellow 1.02 6 30 negative 20 1 3 2.2 moderate mild normal mild normal normal normal normal very severe normal 4 focal proliferative and membranous lupus nephritis classIIIa/V 4 NEPHROTIC (+2) NEPHRI- diffuse proliferative and membranous lupus nephritis class IgG (+3), IgM (+3), IgA (+2), C3 (+3), C1q 55 22 female 21539 1.4 27 yellow 1.01 6 30 negative 20 1 3 2.4 moderate mild normal mild normal normal normal normal very severe normal 4 4 NEPHROTIC IVa/V (+3) 56 35 female 34614 2.9 38 pink 1.01 6 50 negative 30 1 2 1.1 severe moderate mild moderate normal normal normal normal very severe normal NEPHRITIC 4 diffuse proliferative lupus nephritis class Iva IgG (+3), IgA (+1), C3 (+3), C1q (+2) 4 IgG (+3), IgM (+2), IgA (+3), C3 (+2), C1q 57 29 female 78426 9.2 64 pink 1.01 6 50 negative 30 1 3 1.3 severe moderate mild moderate normal normal normal normal very severe normal NEPHRITIC 4 diffuse lupus nephritis class Ivc 4 (+2) 58 13 male 1132 0.8 18 pink 1.015 6 10 negative negative 1 1 0.4 mild normal normal normal normal normal normal normal mild normal NEPHRITIC 2 mesangioproliferative glomerulonephritis IgG (+2), IgM (+2), C3 (+1) 5

59 23 male 66377 0.8 22 yellow 1.015 6 10 negative negative 3 2 1.2 mild mild mild normal normal normal normal normal mild normal NEPHRITIC 2 membranous proliferative glomerulonephritis Negative 5 IgG (+3), IgM (+1), IgA (+3), C3 (+2), C1q 60 18 female 57354 1.4 24 yellow 1.015 6 30 negative negative 1 3 1.8 moderate normal normal normal normal normal normal normal moderate normal NEPHRITIC 3 mesangioproliferative glomerulonephritis 5 (+2) 61 33 male 4578 1.5 32 yellow 1.015 6 30 negative negative 1 2 0.8 moderate normal normal normal normal normal normal normal moderate normal NEPHRITIC 3 mesangioproliferative glomerulonephritis Negative 5

62 18 female 29920 1.5 29 yellow 1.015 6 30 negative negative 1 2 0.6 moderate moderate mild normal normal normal normal normal severe normal NEPHRITIC 3 membranous proliferative glomerulonephritis Negative 5

63 14 male 37117 1.7 24 pink 1.015 6 30 negative negative 1 2 0.4 moderate mild normal normal normal normal normal normal moderate normal NEPHRITIC 3 mesangioproliferative glomerulonephritis Negative 5

64 39 male 4990 1.2 32 yellow 1.015 6 10 negative negative 3 4 3.2 mild normal mild normal normal normal severe moderate severe normal NEPHROTIC 3 focal segmental glomerulosclerosis Negative 6

65 32 male 60658 1.3 29 yellow 1.015 6 10 negative negative 2 3 2.4 mild normal mild normal normal normal moderate moderate moderate normal NEPHROTIC 3 focal segmental glomerulosclerosis Negative 6

66 16 female 43043 1.4 24 yellow 1.015 6 10 negative negative 3 4 2.6 mild normal mild normal normal normal moderate moderate moderate normal NEPHROTIC 3 focal segmental glomerulosclerosis Negative 6

67 15 female 33451 0.8 30 yellow 1.02 6 30 negative negative 2 2 2.8 moderate normal moderate normal normal normal very severe severe very severe normal NEPHROTIC 4 focal segmental glomerulosclerosis Negative 6

68 25 female 2126 2 28 yellow 1.02 6 10 negative negative 2 2 2.9 mild normal moderate normal normal normal severe severe very severe normal NEPHROTIC 4 focal segmental glomerulosclerosis C3 (+2) 6

69 43 male 12797 8.4 56 yellow 1.015 6 10 negative negative 2 2 3.1 mild normal moderate normal normal normal very severe moderate very severe normal NEPHROTIC 4 focal segmental glomerulosclerosis Negative 6

70 32 male 64955 2 40 yellow 1.02 6 10 negative 10 1 1 0.3 mild mild normal mild normal normal moderate normal mild normal NEPHRITIC 2 IgA nephropathy IgA (+3) MESANGIUM 7

71 22 male 23894 2.5 35 yellow 1.015 6 10 negative 20 1 3 1.1 mild moderate normal mild normal normal moderate normal mild normal NEPHRITIC 2 IgA nephropathy IgA (+3)MESANGIUM7

72 37 male 1781 3 46 yellow 1.015 6 10 negative 10 1 2 0.6 mild mild normal mild normal normal mild normal mild normal NEPHRITIC 2 IgA nephropathy IgA (+3),C3 (+3) MESANGIUM 7

73 25 male 30942 1.5 28 yellow 1.015 6 30 negative 20 1 2 1.2 moderate moderate normal moderate normal normal mild normal moderate normal NEPHRITIC 3 IgA nephropathy IgA (+3), C3 (+1)MESANGIUM 7

74 29 male 25426 1.9 30 pink 1.02 6 30 negative 20 1 3 2 moderate moderate normal moderate normal normal moderate normal moderate normal NEPHRITIC 3 IgA nephropathy IgA (+2), C3 (+2)MESANGIUM 7

75 40 female 5234 1 28 pink 1.01 6 10 negative negative 1 2 2.1 mild normal normal normal normal normal normal normal severe normal NEPHROTIC 3 nodular glomerulosclerosis Negative 8

76 35 male 17305 0.6 22 pink 1.01 6 30 negative negative 4 4 4.8 moderate mild normal mild normal normal normal normal very severe normal NEPHROTIC 4 diabetic nephropathy class 3 IgG (+1) linear positivity GBM, TBM 8

77 11 male 21855 3.8 38 pink 1.01 6 60 negative negative 1 2 0.8 severe normal normal moderate normal normal normal normal very severe normal NEPHRITIC 4 crescentic glomerulonephritis C3 (+3)coarse positivity 9

78 29 male 28257 2.8 42 pink 1.01 6 negative negative negative 1 2 0.6 normal normal moderate normal normal moderate normal normal very severe normal ATN 4 acute tubular injury Negative 10

79 30 male 9934 5.8 64 pink 1.01 6 20 negative 30 1 2 1.4 moderate moderate mild normal normal normal normal normal very severe normal OTHERS 4 acute tubulointerstitial nephritis Negative 11

80 30 male 24143 8.3 120 yellow 1.015 6 negative negative 10 1 3 1 normal mild mild normal normal normal normal normal mild normal OTHERS 2 chronic interstial nephritis Negative 12

81 70 male 55396 4.1 153 yellow 1.01 7 negative negative negative 1 2 1.2 normal normal normal normal normal normal normal normal moderate normal CRF 3 mesangioproliferative glomerulonephritis N/A 13

82 50 female 56154 5.5 146 yellow 1.01 7 negative negative negative 1 1 0.8 normal normal normal normal normal normal normal normal severe normal CRF 3 mesangioproliferative glomerulonephritis N/A 13

83 47 male 56900 6.5 44 yellow 1.01 7 negative negative negative 1 1 0.4 normal normal normal normal normal normal normal normal severe normal CRF 3 mesangioproliferative glomerulonephritis N/A 13

84 43 female 55053 7.9 194 yellow 1.01 7 negative negative negative 1 2 0.9 normal normal normal normal normal normal normal normal severe normal CRF 3 lupus nephritis class Ivc N/A 13

85 61 male 56419 9.9 152 yellow 1.01 7 negative negative negative 1 2 1.1 normal normal normal normal normal normal normal normal severe normal CRF 3 lupus nephritis class Iva N/A 13

86 35 female 56617 10.3 124 yellow 1.01 7 negative negative negative 1 1 0.3 normal normal normal normal normal normal normal normal severe normal CRF 3 diabetic nephropathy class 3 N/A 13

87 24 male 56436 10.7 117 yellow 1.01 7 negative negative negative 1 2 1 normal normal normal normal normal normal normal normal moderate normal CRF 3 diabetic nephropathy class 3 N/A 13

88 40 female 56413 11.4 227 yellow 1.01 7 negative negative negative 1 3 1.4 normal normal normal normal normal normal normal normal moderate normal CRF 3 diabetic nephropathy class 3 N/A 13

89 22 male 55377 11.5 189 pink 1.01 7 negative negative negative 1 3 1.6 normal normal normal normal normal normal normal normal severe normal CRF 3 nodular glomerulosclerosis N/A 13

90 68 male 54952 12.1 169 yellow 1.01 7 negative negative negative 1 2 1 normal normal normal normal normal normal normal normal severe normal CRF 3 nodular glomerulosclerosis N/A 13

91 38 male 57324 14 163 yellow 1.01 7 negative negative negative 1 1 0.4 normal normal normal normal normal normal normal normal severe normal CRF 3 nodular glomerulosclerosis N/A 13

92 16 male 56402 14.1 258 yellow 1.01 7 negative negative negative 1 3 1.4 normal normal normal normal normal normal normal normal severe normal CRF 3 nodular glomerulosclerosis N/A 13

93 60 male 55056 14.4 229 yellow 1.01 7 negative negative negative 1 3 1.6 normal normal normal normal normal normal normal normal severe normal CRF 3 crescentic glomerulonephritis N/A 13

94 55 male 56687 17.9 286 yellow 1.01 7 negative negative negative 1 1 0.5 normal normal normal normal normal normal normal normal severe normal CRF 3 crescentic glomerulonephritis N/A 13

95 70 male 56592 11.7 172 yellow 1.01 7 negative negative negative 1 2 1.1 normal normal normal normal normal normal normal normal very severe mild CRF 4 crescentic glomerulonephritis N/A 13

96 47 female 56921 14.2 221 pink 1.01 7 negative negative negative 1 3 1.6 normal normal normal normal normal normal normal normal very severe mild CRF 4 crescentic glomerulonephritis N/A 13

97 45 male 56904 15.5 186 yellow 1.01 7 negative negative negative 1 2 0.9 normal normal normal normal normal normal normal normal very severe mild CRF 4 crescentic glomerulonephritis N/A 13

98 47 male 55787 15.5 166 yellow 1.01 7 negative negative negative 1 2 1.2 normal normal normal normal normal normal normal normal very severe mild CRF 4 crescentic glomerulonephritis N/A 13

99 48 male 53885 16.8 190 pink 1.01 7 negative negative negative 1 3 1.4 normal normal normal normal normal normal normal normal very severe mild CRF 4 crescentic glomerulonephritis N/A 13

100 48 male 57111 17.2 311 pink 1.01 7 negative negative negative 1 3 1.8 normal normal normal normal normal normal normal normal very severe mild CRF 4 crescentic glomerulonephritis N/A 13 SUB GROUP URINARY SEDIMENTS URINE DIPSTICK ANALYSIS CELLS CASTS Cr UREA PROTEIN 24 HRS CASE AGE SEX IP NO COLOR S.G. pH RBCs/uL NITRITE WBCs/uL UPL RBCs WBCs RTECs RBC WBC RTEC FATTY HYALINE GRANULAR BROAD WAXY PATTERN USS RENAL BIOPSY REPORT IMMUNOFLUORESCENCE GROUP mg/dL mg/dL g/L UPL g/day

28 74 male 32658 0.8 38 yellow 1.015 6 30 negative negative 3 4 3.6 moderate normal normal normal normal normal severe normal moderate normal NEPHROTIC 3 membranous nephropathy IgG (+3) 2

32 28 female 2175 0.7 26 yellow 1.015 6 30 negative negative 2 3 3.6 moderate normal normal normal normal normal moderate normal very severe normal NEPHROTIC 4 membranous nephropathy IgG (+3) 2

53 14 male 24962 0.6 19 yellow 1.015 6 30 negative 20 1 3 1.8 moderate mild normal mild normal normal normal normal very severe normal NEPHRI-NEPHROTIC 4 focal proliferative and membranous lupus nephritis classIIIa/V IgG (+3), IgM (+2), IgA (+3), C3 (+3), C1q (+3) 4

54 40 female 37372 0.8 32 yellow 1.02 6 30 negative 20 1 3 2.2 moderate mild normal mild normal normal normal normal very severe normal NEPHRI-NEPHROTIC 4 focal proliferative and membranous lupus nephritis classIIIa/V IgG (+3), IgM (+1), IgA (+3), C3 (+2), C1q (+2) 4

55 22 female 21539 1.4 27 yellow 1.01 6 30 negative 20 1 3 2.4 moderate mild normal mild normal normal normal normal very severe normal NEPHRI-NEPHROTIC 4 diffuse proliferative and membranous lupus nephritis class IVa/V IgG (+3), IgM (+3), IgA (+2), C3 (+3), C1q (+3) 4

73 25 male 30942 1.5 28 yellow 1.015 6 30 negative 20 1 2 1.2 moderate moderate normal moderate normal normal mild normal moderate normal NEPHRITIC 3 IgA nephropathy IgA (+3), C3 (+1)MESANGIUM 7

74 29 male 25426 1.9 30 pink 1.02 6 30 negative 20 1 3 2 moderate moderate normal moderate normal normal moderate normal moderate normal NEPHRITIC 3 IgA nephropathy IgA (+2), C3 (+2)MESANGIUM 7

75 40 female 5234 1 28 pink 1.01 6 10 negative negative 1 2 2.1 mild normal normal normal normal normal normal normal severe normal NEPHROTIC 3 nodular glomerulosclerosis - 8

80 30 male 24143 8.3 120 yellow 1.015 6 negative negative 10 1 3 1 normal mild mild normal normal normal normal normal mild normal OTHERS 2 chronic interstial nephritis - 12

Cr - Creatinine 24 hrs UPL - 24 hours urine protein level RTECs - Renal Tubular Epithelial cells C - Complements S.G. - Specific Gravity RBCs - Red Blood Corpuscles USS - Urine Sediment Store GBM - Glomerular Basement Membrane UPL - Urine Protein Level (Heat Coagulation method) WBCs - White Blood Corpuscles Ig - Immunoglobulin TBM - Tubular Basement Membrane