El Sedimento Urinario Una Vista Integrada

The Urinary
Sediment
An Integrated View
Espero que este libro le ayude en su formación profesional, en él

encontrará un mundo diferente en el estudio de la orina, interpretado
por uno de los grandes líderes del uroanálisis, el Dr. Giovanni
Battista Fogazzi.
Afectuosamente.
QFB. Daniel Arias López.
Mapastepec, Chiapas; México. Julio del 2017.

“This page intentionally left blank"
Giovanni B. Fogazzi
The Urinary
Sediment
An Integrated View
THIRD EDITION
with a Historical Introduction by

J. Stewart Cameron
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2010 - Third edition
ISBN 978-88-214-3016-9
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Contributors
J. Stewart Cameron, CBE, MD, FRCP

Department of Nephrology and Transplantation
Guy’s campus, King’s College
London, United Kingdom
Maria Daniela Croci, technician of biomedical laboratory
Research Laboratory on Urine
Nephrology Unit
Fondazione IRCCS
Ospedale Maggiore Policlinico Mangiagalli e Regina Elena
Milano, Italy
Giovanni B. Fogazzi, MD
Nephrology Unit
Fondazione IRCCS
Milano, Italy
Giuseppe Garigali, ScD
Nephrology Unit
Fondazione IRCCS
Milano, Italy
Barbara Pirovano, ScD
Laboratory Medicine Unit
Ospedale di Romano di Lombardia, Italy
Sandra Secchiero, ScD
Biomedical Research Centre
Castelfranco Veneto, Italy
Simona Verdesca, MD
Nephrology Unit
Fondazione IRCCS
Milano, Italy
Foreword to the third edition
Nephrology, as with all fields of contemporary medicine, is more and more pushed to the limits
to face the huge advancements in basic science research, with the promise and hope of discovering
new diagnostic, prognostic and therapeutic tools.
In the shaded scenario of the new diagnostic proposals coming from the difficult and ever
changig area of the basic research, the study of urinary sediment stands as a solid, valid and
cost-effective diagnostic approach. Giovanni Battista Fogazzi, an outstanding clinical medical
doctor, skilled medical researcher and dear friend, took on for the third time the difficult task of
offering to the Nephrology Community all the knowledge on urinary sediment science, based on
the clinical and laboratory experience collected over 3 decades.
He was consistently helped by a group of hard-working and enthusiastic young doctors operating
in the Nephrology, Dialysis and Renal Transplant Unit which I am proud to direct.
I think that, apart from any other comment, reading this book will not only convince any
nephrologist of the fascinating world of this old medical science but may also give renewed
impetus to further widen interest in this field, possibly enhancing it with new suggestions coming
from the basic reasearch area.
Piergiorgio Messa
Preface to the third edition
This third edition of “The urinary sediment. An integrated view” appears ten years after the
previous one, a fact which explains the many changes which this book contains.
First of all, there is a new panel of contributors, Professor Stewart Cameron and myself being
the only ones left from the previous edition. The new contributors are persons who, with different
roles, currently work on urinalysis and urinary sediment, and together form what I call a dedicated
and enthusiastic “urine group”.
All parts of the book have been expanded and updated, including the historical introduction.
We have added more than 60 new images and replaced more than 110 of the second edition.
We have also given room to urinary sediment particles which we did not describe before, such
as macrophages and decoy cells, and we have added images and data on unusual crystals and
crystals due to drugs. Another distinguishing feature of this edition is the presentation of original
and personal urinary sediment data on various renal diseases, but especially some primary and
secondary glomerulopathies. I consider this fact a contribution to evidence-based medicine in the
field of urine microscopy. Finally, a new chapter has been added (Chapter 8) on quality control
programs, which are aimed at improving the overall quality of urinary sediment examination.
I cannot close this preface without thanking openly the company “A. Menarini Diagnostics” for
the support given to this new enterprise.
G.B. Fogazzi
Milan, October 2009
Dedicated to those who are still fascinated
by the examination of the urinary sediments
Contents
Foreword to the third edition VI

Preface to the third edition VII
Historical introduction (J.S. Cameron) 1

References and notes 16
Chapter 1. G.B. Fogazzi and G. Garigali

Collection, preparation and examination of the samples,
and report of the urinary findings 19
Urine collection 19
Preparation of the samples 21
Inspection 21
Preservation of samples 21
Centrifugation 22
Resuspension 24
Preparation of slides 24
Microscopic examination 24
Report of findings 26
The microscope for the analysis of urinary sediments 32
The phase contrast microscope 32
The polarized light 35
The bright field microscope 35
Other microscopic techniques 35
The stains for urinary sediments 37
References 38
Chapter 2. G.B. Fogazzi, G. Garigali, M.D. Croci and S. Verdesca

The formed elements of the urinary sediment 41
Cells 41
Erythrocytes 42
Leukocytes 49
Macrophages 54
XII Contents
Renal tubular epithelial cells 57

Transitional epithelial cells 63
Squamous epithelial cells 69
Lipids 71
Casts 77
Hyaline casts 80
Granular casts 83
Waxy casts 85
Cellular casts 88
Fatty casts 92
Casts containing crystals and amorphous salts 92
Casts containing microorganisms 92
Pigmented casts 95
Mixed casts 97
Cylindroids 99
Pseudocasts 101
Mucus 103
Crystals 105
Common crystals 108
Uric acid 108
Amorphous urates and amorphous phosphates 113
Calcium oxalate 115
Calcium phosphate 120
Triple phosphate 123
Pathologic crystals 127
Cholesterol 127
Cystine 128
Leucine 130
Tyrosine 130
2,8-dihydroxyadenine 130
Crystals due to drugs 133
Other crystals 133
Hippuric acid 133
Calcium carbonate 134
Ammonium biurate 135
Organisms 136
Bacteria 136
Yeasts 139
Trichomonas vaginalis 141
Schistosoma haematobium (urinary schistosomiasis) 142
Enterobius vermicularis 143
Contaminants 144
Contaminants originating from the patient 144
Contaminants originating in the laboratory 148
Contaminants originating in the environment 150
Possible misidentifications 152
References 153
Contents XIII
Chapter 3. G.B. Fogazzi and S. Verdesca

Changes of urinary sediment caused by drugs 159
Drug-related crystalluria 159
Other changes induced by drugs 168
Diuretics 168
Drugs influencing urinary pH 168
References 168
Chapter 4. G.B. Fogazzi
The urinary sediment of the normal subject 173
References 176
The urinary sediment in the main diseases of the kidney and of urinary tract 177
Minimal change disease and focal segmental glomerulosclerosis 177
Urinary findings 178
Membranous nephropathy 178
IgA nephropathy 180
Membranoproliferative glomerulonephritis 183
Acute post-streptococcal glomerulonephritis 184
Extracapillary glomerulonephritis 185
Lupus nephritis 186
Schönlein-Henoch purpura nephritis 187
Diabetic nephropathy 189
Nephropathies due to plasma cell dyscrasias 190
Acute interstitial nephritis 191
Eosinophiluria – is it a specific marker of ain? 193
Erythrocytic casts – can they be found in the urine of patients with ain? 194
Chronic interstitial nephritis 194
Acute tubular necrosis 195
Renal transplantation 197
Acute cellular rejection 197
Polyomavirus BK infection 198
XIV Contents
De novo or recurrent glomerulopathy 203

Urinary tract infection 203
Urological disorders 204
References 206

Interpretation of the urinary sediment findings 211
The nephrotic sediment 211
The nephritic sediment 214
The nephrotic and nephritic sediment 216
The urinary sediment containing many renal tubular epithelial cells 216
The urinary sediment containing increased numbers of erythrocytes 217
The urinary sediment containing bacteria and leukocytes 218
Minor urinary abnormalities 218
References 219
Chapter 7. B. Pirovano and G.B. Fogazzi

Automated systems for urinary sediment analysis 221
Automated intelligent microscopy 221
Flow cytometry 224
Main performances of automated analyzers 226
Carry over 226
Precision 229
Accuracy 229
Advantages, limits and role of automated instruments 230
References 232
Chapter 8. S. Secchiero and G.B. Fogazzi

Quality control programs for urinary sediment 233
Internal Quality Control 233
External Quality Control 234
Features of the Italian EQA Program “Urinalysis Performance” 234
Results of “Urinalysis Performance” 238
Comments on “Urinalysis Performance” 244
References 244
Appendix. G.B. Fogazzi

Adjustment of the microscope 247
1. Adjustment according to the Köhler principle 247
2. Centering of the annular diaphragm of the condenser
with the phase ring of the objective 249
Index 251
introduction
HISTORICAL INTRODUCTION
J. Stewart Cameron
«When the patient dies the kidneys may go to the pathologist, but while he lives the urine
is ours. It can provide us day by day, month by month, and year by year with a serial story of
the major events within the kidney. The examination of the urine is the most essential part of
the physical examination of any patient...» (Thomas Addis, 1948 [1]).
Some years have passed since the earlier editions of this book, but this brief extension
in time has not blunted the truth of this statement by one of the great clinical nephrologists
of the last century. Doctors have been looking at the urine for diagnostic information for at
least one and a half millennia [2,3], and the examination of the urine was already part of the
Hippocratic system 500 years before the beginning of the past millennium. However, for the
first thousand years or more, this enquiry was pursued by eye, unaided by either microscopy
or chemistry (Figure 1).
FIGURE 1 Most medieval illustrations of physicians show

the practitioner either carrying a urine glass, or engaged
in the art of uroscopy, as in this 15th century illustration.
This was, in effect, divination by uroscopy, and the asso-
ciations of the various appearances of the urine are fan-
ciful [2]. (Bartolomeus Anglicus, De proprietatibus rerum,
Cambridge, Fitzwilliam Museum, MS 254, Folio S49; from
MacKinney, L. Medical Illustrations in Medieval Manu-
scripts, London, 1965, S, 14).
2 J. Stewart Cameron
This third edition of this textbook and atlas on the urinary sediment reflects a renewed interest
in microscopy of the urine in recent years, after a period of relative neglect since the days
when Addis wrote. One clue to the origins of that temporary neglect is given in the quotation:
“when the patient dies his kidneys may go to the pathologist...”. Addis wrote just after Nils
Alwall (1904-1986) had attempted unsuccessfully to add renal biopsy to autopsy examination
[4,5], and just before its successful application by Claus Brun (1914-) [6], and Bob Muehrcke
(1921-2004) and his mentor Robert Kark (1910-2003) [7]. In the excitement of actually
following renal histology during life, many clinicians – myself included – partially forgot the
powerful lessons the urine can teach us. Just how far this neglect was allowed to progress has
been demonstrated by one of the authors of this book [8]. This has happened before. In 1844,
Golding Bird (1814-1854), yet another of the amazing run of great Guy’s physicians on the
mid-19th Century, writes [9] of the “rediscovery” of the art of microscoping the urine. He was
referring to the fact that after the assembly of the first simple microscopes in the 16th century
and compound microscopes in the early 17th, urine was examined by observers as early as the
Provençal astronomer and polymath Nicolas-Claude Fabricius de Peiresc (1580-1637) (Figure
2) in 1630, who described urinary crystals as resembling “a heap of rhomboidal bricks” [10].
This may well be the first record of urine microscopy but, despite the continuing popularity
of divinative uroscopy [2] (Figure 1), it was not until about the time of the first microscopic
observations of kidney tissue itself [11,12] in the late 1830s that observers began to examine the
urine by the microscope with any regularity. This may have been the result of the availability of
better microscope objectives at about this time (see below).
FIGURE 2 Nicolas Fabricius de Peiresc (1580-1637) who

described in 1630 the first observations on microscoping
the urine. (From Gassendus P. Vir illustris Nicolai Clau-
dii Fabricii de Peiresc, senatoris aquisextiensis vita, per
Petrum Gassendum, philosophum et matheseos profes-
sorem parisiensem. Hagae Comitis, Adriani Vlach, 1651).
Historical introduction 3
Although bloody urine had been noted in scarlatinal nephritis throughout the 17th
and 18th centuries [13-15], most notably by the astute Swedish paediatrician Rosén von
Rosenstein (1706-1773) [16], I have been unable to find any record during this period of
the confirmation of the presence of red cells in the urine by microscopy; that may come as
a result of further enquiry, and it would be surprising if none of the enquiring 18th century
minds did not think to look for Leuwenhoek’s corpuscles in urine which appeared to contain
blood. Neither Domenico Cotugno (1736-1780) [17], who discovered and named albuminuria
in the 1770s, nor Richard Bright (1789-1858) and John Bostock (1773-1846) (although
they made many chemical observations on their patients’ body fluids [18]) actually used a
microscope on the urine, although by 1840 Bright’s student Joseph Toynbee (1815-1866) was
microdissecting kidneys and examining them microscopically [19]. Interestingly, William
Bowman (1816-1892) himself – although he made no microscopical observations on the
urine – clearly appreciated that red cells could pass through the Malpighian corpuscles in
disease. He describes, in a footnote to his 1842 paper, kidneys from patients with Bright’s
disease that he examined under the microscope [20, pp. 67-8]:
«It is well known that blood is often passed in the urine during the course of the disease,
especially at the earlier periods of it, when many circumstances contribute to prove that the
kidneys are in a state of sanguineous turgescence. How does this blood escape into the ducts
of the gland? The organ examined at this time presents on its surface and throughout its
cortical substance, scattered red dots, of somewhat irregular shape, not accurately rounded,
and generally as large as pins’ heads, that is very many times larger than the Malpighian
bodies... they are nothing less than the convolutions of the tube filled with blood that has
burst into it from the gorged Malpighian tuft at its extremity».
However, it seems credit must be given to the founder of French nephrology, Pierre Rayer
(1793-1867), and his young associate Eugène Napoléon Vigla (1813-1872) (Figure 3) for
the introduction of regular urine microscopy to clinical practice, described in 1837 [3]. Vigla
describes how impressed Rayer was by the results of microscopic examination of a urine
from one of his patients, supposedly full of pus, but which in fact contained crystals, by the
young Gottlieb Gluge (1812-1898) of Brussels – probably the first physician to examine
renal tissue microscopically (in 1839). Rayer vowed to make microscopic examination of the
urine a regular feature of his clinical practice. In the preface of his magnificent “Traité des
Maladies des Reins” published in three volumes between 1839 and 1841 [21], he writes:
«It is to be regretted that another method of investigation, microscopical examination, is

not yet generally employed to examine the matter suspended in the urine, thrown down by
cooling, or which one may precipitate by various reagents... I cannot thus understand the
lack of urgency in the majority of physicians to avail themselves of the microscope in the
examination of the urine».
As Gabriel Richet expressed it “la microscopie de l’urine, en revanche, fut crée par Rayer
lui-même” [22] and he notes that Rayer made a microscope available at all hours for his
associates to microscope the urine (it was probably one made by Chevalier himself (see
FIGURE 3 Left: Pierre Olive François Rayer (1793-1867), founder of French Nephrology. Right: his pupil and col-
league Eugène Napoléon Vigla (1813-1872). Between them, they introduced urine microscopy into clinical medicine
in 1837 (courtesy of the Wellcome Foundation library).
below) or Oppolzer). Rayer and Vigla examined and analysed the crystals present in the
urine, as many had before them, but – and this was new – they also noted the red cells, the
pus cells, the epithelial cells, fatty bodies and sperm. They realized that otherwise normal
(clear) urine might nevertheless contain an excess of red cells – the first description I can
find, surprisingly late, of microscopic haematuria. It must be noted, however, that Rayer
and Vigla’s priority in introducing clinical urine microscopy was hotly disputed by fellow
Parisian Alfred Donné (1801-1878), who ran courses in microscopy not only for doctors
but for the general public, and took and exhibited the first photomicrographs, using Louis
Daguerre’s new technique in 1840 the same year the new method was described, although
these pictures were only published 5 years later [3,22].
Thus in 1841, in his classic book on the analysis of urine, Alfred Becquerel (1814-1866)
could refer casually [23] to “examen microscopique” of the urine: and he notes again that in
perfectly clear urine, one sees only sheets of epithelium, in urine with mucus, globules of this
substance, closely resembling globules of pus, and also red blood cells “plus souvent déformés
et irreguliers” – the first description of dysmorphic red cells? He noted also sperm, and of
course crystals: calcium and magnesium carbonate, and phosphates, including ammonium
magnesium phosphate. Animal chemistry had developed rapidly since the beginning of the
19th century [24] and the chemical approach to disease was being applied in many centres
throughout Europe.
The years 1842-1844 saw a number of workers in Germany describing casts in the urine
almost at the same time. Jacob Henle (1809-1885) [25] recognized the tubular casts seen in
histological sections as identical to those found in the urine in 1842, and Theodor Frerichs
(1819-1885), writing his influential book in Breslau (now Wroclaw) in 1851, also credits
Hermann Nasse (1807-1892) [26] as reporting them at about the same time. The following
year, Johann Joseph Scherer (1814-1869) [27], Julius Vogel (1814-1880) [28] and Johann
Franz Simon (1807-1843) [29] all described casts in the urine, and this observation was so
striking that Golding Bird (Figure 4) reproduced Simon’s illustration of them in his own
book “Urinary Deposits: Their Pathology and Therapeutical Indication” [9]. This was
published in 1844, a year after Simon’s premature death, following a paper on the subject
in Guy’s Hospital Reports of 1842 [30], which indicates Bird had been studying the urinary
sediment for some time. This book of Bird’s was the first comprehensive description of
the presence and significance of urinary crystals and sediments, which ran to five English
and two American editions over the next decade, and placed urine microscopy firmly in the
realm of routine clinical examination in the Anglo-Saxon world, as Rayer’s had previously
in France.
Bird notes that his copy of Simon’s picture (Figure 5) is “the common appearance of
deposits in the urine of morbus Brightii” and that “a tubular mass of coagulated albumen,
probably the cast of a uriniferous tubule, entangling granules and blood discs, occupies the
FIGURE 4 Golding Bird (1814-1854), physician to Guy’s Hospital, London. His book published first in 1844 was
hugely influential in introducing microscopic examination of the urine into general clinical practice in Anglo-Saxon
countries. In 1840, he prepared a large series of permanent microscopic slides to be used for practical demonstra-
tions for student teaching (Bird G. Lectures on the physical and pathological characters of urinary deposits, delivered
at Guy’s Hospital. London Med Gaz 1843 [lecture III, pp. 761-8]). The hand-written descriptions of the slides, still to
be found in the Gordon Museum at Guy’s Hospital, are in Bird’s own script. (Slides provided by Mr J. Dawes, curator,
Gordon museum, Guy’s Hospital, courtesy of Dr G.B. Fogazzi).
FIGURE 5 One of the earliest illustrations of a urinary

cast, taken from Simon’s paper of 1843 [29] and repro-
duced in the first edition of Golding Bird’s book [9] the
following year.
centre of the figure”. Present also are red cells, epithelial cells and “large organic globules”
which contain “nuclei” (although not cell nuclei). I am unsure what these particles may
represent in contemporary terms. Golding Bird also illustrates fresh blood in the urine (Figure
6 left), with round cells and rouleaux, which he contrasts with older bleeding, some of the
dispersed cells in Figure 6 right showing clear evidence of membrane “spikes” and “hooks”
of the type illustrated using electron microscopy recently. Bird shows also epithelial cells
(Figure 7) and pus cells (Figure 8).
Bird tells us that he ordinarily used “a good achromatic objective of a quarter of an inch
(6 mm) focus”, but that one of one-seventh (3.6 mm) or one-eighth (3.2 mm) focus was used
occasionally. The achromatic lens had been perfected around 1830 by combining more than
one lens of flint and crown glass together, principally by the brilliant amateur microscopist and
innovator, the wine merchant Joseph Jackson Lister (1786-1869) in London and Charles Louis
Chevalier (1804-1850) in Paris [31]. Rayer and Bird both depended upon this advance for their
ability to describe the urinary sediment with accuracy. Chevalier’s microscope had a resolution
of 1.7 µm and a magnification of x 280-x 540 and, as early as 1835, Oberhauser had made a
microscope capable of magnifications up to 1000 diameters and a resolution of 0.7 µm, a figure
scarcely surpassed since then, although distortion has been reduced dramatically [31].
In the later editions of his book, Bird refers to the important work of George Johnson
(1818-1896) [32] on fatty deposits in the urine, published in 1846. Although it was known
that the kidneys of nephrotic patients contain an excess of fat [11,12], Johnson was unaware
of these papers; and when he demonstrated fat in nephrotic kidneys as well as an excess of fat
both microscopically (Figure 9) and chemically in both epithelial cells and casts contained
in the urine, he claimed this as an original observation; when he presented this work to the
FIGURE 6 Left: normal red cells in fresh urine associated with rouleaux. Right: altered red cells in urine (from ref.
9). Golding Bird thought that altered red cells resulted from the urine standing for a while in the bladder or in the
urine jar.
FIGURE 7 Epithelial cells in the urine, from Bird (1844) FIGURE 8 Pus cells as illustrated by Bird (9). Again
[9]. These had been observed by previous microscopists, these had been observed and correctly identified by
including Rayer in 1839 [21] and Becquerel in 1841 [23]. Rayer (21).
FIGURE 9 The urinary sediment as illustrated in John-

son’s article of 1846 [32]. He drew attention to the fatty
(oily) casts (bottom right) in the urine of nephrotic pa-
tients (magnification x 400) and noted that this corre-
sponded to fatty deposits in the renal tubule, seen in an
unstained, isolated tubule in the centre of the picture.
Johnson also noted tubular cells gorged with lipids (bot-
tom left).
Medical Chirurgical Society in London in 1845, he was tartly reprimanded by an anonymous

correspondent in the Lancet [33], who used the pseudonym “One who reads before he
discovers”, drawing his attention to the prior publications from Europe [11,12]. The idea of a
fatty degenerative parenchymatous nephritis gained strength from Fritz Munk’s (1879-1945)
use of polarized light on the urinary sediment for the first time in 1911 [34], showing the
beautiful and now familiar “achsenkreuz” of the fatty casts in the urine (Figure 10); these
observations led to the term “lipoid nephrosis” which remained current until the 1970s.
Thus, during the second half of the 19th century, urine microscopy became standard practice
world-wide, through the writings of Golding Bird, and also of Lionel Beale (1829-1906),
who although English in origin was particularly influential through the American editions of
his books [35-37], along with those of the American James Tyson (1841-1919) [38]. Other
manuals and atlases were published in English during the second half of the nineteenth century
(Table 1). Throughout the next century until the introduction of renal biopsy, examination of
the urine was the main route by which the nature and activity of the disease could be judged.
In addition, a notable series of fine atlases illustrating the urinary sediment appeared from
German-speaking countries during the second half of the 19th century and the first half of
the 20th century (Table 2). In all these works, great care was taken with the preparation and
printing of the illustrations, which took advantage of advances in printing technology about
that time, especially chromolithography. The first of these German language atlases was that of
surgeon Robert Ultzmann (1842-1889) and his colleague, professor of medical chemistry Karl
Berthold Hofmann (1842-1922) [39] of Vienna, published in 1871 [40], which to some extent
set the model for subsequent publications including those in other countries (Figure 11).
FIGURE 10 An illustration from Munk’s paper of 1913 [34]. Polarized light was used for the first time to show the
presence of lipid.
TABLE 1 The main monographs on urinary sediment published in the United Kingdom in the
second half of 19th century and in the first half of 20th century.
Year Author(s) Country Title
Illustration of the constituents of urine,

1858* Lionel Smith Beale United Kingdom
urinary deposits and calculi
Practical treatise on urinary and renal

diseases, including urinary deposits
1865** William Roberts United Kingdom
illustrated by numerous cases and
engravings
1872 George Harley United Kingdom The urine and its derangements
The clinical examination of urine with

1900 Lindley Scott United Kingdom
an atlas of urinary deposits
* 1869 (3rd and last edition); ** 1884 (4th and last edition).
TABLE 2 The main monographs on urinary sediment published in the German-speaking area
in the second half of 19th century and in the first half of 20th century.
Robert Ultzmann und Atlas der physiologischen und

1871 Austria
Karl Berthold Hofmann pathologischen Harnsedimente
Robert Ultzmann and Karl Anleitung zur Untersuchungen
1878 Austria
Berthold Hofmann der Harnes
1894 Albert Daiber Switzerland Chemie und Mikroskopie der Harnes
1896* Albert Daiber Switzerland Mikroskopie der Harnsedimente
Atlas der klinischen Mikroskopie

1898 Hermann Rieder Germany
des Harnes
F. Kratschmer Mikroskopische und mikrochemische
1901** Germany
and E. Senft Untersuchungen der Harnsedimente
1934 C. Lutz C and P. Schugt Germany Atlas der Mikroskopie der Harnsedimente
nd nd
* 1906 (2 and last edition); ** 1909 (2 and last edition).
FIGURE 11 Various types of uric acid crystals (left, bottom and top) and various types of casts (right, bottom and
top) as shown in plate VII and plate XXXV respectively of the monograph by Robert Ultzmann and Karl Hofmann [40].
Note the very high quality of the images which could be obtained thanks to the technique of chromolithography
(reproduced with permission from reference [39]).
In the same period, another source which contributed to the diffusion of urine microscopy
was the publication of books on clinical microscopy by several authors from different
countries (Table 3). Some of these books were largely based on images and contained
detailed microscopical descriptions of various body components such as blood, exudates,
liquor, pus, sputum, genital secretions, faeces, milk, and urine both in health and disease.
Among these books it is worth mentioning, for the historical importance of its author, that of
the Italian Giulio Bizzozero (1846-1901), which ran to five editions and was translated into
many languages.
During this time, the significance of the “fragmented” red cells in the urine was clearly
worked out, first in Germany [41-44] and in the UK [45], although forgotten for another century.
Thus in 1898 in one of the finest of these German Atlases, Hermann Rieder (1858-1932) [44]
published beautiful illustrations of casts in his “Atlas der klinische Mikroscopie des Harnes”
(Figures 12-13), and noted:
«in renal bleeding... (the erythrocytes) differ greatly in both size and shape, some being
small and contracted whilst others look like a thorn apple. At times (the erythrocytes) are
swollen, deprived of their pigment... or they are dismantled in granules or spheres containing
haemoglobin».
TABLE 3 Principal atlases of clinical microscopy in the second half of 19th century and in the
first half of 20th century. All these atlases contain interesting and detailed chapters
on urine microscopy.
1880* Giulio Bizzozero Italy Manuale di microscopia clinica
Atlas der Mikroskopie

1884** Alexander Peyer Switzerland
am Krenkenbette
Atlante
1886 Gaetano Primavera Italy
di microscopia clinica
Mikroskopie und chemie

am Krankebett.
1893*** Hermann Lenhartz Germany
Fur Studirende
and aerate bearbeit
M. Deguy, A.
1906 France Traité de microscopie clinique
Guillaumin
1914 Ch. Lesieur, M. Favre France Précis de microscopie clinique
* 1901 (5th and last edition); ** 1897 (4th and last edition); ***1919 (9th and last edition).
FIGURE 12 Urinary sediment in a fatal case of nephritis illustrated in Rieder’s atlas of the urinary sediment, pub-
lished at the end of the 19th Century [44]. The original caption reads: “contracted kidney with fatal outcome. Hyaline
cylinders, covered with numerous globules of albumin, thus seeming fragmented, cylinders with fatty droplets, cells
with fatty globules, partly free and partly enclosed in the cylinders, innumerable scattered globules of albumin”.
FIGURE 13 Rieder’s illlustration of red cells in

the urine [44]; again as in Bird’s work, there is
awareness of the fact that some red cells may
be grossly dysmorphic in haematuric patients.
In Italy, Carlo Leopoldo Rovida (1844-1877) [46] of Pavia, working in the Ospedale
Maggiore of Milan undertook painstaking studies on the nature of urinary casts during the 1860s
and 1870s [46,47], noting that particularly the colourless casts contained a hitherto unknown
protein, which he would later call “cilindrina” and which was only identified a century later (see
below). His views on the origin of casts were also in advance and at variance with those popular
at the time in England, Germany and Scandinavia: the conventional idea was that they mainly
consisted of fibrin (following the suggestion of Jacob Henle), or had their origin as tubular cells
themselves. Rovida, again using microchemical staining techniques, showed that in yellow
casts another unknown protein was present, which was also found in the tubular cells.
The study of the urinary sediment perhaps reached its apotheosis in the studies of Thomas
Addis (1881-1949) (Figure 14) [48], quoted at the beginning of this introduction. For more
than two decades from 1920, he examined the urine of countless patients with all varieties of
renal diseases and noted their appearances; year in, year out, correlating these data with the
inevitable post mortem findings in some patients when available. He noted the appearance of
broad “renal failure casts” [49] for the first time in uraemia from any cause. In addition, for
the first time since Donné’s pioneer efforts 80 years before, photomicrographs of the urinary
sediment are used to illustrate the articles. All this work was first expounded in detail in his
joint book with Jean Oliver (1889-1976) published in 1931 [50], which was based on a core
study of 72 patients in whom Addis had examined serial urinary samples, and in whom they
also had performed post mortems. Addis remained suspicious of functional analysis of the
kidney, preferring, as he put it, the morphological tradition epitomized by the book “Die
Brightsche Nierenkrankheit” of Franz Volhard (1872-1950) and Theodor Fahr (1877-1945),
published in 1914; nevertheless, he advocated both urinary sediment quantitation in a
FIGURE 14 Thomas Addis (1881-1949), who through-

out his career emphasized the value of examining and
quantitating the urinary sediment in glomerulonephritis.
He felt strongly that morphological, rather than func-
tional, aspects of renal diseases needed emphasis [1]. He
pointed to the significance of broad, “renal failure casts”
[49], although noting that they had been described previ-
ously as early as 1868, by Wyss. In this 1925 article, Ad-
dis writes: “It is an index of the preoccupation of clinical
investigators with functional measures to the exclusion
of methods of morphology, and their quite unwarranted
faith in the dictum of Schlayer and others to the effect
that little or nothing can be learned from the microscopic
examination of the urine” (picture courtesy of Dr Belding
H. Scribner, a pupil of Addis).
concentrated acid overnight urine, together with quantitation of proteinuria. A lifetime’s study
was summarized in his book of 1948 quoted at the beginning of this introduction [1], one of
the few books devoted exclusively to the study of kidney disease at that time. However, after
his death the following year it proved that his method of quantitating 12 or 24 hour cell and
cast excretion rates had fallen on deaf ears. The practical problems of obtaining such samples
in busy hospital or office practices, and the undoubted fact that even in acid concentrated
urines the majority of the cells lysed overnight in a variable fashion in the bladder, militated
against its use. Although the “Addis count” was known by this time everywhere, and I saw it
used during the 1950s in England, by 1960 it was performed regularly almost nowhere and
died out completely by the end of the following decade.
In the United States at least however, perhaps as a result of Addis’ influence, urine
microscopy of fresh urine continued to be performed, as again I can testify from experience
in New York in the early 1960s. In the renal unit at Cornell a centrifuge and a microscope
were available on each ward for in-out patients – and were regularly used by young fellows
and staff alike at each consultation.
Richard W. Lippman (1916-1959), New York trained but a pupil of Addis’ (and also of
Addis’ collaborator Jean Oliver [1889-1976]), who worked in Los Angeles, managed to
achieve good colour photography of urine sediment for the first time, which is the major
achievement of his monograph published in 1952 (Figure 15) [51]. Unfortunately he says
little about how these images were achieved: in the introduction he writes “these photographs
of material in an ordinary hemocytometer chamber were taken with simple equipment and
with the high dry (4 mm) objective.” Much progress has been made since, as a comparison of
the photographs in this volume and in Lippman’s from half a century ago, shows vividly.
Colour microphotography had been realized early in the first decade of the twentieth
century, but usually involved multiple exposures using filters and combining the images. In
1935, colour film became generally available in 35 mm format and led to an explosion in this
type of photography. Even so, until well after the Second World War most photomicrographic
slides and all almost book illustrations were in black and white – paintings and drawings were
used when colour was wanted for books, as in Volhard and Fahr’s magnificent textbook.
The high cost of using colour printing remained a problem for several decades, and
Lippman notes he had grants for this from the John Simon Guggenheim Memorial Foundation
and from CIBA Pharmaceutical products, which first published some of this material in a
November 1950 number (vol 2 no 9) of their “CIBA Clinical Symposia” series (pp 287-298).
The problem was eventually solved by advances in print technology which made the process
cheaper. The second edition in 1957 ran to no less than 9 printings, the last in 1977 which
alone sold 1400 copies! – despite the death of the author in 1959. This monograph must have
been widely available in US laboratories and renal units from the 1960s to 1980s.
Lippman’s early death meant that there was no updating of the knowledge available in
1957 in the text. Thus there was no mention in it of the technical advances of the subsequent
quarter century, such as phase contrast microscopy, whose advantages over bright field
microscopy for urine sediment examination were emphasized as early as 1968 by Robert
Kark’s group in Chicago [52] and again by Edwin Spencer (1934-) and Ib Pedersen in Aarhus,
Denmark in 1971 [53]; and the discovery of the nature of the matrix protein of casts as Tamm-
Horsfall protein [54] and the composition of the granules [55] both by immunofluorecence
FIGURE 15 The front cover of Richard

W. Lippman monograph [51].
techniques, in spite of the fact that these discoveries were published in 1966 [54] and 1971
respectively [55].
Why microscopy of fresh urine is undoubtedly less commonly practiced today than half a
century ago is a complex subject [8]. From the 1950s onwards, renal biopsy seemed to provide
a more precise court of appeal than urinary microscopy could ever provide, but is much more
expensive, cannot be done daily nor can it be available at midnight on demand when an
anuric or other acutely ill patients present. The renewed debate on red cell morphology in
relation to the origin of bleeding, begun in the early 1980s by work in Australia [56,57] did
bring urine microscopy back into the spotlight to some extent, but most nephrologists today
still do not know how to exploit it, and are not taught to perform it adequately.
We can always return to the simple optical microscopy of our ancestors for most of our
work, allowing the urine to give us a free and easily available sample of what is happening in
the kidney and urinary tract day-by-day. This book beautifully and lucidly tells us what we
can learn from that examination.
Acknowledgement. I am very grateful to Dr G.B. Fogazzi for much valuable help in

preparing this revised manuscript.
References and Notes

[1] ADDIS T. Glomerular Nephritis. Diagnosis and Treatment. New York: Macmillan, 1948; p. 2.
[2] Uroscopy has been the subject of many articles and reviews. See the following: for details: FINE L.
Circle of urine glasses: art of uroscopy. Am J Nephrol 1986; 6: 307-11; HABER M.H. Pisse prophecy: a
brief history of urinalysis. Clin Lab Med 1988; 8: 415-30; DAL CANTON A., CASTELLANO M. Theory of
urine formation and uroscopic diagnosis in the medical school of Salerno. Kidney Int 1988; 34: 273-7;
VOSWINCKEL P. From uroscopy to urinalysis. Clin Chim Acta 2000; 297: 5-16.
[3] FOGAZZI G.B., CAMERON J.S. Urinary microscopy from the seventeenth century to the present day.
Kidney Int 1996; 50: 1058-68. The sources of many of the unreferenced statements in this introduction
will be found in this article. Vigla’s paper was published in two parts in 1837-8: Vigla, M(onsieur:
E.N.) Etude microscopique de l’urine, éclairée par l’analyse chimique. L’Expérience etc. 1837; Dec,
No 12 : 177- 90; 1838; Jan, No 1: 193-204.
[4] ALWALL N. On the organization of treatment with the artificial kidney and clinical nephrology in the
1940s and following decades. A contribution to the history of medicine. I. The nineteen-forties. Dial
Transplant 1980; 9: 307-11.
[5] CAMERON J.S., HICKS J.A. The introduction of renal biopsy into nephrology from 1901 to 1961: a
paradigm of the forming of nephrology by technology. Am J Nephrol 1997; 17: 347-59.
[6] IVERSEN P., BRUN C. Aspiration biopsy of the kidney. Am J Med 1951: 11: 324-30.
[7] MUEHRCKE R.C., KARK R.M., PIRANI C.L. Technique of percutaneous kidney biopsy in the prone
position. J Urol 1955; 74: 267-77.
[8] FOGAZZI G.B., GRIGNANI S. Urine microscopic analysis – an art abandoned by nephrologists? Nephrol
Dial Transplant 1998; 13: 2485-7.
[9] BIRD G. Urinary Deposits. Their Diagnosis, Pathology, and Therapeutical Indications. London: Churchill
(1st edition), 1844. For details of this remarkable man, see: WILKS S., BETTANY G.T. A Biographical
History of Guy’s Hospital. London: Ward, Lock Bowden & Co., 1892; pp. 245-50; STEPHEN L., Lee S.
(eds), Dictionary of National Biography. London: Smith, Elder & Co., 1908; Vol II, pp. 536-7.
[10] VAN SWIETEN J. Commentarius. Edinburgh, 1776; xvi, p 81. Van Swieten quotes Pierre Gassendi,
Peiresc’s great philosopher friend, biographer (Viri illustris Nicolai Claudii Fabricii de Peiresc ...vita.
Paris 1641) and collaborator, as writing that Peiresc microscoped the urine around 1630. For details of de
Peiresc, who was born and lived in Aix-en-Provence, see: BROWN H. Peiresc, Nicolas Claude Fabri de. In:
Dictionary of Scientific Biography. Gillespie C.C., ed. New York: Scribner’s, 1974; Vol 10, p. 488-92.
[11] VALENTIN G. Repertorium für Anatomie und Physiologie. Bern und St Gall: Huber, 1837; 11(2) pp.
290-1.
[12] GLUGE G. Anatomisch-microskopische Untersuchung zur Allgemeinen und Speziellen Pathologie.
Jena: 1842; II, pp. 126-31.
[13] STORCH J. Praktischer und Theoretischer Traktat vom Scharlachfi eber. Gotha: C Mevius, 1742; pp.
238-42.
[14] NAVIER P.T. Dissertation en Forme de Lettres sur Plusieurs Maladies Popularies qui ont Regné à
Chalons sur Marne. Paris: Cavelier, 1753; pp. 308, 438.
[15] REIL J. Ch. Über die Erkentniss und Cur der Fieber. Halle: Cursche Buchhandlung, 1799-1815; bk 5,
pp. 123-5.
[16] ROSÉN VON ROSENSTEIN N. Underratleser on bjarnsjukdomar och deras botmedel. Stockholm, 1765
(translated into English by SPARRMAN A., London: T Cadel, 1776; pp. 158-9).
[17] COTUGNO D. De ischiade nervosa commentarius. Vienna: Graeffer, 1778.
[18] BRIGHT R. Reports of Medical Cases. London: Longman, Green Orme, etc. 1827.
[19] CAMERON J.S., BECKER E.L. Richard Bright and observations in renal histology. Guy’s Hosp Rep 1964; 114:
159-71. The original paper is: Toynbee J. On the intimate structure of the human kidney and on the changes
which its several component parts undergo in “Bright’s disease”. Med Chir Trans 1843; 29: 304-26.
[20] BOWMAN W. On the structure and use of the Malpighian bodies of the kidney with observations on the
circulation through that gland. Philos Trans R Soc Lond 1842; 132: 57-80.
[21] RAYER P.F.O. Traité des Maladies des Reins, etc. 3 vols and Atlas. Paris: J.B. Ballière, 1839-41.
Preface, pp. viii-ix; and pp. 58, 99, 105, 114, 116-7, 122, 207 for comments on urine microscopy.
[22] RICHET G. Pierre Rayer, createur de la méthodologie néphrologique. Histoire Sci Méd 1991; 25:
285-92. A brief biography of Vigla is in: Gurlt E., Wernich A., Hirsch A. (eds). Biographisches
Lexicon der Hervorragenden Artze aller Ziet und Volker. Zweite Aufl age. Berlin und Wien: Urban
and Schwartzenberg, 1934; Band V, pp. 756-7. For more on Donné, see: Richet G. Daguerre, Donné
et Foucault, trois franc-tireurs créent la microphotographie. Histoire Méd Sci 1997; 13: 45-8. Also :
Donné A, Foucault L. Cours de microscopie complémentaire des études médicales. Paris, J-B Ballière
1844-5. Donné appears to have published a Tableau des sédiments des urines in 1838, but we have
been unable to locate copy of this so far.
[23] BECQUEREL A. Séméiotique des Urines; Ou Traité des Altérations des Urines dans les Maladies; Suivi
d’un Traité de la Maladie de Bright au Divers Ages de la Vie. Paris: Fortin et Masson, 1841; p. 172.
[24] FOSTER W.D. The early history of chemical pathology in Great Britain. Med Hist 1959; 3: 173-87;
see also: Brock W.H. The life and work of Wiliam Prout. Med Hist 1965; 9: 101-26; Richet G. The
chemistry of urinary stones around 1800; a first in clinical chemistry. Kidney Int 1995; 48: 876-86.
[25] HENLE F.G.J. In: PFEUFER C. Morbus Bright. Klinische Mitteilungen. Z Nat Med 1844; 1: 57-60 (This
article includes a note by Henle on pp. 60-61, in which he describes casts for the first time).
[26] NASSE H. Schmidt’s Jahrbucher, 1843, 356. Quoted in: Frerichs F.T. Die Bright’sche Neirenkrankheit
und deren Behandlung. Braunschweig: Bieweg; p. 9.
[27] SCHERER J.J. Chemische und Mikroskopische Untersuchungen zur Pathologie. Heidelberg 1843.
Quoted ibid. p. 9
[28] VOGEL J. Icones Histologiae Pathologicae. Tabulae Histologiam Pathologicam Illustrantes. Lipsiae:
Voss; 1843; pp 108-9.
[29] SIMON J.F. Beitrage zur Physiologische und Pathologische Chemie und Microskopie. Berlin, 1843. B.s
190; see also: Ueber eigentumliche Formen in Harnsediment bei Morbus Bright. Arch Anat Physiol
Wissenschaft Med 1843; 28-30.
[30] BIRD G. Note on the microscopic globules found in the urine. Guy’s Hosp Rep 1842; 7: 336-40.
[31] BRACEGIRDLE B. The microscopical tradition. In: Companion Encyclopaedia of the History of
Medicine. Bynum W.F., Porter R., eds. London: Routeledge, 1993; pp. 102-19. For more on the
history of microscopy and achromatic lenses in particular, see also: Hughes A. Studies in the history
of microscopy. I – The influence of achromatism. J R Microscop Soc 1955; 75: 1-21. Hughes questions
how great the improvement in performance the achromatic lenses delivered was, and to what extent the
huge expansion in microscopical studies from the 1840s onwards was powered by technical advances
or how much by changes in concepts of physiology and disease. He points out also that others, in
particular C.R. Goring (1792-1840), a physician in London, played a major role in development of the
new lenses in the 1820s and later.
[32] JOHNSON G. On the minute anatomy and pathology of Bright’s disease of the kidney and on the relation
of the renal disease to those diseases of the liver, heart and arteries with which it is associated. Med
Chir Trans 1846, 29: 1-43.
[33] One who reads before he “discovers”. On granular degeneration of the kidneys. Lancet 1846; 1: 239 (letter).
[34] MUNK F. Klinische Diagnostik der degenerativen Nierenkrankungen. Klin Med 1913; 78: 1-52.
[35] BEALE L.S. On casts composed of mucus formed in the straight portion of the uriniferous tubules. Arch
Med 1867; IV: 56.
[36] BEALE L.S. Tables for the Clinical and Microscopical Examination of Urine in Health and Disease.
London: Churchill, 1856
[37] BEALE L.S. Kidney Diseases, Urinary Deposits and Calcolous Disorders. Their Nature and Treatment.
3rd ed. London: Churchill, 1869.
[38] TYSON J. A Guide to the Practical Examination of the Urine for the Use of Physicians and Students.
Philadelphia: Lindsay & Blakiston, 1875.
[39] FOGAZZI G.B. An atlas on urinary sediment written by a surgeon and a chemist still of interest today.
Nephrol Dial Transplant 1999; 14: 2038-40.
[40] ULTZMANN R., HOFMANN K.R. Atlas der physiologische und pathologische Harndsedimente.
Braumüller: Vienna 1871.
[41] KÜSTER S., RITZ E. Fragmentocytes in the diagnosis of renal hematuria- observations in the 19th
century. Nephrol Dial Transplant 1994; 9: 569-70.
[42] FRIEDRIECH N. Eine Betrag zur Lebensgeschichte der rothen Blutkörperchen. Virchows Arch 1867; 41:
395-411.
[43] GUMPRECHT F. Die Fragmentation der rothen Blutkörperchen und ihre bedeutung fur die Diagnose der
Hämaturie. Dtsch Arch Klin Med 1894; 11: 53-5.
[44] RIEDER H. Atlas der Klinischen Mikroscopie des Harnes. Lepizig: Vogel 1898.
[45] HARLEY G. The urine and its Derangements etc. London: Churchill, 1872; pp. 178-179.
[46] FOGAZZI G.B., TESTANERA G. The farsighted studies of the Italian Carlo L. Rovida (1844-1877) on the
nature of urinary casts. Am J Nephrol 2002; 22: 300-8.
[47] ROVIDA C.L. I cilindri e i loro rapporti colle lesioni delle reni. Arch Sci Med. 1876-7; 1: 279-314;
365-419.
[48] PEITZMAN S.J. Thomas Addis (1881-1949): mixing patients, rats and politics. Kidney Int 1990; 37:
833-40.
[49] ADDIS T. Renal failure casts. J Am Med Assoc 1925; 84: 1013-5.
[50] ADDIS T., OLIVER J. The renal lesion in Bright’s disease. New York: Paul Hoeber 1931. Chapter IV and
Table.
[51] LIPPMAN R.W. Urine and the urinary sediment, Springfield Ill: Charles C. Thomas, 1952; 2nd ed. 1957
- 1977 (9th printing).
[52] BRODY L., WEBSTER M.C., KARK R.M. Identification of the elements of urinary sediment by phase
contrast microscopy. J Am Med Assoc 1968; 206: 1777-81.
[53] SPENCER E.S., PEDERSEN I. Hand atlas of the urinary sediment. Bright field, contrast and polarized
light. Munksgaard: Copenhagen, 1971.
[54] MCQUEEN E.G. Composition of urinary casts. Lancet 1966; i: 397-8.
[55] RUTECKY G.J., GOLDSMITH C.G., SCHREINER G.E. Characterization of proteins in urinary casts. N Engl
J Med 1971; 284: 1049-52.
[56] FAIRLEY K., BIRCH D.F. Hematuria: a simple method for identifying glomerular bleeding. Kindey Int
1982; 21: 105-8.
[57] FASSET R.G., HORGAN B.A., MATHEW T.H. Detection of glomerular bleeding by phase contrast
microscopy. Lancet 1982; i: 1432-4.
CHAPTER 1
COLLECTION, PREPARATION
AND EXAMINATION OF THE SAMPLES,
AND REPORT OF THE URINARY FINDINGS
G.B. Fogazzi and G. Garigali
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 [1,2]. However, reliable results can be obtained only by using the correct
methodology.
This chapter describes the methodological aspects related to urine microscopy, which
include collection, preparation and examination of the samples, and the report of the
findings.
urine collection
International guidelines suggest the procedures for urine collection [3,4]. In our
laboratory, we adhere to most of the recommendations of such guidelines.
We give the patient a disposable, cylindrical, and capped container with a capacity of
100 mL, a diameter of 5.8 cm, and a height of 7 cm. This allows easy collection of urine
by both female and male and avoids accidental spillage. The date of the test, the name
and the date of birth of the patient as well as the hour of collection are reported on the
label of the container. We firmly discourage the use of recycled bottles or jars since these
may not be clean or may be contaminated with substances which may interfere with the
examination.
We ask the patient to deliver the second urine of the morning, which 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 [5,6]. Unless we have to investigate
a post-physical exercise haematuria [7], we ask the patient to avoid strenuous physical
activity, e.g. jogging, in the 48 hours preceding urine collection. This activity, in fact,
can cause urinary changes including haematuria and cylindruria [7]. It is preferable
to examine the sample while the patient is not undergoing excessive diuresis, since in
diluted urine several types of elements are reduced in number because of lysis. It is also
undesirable to examine highly alkaline urine, because this favours the lysis of leukocytes
20 G.B. Fogazzi and G. Garigali
[8] and casts [9], as well as the precipitation of phosphates. These latter can mask other
urinary elements.
In order to minimize contamination, hands and external genitalia must be cleaned. We
suggest conventional cleaning with water, avoiding disinfectants, which can favour the lysis
of cells. We do not suggest more scrupulous techniques, because these frequently are poorly
adhered to by patients. For females, we also suggest the spreading of the labia of vagina, for
males the retraction of the foreskin of the glans.
The midstream technique is another recommended procedure. This implies that 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. However, the very first portion of
the urine may be of diagnostic interest when urethral disorders are suspected. In such cases,
comparison of the first voided portion and the midstream portion of urine may distinguish
between urethral and bladder infection.
For women, urine sediment should not be examined during menstruation, since
contamination by erythrocytes is very likely. In such cases, a possible alternative is the use
of internal tampons but, even with these, contamination can occur.
When there is doubt about contamination, or if contamination cannot be avoided and
urinary sediment examination is indispensable for clinical reasons, suprapubic puncture of
the bladder may be considered (Figure 1.1). If properly done, this procedure causes mild
discomfort and is safe. In contrast, bladder catheterization is not bacteriologically safe and
may cause erythrocyturia in itself [10]. Therefore, it should be avoided.
Plastic bags attached to clean genitalia are frequently used for infants and small children
who are unable to control micturition. If properly handled, adequate urine samples can be
obtained. However, there may be faecal contamination and spurious bacteriuria, and one may
still have to resort to suprapubic puncture.
Pubic
symphysis Peritoneum
FIGURE 1.1 Suprapubic bladder puncture. The patient

is in the supine position with a full bladder, and a small
pubic area shaved. A 3.5 inch 22-gauge spinal needle is
attached to a syringe and is introduced slowly at a right
angle through the skin just above the pubic symphysis.
The bladder is then pierced quickly and the urine aspi-
rated.
Collection, preparation and examination of the samples, and report of the urinary findings 21
preparation of the samples

inspection
Macroscopic inspection of the sample is a useful step since it may reveal the presence of
turbidity and of abnormal changes of the urine colour.
Most frequently, turbidity is caused by large numbers of squamous epithelial cells of
vaginal origin, leukocytes, bacteria, amorphous phosphates or urates [11]. However, it is
important to remember that pathological samples are often perfectly clear. Therefore, the
absence of turbidity in itself is not a reliable criterion to judge a urine sample [12].
Abnormal urine colour can be due to a large number of causes (Tables 1.1 and 1.2).
It must be remembered that in some conditions such as gross haematuria, bilirubinuria,
or chyluria [13], the examination of the urinary sediment allows us to identify the cause of
abnormal urine colour.
preservation of samples
To avoid bacterial overgrowth, dissolution of casts and cells, and contamination from the
environment, we do our best to examine the samples within 3 hours of collection. This is
because in some instances leukocytes can lyse in less than 1 hour [14].
Lysis upon standing may be prevented by refrigeration of the samples at + 2 °C to + 8 °C
[3,4]. However, it is a common experience that at these temperatures phosphates and urates
may precipitate with a masking of diagnostically important elements.
A possible alternative is the use of preservatives such as thimerosal [15], formaldehyde,
glutaraldehyde [16] or CellFIX [17]. The latter has been proposed for the preservation of
erythrocytes, so that their glomerular or non glomerular origin (see Chapter 2) can be analyzed
by experts even weeks after collection. We use CellFIX when we want to preserve samples
for practical courses on urine microscopy, and we find that it preserves most urine particles
well, even though it tends to alter somewhat the appearance of casts and it dissolves crystals
which precipitate in acid urine due to its alkaline pH [18]. Due to the tendency of centralizing
tests in large laboratories, preservatives are becoming more and more important. This aspect
TABLE 1.1 The main causes of abnormal urine colour.
Endogenous causes Exogenous causes
Haemoglobin Vegetable-derived substances

Myoglobin Alimentary pigments
Bilirubin Drugs
Biliverdin Chemicals
Melanogen
Homogentisic acid (alcaptonuria)
Fat (chyluria)
TABLE 1.2 Differentiation of the causes of the most important changes in the colour of the urine.
Colour Causes
White Chyluria
Pink Massive uric acid crystalluria in morbidly obese patients after

gastric by-pass procedures
Red Haematuria, haemoglobinuria, myoglobinuria, porphyrinuria
Rhubarb, senna, beetroot
Aminophenazone, aminopyrine, antipyrine, bromsulphthalein,
chrysarubin, metronidazole, nitrofurantoin, phenacetin,
phenothiazine, phenytoin, salazosulfapyridine, rifampicin
Yellow-orange Bilirubin
Rhubarb, senna
Phenacetin, pyridine and its derivatives, rifampicin
Yellow-green Riboflavin, thymol
Brown Bilirubin, haemoglobin, myoglobin, homogentisic acid,

melanogen
Rhubarb, carotene
Aniline derivatives, bromsulphthalein, cascara, chinin,
chloroquine, hydroquinone, naphtol, nitrite, nitrofurantoin,
thymol, phenacitin, phenolphthalein
Green Biliverdin
Arbutin, creosot, chlorophyll-containing breath mints, guaiacol,
flavin, methylene blue, triamterene, blue dyes of enteral feeds
Blue Methylene blue, indigo blue
Black Haemoglobin, melanogen, homogentisic acid
Darkening upon standing Porphyrin, melanogen, homogentisic acid

Serotonin, cascara, chlorpromazine, methyldopa, metronidazole,
phenacetin, imipenem-cilastatin
has recently been investigated by comparing different preservation procedures on different

urine particles for a period of up to 3 days after collection [19]. Unfortunately, different
degrees of preservation were found with different preservatives on different particles, which
leads to the conclusion that an ideal preservative for all particles does not yet exist.
Thus, whenever possible, the samples should be examined without preservatives within a
few hours of collection.
One should always remember that lysis upon standing occurs especially in samples with
a high pH (e.g., > 7.0) and/or a low specific gravity (e.g., < 1.010). On the contrary, we have
seen samples with a low pH and/or a high specific gravity which were well preserved even
several hours after collection.
centrifugation
International guidelines recommend standardized procedures for centrifugation and the
subsequent steps of urine preparation and examination (Table 1.3).
TABLE 1.3 Some recommendations of two international guidelines for urine sample prepara-
tion and examination.
European urinalysis
NCCLS 2001
Procedure guidelines 2000
[4]
[3]
Centrifugation Yes Yes
Volume of urine Standardized Standardized
to be centrifuged (e.g., 5 or 15 mL) (e.g., 10 or 15 mL)
Time and speed
5 minutes at 400 g 5 minutes at 400 g
of centrifugation
Concentration Standardized
Standardized
after centrifugation (e.g., 0.5 or 0.6 mL)
Volume of resuspended
Standardized
urine to be transferred Standardized
(e.g., 0.13 mL)
to the slide
Standardized
Size of the coverslip Standardized
(e.g., 18 x 18 or 22 x 22 mm)
Phase contrast
enhances identification
of particles
Phase contrast and polarized
Type of microscope
light recommended
Polarized light strongly
recommended for lipids
and crystals
Expression of results Number of particles/L Number of particles/mL
Centrifugation is used to concentrate the figured elements of the urine, but if it is not done
properly, it may introduce relevant biases. These are due mainly to partial recovery of the
elements present in the urine. Using a centrifuge with a radius of 16 cm and a centrifugation
time of 5 min, Gadeholt [20] 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. Therefore, the yield is strongly influenced by the speed of centrifugation. However,
other factors also influence the yield. These are the duration of centrifugation and the volume
of urine centrifuged. Consequently, one should always use the same combination of speed
and duration of centrifugation, as well as the same volume of urine. Only in this way are
reproducible results possible [21]. For this reason, in our laboratory, we always centrifuge an
aliquot of 10 ml at 400 g for 10 min.
The speed of centrifugation is most often expressed as rotations per minute (rpm).
However, it should more correctly be expressed 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
where: r = the radius in cm from the center of the spindle to the bottom of the tube; N =
rotations per minute.
In some laboratories, to avoid the biases caused by centrifugation, samples are not
centrifuged. However, in this way the particles are not concentrated in the bottom of the tube,
a fact which can lead to the missing of elements of diagnostic importance. This is important,
for instance, for erythrocytic casts which, in patients with isolated microscopic haematuria,
we find in low numbers even in centrifuged samples [22]. Therefore, we always centrifuge
our samples even in the case of gross haematuria or highly cloudy urine. In such cases,
after centrifugation, we put a smaller volume of resuspended urine under the coverslip (e.g.,
20 μL instead of 50 μL), which reduces the crowding of the particles and allows a proper
examination of the sample.
resuspension
After centrifugation, the supernatant is discarded. While in many laboratories this is still
done by pouring off, international guidelines recommend removing a standardized volume
of urine (Table 1.3). In our laboratory we do this by a water pump, with which we remove
a fixed volume of 9.5 mL of supernatant urine. This helps to standardize the procedure and
increases its reproducibility [21].
We do not resuspend the sediment by shaking the tube or finger flipping, but by gently
pulling into a Pasteur pipette several times until the entire precipitate is in suspension. If
too much precipitate is present, we resuspend only a portion of the pellet. This procedure
avoids the packing of too many elements onto the slide. In this case, obviously, we report that
resuspension is partial and that the results are only qualitative.
preparation of slides
A standardized volume of the resuspended urine should be transferred to the slide (Table
1.3). We transfer 50 μL of urine with a precision pipette, since this volume fits under a
coverslip of 24 x 32 mm without spilling [21].
microscopic examination
Before starting the examination of the sample, we always make sure that the microscope
is well regulated (see Appendix).
Then, we examine the urinary sediment having in front of us the results obtained by
dispstick for pH, specific gravity, haemoglobin, leukocyte esterase, nitrites, and albumin.
We do this because specific gravity and pH values of the specimen may influence the
urinary findings in several ways:
• at high specific gravity, erythrocytes and leukocytes become smaller (Figure 1.2), which
may render their identification somewhat difficult;
• at a specific gravity of Φ 1.010, erythrocytes and leukocytes swell and can undergo
considerable lysis [23]. In addition, cytoplasmic Brownian movements may appear, and
loss of nuclear segmentation of leukocytes may develop [24];
Cell diameter
(μm)
13
11
9 (9.2-16.8)
(8.2-14)
7 (7-12.6)
5 (5.2-11.8)
(4.2-10.2)
3 (2.8-8.4) FIGURE 1.2 Changes in cellular size with variations of the

density of the urine. Urine sediments from five different pa-
tients were evaluated for each range of specific gravity. A
1 total of 40 isomorphic erythrocytes and 40 leukocytes were
measured for each sediment. Each mean and standard de-
viation was therefore obtained from 200 measurements.
1025-1030 1013-1017 1005-1010 Circles represent erythrocytes, squares represent leuko-
Specific gravity cytes. The numbers in parentheses indicate the ranges of
the size measurements.
• at high pH, leukocyte survival is shortened [8], casts are reduced in number [9], and the
precipitation of phosphates is favoured. Low pH, on the contrary, favours the precipitation
of urates.
The knowledge of the dipstick results for other analytes is also important. This is because:
• it helps to direct the examination of the urinary sediment. 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;
• it reduces the possibility of false negative or false positive results, which are always possible
when the urine is analyzed only by dipstick or only by microscopy. In fact, if it is true that in
the majority of cases dipstick and microscopy give comparable results, in a number of samples
there may be disagreement (Table 1.4). This may be due to different causes. For instance, a
case with dipstick clearly positive for 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) [1,2]. In such a case, dipstick without microscopy gives a false negative result.
TABLE 1.4 Dipstick results for haemoglobin and leukocyte esterase compared to the results
obtained by phase contrast microscopy for erythrocytes and leukocytes on 2305
samples analyzed in our laboratory during the year 2006 (* = all samples positive
from + to +++).
Dipstick versus Haemoglobin Leukocyte esterase

microscopy (No = 2305) (No = 2305)
Overall concordance 1755 (76.1%) 1913 (82.9%)

Positive by both methods 920 (52.4%) 420 (22.0%)
Negative by both methods 835 (47.6%) 1493 (78.0%)
Disagreement 550 (23.9%) 392 (17.1%)
Positive dipstick*
versus 295 (12.8%) 101 (4.4%)
Negative microscopy
3+ dipstick
versus 159 (6.9%) 37 (1.6%)
1+ microscopy
Negative dipstick
versus 96 (4.2%) 254 (11.0%)
Positive microscopy
From such examples it is clear that in a number of cases reliable results can be obtained
only by coupling dipstick with microscopy.
Once we put the slide under the microscope, we examine it without delay to avoid changes
due to the heat caused by the light beam of the microscope or the drying up of the sample.
We start examination at low magnification (160x). This is indispensable for an overview of
the sample, and to analyse the distribution of the elements, which very frequently is uneven
[25]. First, we examine the edges of the sample where casts tend to collect, and then the rest
of the specimen. Then, we proceed to higher magnification (400x) to identify the elements
properly. Since these usually lie on different planes, frequent adjustments of focus are
necessary. For every case, we examine at least 20 random low and high microscopic fields.
report of findings
An appropriate report of the urinary sediment findings is also necessary. Our report
includes the following information (Figure 1.3):
• date
• patient’s surname and first name and date of birth
• the following physico-chemical analytes by dipstick:
- pH
- specific gravity
Collection, preparation and examination of the samples, and report of the urinary findings
DATE ...............................................................................................................................................
SURNAME ....................................................................... NAME ................................................................... DATE OF BIRTH ...................................................
pH:.......... SPECIFIC GRAVITY: .............. HAEMOGLOBIN: ............ LEUKOCYTE ESTERASE: .................... NITRITES:............ ALBUMIN: .............
ERYTHROCYTES: ...............................................................................................................................................................................................................................
ISOMORPHIC (%): ............................................ DYSMORPHIC (%): .................................................. ACANTHOCYTES(%): .................................................
LEUKOCYTES: ....................................................................................................................................................................................................................................
RENAL TUBULAR EPITHELIAL CELLS: ........................................................................................................................................................................................
TRANSITIONAL CELLS. SUPERFICIAL: .................................................................................... DEEP: ......................................................................................
SQUAMOUS CELLS: ...........................................................................................................................................................................................................................
CASTS: ......................................................................................................... TYPES: ........................................................................................................................
................................................................................................................................................................................................................................................................
LIPIDS: ..................................................................................................................................................................................................................................................
CRYSTALS:...........................................................................................................................................................................................................................................
BACTERIA: ......................................................................................... YEASTS: ..............................................................................................................................
OTHERS: ...............................................................................................................................................................................................................................................
COMMENT: ..........................................................................................................................................................................................................................................
................................................................................................................................................................................................................................................................
SIGNATURE
......................................................
27
FIGURE 1.3 The urinary sediment report we use in our laboratory. pH, specific gravity, haemoglobin, leukocyte esterase, nitrites and albumin are evaluated by dipstick.
- haemoglobin
- leukocyte esterase
- nitrites
- albumin
• the following microscopic particles:
- erythrocytes (we report glomerular or non glomerular appearance on request only)
- leukocytes
- renal tubular epithelial cells
- transitional epithelial cells, which we distinguish as superficial and deep
- squamous epithelial cells
- casts with their subtypes
- lipids
- crystals
- bacteria
- yeasts
- other elements
Comment. This is useful to summarize the main findings and to give an interpretation of them.
An important aspect of the report is the use of a correct nomenclature. Terms such as cells
“of the upper”, “middle” or “lower urinary tract” are no longer acceptable, since they are
obsolete and misleading. These terms must be replaced by “renal tubular epithelial cells”,
“transitional epithelial cells (either superficial or deep)”, and “squamous epithelial cells”
respectively. Also the description of erythrocytes and leukocytes as “pale”, “degenerated”,
or “poorly preserved” must be avoided, since it is deprived of clinical implications. The only
adjectives which can be used for erythrocytes are “dysmorphic or isomorphic” to indicate
their glomerular or non glomerular origin respectively (see Chapter 2)
Examination of the urinary sediment is not only qualitative but is also quantitative. Thus,
one should always quantitate the elements found.
International guidelines recommend the expression of particles as number/L or number/
μL [3,4]. This is done by counting the elements in counting chambers (or haemocytometers).
Kesson and co-workers [26], using centrifuged spot urine samples and a Fuchs–Rosenthal
counting chamber, found that there was a good correlation between the count/mL and the
excretion rate for leukocytes obtained in timed samples. In addition, the count/mL was by far
more sensitive than the count per high-power field. However, it should be noted that the latter
had been done on samples which had been handled in a very non-standardized way.
In spite of guideline recommendations, in the vast majority of laboratories including our
own, the amount of particles is still expressed as mean number or as lowest-highest number per
microscopic field [27] (e.g., 3 erythrocytes/high-power field (HPF), or 3-12 erythrocytes/HPF).
We express the amount of casts at low magnification, while all the other elements are semi-
quantitated at high power magnification (Table 1.5). In our laboratory, for scientific investigation
we quantitate the cells as total number found over 20 high power fields at 400x [21,28].
It should also be noted that even with counting chambers, errors are possible. These are
caused by the tendency of cells to clump and to move towards the lines of the grid, and by
the entrapping of cells and casts by mucus [25]. Therefore, whatever method is used, it is
TABLE 1.5 The method used in our laboratory to express the results of the examination of the urinary sediments.
Mild Moderate Severe Very severe

Particle Magnification Normal
(+) (++) (+++) (++++)
< 1 every 1 every 1 every 1 every

Γ 1/
8-10 microscopic fields 8-10 4-7 2-3
Casts 160 x microscopic
(hyalin and hyalin- microscopic microscopic microscopic
field
granular) fields fields fields
Erythrocytes 11-30/ 31-50/ > 50
> 1-10/
400 x Φ 1/microscopic field microscopic microscopic microscopic
Leukocytes microscopic field
field field field
Renal tubular 1 every
epithelial cells 1 every 1 every Γ1
2-3
400 x Absent 8-10 microscopic 4-7 microscopic microscopic
Transitional microscopic
fields fields field
epithelial cells fields
Crystals
Squamous
epithelial cells 400 x Absent + ++ +++ ++++
Bacteria
Yeasts
29
30
DATE ...............................................................................................................................................
pH: 7.0 SPECIFIC GRAVITY: 1.005 HAEMOGLOBIN: +++ LEUKOCYTE ESTERASE: +++ NITRITES: Absent ALBUMIN: Absent
ERYTHROCYTES: .........1-3/HPF .......................................................................................................................................................................................................
ISOMORPHIC (%): ..............// ............................... DYSMORPHIC (%): // ............................................. ACANTHOCYTES(%): // ..............................................
LEUKOCYTES: ..............1-2/HPF .......................................................................................................................................................................................................
RENAL TUBULAR EPITHELIAL CELLS:.................// .....................................................................................................................................................................
TRANSITIONAL CELLS. SUPERFICIAL:............................// ..................................................... DEEP:....................................// ................................................
SQUAMOUS CELLS:..........................................................................// ...............................................................................................................................................
CASTS:....................................................// ................................................... TYPES: ........................................................................................................................
................................................................................................................................................................................................................................................................
LIPIDS:....................................................// ............................................................................................................................................................................................
CRYSTALS:............................................// ............................................................................................................................................................................................
BACTERIA:............................................//........................................... YEASTS:............................................//................................................................................
OTHERS:................................................// .............................................................................................................................................................................................
G.B. Fogazzi and G. Garigali

COMMENT: Mild erythrocyturia and mild leukocyturia with a +++ haemoglobin and +++ leukocyte esterase. This discrepancy might be due to cell lysis
caused by high pH and/or low specific gravity.
SIGNATURE
......................................................
FIGURE 1.4 An example of how the findings obtained by urinary microscopy can be reported. Note the comment about the discrepancy between the microscopic
findings and the results obtained by dipstick for haemoglobin and leukocyte esterase. HPF = high power field (x 400); LPF = lower power field (x 160).
DATE ...............................................................................................................................................
pH: 5.0 SPECIFIC GRAVITY: 1.025 HAEMOGLOBIN: +++ LEUKOCYTE ESTERASE: + NITRITES: Absent ALBUMIN: +++
ERYTHROCYTES: .....40-50/HPF .......................................................................................................................................................................................................
ISOMORPHIC (%):........15% ....................... DYSMORPHIC (%):................85% .............................. ACANTHOCYTES(%): ......................10% ..................
LEUKOCYTES: ..............2-4/HPF .......................................................................................................................................................................................................
RENAL TUBULAR EPITHELIAL CELLS:.........................1-2/HPF .................................................................................................................................................
TRANSITIONAL CELLS. SUPERFICIAL:............................// ..................................................... DEEP:....................................// ................................................
SQUAMOUS CELLS:..........................................................................// ...............................................................................................................................................
CASTS..................>1/LPF ........................................................................... TYPES: Hyaline, hyaline – granular, granular, erythrocytic .................................
................................................................................................................................................................................................................................................................
LIPIDS:....................................................// ............................................................................................................................................................................................
CRYSTALS:............................................// ............................................................................................................................................................................................
BACTERIA:............................................//........................................... YEASTS:............................................//................................................................................
OTHERS:................................................// .............................................................................................................................................................................................
COMMENT: Severe dysmorphic erythrocyturia associated with mild leukocyturia, very severe shedding of renal tubular epithelial cells, and very severe
cylindruria (which also includes erythrocytic casts). All these findings are consistent with the presence of an active proliferative glomerular disease.
SIGNATURE
......................................................
FIGURE 1.5 Another example of urinary sediment report.
31
extremely important to remember that (i) standardized procedures are mandatory and (ii) all
methods have limitations.
Figures 1.4 and 1.5 show two examples of reports as filled in our laboratory.
Table 1.6 summarizes the methods and the equipment used by the authors to collect,
handle and analyse the urine samples.
the microscope for the analysis of urinary sediments

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, x 160 or x 200) and a high
magnification (i.e., x 400).
International guidelines recommend the use of phase contrast microscopy and of polarized
light (Table 1.3).
the phase contrast microscope

The Dutchman Frits Zernike (1888-1966) received the 1953 Nobel Prize for Physics
for discovering the principle of phase contrast microscopy. This is based on a special
condenser and objective (Figure 1.6).
TABLE 1.6 Procedure and equipments for the preparation and analysis of the urinary sedi-
ments used in our laboratory.
Collection in disposable containers of the second urine of the morning produced
over 2 h after discarding the first few mL (~10) of urine
Sample preparation and analysis within 3 h from the collection
Standardized centrifugation of a 10 mL aliquot of urine at 400 g for 10 min
Removal by a water pump of 9.5 mL of the supernatant urine

Gentle but thorough resuspension with a Pasteur pipette of the precipitate
in the remaining 0.5 mL of urine
Transfer by a precision pipette of 50 μL of the resuspended urine to a grease-free slide
Covering of the sample with a 24 x 32 mm coverslip

Examination of the urinary sediment by a phase contrast microscope equipped
with two objectives (x 16 and x 40) and a x 10 binocular
Use of polarized light to identify doubtful lipids and crystals
Semi-quantitation of the particles per microscopic field after examining
at least 20 microscopic fields
Matching of the microscopic findings with dipstick findings for pH, specific gravity,
haemoglobin, leukocyte esterase, nitrites, and albumin
¼λ
E
D
C A
B
FIGURE 1.6 Schema of the phase contrast micro-
scope. A = condenser; B = annular diaphragm of the
condenser; C = hollow cone of light; D = objective; E
= phase ring with the layer of translucent silver (dot-
ted grey area);
= direct light; = diffracted light.
The condenser contains an annular diaphragm which transforms the incident light into
a hollow cone of light. The objective in its posterior focal plane contains a circular etched
ring, the so-called phase ring, which is covered by a translucent layer of silver.
After penetrating the object under study, the light beam is composed of both direct light
and diffracted light, the photons of which have interacted with the object. While the direct
light passes through the phase ring, the diffracted light passes only through the surrounding
thicker areas. This difference in the lengths of the light paths results in a phase difference
between the two light beams of one-quarter of the wavelength. In this way a clear-cut
contrast between the background and the elements under investigation is obtained (Figures
1.7 and 1.8), which is the distinguishing feature of phase contrast microscopy.
Another peculiar feature of the phase contrast microscope is the presence of a halo, i.e.
a clear zone around dark details and a dark zone around clear details.
Phase contrast microscopy is far better than bright field microscopy in the identification
of hyaline casts, erythrocytes with low haemoglobin content [29], and cellular details. For
this last reason, it is the best instrument for the differentiation of dysmorphic and isomorphic
erythrocytes, and of renal tubular epithelial cells from deep transitional epithelial cells. In
our experience, it also allows the identification of cells infected by polyomavirus BK, the
so-called decoy cells [30] (see Chapter 5). Therefore, phase contrast microscopy makes
the use of stains for general purposes unnecessary, the latter being needed only for the
identification of eosinophils and lymphocytes (see below).
The use of a phase contrast microscope implies the centering of the ring of the condenser
on the phase ring of the objective (Figure 1.6). In the newest types of microscopes this
is easily done by the regulating of knobs placed in the condenser, while for less recent
microscopes this is done by the so-called phase telescope or auxiliary microscope, which
FIGURE 1.7 An example of the superiority of phase contrast microscopy (left) over bright field microscopy (right).
While cells, casts and morphological details are easily seen with phase contrast microscopy, they are hardly discern-
ible with traditional microscopy (× 400).
FIGURE 1.8 Another example of the advantage of using phase contrast microscopy. The cast in the left side of the
image would be identified as hyaline-granular by bright field microscopy. However, phase contrast microscopy (right)
shows that also erythrocytes (arrows) are present in the cast, a fact which can have relevant clinical implications.
is used by removing one eyepiece (see Appendix). The maneuver of centering has to be
done frequently, but it takes only a few seconds.
When changing to an objective with a different magnification, the annular condenser
has to be changed in parallel, since the ring of the annular diaphragm and the ring in the
objective must match. For this purpose, the best microscopes have a “universal condenser”
with several different annular diaphragms which match the different objectives.
With a phase contrast microscope, examination with polarized light is also possible, but
the results are not as good as when polarization is performed in connection with bright
field optics. However, since phase contrast objectives can also be used with bright field
condenser lenses, it is sufficient to switch the condenser from the phase contrast to the
bright field position to obtain a perfect polarization.
the polarized light

The microscope must also be equipped with filters for polarized light (Table 1.3). The
rotation of one filter placed below the condenser by 90° causes the microscopic field to
darken. When an anistotropic object is placed between the two filters, the plane of oscillation
of the light beam is shifted by interaction with the anisotropic particle under study and a
fraction of light reaches the eye (Figure 1.9). The elements which polarize light are seen as
shining particles against a dark background. This phenomenon is known as birefringence.
Polarized light is useful, and sometimes indispensable, for the identification of lipids
containing cholesterol esters and free cholesterol (which give “Maltese crosses”), crystals,
and some contaminants, such as starch and synthetic fibers (see Chapter 2).
the bright field microscope

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 (Figures 1.7 and
1.8), 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 or by the use of stains (see below). However, even with these
devices, the results are not as good and consistent as with phase contrast microscopy.
other microscopic techniques

Interference contrast microscopy, immunofluorescence microscopy, electron microscopy
and new types of microscopy have been applied to the analysis of the urinary sediment.
Interference contrast microscopy provides a three-dimensional image of the object under
study [31]. It offers nice images, but its diagnostic value is not superior to that of phase contrast
microscopy. Moreover, it has a limited resolution in depth.
Filter 1 Filter 2 Microscopic field
90°
Anisotropic object
90°
FIGURE 1.9 Principle of the microscope with polarized light. (A) The two polarization filters are in parallel: the light
passes through them both and the microscopic field is visible. (B) Filter 2 is rotated by 90°: the light is absorbed by
filter 2 and the microscopic field darkens. (C) An anisotropic object is placed between the two filters: the plane of
vibration of the light which has crossed filter 1 is changed and only a fraction of the light reaches the eye, showing a
shining birefringent object against a dark background.
Immunofluorescence microscopy historically allowed the identification of the nature

of the matrix [32] and granules [33] of casts. It has been used to differentiate non-
pathological casts from casts caused by glomerulonephritis [34], and to identify patients
with monoclonal gammopathies [35]. Immunofluorescence utilizing the antiserum against
Tamm–Horsfall protein has been proposed to differentiate glomerular haematuria from
non-glomerular haematuria [36]. All these applications, however, are time consuming and
difficult to use in everyday work.
In recent times, immunofluorescence with monoclonal antisera against podocalyxin has
been used to investigate the role in the urine of podocytes as a possible marker of an active
glomerular disease [37,38].
Scanning electron microscopy has helped to define further various types of casts [39] and
dysmorphic erythrocytes [40].
Transmission electron microscopy can demonstrate necrotic tubular cells in the urine of
patients with acute renal failure [41]. It also shows myelin bodies within tubular epithelial
cells in patients with aminoglycoside nephrotoxicity [42] and Fabry’s disease [43]. We used
this technique to elucidate the mechanisms of lipiduria [44]. Several transmission electron
microscopy images are presented in Chapter 2.
Confocal scanning laser microscopy, has been used to study urinary erythrocytes [45].
the stains for urinary sediments

If phase contrast microscopy is not available, the use of general supravital stains is
recommended [3]. Of these, Sternheimer’s stain is the most popular [46]. This stain is
based on a mixture of a copper-phthalocyanine dye, national fast blue and a xanthene dye,
pyronin B. After one drop of stain is added to the sediment, staining develops and increases
in intensity in 5-10 min.
Cells show a pink to red cytoplasm, while nuclei are blue. Casts show a bluish matrix, while
the tinge of inclusions varies according to their nature. A good differentiation of erythrocytes,
polymorphonuclear leukocytes, lymphocytes, transitional epithelial cells, casts, bacteria and
malignant uroepithelial cells can thus be obtained. However, renal tubular epithelial cells are
not always distinguishable, and a poor staining may be observed, especially when there is a
reduced viability of cells. Besides Sternheimer’s stain, other supravital stains are available
on the market (Figure 1.10).
Eosinophils (which were once considered a marker of acute interstitial nephritis caused
by antibiotics) and lymphocytes (which are considered a marker of acute cellular rejection of
the renal allograft) always need stains to be seen.
For those who still believe in the utility of the search for urinary eosinophils, Hansel’s
stain is the most sensitive and simple to use [47,48]. Other possible stains are Wright’s stain
(which was the gold standard before the introduction of Hansel’s stain), Papanicolaou’s stain
and May-Grünwald-Giemsa.
For recognition of lymphocytes, one may use Wright’s stain, a supravital stain based on methy-
lene blue [49], Papanicolaou’s stain [50,51] or May-Grünwald-Giemsa stain. Activated lymphocytes
may be identified by methylene green pyronin, which recognizes RNA-rich cells [52].
FIGURE 1.10 A renal tubular epithelial cell cast and squamous epithelial cells as shown by a supravital stain made
of crystal violet and saphranine (x 400).
Specific stains may also be used for other elements. For instance, lipids are stained with
oil-red O or Sudan III, while haemosiderin is stained with Prussian blue [4]. These stains,
however, can hardly be applied to routine work, and have mostly an historical interest.
Staining with monoclonal antibodies specific for several types of cells of the urinary
sediment (granulocytes, monocytes, lymphocyte subpopulations, glomerular epithelial cells,
different subtypes of renal tubular epithelial cells and urothelial cells) is also possible. With
this technique, specific cytopathology profiles have been identified in acute cellular rejection
[53,54], crescentic glomerulonephritis, acute interstitial nephritis and acute tubular necrosis
[55]. These stains, however, are used in specialized laboratories and more for research
purposes than for routine work.
Several other novel technologies have recently been applied to the analysis of urinary
sediments. These technologies include extraction of DNA for polymerase chain reaction
(PCR) and the investigation of microsatellite instability, urinary cytokine level, and urinary
mRNA expression [56,57]. All these techniques are beyond the aims of this book.
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CHAPTER 2
THE FORMED ELEMENTS
OF THE URINARY SEDIMENT
G.B. Fogazzi, G. Garigali, M.D. Croci and S. Verdesca
This chapter describes the particles of the urinary sediment which can be identified with
phase contrast microscopy and polarized light when indicated.
For this reason, the images shown were obtained with these two techniques, which are
recommended by international guidelines for routine work [1,2]. For some particles, also
images obtained with electron microscopy are shown for a better understanding of their
structure. A few images were obtained with bright field microscopy.
cells
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 (Tables 2.1 and 2.2). Most cells
include several subtypes, some of which can be identified only by using general or specific
TABLE 2.1 The cells of the urinary sediment which can be identified by conventional micro-
scopy.
Type Subtype
Erythrocytes Dysmorphic or glomerular (including acanthocytes
or G1 cells)
Isomorphic or non glomerular
Others (sickled erythrocytes, anysocytes,
poikilocytes)
Leukocytes Polymorphonuclear leukocytes
Eosinophils
Lymphocytes
Macrophages --
Renal tubular epithelial cells --
Transitional epithelial cells From the deep layers of the uroepithelium
From the superficial layers of the uroepithelium
Squamous epithelial cells --
42 G.B. Fogazzi, G. Garigali, M.D. Croci and S. Verdesca
TABLE 2.2 Size of the cells found in the urinary sediment (measurements performed with the
“Measurement” module of Leica IM 1000 “Original Image” system).
Nucleus
Cell diameter
Number of Number of cells diameter (μm)
Cell (μm) mean ± sd
subjects measured mean ± sd
(range)
(range)
6.6 ± 1.1
Erythrocytes 21 1100 --
(4.0-10.2)
10.6 ± 1.2
Neutrophils 11 600 --
(7.2-15.0)
39.6 ± 13.9 15.9 ± 5.9
Macrophages 55 140
(13.0-95.0) (7.5-27.5)
Renal tubular 14.3 ± 3.4 7.6 ± 1.4
8 66
epithelial cells (9.4-25.1) (5.1-12.4)
Deep
transitional 22.1 ± 5.8 9.9 + 2.1
11 114
epithelial (10.7-37.8) (5.6-17.5)
cells
Superficial
25.0 ± 4.9 10.3 ± 2.2
transitional 15 140
(17.0-42.8) (6.4-18.8)
epithelial cells
Squamous 53.2 ± 14.7 9.3 ± 2.6
18 500
epithelial cells (16.8-118.0) (2.2-15.4)
stains. Other cells, such as podocytes [3], basophilic leukocytes [4], platelets [5] or monocytes
[6] have also been described in urine. However, they were identified with sophisticated
techniques, often by single groups of investigators, and/or their clinical meaning is not yet
definitely clarified. Therefore, they are not described in this book.
erythrocytes
Erythrocytes are a frequent finding in urine, in which they are often present as contaminants
deriving from genital secretions, especially in women. Erythrocyte diameter, refractivity
index and morphology can vary under various conditions.
The diameter of erythrocytes ranges from 4.0 to about 10 μm (Table 2.2), but this is
influenced by changes in specific gravity (or osmolality), increasing as specific gravity
decreases and vice versa (see Chapter 1, Figure 1.2). It is important to remember that at
a specific gravity of about Φ 1.010, the erythrocytes tend to undergo lysis, a fact which
can cause false negative results and discrepancies between microscopy and dipstick for
haemoglobin (see Chapter 1, page 26).
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”) (Figures 2.1 and 2.6).
The morphology of urinary erythrocytes ranges from perfectly round cells to particles
with very changed shape. This was already noted in the second half of the 19th century
(see Historical Introduction, page 11), and the old observation that altered erythrocytes
were typical of patients with Bright’s disease (i.e., glomerulonephritis) was confirmed by
Fairley and Birch in 1982 [7]. 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”) (Figures 2.2-2.5), 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”) (Figures 2.5-2.10).
After the seminal paper by Fairley and Birch [7], many other studies were published by
different investigators on the same subject in the 1980s, and all of them, with very few
exceptions, confirmed the utility of examining urinary erythrocyte morphology [8-13].
However, over the years it has become clear that the evaluation of urinary erythrocytes
morphology is associated with three types of limitation [14].
1. The lack of univocal criteria to define a haematuria as glomerular or non glomerular.
In fact, while for some investigators a haematuria was of glomerular origin if >80%
erythrocytes were dysmorphic [8,10,11], for others discriminating cut off was as low as
10% [15] or 14% [16]. Other investigators, instead, defined a haematuria as glomerular
when more than two [17] or three [18] erythrocyte subtypes were found in the same
sample, or when this contained Γ 5% “acanthocytes” or “G1 cells” [19-23]. The latter
are doughnut-shaped dysmorphic erythrocytes with one or more vesicle-like protrusions
(Figures 2.11-2.14), which can be identified easily and with much less subjectivity than
other dysmorphic erythrocytes.
2. There may be a low inter-observer reproducibility [24], a fact which is explained by the
very wide spectrum of appearances that erythrocytes may have in the urine, in the category
of both dysmorphic and isomorphic erythrocytes [25,26].
3. A non glomerular haematuria can be found in patients with a glomerular disease
due to gross haematuria [27], renal insufficiency [28], increased diuresis after furosemide
administration [29] or necrotizing glomerulonephritis [30].
In spite of all these uncertainties, in our laboratory we do evaluate the morphology of urinary
erythrocytes upon request for patients with isolated microscopic haematuria of unknown origin.
For each urine sample, we calculate the percent of isomorphic and dysmorphic erythrocytes
as well as of acanthocytes/G1 cells. In addition, we analyse 50 microscopic fields at low
magnification (x 160) for the search of erythrocytic casts. As suggested by others, we define
a haematuria as glomerular when we find Γ 40% dysmorphic erythrocytes [31] and /or Γ 5%
acanthocytes (also defined as G1 cells) [19-23] and/or Γ 1 erythrocytic casts/50 low power
fields. Since in some patients we have observed some variability of the urinary erythrocyte
pattern (= from glomerular to non glomerular haematuria and vice versa), we ask each patient
to deliver three samples over a period of few weeks. We consider a haematuria as glomerular
when at least 2 out of 3 samples show dysmorphic erythrocytes and/or acanthocytes/G1 cells
and/or erythrocytic casts as described above. With this approach, we have been able to find
a close correlation with the presence of glomerular changes at renal biopsy [32] (see also
Chapter 6, page 218).
Alternative approaches for evaluation of glomerular or non glomerular haematuria have

been proposed. These include the measurement of erythrocyte volume by coulter counter
[33] or automated sediment analyzers [34,35] (See Chapter 7, page 226), or the search of
Tamm-Horsfall glycoprotein on the surface of the urinary erythrocytes [36]. However, none
of these methods is widely used today.
The cause of glomerular erythrocyte dysmorphism is not entirely clear. In vitro experiments
have shown that neither osmolality nor pH changes of solutions in which erythrocytes are
suspended are sufficient to cause dysmorphic morphology [37]. 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 [38]. 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 [38]. These data led to the
hypothesis that in vivo erythrocytes become dysmorphic as a consequence of a dual injury
[39]. 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 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. This may happen in patients with haematuria caused by sickle cell
disease, whose urine can show sickle erythrocytes (Figure 2.15) [40,41], and in patients with
urological haematuria and concomitant iron deficiency anaemia, whose urine can contain
anysocytes and poikilocytes (Figure 2.16) [42].
FIGURE 2.1 “Ghost” erythrocytes (phase contrast, x FIGURE 2.2 Several dysmorphic erythrocytes, which
400). are easily identifiable due to their membranes and con-
tour irregularities (phase contrast, x 400).
FIGURE 2.3 Different types of dysmorphic erythro- FIGURE 2.4 Other types of dysmorphic erythrocytes
cytes as seen at high magnification (phase contrast, as seen at high magnification (phase contrast, original
original magnification x 500). magnification x 500).
FIGURE 2.5 Two dysmorphic erythrocytes (bottom) FIGURE 2.6 Spherical shaped isomorphic erythro-
compared to two isomorphic erythrocytes (top) (phase cytes. Many of them have lost their haemoglobin con-
contrast, x 640). tent (the so-called “ghost” erythrocytes)(phase con-
trast x 400).
FIGURE 2.7 Isomorphic erythrocytes with well pre- FIGURE 2.8 Isomorphic erythrocytes with a central
served haemoglobin content (phase contrast, x 400). clear halo (phase contrast, x 400).
FIGURE 2.9 Isomorphic erythrocytes with various de- FIGURE 2.10 Isomorphic erythrocytes (discs and cre-
grees of crenation (= with spike protrusions from the nated cells) as seen by scanning electron microscopy
body of the cell)(phase contrast, x 400). (x 4,000).
FIGURE 2.11 An acanthocyte or G1 cell with the typi- FIGURE 2.12 Several acanthocytes or G1 cells (ar-
cal appearance of a doughnut with blebs as seen by rows) as seen by phase contrast microscopy (x 400).
scanning electron microscopy (x 12,000).
FIGURE 2.14 Other morphological types of acan-

thocytes or G1 cells at high magnification. Note the cell
top right without haemoglobin content (phase contrast,
original magnification x 500).
FIGURE 2.13 Three acanthocytes or G1 cells com-

pared to two dysmorphic erythrocytes without blebs at
high magnification (phase contrast, original magnifica-
tion x 500).
FIGURE 2.15 Sickled erythrocytes (arrows)(phase con- FIGURE 2.16 Schistocytes and poikilocytes (arrows)
trast, original magnification x 400). (phase contrast, x 400).
leukocytes
Three types of leukocytes can be found in the urine.
Neutrophils
Neutrophils are the leukocytes most frequently found in the urinary sediment. Typically
they appear as round granular cells (Figure 2.17), granules representing cytoplasmic
organelles. Their diameter ranges from about 7.0 to 15.0 μm (Table 2.2). However, substantial
differences in diameter and morphology may be caused by differences in urine specific
gravity or osmolality (see Chapter 1, Figure 1.2). In fact, in diluted urine, the cell is larger
and swollen, and both nucleus and cytoplasmic organelles are easily identifiable (Figures
2.18 and 2.19), the latter often showing Brownian movement. In contrast, in concentrated
urine, the cytoplasmic organelles are packed and the identification of the lobulated nucleus
may be difficult. Occasionally, for unknown reasons, neutrophils may show blebs protruding
from the cell body (Figure 2.20) or may have an elongated shape (Figure 2.21). It may
even happen that, during the microscopic examination of the sample, neutrophils with usual
appearance transform into larger cells with irregular shape and a thin transparent granular
cytoplasm, hardly distinguishable from the background (Figure 2.22). Interestingly, these
cells were considered in the past as histiocytes, while now we consider them as neutrophils
which have undergone degenerative processes probably favoured by some physico-chemical
features of the urine.
Neutrophils may also appear in “clumps” (Figure 2.23), which is seen especially in urinary
tract infection.
In doubtful cases, the identification of neutrophils may be helped by addition to the
sediment of a few drops of acetic acid, after which the nuclear membranes become more
clearly visible.
Bacterial urinary tract infection is the most frequent cause of neutrophiluria. However, it
should not be forgotten that neutrophils may be found in a wide spectrum of non-infectious
renal diseases including glomerulonephritis [43], acute and chronic interstitial nephritis,
polycystic kidney disease, or urologic disorders. It has been claimed that neutrophiluria can
also be observed in inflammatory diseases affecting organs adjacent to the urinary tract such
as acute appendicitis or acute adnexitis [44]. However, we do not have personal data to
support, or not, this view.
It is important to remember that, especially in women, neutrophils may be found in the
urine because of contamination by genital secretions. This condition is suggested by the co-
presence in the urine of large amounts of squamous epithelial cells deriving from the vagina,
with or without bacteria, Candida, or Trichomonas vaginalis.
Eosinophils
Eosinophils too may be present in the urine. They can definitely be identified only by
using May-Grünwald-Giemsa, Wright’s or Hansel’s stains, the latter being considered as the
most sensitive [45,46].
Eosinophils have a bilobar nucleus and well-defined granules, which occupy the entire
cytoplasm. With May-Grünwald-Giemsa, the granules have a purple colour (Figure 2.24),
with Wright’s stain the granules range from deep blue to faint pink, while with Hansel’s stain
they are pink.
The finding of eosinophiluria was considered in the past as a specific marker of acute
interstitial nephritis caused by methicillin [47]. However, subsequent studies have shown
that eosinophiluria can be found in a wide spectrum of disorders including urinary tract
infection, prostatitis, extracapillary glomerulonephritis, Schönlein-Henoch purpura
nephritis, acute allograft rejection, urinary schistosomiasis or cholesterol embolism of the
kidney [48-52].
One should also be aware that in acute interstitial nephritis, eosinophiluria may be either
mild or even absent, which is partly due to the heterogeneous nature of this condition
[45,46,52]. Therefore, today, eosinophiluria is considered as an unspecific finding of much
less diagnostic importance than thought in the past [52].
Lymphocytes
Lymphocytes can be identified with certainty only with specific or general stain
preparations, such as Papanicolou’s stain. (Figure 2.25). However, it is our experience that
they can also be identified with phase contrast microscopy only. Irrespective of the method
used, they appear as small round cells with a large nucleus and a very thin pale cytoplasmic
rim (Figure 2.26).
Lymphocyturia is considered as an early and sensitive marker of acute cellular rejection
in renal allograft recipients [53-55]. It is also common in chyluria, a condition which is
characterized by “milky” urine. [56]. Lymphocyturia in other conditions, such as acute
interstitial nephritis or viral infection, has been much less studied and documented.
FIGURE 2.17 Polymorphonuclear leukocytes with FIGURE 2.18 Polymorphonuclear leukocytes with
their lobated nucleus and granular cytoplasm (phase very evident lobated nucleus and cytoplasmic granules
contrast, x 400). (phase contrast, x 400).
FIGURE 2.19 Swollen polymorphonuclear leukocytes FIGURE 2.20 Polymorphonuclear leukocytes which
with apparently scant cytoplasm granules as seen in have lost the differentiation between the nucleus and
urine with low specific gravity (phase contrast, x 640). the cytoplasm. Also note that some leukocytes have
multiple blebs (arrows)(phase contrast, x 400).
FIGURE 2.21 Elongated and round polymorphonu- FIGURE 2.22 Degenerated polymorphonuclear leu-
clear leukocytes (phase contrast, original magnification kocytes (phase contrast, x 400).
x 400).
FIGURE 2.23 A clump of polymorphonuclear leuko-

cytes (phase contrast, x 400).
FIGURE 2.24 Eosinophils with their abundant purple FIGURE 2.25 Lymphocytes (arrows) intermingled with
granules (arrows) as seen with May-Grünwald-Giemsa erythrocytes found in the urine of a patient with chyluria
stain (x 1,000). due to lymphangioma (Papanicolaou stain on a cytocen-
trifuged smear, x 1,000).
FIGURE 2.26 A lymphocyte with its typical thin cyto-

plasmic rim as seen by phase contrast microscopy with-
out stain (original magnification x 400).
macrophages
Urinary macrophages are roundish cells with very variable diameter (from about 13.0 to
95.0 μm in our experience) and variable appearance. They contain one or more nuclei (up
to >10) [57], which can be either in central or peripheral location. Quite often, however, the
nucleus is not visible, which can be caused by the masking effect of granules, vacuoles or
other particles contained in the cytoplasm (Figures 2.27-2.31).
Morphologically, we classify them according to Ito et al. [57], who described four different
types of macrophages in unstained and Sternheimer-stained urine specimens:
- granular macrophages, whose cytoplasm contains variable amounts of granules;

- vacuole-forming macrophages, whose cytoplasm contains variable numbers of vacuoles;
- macrophages with phagocytic activity, whose cytoplasm contains bacterial debris, cell
fragments, destroyed erythrocytes, etc.;
- macrophages with a homogeneous or hazy appearance, whose cytoplasm does not
contain granules or other particles.
To these four types we add fatty macrophages, whose cytoplasm is gorged with packed
small lipid droplets and correspond to the urinary sediment particles known as “oval fat
bodies” (Figures 2.68-2.71).
The macrophagic nature of these last particles was demonstrated by Hotta and coworkers
[58], who found that oval fat bodies staining for Oil Red O (a specific stain for lipids) were
also positive for the monoclonal antibody to human macrophages CD68. Not surprisingly,
these fatty macrophages were significantly increased in the urine of patients with non selective
and heavy proteinuria. Moreover, they correlated with the decline of renal function over
time. Other studies performed by the same investigators using flow cytometry and another
monoclonal antibody to macrophages, CD14, showed that macrophages also are a marker of
proliferative glomerulonephritis in active phase [58].
Other investigators used immunofluorescence microscopy and the monoclonal antibody
to human macrophages CD68, clone PG1 (Figure 2.32) and found that the number of
macrophages in the urine was increased in the urine of patients with IgA nephropathy and that
the number of urinary macrophages significantly correlated with: macrophage infiltration in
the kidney; histologic activity index at renal biopsy; urinary leukocyte excretion and protein/
creatinine ratio in the morning urine [59].
In our experience, based on phase contrast microscopy alone, macrophages are most
frequently found in polyomavirus BK infection observed in kidney transplant recipients,
whose urinary microscopic marker is represented by “decoy cells” (see Chapter 5, page 198).
In a retrospective unpublished investigation, we found macrophages in 12 out of 13 sediments
(92.3%) from 7 patients with decoy cells in the urine (5 patients with BK virus nephropathy,
2 with BK virus reactivation but without nephropathy). The number of macrophages counted
over 50 microscopic fields at x 400 ranged from 1 to 23, with a mean of 8.1 ± 6.7. Interestingly,
the number of macrophages correlated positively with the number of decoy cells.
Based on the findings described above, we think that further studies are needed to clarify
the role of macrophages in the urinary sediment.
FIGURE 2.27 Left. A binucleated granular macrophage with two vesicles, due to degenerative processes, protrud-
ing from the cell body (phase contrast, original magnification x 400). Right. A binucleated granulo-vacuolar macro-
phage (phase contrast x 400).
FIGURE 2.28 Left. A phagocytic macrophage containing particles with various sizes and shapes (phase contrast x
400). Right. Polarized light shows that the phagocytosed particles are fragments of crystals of undefined nature (x
400).
FIGURE 2.29 A granulo-vacuolar macrophage whose FIGURE 2.30 A mononucleated vacuolar macrophage
nucleus is not evident (phase contrast, x 400). (phase contrast, original magnification x 400).
FIGURE 2.31 A mononuclear macrophage with cy- FIGURE 2.32 Macrophages (stained in red) as seen
toplasm with scanty cytoplasmic organelles (original with monoclonal antibody CD68-PG1 (cytocentrifuged
magnification x 400). smear, x 1,000).
renal tubular epithelial cells

The different segments of the renal tubules are lined by different types of epithelial cells.
These differ with respect to: (i) shape (flat, cuboidal or even columnar); (ii) contours (with
or without a brush border; with few or many microvilli; with scarce or extensive infolding
or interdigitation of the basolateral membrane); (iii) cytoplasmic organelles (scarce or
abundant); and (iv) location of the nucleus (basal, central or even apical) (Figures 2.33-2.36).
Their size ranges from about 9.0 to 25.0 μm (Table 2.2).
Using specific monoclonal antibodies or lectin staining, the cells of well-defined tubular
segments can be identified in the urine [60,61]. With conventional techniques, however,
precise categorization of these cells is difficult and can only be approximate.
The renal tubular epithelial cells (RTECs) we most frequently find in the urinary sediment
probably derive 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 abundant organelles, and a mean diameter of about 14.0 μm (Figures 2.37-2.40).
Other RTECs, probably deriving from distal tubules, are polygonal with a central nucleus
and are smaller (Figure 2.41). RTECs probably deriving from the collecting ducts have a
columnar shape with a nucleus in the basal position containing prominent nucleoli (Figures
2.42).
At times, RTECs show degenerative changes (Figure 2.43) or appear in aggregates (Figures
2.44 and 2.46), which indicates a particularly severe tubular damage. RTECs can also be
embedded in casts (see Figures 2.109-113), which are therefore defined as “RTEC casts”
or “epithelial” casts (see page 88). Such casts offer a good opportunity to the inexperienced
urine microscopist to learn about the appearance of RTECs.
At times, it can be difficult to differentiate the round or ovoid RTECs from the small
ovoid cells of the deep layers of the uroepithelium (Figures 2.48-2.53). Admittedly, deep
uroepithelial cells are usually larger than tubular epithelial cells (Table 2.2), have frequently
a club-like or ovoid shape and have a higher nucleus to cytoplasm ratio, with a thin cytoplasm
rim. However, the most useful clue to differentiate the two types of cells is the urinary
context in which they are seen. RTECs are usually accompanied by elements indicative of
parenchymal renal disease such as casts, dysmorphic erythrocytes or lipids. Deep transitional
cells, on the contrary, are commonly associated with isomorphic erythrocytes, leukocytes
and superficial transitional cells [62].
RTECs are found in disorders that primarily involve the tubules, such as acute tubular
necrosis [63-65], acute interstitial nephritis [66] or acute rejection of a renal allograft
[67]. 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. In
fact, we found RTECs in 43 out of 52 patients (82.7%) with various types of proliferative
glomerulonephritis [43] and, more recently, in 34 out of 52 patients (65.4%) with nephrotic
syndrome (see Table 6.1, page 213). In patients with proliferative glomerulonephritis, the
number (mean ± SD) of RTECs counted over 20 high power fields (x 400) was 8.4 ± 8.9,
while in patients with nephrotic syndrome it was 4.0 ± 3.2.
FIGURE 2.33 Ultrastructure of cells of the proximal

tubule. (a) Cell of the S1 segment, with very extensive
cell interdigitation and a high and dense brush border.
(b) Cell of the S2 segment, with reduced interdigitation
and a less dense brush border.(c) Cells of the S3 seg-
ment, whose cellular interdigitation and brush border
are reduced.
Short Long
loop loop
1 2
3 4
Complex
2
FIGURE 2.34 Ultrastructure of cells of the loop of

Simple
Henle’s loop. (1) Cells of the thin limb of short loops. (2)
Cells of the thin limb of long loops (the complex type
being found in rabbit and guinea pig). (3) Cells of the 3
lower part of the descending thin limb of long loops. (4)
Cells of the ascending thin limb. Courtesy of Drs. Kritz
W. and Kaissling B., Heidelberg, Germany. Reproduced
with permission from Seldin D.W. and Giebisch G. (eds) 4
The Kidney: Physiology and Pathophysiology, 2nd edn.
New York, Raven Press, 1992.
FIGURE 2.35 Ultrastructure of the cells of the distal

straight tubule including the macula densa. (a) The
medullary part. (b) The cortical part. (c) The macula
densa. Courtesy of Drs. Kritz W. and Kaissling B., Hei-
delberg, Germany. Reproduced with permission from
Seldin D.W. and Giebisch G. (eds) The Kidney: Physiol-
ogy and Pathophysiology, 2nd edn. New York, Raven
Press, 1992.
FIGURE 2.36 Ultrastructure of cells of: (a) The distal

convoluted tubule. (b) The connecting tubule. (c) The
collecting duct (principal cell). (d) The inner medullary
collecting duct. Courtesy of Drs. Kritz W. and Kaissling
B., Heidelberg, Germany. Reproduced with permission
from Seldin D.W. and Giebisch G. (eds) The Kidney:
d
Physiology and Pathophysiology, 2nd edn. New York,
Raven Press, 1992.
FIGURE 2.37 Roundish RTECs from the proximal tubu- FIGURE 2.38 An ovoid proximal RTEC (phase con-
lar segments with well evident nucleus in central loca- trast, x 400).
tion and granular cytoplasm (phase contrast, x 500).
FIGURE 2.39 A rectangular proximal RTEC (phase FIGURE 2.40 An ovoid proximal RTEC (arrow) and
contrast, x 640). polymorphonuclear leukocytes. Note the different size
of the two types of cells (phase contrast, x 400).
FIGURE 2.41 A polygonal RTEC probably deriving FIGURE 2.42 A columnar RTEC with the nucleus in
from the distal renal tubules (phase contrast x 500). basal location, probably deriving from collecting renal
ducts (phase contrast, x 800).
FIGURE 2.43 An ovoid proximal RTEC showing de-

generative changes (i.e., condensation of cytoplasmic
organelles) found in the urine of a patient with acute tu-
bular necrosis due to rhabdomyolysis (phase contrast,
x 400).
FIGURE 2.44 A clump of ovoid proximal RTECs found FIGURE 2.45 Another clump of RTECs, probably from
in the urine of a patient with acute tubular necrosis proximal renal tubules (phase contrast, x 400).
(phase contrast, x 400).
FIGURE 2.46 A fragment of renal tubular epithe-

lium found in the urine of a patient with acute tubular
necrosis associated with extracapillary/necrotizing
glomerulonephritis (phase contrast, x 400).
transitional epithelial cells

Transitional epithelial cells derive from the uroepithelium, which lines the urinary tract from
the calyces to the bladder in women, and to the proximal urethra in men. It is a multilayered
epithelium, consisting of several types of cells which form a continuum from the deep to the
superficial layers (Figure 2.47). The cells of all layers can be found in the urine, but the cells
from the deep layers and from the superficial layers are the best identifiable.
Transitional cells of the deep layers of the uroepithelium 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 (Figures 2.48-2.53). Their longitudinal diameter
ranges from about 10.7 to 38.0 μm (Table 2.2).
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.
In our experience, deep transitional cells are seen in large quantities (i.e. one or more
per high power field) in conditions characterized by damage to the deep layers of the
uroepithelium such as urolithiasis, bladder carcinoma, or hydronephrosis [62]. They are
also frequently found in the urine of patients with ureteric stents or bladder catheters for
prolonged periods of time (Table 2.3).
The cells of the superficial layers of the uroepithelium are larger than those of the deep
layers (Figure 2.53). Their size ranges about from about 17.0 to 43.0 μm (Table 2.2). They
are round to oval, with round or oval nuclei located in a central or only slightly eccentric
position. Occasionally, binucleated cells are seen. The cytoplasmic granules are usually
scarce around the nucleus, but abundant in the periphery, resulting in a perinuclear halo
(Figures 2.54-2.59).
These cells are easily identified and are much more frequent than deep transitional cells,
since even mild injury to the uroepithelium causes their exfoliation. In our experience, they
are seen most frequently in patients with cystitits.
Transitional cells with morphological atypias such as those caused by cancer of the excretory
system (renal pelvis, ureters and bladder) can be found in the urine. In our experience, as well
as in others’, these cells can also be identified by phase contrast microscopy on unfixed and
unstained samples [68] (For details see Chapter 5, page 204).
64
TABLE 2.3 Urinary sediment features and clinical conditions associated with the finding of ≥ 1 deep transitional epithelial cell/high
power field in authors’ experience.
Patient Gender Age RBC WBC STC Bacteria Disease
1 M 71 + -- -- -- Bladder carcinoma
2 F 65 + + -- -- Bladder carcinoma
3 M 57 + + -- + Urolithiasis*
4 M 60 + + -- -- Urolithiasis*
5 M 69 + -- -- -- Urolithiasis*
6 F 21 + + -- -- Urolithiasis*
7 M 63 + -- + + Urolithiasis*
8 M 40 + + -- -- Hydronephrosis
9 F 78 + + -- -- Ureteric stenosis
10 M 37 + + -- -- Bladder carcinoma
G.B. Fogazzi, G. Garigali, M.D. Croci and S. Verdesca

UTI + bladder catheter
11 F 73 + + + +
(30 days)
12 F 51 + + + -- Ureteric stent (60 days)**
13 M 45 + + -- -- Ureteric stent (68 days)**
14 M 40 + -- + -- Ureteric stent (30 days)**
15 F 59 + -- + -- Ureteric stent (34 days)**
16 F 31 -- -- -- -- Ureteric dilatation
17 M 61 + + + -- Bladder catheter (26 days)
16/17 11/17 6/17 3/17
M: 10; F: 7 54.2±16.1
(94.1%) (64.7%) (35.2%) (17.60%)
RBC = Red blood cells (+ present; -- absent); WBC = white blood cells; STC = superficial transitional epithelial cells; * associated with ureteric stent; ** ureteric stent be-
cause of retroperitoneal fibrosis (patient 12) or prevention of ureteric stenosis in post kidney transplant period (patients 13-15).
FIGURE 2.47 The multilayered epithelium of the human ureter (left). Note the marked morphological differences
between the cells of the deep layers (bottom right) and the cells of the superficial layers (top right)(haematoxylin &
eosin, x 400).
FIGURE 2.48 Club-like and ovoid cells from the deep FIGURE 2.49 A tailed and an ovoid cell from the deep
layers of the uroepithelium (phase contrast, x 400). layers of uroepithelium (phase contrast, x 400).
FIGURE 2.50 Club-like and ovoid cells from the deep FIGURE 2.51 A small clump of ovoid cells from the deep
layers of the uroepithelium (phase contrast, x 400). layers of the uroepithelium (phase contrast, x 400).
FIGURE 2.52 Another clump of cells from the deep FIGURE 2.53 A cell from the superficial layers of the
layer of the uroepithelium (phase contrast, x 500). uroepithelium (top) and two cells from the deep layers.
Note the different size and shapes of the two types of
cells (phase contrast, x 400).
FIGURE 2.54 A kidney-shaped transitional cell from FIGURE 2.55 An ovoid transitional cell from the su-
the superficial layers of the uroepithelium (phase con- perficial layers of the uroepithelium (phase contrast,
trast, original magnification x 400). original magnification x 400).
FIGURE 2.56 Another ovoid transitional cell from the

superficial layers of the uroepithelium. Note the clear
halo around the nucleus (phase contrast, original mag-
nification x 400).
FIGURE 2.57 Two ovoid and one round transitional FIGURE 2.58 A cluster of ovoid transitional cells from
cells from the superficial layers of the uroepithelium. the superficial layers of the uroepithelium (phase con-
Note the different size between these cells and the pol- trast, original magnification x 400).
ymorphonuclear leukocytes in the background (phase
contrast, original magnification x 400).
FIGURE 2.59 A large cluster of ovoid transitional cells

from the superficial layers of the uroepithelium (phase
contrast, x 256).
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) (Table 2.2). These cells are quadrangular to polygonal in
shape, and have a broad cytoplasm containing few granules and a small central nucleus
(Figure 2.60). Frequently, squamous cells are folded or are aggregated in “clumps” (Figure
2.61). Occasionally, bacteria are attached to their cell membrane (Figure 2.62), 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 (Figure 2.63).
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 (Figure 2.64). In women with vaginitis,
large numbers of squamous epithelial cells are often associated with Candida, Trichomonas
vaginalis and/or bacteria and polymorphonuclear leukocytes (Figure 2.65).
FIGURE 2.60 A squamous epithelial cell with a few FIGURE 2.61 A clump of squamous epithelial cells
rods atthached to its cell membrane (phase contrast, x (phase contrast, x 400).
320).
70
FIGURE 2.62 A squamous epithelial cell almost com- FIGURE 2.63 Free nuclei deriving from disrupted
pletely masked by rods attached to the cell membrane. squamous epithelial cells (arrows)(phase contrast, x 400).
In the background a clump of other squamous epithe-
lial cells (phase contrast, x 400).
FIGURE 2.64 A massive amount of squamous epithe- FIGURE 2.65 A massive amount of squamous epithe-
lial cells as can be observed in women when the urine lial cells associated with Candida, a frequent finding in
is not properly collected or there is a vaginal discharge the urine of women with candidal vaginitis (phase con-
(phase contrast, x 160). trast, x 400).
lipids
In the urine sediment lipids appear as:
- free lipid droplets (isolated or in aggregates)
- oval fat bodies
- fatty casts
- cholesterol crystals.
Free lipid droplets, isolated or in aggregates, appear as translucent round particles of
very variable size, with a bright yellow colour. Under polarized light, they show the typical
appearance of “Maltese crosses”, with perfectly symmetrical arms (Figures 2.66-2.67).
The term “oval fat bodies” defines macrophages [58] or renal tubular epithelial cells [69]
when they are so much gorged with lipid droplets that the underlying cellular structures
cannot be identified (Figures 2.68-2.71). Intracellular lipid particles can also be in small
amounts, in which case the underlying cellular details can be seen (Figures 2.72 and 2.73).
Fatty casts are cylinders which contain lipid droplets in their matrix. The amount of lipids
can vary from a few and isolated droplets (Figure 2.75) to tightly packed droplets, which
mask the matrix of the cast (Figures 2.76 and 2.77; 2.114 and 2.117).
Cholesterol crystals are thin, colourless and transparent plates with well-defined edges,
which can be isolated or in aggregates (Figures 2.78 and 2.79; 2.195 and 2.196).
Lipid droplets can usually be identified without difficulty. However, larger fat globules
may be confused with isomorphic erythrocytes, yeasts or round calcium oxalate crystals.
On the other hand, if scarce and tiny, they may be overlooked. For such doubtful cases,
polarized light must be used (see Chapter 1, page 35), under which lipid droplets appear as
“Maltese crosses”. However, this is true only for lipids containing cholesterol esters and free
cholesterol, not for other lipids.
A birefringence somewhat similar to that caused by lipids may be due to starch particles,
a contaminant of the urinary sediment. However, starch causes a “cross” with asymmetrical
arms (see Figures 2.235 and 2.236).
Lipids can also be identified by stains such as Oil-Red O or Sudan III [2]. However, the
use of phase contrast microscopy coupled with polarized light, is much less laborious than
these stains.
Lipiduria can be found in several renal diseases, but especially in nephrotic syndrome
[70]. In this condition, lipiduria is due to free cholesterol, cholesterol esters, triglycerids,
free fatty acids and phospholipids, the main lipoprotein being represented by HDL [71].
However, they may also be found in patients with non-nephrotic proteinuria, in some patients
with non glomerular diseases [72] or in patients with polycystic kidney disease and low
grade proteinuria [73]. In the latter condition, the lipid droplets are thought to derive from
renal cysts containing degraded blood.
Lipid droplets, free or within the cytoplasm of renal tubular epithelial cells, are also found
in the urine of patients with Fabry’s disease, even in the absence of proteinuria [74,75]. This is
a condition due to the hereditary deficiency of the enzyme | -galactosidase A, resulting in the
intracellular accumulation of the neutral glycosphingolipid globotriaosylceramide in several
organs, including the kidney. In the experience of some authors, the fatty particles found
in this condition show an irregular membrane protrusion [76], while for other authors they
are identical to those seen in the urine of patients with nephrotic syndrome [77]. However,
at electron microscopy these particles have a peculiar structure, which is characterised by
both intracellular and extracellular electrondense lamellae, alternating dark and clear layers
arranged in concentric whorls [74,75,78], which are not seen in lipiduria caused by other
diseases [79]. Thus, the use of electron microscopy for the search of such urinary particles
has been proposed as a non invasive method to diagnose Fabry’s disease [74,75,78], to
reveal its recurrence after renal transplantation [80], or to monitor the efficacy of the enzyme
replacement therapy [81].
In patients with glomerular diseases, lipids enter the urine because of abnormal glomerular
ultrafiltration. Within the tubules, they are partially reabsorbed by proximal tubular cells
[69,71] and transported for hydrolysis into lysosomes [82,83]. Then, they re-enter the tubular
urine via regurgitation i.e., active expulsion [69], or as a result of cellular breakdown.
FIGURE 2.66 An aggregate of lipid droplets of differ- FIGURE 2.67 The same lipid droplets shown in Figure
ent size. Note the shining yellow colour (phase contrast, 2.66 as seen by polarized light. Note the typical Maltese
original magnification x 400). crosses, with perfectly symmetrical arms (original mag-
nification x 400).
FIGURE 2.68 A large oval fat body (gorged with lipid FIGURE 2.69 The same oval fat body shown in image
droplets of different size (phase contrast, original mag- 2.68 as seen by polarized light (original magnification
nification x 400). x 400).
FIGURE 2.70 A cluster with five oval fat bodies of dif- FIGURE 2.71 The same oval fat bodies shown in Fig-
ferent size, four of which are entirely gorged with lipids ure 2.70 as seen by polarized light (original magnifica-
(phase contrast, original magnification x 400). tion x 400).
FIGURE 2.72 A proximal renal tubular epithelial cell

whose cytoplasm contains several fatty lysosomes of
various size (phase contrast, x 400).
FIGURE 2.73 A renal tubular epithelial cell (prob- FIGURE 2.74 The same renal tubular epithelial cell
ably from the distal tubule or from the collecting duct) shown in Figure 2.73 as seen by polarized light (x 400).
gorged with lipids, whose nucleus can still be identified
FIGURE 2.75 A fatty cast containing some lipid drop-

lets of different size (phase contrast, x 400).
FIGURE 2.76 A fatty cast whose matrix is completely FIGURE 2.77 The same fatty cast shown in Figure
masked by tightly packed lipid droplets (phase con- 2.76 as seen by polarized light (x 400).
trast, x 400).
FIGURE 2.78 A cholesterol crystal made of several FIGURE 2.79 A cholesterol crystal with very sharp
plates clumped together (phase contrast, x 400). and straight edges (phase contrast, x 400).
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 (Figures 2.80 and 2.93).
The matrix of casts is made of Tamm-Horsfall glycoprotein (THG) [84-87] (Figure 2.80),
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 [86,87]. Electron microscopy shows that this protein has a fibrillar structure, with
unbranched fibrils of variable length and 9-15 nm in diameter (Figures 2.81 and 2.82).
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
(Figure 2.83). 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 [88].
Casts may be hyaline, if they consist of THG only, or complex if they also contain other
elements (Figure 2.83). 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 significance (Table 2.4). 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.
It is important to remember that 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 [89-91].
FIGURE 2.80 A branched cast stained with Tamm-

Horsfall antiserum (immunofluorescence microscopy,
x 250).
FIGURE 2.81 The fibrillar meshwork of the surface of FIGURE 2.82 The fibrillar nature of the matrix of a
a loose hyaline cast as seen by scanning electron micro- cast as seen by transmission electron microscopy (x
scopy (x 3,000). 15,000). Note the haphazard arrangement of the fibrils
within the casts and the bundles of fibrils on the surface
of the casts.
Conditions reducing colloid stability

of Tamm-Horsfall Glycoprotein (THG)
Urinary concentration of:
electrolytes, hydrogen ions,
ultrafiltered proteins, THG
Interaction of THG with:
haemoglobin, myoglobin,
Bence-Jones protein,
radiocontrast media
Intratubular
cylindrical gel
No particles in the Particles in the tubular

tubular lumen lumen (cells, proteins, lipids, etc.)
HYALINE CASTS COMPLEX CASTS
FIGURE 2.83 Factors involved in the formation of casts.
TABLE 2.4 Classification of casts.

Type Subtype
Hyaline --
Finely granular
Granular
Coarsely granular
Waxy --
Erythrocytic
Cellular Leukocytic
Containing renal tubular epithelial cells (epithelial casts)
Fatty --
Containing crystals --
Bacterial
Containing microorganisms
Candidal
Haemoglobinic
Pigmented Myoglobinic
Bilirubinic
Hyaline-granular
Granular-waxy
Mixed Granular-cellular
Granular-fatty
Etc.
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.
There are several morphological types of hyaline casts: compact, fibrillar, convoluted or
wrinkled (Figures 2.84-2.89).
Scanning electron microscopy has shown that these different types of casts reflect different
patterns of interweaving fibrils [92]. In fibrillar casts, the interweaving is loose, while in
compact casts it is tight. Wrinkling, instead, seems to be caused by contraction of the fibrils
or by degenerative processes.
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. [93] found
transient abundant hyaline cylindruria after a single oral dose of furosemide (80-160 mg) or
ethacrynic acid (50-100 mg). However, it should not be forgotten that hyaline casts are also
found in renal diseases, mostly in combination with other types of casts (Table 2.5) [43].
TABLE 2.5 Prevalence of the main types of casts in 100 patients with glomerular diseases of
proliferative type (mainly: IgA nephropathy, class III and IV lupus nephritis, extracap-
illary and/or necrotizing glomerulonephritis) and non proliferative type (mainly:
membranous nephropathy, amyloidosis, focal and segmental glomerulosclerosis,
minimal change disease). For further details see ref [43].
Non
Total Proliferative
Cast proliferative P
(N = 100) (N = 52)
(N = 48)
Hyaline 100 (100%) 52 (100%) 48 (100%) 1.000
Granular 51 (51%) 27 (51.9%) 24 (50%) 1.000
Waxy 16 (16%) 10 (19.2%) 6 (12.5%) 0.421
Erythrocytic 63 (63%) 44 (84.6%) 19 (39.6%) <0.001
Leukocytic 8 (8%) 6 (11.5%) 2 (4.2%) 0.272
Epithelial 87 (87%) 49 (94.2%) 38 (79.2%) 0.036
Fatty 83 (83%) 42 (80.8%) 41 (85.4%) 0.601
Hyaline-granular 100 (100%) 52 (100%) 48 (100%) 1.000
FIGURE 2.84 A compact hyaline cast (phase contrast, FIGURE 2.85 A hyaline cast clearly showing the cylin-
x 400). drical shape, which is the replica of the renal tubular
lumen (phase contrast, x 200).
FIGURE 2.86 A compact hyaline cast (top) and a fi-

brillar hyaline cast (bottom)(phase contrast, original
magnification x 400).
FIGURE 2.87 A convoluted hyaline cast (phase con- FIGURE 2.88 A partially wrinkled hyaline cast (phase
trast, original magnification x 400). contrast, x 400).
FIGURE 2.89 A tortuous (snake-like) hyaline cast

granular casts
Typical granular casts have their surface covered by granules (Figures 2.90-2.94 and 2.125),
which vary in number and size. Granules can be fine or coarse, and clear, dark or pigmented. In
our experience, finely granular casts are common, while coarsely granular casts are infrequent.
Immunofluorescence studies performed on the urine of patients with proteinuria have
demonstrated that fine granules contain several types of proteins [94], and electron microscopic
studies have shown that granules resemble the lysosomes seen in cytoplasm of tubular cells
[95]. Therefore, we can hypothesize that 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 [96], it is accepted that granules might also derive from cellular degeneration.
Coarse granules also are thought to derive from degenerated cells such as leukocytes or
renal tubular epithelial cells [88].
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 (see Chapter 5, page 195). However, they are also
frequent in patients with glomerulonephritis (Table 2.5) [43].
FIGURE 2.90 A granular cast as seen by transmission FIGURE 2.91 A finely granular cast (phase contrast,
electron microscopy: the granules surround the fibrillar original magnification x 400).
matrix of the cast like a crown (x 5,000).
FIGURE 2.92 A finely granular cast with both clear FIGURE 2.93 A pigmented (brownish) finely granular
(top) and dark (bottom) granules (phase contrast, origi- cast. Note that it also is a branched cast (phase con-
nal magnification x 400). trast, x 400).
FIGURE 2.94 A coarsely granular cast (phase con-

trast, original magnification x 400).
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
(Figures 2.95-2.100). By scanning electron microscopy, their surface is characterized by the
appearance of “plates” of unknown composition [88] (Figure 2.101).
It has been claimed that waxy casts may derive from hyaline casts that have been altered
by urine products [88]. However, in a study based on immunofluorescence microscopy, we
were unable to find Tamm-Horsfall protein on the surface of waxy casts, while it was present
on the surface of all other casts [97]. Thus, we believe that the origin and composition of
waxy casts still remains unknown.
Waxy cast are a frequent finding in patients with renal failure, both acute and chronic. In
patients with glomerular diseases, we found waxy casts only in 16% of cases without significant
differences between proliferative and non proliferative histologic types (Table 2.5) [43].
FIGURE 2.95 A waxy cast with typical indented edges FIGURE 2.96 A waxy cast with hard edges without
(phase contrast, x 160). indentations (phase contrast, x 200).
FIGURE 2.97 A waxy cast with indented and cracked FIGURE 2.98 A waxy cast with a longitudinal crack
edges (phase contrast, original magnification x 400). involving the whole length of the cast (phase contrast,
x 160).
FIGURE 2.99 A waxy cast with indented edges and a FIGURE 2.100 A waxy cast with very irregular con-
slightly irregular surface (phase contrast, x 200). tours and a surface showing various plates of different
size (phase contrast, x 200).
FIGURE 2.101 The hard and fractured surface of a

waxy cast as seen by scanning electron microscopy (x
1,100).
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 (Table 2.6).
Erythrocytic 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 (Figures 2.102 and 2.103). Alternatively,
only a few erythrocytes may be trapped in the hyaline matrix (Figures 2.104 and 2.105).
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. By scanning
electron microscopy we have even found acanthocytes trapped within the cast matrix [98].
Erythrocytic casts are a marker of glomerular bleeding. For this reason, their systematic
search should be carried out in all patients with isolated microscopic haematuria of unknown
origin [32]. In patients with overt glomerulonephritis, erythrocytic casts are found in 22%
[9] to 85% of cases [17], which depends on the types of glomerular disease investigated and
on the methodology used to search them. In our experience, erythrocytic casts are found in
63% of patients with overt glomerular disease, the prevalence being significantly higher in
proliferative disorders than in non proliferative ones (Table 2.5) [41]. Together with dysmorphic
erythrocytes, erythrocytic casts are a distinguishing feature of the nephritic sediment (see
Chapter 6, page 214), without forgetting that very rarely they may be found in patients with
haematuria caused by acute interstitial nephritis [99] (see Chapter 5, page 194).
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
Leukocytic casts can contain variable amounts of leukocytes, from few to so many that the
matrix of the cast is completely masked (Figures 2.106-2.108). Leukocytes may be well preserved
or degenerated, in which case they are hardly distinguishable from renal tubular epithelial cells.
Leukocytic casts were once considered pathognomonic of acute bacterial infection involving
the kidney. However, we know today that they can also be found in patients with active proliferative
lupus glomerulonephritis [100], other glomerular diseases (Table 2.5) [43] or acute interstitial
nephritis.
Renal tubular epithelial cell casts

Renal tubular epithelial cells (RTEC) casts, which are also known as epithelial casts, can
contain variable amounts of RTECs, from few to many (Figures 2.109-2.113). 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 [90] and acute interstitial nephritis of whatever cause. However, in our experience,
these casts are a very frequent finding also in patients with glomerular diseases (Table 2.5) [43].
FIGURE 2.102 An erythrocytic cast containing un- FIGURE 2.103 An erythrocytic cast made of tightly
countable packed erythrocytes which have lost their packed erythrocytes. Note the reddish hue due to hae-
haemoglobin content (phase contrast, x 500). moglobin (phase contrast, original magnification x 400).
FIGURE 2.104 An erythrocytic cast in which the hya- FIGURE 2.105 An erythrocytic cast in which few eryth-
line matrix is still visible (phase contrast, x 400). rocytes are plunged into the hyaline matrix. Note that
some erythrocytes have lost their haemoglobin content
while others have not (phase contrast, x 500).
FIGURE 2.106 A leukocytic cast containing some leu- FIGURE 2.107 A leukocytic cast containing many poly-
kocytes (phase contrast, x 400). morphonuclear leukocytes with their lobated nucleus
FIGURE 2.108 A leukocytic cast containing packed FIGURE 2.109 A renal tubular epithelial cell cast. Note
and well preserved polymorphonuclear leukocytes (also in the 4 following figures) the large central nucleus
(phase contrast, x 400). of the cells (phase contrast, x 400).
FIGURE 2.110 A renal tubular epithelial cell cast (phase FIGURE 2.111 A renal tubular epithelial cell cast at
contrast, x 400). high magnification (phase contrast, x 600).
FIGURE 2.112 A renal tubular epithelial cell cast (phase FIGURE 2.113 A renal tubular epithelial cell cast in
contrast, x 400). which the cells are so packed that the matrix of the cast
is not visible (phase contrast, x 400).
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. In this last case, cholesterol plates
may at times protrude from the edges of the cast. In all cases, polarized light shows lipid
droplets as “Maltese crosses” (Figures 2.114-2.117; 2.75-2.77).
Cholesterol crystals (see page 127) within casts are, in our experience, a rare finding
(Figure 2.118).
Fatty casts are usually associated with other fatty particles in the urine sediment. Therefore,
they are typical of patients with nephrotic range proteinuria. In our experience, they are a
very frequent finding also in patients without nephrotic range proteinuria (Table 2.5) [43].
casts containing crystals and amorphous salts

Isolated or aggregated crystals and amorphous salts are occasionally seen in the casts.
All types of crystals may be entrapped by casts but, in our experience, calcium oxalate-
containing casts are the most frequent (Figure 2.119). Occasionally, we have also seen casts
containing amorphous phosphates.
The presence of these casts indicates that crystals or salts have precipitated within the
renal tubules where casts form.
casts containing microorganisms

Both bacteria and yeasts can be observed in casts.
Bacterial casts are observed in patients with infection of the kidney. These casts have a
granular or a mixed (granular and cellular) composition, and can be best detected by phase
contrast microscopy [101]. We have seen bacterial casts only once, in a patient with acute
pyelonephritis (Figure 2.120).
Fungal casts have been found in the urine of patients with visceral candidiasis [102,103].
Their presence strongly suggests renal parenchymal involvement.
FIGURE 2.114 A fatty cast containing both individual

and aggregated lipid droplets of different size and an
oval fat body (upper left)(phase contrast, x 400).
FIGURE 2.115 A fatty cast containing several aggre- FIGURE 2.116 The same fatty cast shown in Figure
gates of lipid droplets of different size and a large oval 2.115 as seen by polarized light (x 400).
fat body (in central location)(phase contrast, x 400).
FIGURE 2.117 A fatty cast containing tightly packed FIGURE 2.118 A cast containing cholesterol crystals
lipid droplets. Note the cholesterol plates protruding clumped together (phase contrast, x 400).
from the edges of the cast (phase contrast, x 400).
FIGURE 2.119 A cast containing a large monohy- FIGURE 2.120 A bacterial cast containing many rods
drated calcium oxalate crystal (phase contrast, original (phase contrast, x 400).
pigmented casts
This category of casts includes haemoglobin, myoglobin and bilirubin casts.
Haemoglobin casts
In most instances, these casts derive from erythrocytes which have undergone degeneration.
Haemoglobin casts have a typical brownish to reddish-brown colour and a granular appearance
(Figures 2.121 and 2.122). 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. More
rarely, haemogobin casts derive from intravascular haemolysis, in which case no haematuria
is observed and no remnants of erythrocytes can be identified within the cast.
Myoglobin casts
Myoglobin casts have a brown to reddish-brown colour similar to that of haemoglobin

casts. The surface can be either smooth or granular (Figure 2.123), 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 (see Chapter 5, page 197) [104,105].
Bilirubin casts
Casts of any type i.e., hyaline, granular, waxy or cellular, may be stained by the typical
yellow colour of bilirubin (Figure 2.124). Bilirubin casts are observed in the urine of patients
with jaundice associated with increased direct (conjugated) bilirubin.
FIGURE 2.121 A haemoglobin cast with its typical FIGURE 2.122 A haemoglobin cast with granular surface
reddish hue. Note the granular surface (phase contrast, and irregular contours. Note, in the lower part of the cast,
original magnification x 400). some remnants of erythrocytes (phase contrast, x 400).
FIGURE 2.123 A myoglobin cast whose colour is simi- FIGURE 2.124 A bilirubin cast with the typical yellow
lar to that of haemoglobin casts (phase contrast, x 400). colour containing granules and tubular cells (phase
contrast, original magnification x 400).
mixed casts
Different components may be present simultaneously in the same cast, giving rise to mixed
casts such as hyaline-granular casts, granular-waxy casts, granular-cellular casts, granular-
fatty casts, etc. (Figures 2.125-2.128).
In our experience, hyaline-granular casts are among the most frequent casts found in the
urine (Table 2.5) [43].
The clinical significance of these casts is the same as that of the pure types of casts of
which mixed casts contain some components.
FIGURE 2.125 A hyaline-granular cast (bottom) and a FIGURE 2.126 A hyaline-granular cast containing
finely granular cast (top)(phase contrast, x 400). more granules than the cast shown in Figure 2.125
FIGURE 2.127 A mixed coarsely granular-waxy cast FIGURE 2.128 A mixed erythrocytic-waxy cast (phase
(phase contrast, x 160). contrast, original magnification x 400).
Cylindroids
There is no agreement about the definition and nature of cylindroids. According to Schreiner
[106] and Graff [44], cylindroids are elongated elements with one rounded extremity which
resembles that of a cast, and the other extremity which resembles a mucus thread.
By adhering to this definition, in a prospective study of 600 sediments over a period of 4
months, we found cylindroids in 90 samples from 79 patients.
We consider cylindroids as one morphological variant of casts because we found that
they:
- were almost always associated with casts (85 out of 90 samples, 94.4%)
- could have the same pleomorphic appearances as casts (i.e., hyaline, granular,
cellular or fatty) (Figures 2.129-2.131)
- contained Tamm-Horsfall protein on their surface [97], a finding which has been
observed also by other investigators [85] (Figure 2.132).
FIGURE 2.129 A hyaline cylindroid (phase contrast, FIGURE 2.130 A hyaline-granular cylindroid (phase
x 160). contrast, x 400).
FIGURE 2.131 An erythrocytic cylindroid (phase con- FIGURE 2.132 Staining with an antiserum specific
trast, x 400). for Tamm-Horsfall glycoprotein shows that the matrix of
cylindroids is the same as that of casts (see Figure 2.80)
(immunofluorescence microscopy, x 160).
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 (Figures 2.133-2.137; 2.227, 2.229, 2.230 and 2.234).
Compared to casts, pseudocasts may show:
- harder edges
- more irregular contours
- more variable size
- unusual colours, differing from the colour due to haemoglobin, myoglobin or bilirubin.
Careful observation and experience are necessary to avoid misidentifications.
FIGURE 2.133 A calcium phosphate plate resembling FIGURE 2.134 A cloth fibre resembling a haemoglob-
a hyaline cast (phase contrast, x 400). in cast (phase contrast, x 256).
FIGURE 2.135 A particle with cracked edges resem-

bling a waxy cast. The unusual colour suggests that it is
not a true cast (phase contrast, x 400).
FIGURE 2.136 A synthetic cylindrical fibre resembling FIGURE 2.137 The same particle shown in Figure
a cast (phase contrast, x 160). 2.136 as seen by phase polarized light. The strong bi-
refringence clearly indicates that the particle cannot be
a cast (x 160).
mucus
Mucus is a substance derived from the secretion of the accessory glands (Cowper’s or
Littré’s glands in men; Skene’s ducts in women). It has a low refractive index and is therefore
best seen using phase contrast microscopy.
Usually, mucus appears as ribbon-like threads with irregular contours and fibrillar
structure (Figure 2.138). 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 (Figure
2.139) or networks of fine fibrils. Occasionally, threads of mucus resemble cylindroids or
even hyaline casts (pseudocasts). Cells may be trapped in mucus (Figure 2.140), which leads
to a grossly inhomogeneous distribution across the slide and may interfere with quantitation
of particles.
Mucus is a frequent finding in urinary sediments. We found it in 760 of 1000 consecutive
samples from subjects mostly with renal diseases. Half of the samples contained mild to
moderate amounts (± to ++) of mucus, while the remainders contained large quantities (+++
to ++++).
It has been claimed that mucus is seen more frequently in the urine of women and
that large quantities of mucus point to inflammation of the lower urinary tract or genital
apparatus. However, in our series, large amounts of mucus were more frequent in men,
and were present in more than one-third of the samples despite no evidence of diseases
of the lower urinary tract or genital apparatus. In addition, we found a clear-cut inverse
correlation between urinary specific gravity and the presence (and amount) of mucus. At
specific gravity values of Φ 1.010, mucus is usually absent or only in very small quantities,
while it is in large amounts in samples with specific gravity of Γ1.025.
FIGURE 2.138 Ribbon-like mucus threads (phase FIGURE 2.139 A large mass of mucus (phase con-
contrast, x 256). trast, x 160).
FIGURE 2.140 Squamous epithelial cells clumped by

mucus (phase contrast, x 160).
crystals
In our laboratory, crystals are a frequent finding, being observed in about 8% of specimens
[107].
There are many types of urinary crystals (Table 2.6), which we arbitrarily classify as:
- common crystals: uric acid, amorphous urates and amorphous phosphates, calcium
oxalate, calcium phosphate, triple phosphate
- pathological crystals: cholesterol, cystine, leucine, tyrosine, 2,8-dihydroxyadenine
- crystals due to drugs: described in details in Chapter 3
- other crystals: hippuric acid, calcium carbonate, ammonium biurate.
TABLE 2.6 Classification of crystals.

Category Crystals
Uric Acid
Amorphous urates and amorphous phosphates
Common Calcium oxalate (monohydrated and bihydrated)
Calcium phosphate (crystals and plates)
Triple phosphate
Cholesterol
Cystine
Pathologic Leucine
Tyrosine
2,8-dihydroxyadenine
Sulfadiazine
Amoxycillin
Ciprofloxacin
Acyclovir
Indinavir
Triamterene
Due to drugs
Piridoxilate
Primidone
Naftidrofuryl oxalate
Vitamin C
Orlistat
Felbamate
Hippuric acid
Others Calcium carbonate
Ammonium biurate
For the correct identification of crystals, the combined knowledge of:

- their most common appearances
- their birefringence features
- the urinary pH
is mandatory [108,109].
The knowledge of the most common appearances of crystals. This knowledge is usually
sufficient for the correct identification of most crystals. However, all types of crystals can
show a very wide spectrum of appearances, some of which can be very unusual. For such cases
the knowledge of birefringence features and of urinary pH is of the highest importance.
The birefringence features of crystals. These can be known only with the use of polarized
light (see Chapter 1, page 35), which allows the differentiation between birefringent and non
birefringent crystals (Table 2.7). This knowledge is useful not only to confirm the identification
based on morphology, but also to distinguish crystals with identical morphology but different
composition. This happens, for instance, with amorphous urates and amorphous phosphates
(while the former polarize light the latter do not) and with hexagonal crystals, which may
be due to either uric acid or cystine (while uric acid exhibits a polychromatic birefringence,
cystine is birefringent without being polychromatic) [110].
The urinary pH. Some crystals tend to precipitate in acidic urine, while others precipitate
in an alkaline milieu (Table 2.7). Uric acid and amorphous urates are found exclusively
in acidic urine (pH < 5.4-5.8), while amorphous phosphates, calcium phosphate and triple
phosphate are observed in urine pH of 6.2 to > 7.0. Calcium oxalate crystals can be found
at a wider range of pH values (< 5.4-6.7), although they tend to be more frequent in acidic
urine.
Testing the solubility features of crystals is an additional tool for their identification in
doubtful cases. This is done by adding to the sample a few drops of a chemical reagent which
is known to dissolve the crystals under investigation or, for some crystals, by heating the
sample (Table 2.8). If the crystals do not dissolve, they belong to another category of crystals.
However, this procedure, which was commonly done in the past, is very rarely performed
today.
Occasionally, crystals cannot be identified with certainty in spite of the combined
knowledge mentioned above. For such cases, more sophisticated techniques are needed
such as Fourier transform infrared microscopy (FTIRM) [111]. We turn to this technique
whenever we come across crystals with atypical features which cannot be identified with the
conventional approach*. Over a 52-month period, we have used FTIRM for 14 samples out
of 807 samples containing crystals (1.7%) [107].
* FTIRM is kindly performed for us by Professor Michel Daudon, Laboratoire de Biochimie A, Hôpital Necker-Enfants
Malades, Paris, France.
TABLE 2.7 Birefringence features and urinary pH (by pH Indicatorpapier pH 1-10, E. Merk, Ger-
many) of common crystals of the urine sediment found in authors’ laboratory over
a 2-year period.
Number of Birefringence pH < 5.8 pH > 7.0
Crystal pH range
samples (%) (%) (%)
Uric acid 36 100 <5.4-5.8 100 0
Amorphous urates 7 100 <5.4-5.8 100 0
Amorphous
27 0 6.2->7.0 0 78
phosphates
Calcium oxalate
18 100 <5.4-6.7 89 0
monohydrated
Calcium oxalate
67 25* <5.4-6.7 82 0
bihydrated
Calcium phosphate
2 100 Γ7.0 0 100
(crystals)
Calcium phosphate
3 0 6.7->7.0 0 67
(plates)
Triple phosphate 25 100** 6.2->7.0 0 73
* The reasons why some crystals are birefringent are not clear.
** Recent observations in our laboratory showed that not all triple phosphate crystals are birefringent (see Figures 2.184
and 2.192).
TABLE 2.8 Solubility features of most urinary crystals.

Crystal Soluble Unsoluble
Uric acid and amorphous

Heat, alkali Alcohol, HCl, CH3COOH
urates
Calcium oxalate HNO3, NaOH, HCl CH3COOH
Calcium phosphate HCl, CH3COOH NaOH, heat
Triple phosphate and
HCl, CH3COOH NaOH, heat
amorphous phosphates
Dilute acids, dilute alkali,
Cholesterol CHCl3, ether, hot alcohol
alcohol, H2O
CH3COOH, ether, alcohol,
Cystine HCl, NaOH, NH4OH
boiling H2O
NaOH, hot alcohol,
Leucine Ether, HCl, CH3COOH
hot CH3COOH
Tyrosine HCl, NaOH, NH4OH, heat Alcohol, ether, CH3COOH
NaOH + NH3 , HCl,
Ammonium biurate
CH3COOH, heat
common crystals
uric acid
Uric acid crystals are invariably found in acidic urine with a pH ranging from < 5.4 to 5.8
(Table 2.7). These crystals come in a wide variety of sizes and shapes (rhomboids, barrels,
rosettes, needles, six-sided plates, etc.). They have a typical amber colour, and under polarized
light, they always show a strong birefringence, which is very frequently polychromatic
(Figures 2.141-2.155).
Occasionally, uric acid crystals may resemble cystine crystals (Figures 2.197-2.200). In
this case, the use of polarized light is useful in distinguishing the two types of crystals, since
uric acid is polychromatic while cystine is not. Other potentially misleading particles are
glass slivers (Figure 2.237), which can be recognized because they are not birefringent under
polarized light.
Uric acid crystals can occasionally be found in normal subjects as well as in patients with
uric acid urolithiasis. Massive uric acid crystalluria with or without uric acid-containing
casts can be found in patients with acute uric acid nephropathy. This is a condition seen in
patients with aggressive lymphoproliferative disorders or solid tumours, in whom severe
hyperuricaemia can develop as a consequence of tumour lysis, either spontaneous or induced
by chemotherapy. In this condition, acute renal failure is caused by the precipitation of uric
acid crystals within the lumen of the distal tubules and collecting ducts, and in peritubular
capillaries. In the urine of these patients, besides uric acid crystals, amorphous urates and
crystallized xanthine can also be found [112,113].
It is important to remember that in acute uric acid nephropathy, uric acid crystalluria is not
invariably present [112]. Conversely, it may occur in patients with tumour lysis but without
acute renal failure [113].
Uric acid crystals can also precipitate after urine collection if the urine is stored in the
refrigerator at 4 °C before microscopic examination.
FIGURE 2.141 Rhomboid uric acid crystals. Note the FIGURE 2.142 Biconvex uric acid crystals mimicking
amber colour (phase contrast, x 400). plates due to the front view (phase contrast, x 400).
FIGURE 2.143 A barrel-shaped uric acid crystal FIGURE 2.144 A uric acid crystal whose shape resem-
(bright field, x 400). bles a little that of an axe (phase contrast, x 160).
FIGURE 2.145 A large cluster of uric acid crystals FIGURE 2.146 A cluster of triangular uric acid crys-
with irregular shape (phase contrast, x 160). tals (phase contrast, x 256).
FIGURE 2.147 Irregular uric acid crystals resembling

glass slivers (bright field, x 400).
FIGURE 2.148 Two overlapping rhomboid uric acid FIGURE 2.149 The same crystals shown in Figure 2.148
crystals (phase contrast, original magnification x 400). as seen by polarized light. Besides the beautiful polychro-
matic birefringence, also note that the round particle, which
by phase contrast microscopy could have been identified as
a possible macrophage, is birefringent. Therefore, it also is
an uric acid crystal (original magnification, x 400).
FIGURE 2.150 Two clusters of rhomboid uric acid FIGURE 2.151 The same clusters of crystals shown in
crystals (phase contrast, x 400). Figure 2.150 as seen by polarized light (x 400).
FIGURE 2.152 A large cluster of rhomboid uric acid FIGURE 2.153 The same cluster of crystals as seen by
crystals (phase contrast, x 400). polarized light (x 400).
FIGURE 2.154 A large uric acid crystal with a diamond FIGURE 2.155 The same crystal shown in Figure
shape (phase contrast, original magnification x 400). 2.154 as seen by polarized light. Note that birefringence
is polychromatic only in some peripheral parts of the
crystal (x 400).
amorphous urates and amorphous phosphates

Amorphous urates are tiny birefringent granules with irregular shape, which can be
observed either singly or, more often, as aggregates in acidic urine (Table 2.7) (Figures 2.156
and 2.157).
Amorphous phosphates are morphologically identical to urates, but they are not birefringent
under polarized light and precipitate in alkaline urine (Table 2.7).
Another difference between the two types of particles is that if the urine contains massive
amounts of amorphous urates, the bottom of the tube after centrifugation shows a macroscopic
sediment of pink to reddish colour (the so-called sedimentum lateritium, which indicates
a colour similar to that of bricks). On the contrary, with massive amounts of amorphous
phosphates the macroscopic sediment is white to beige (Figure 2.158).
Amorphous urates may be found in the urine of the normal subject, but they can also be
found in the same pathological conditions associated with uric acid crystalluria as described
above. Moreover, their precipitation can be induced by storing the sample in the refrigerator
at 4 °C before examination.
Amorphous phosphates are frequently found in association with calcium phosphate
crystals [114].
Large amounts of amorphous urates or phosphates may mask other particles which may be
in the urine, and both may be confused with cocci. However, cocci usually display movement,
which lacks with amorphous material.
FIGURE 2.156 Amorphous urates (phase contrast, FIGURE 2.157 The same urates shown in Figure 2.156
original magnification x 400). as seen by polarized light (x 400).
FIGURE 2.158 The different colour of urates (left) and

phosphates (right) after centrifugation.
calcium oxalate
Calcium oxalate crystals can be found in urine at pH values ranging from < 5.4 to 6.7, but
mostly in urine with pH < 5.8 (Table 2.7).
There are two main types of calcium oxalate crystals, monohydrated (or Whewellite) and
bihydrated (or Weddellite).
Monohydrated crystals are colourless and pleomorphic. Most frequently they appear as
ovoid structures, biconcave disks, dumb-bells, rods, etc. (Figures 2.159-2.166), and they are
always strongly birefringent (Table 2.7). When ovoid or roundish, they might be confused
with isomorphic erythrocytes (Figure 2.7). The latter, however, are not birefringent under
polarized light.
Typical bihydrated crystals appear as bipyramidal colourless structures of highly variable
size (Figures 2.167-2.170), which in most instances do not polarize light. However, when large
or in aggregates, some crystals may show some birefringence (Table 2.7). Less frequently
these crystals have a star-like appearance (Figure 2.171).
Usually, only one type of calcium oxalate crystals is found in the same urine sample, even
though occasionally both types are seen (Figure 2.172).
Calcium oxalate crystals may be found in normal subjects [115], often as a consequence of
ingestion of foods such as chocolate, beetroot, peanuts, rhubarb, or spinach. [116]. Recently,
it has been shown that also the ingestion of star fruit (Averrhoa carambola, which grows in
Taiwan, Thailand, Brazil) or its juice (which has now a worldwide distribution) can cause
no better defined calcium oxalate crystalluria. This can be associated with acute renal failure
due to the intrarenal precipitation of calcium oxalate crystals [117,118].
Calcium oxalate crystals can also be seen in stone formers [115], in patients with primary
or secondary hyperoxaluria [119], or in subjects treated with large intravenous doses of
vitamin C, naftidrofuryl oxalate, or orlistat (see Chapter 3, page 167).
Another important cause of calcium oxalate crystalluria is the accidental or deliberate
ingestion of ethylene glycol. This is a compound contained in antifreeze agents which,
after ingestion is transformed by the liver into glycolate, glyoxalate and then oxalate. These
metabolites cause a multisystem disease due to the precipitation of calcium oxalate crystals
in the brain, lungs, and heart. In the kidneys, the crystals precipitate in the tubules, both
in the cells and in the lumen, causing acute kidney injury. The typical laboratory findings
are those of increased serum creatinine, metabolic acidosis, high anion gap, high osmolal
gap and crystalluria [120]. The latter is characterised by massive amounts of birefringent
monohydrated calcium oxalate crystals with unusual shape, such as short prisms, needles,
spindles or elongated hexagons (Figures 2.173 and 2.174) [120,121]. However, also
bipyramidal bihydrated crystals can be found, especially in the early phases. Crystalluria can
be prevented by early treatment of the intoxication, and disappears when ethylene glycol is
removed from blood by dialysis.
FIGURE 2.159 Clumps of ovoid and biconcave mono- FIGURE 2.160 The same crystals shown in Figure
hydrated calcium oxalate crystals (phase contrast, orig- 2.159 as seen by polarized light (original magnification
inal magnification x 400). x 400).
FIGURE 2.161 Ovoid monohydrated calcium oxalate FIGURE 2.162 The same crystals shown in Figure
crystals (phase contrast, x 400). 2.161 as seen by polarized light (x 400).
FIGURE 2.163 A biconcave monohydrated calcium FIGURE 2.164 The same crystal shown in Figure
oxalate crystal (original magnification x 400). 2.163 as seen by polarized light (original magnification
x 400).
FIGURE 2.165 Unusual rectangular monohydrated FIGURE 2.166 The same crystals shown in Figure
calcium oxalate crystals (phase contrast, x 400). 2.165 as seen by polarized light (x 400).
FIGURE 2.167 A cluster of bihydrated calcium oxalate FIGURE 2.168 The same crystals shown in Figure
crystals with different sizes (phase contrast, original 2.167 as seen by polarized light. The fact that all crys-
magnification x 400). tals are birefringent is unusual (x 400).
FIGURE 2.169 A large cluster of bihydrated calcium FIGURE 2.170 The same crystals shown in Figure
oxalate crystals (phase contrast, x 400). 2.169 as seen by polarized light. Note that in this cluster
not all crystals are birefringent (x 400).
FIGURE 2.171 Bihydrated calcium oxalate crystals FIGURE 2.172 Monohydrated and bihydrated cal-
with a star-like appearance (phase contrast, original cium oxalate crystals in the same sample (phase con-
magnification x 400). trast, original magnification x 400).
FIGURE 2.173 A massive crystalluria due to ethylene FIGURE 2.174 Another example of atypical monohy-
glycol intoxication. Note the spindle-like appearance of drated calcium oxalate crystals caused by ethylene gly-
these monohydrated calcium oxalate crystals (bright field, col intoxication as seen by polarized light (x 400).
x 400). Courtesy of Dr. Carlo Massimetti, Viterbo, Italy.
calcium phosphate
Calcium phosphate, which can appear either as crystals or plates, is found in alkaline urine
(Table 2.7).
Crystals can show a very wide spectrum of morphologies, such as prisms, stars, rosettes,
sticks or needles, which can occur in isolation or in aggregates. They are always strongly
birefringent (Figures 2.175-2.180).
Plates have well-defined edges, which can be either linear or irregular, and a granular
surface. They are non-birefringent (Figures 2.181 and 2.182).
Calcium phosphate crystals can be found in normal subjects as well as in stone formers
[122,123].
FIGURE 2.175 A calcium phosphate crystal with the FIGURE 2.176 Thin and elongated crystals of calcium
shape of a cross (phase contrast, x 400). phosphate associated with some bihydrated calcium
oxalate crystals (phase contrast, x 400).
FIGURE 2.177 A calcium phosphate crystal with a FIGURE 2.178 The same crystal shown in Figure 2.176
star-like appearance (phase contrast, original magnifi- as seen by polarized light (x 400).
cation x 400).
FIGURE 2.179 Aggregated calcium phosphate crys- FIGURE 2.180 The same crystals shown in Figure
tals with the shape of sticks (phase contrast, original 2.179 as seen by polarized light (original magnification
magnification x 400). x 400).
FIGURE 2.181 A calcium phosphate plate (phase FIGURE 2.182 Another example of calcium phos-
contrast, x 400). phate plate (phase contrast, x 400).
triple phosphate
These crystals contain magnesium ammonium phosphate, and are typically found in urine
with alkaline pH (Table 2.7).
In most instances, they have an easily identifiable “coffin-lid” appearance, while
occasionally they may have the shape of trapezoids, elongated prisms, feather-like, or others.
Under polarized light, they show a weak to strong birefringence (Figures 2.183-2.194).
Triple phosphate crystals are typical of urine with bacterial infection caused by urea-
splitting microorganisms such as Ureaplasma urealyticum and Corynebacterium urealyticum
[109].
FIGURE 2.183 The typical “coffin lid” appearance of FIGURE 2.184 The same crystals shown in Figure
triple phosphate crystals (phase contrast, original mag- 2.183 as seen by polarized light. Note the different de-
nification x 400). grees of birefringence (from negative to strongly posi-
tive) of the three crystals (original magnification x 400).
FIGURE 2.185 Two different morphological variants FIGURE 2.186 The same crystals shown in Figure
of triple phosphate crystals (phase contrast, original 2.185 as seen by polarized light (original magnification
magnification x 400). x 400).
FIGURE 2.187 Another type of triple phosphate crys- FIGURE 2.188 The same crystal shown in Figure 2.187
tal (phase contrast, original magnification x 400). as seen by polarized light. Note the weak birefringence
(original magnification, x 400).
FIGURE 2.189 A triple phosphate crystal with the shape FIGURE 2.190 The same crystal shown in Figure 2.189
of a prism (phase contrast, original magnification x 400). as seen by polarized light (original magnification x 400).
FIGURE 2.191 A cluster of triple phosphate crystals FIGURE 2.192 The same crystals shown in Figure
with different shapes and sizes (phase contrast, origi- 2.191 as seen by polarized light. Note the different de-
nal magnification x 400). grees of birefringence of the different crystals (original
FIGURE 2.193 A feather-like triple phosphate crystal FIGURE 2.194 The same crystal shown in Figure 2.193
(phase contrast, original magnification x 400). as seen by polarized light (original magnification x 400).
pathologic crystals
cholesterol
Cholesterol crystals occur mostly in acidic urine (Table 2.7). They are transparent and thin
plates, quite often heaped one upon another, with well-defined edges. The latter are usually
straight, but occasionally they are rounded (Figures 2.195 and 2.196; 2.78 and 2.79). Rarely
these crystals may be seen within casts (Figure 2.118).
Usually they do not polarize light, even though occasionally they show a weak
birefringence.
Cholesterol crystals occur together with other lipid particles in the urine of patients
with severe proteinuria. In our experience, however, they are less frequent than other lipid
particles.
FIGURE 2.195 A cholesterol crystal (phase contrast, FIGURE 2.196 Another example of cholesterol crystal
original magnification x 400). (phase contrast, original magnification x 400).
cystine
Cystine crystals are thin hexagonal colourless plates with irregular sides. Occasionally,
however, they may have a rosette-like appearance. They can be either isolated, heaped one
upon another, or they may form aggregates. Under polarized light they are birefringent
(Figures 2.197-2.200).
Cystine crystals are typical of patients with cystinuria. This is a recessive inherited
disease characterised by the deficient absorption, at renal tubular level, of cystine and
dibasic amino acids lysine, arginine and ornithine. The consequences are urolithiasis and
obstructive uropathy. The identification of typical crystals in the urine is a clue to diagnosis.
High performance liquid chromatography allows the quantification of the amino acids in the
urine.
The possibility to find cystine crystals in the urine is increased by acidic pH, since cystine
has a high solubility at alkaline pH [124]. Recently, it has been demonstrated that the serial
measurement of cystine volume is a useful tool in the follow-up of patients with cystinuria at
risk of developing stone disease [125].
FIGURE 2.197 Cystine crystals heaped one upon an- FIGURE 2.198 Cystine crystals showing some poly-
other (phase contrast, x 500). chromatism (phase contrast, x 400).
FIGURE 2.199 An unusual cystine crystal with a FIGURE 2.200 Cystine crystal as seen by polarized
rosette-like structure within the typical hexagonal con- light. Note the non polychromatic birefringence (x 400).
tours (phase contrast, x 640).
leucine
Leucine crystals appear as yellow-brown spheres resembling oil drops with concentric
striations (Figure 2.201). Under polarized light, they form pseudo-Maltese crosses. The
presence of leucine crystals is typical of patients with liver failure.
tyrosine
Tyrosine crystals appear as thin needles often aggregated in bundles or rosettes. Tyrosine
crystals too are typical of patients with liver failure.
2,8-dihydroxyadenine
2,8-dihydroxyadenine (2,8-DHA) crystals are round and reddish-brown particles with a
dark outline and central spicules, which under polarized light appear as “Maltese crosses”
(Figures 2.202 and 2.203). However, especially in patients with severe renal function
impairment, 2,8-DHA crystals may have atypical appearances with unusual birefringence
features [126] (Figures 2.204 and 2.205).
2,8-DHA crystals are found in the urine of patients with adenine phosphoribosyltransferase
(APRT) deficiency, an enzyme which transforms adenine into adenosine monophosphate. Due
to the enzyme deficiency, adenine is transformed by xanthine oxidase into 2,8-DHA (Figure
2.206), which is highly insoluble at any pH, a fact which is responsible for crystalluria.
Other renal clinical manifestations of APRT deficiency include: recurrent radiolucent
stone disease (65%), acute renal failure (26%) due to intratubular and interstitial 2,8-DHA
precipitation, and chronic renal failure (17%) probably due to interstitial fibrosis [127].
The diagnosis of APR deficiency is made by the measurement of the residual APRT activity
in peripheral red blood cell lysate, the dosing of 2,8-DHA in the urine and the recognition
of 2,8-DHA crystals in the urine sediment. These are present in virtually all patients without
treatment [126,127].
FIGURE 2.201 A leucine crystal (bright field, x 400).
FIGURE 2.202 A crystal of 2.8-DHA (bright field, origi- FIGURE 2.203 The same crystal shown in Figure
nal magnification, x 400). Courtesy of Prof. Michel Dau- 2.202 as seen by polarized light (original magnification,
don, Paris, France. x 400). Courtesy of Prof. Michel Daudon, Paris, France.
FIGURE 2.204 2.8-DHA crystals with atypical appear- FIGURE 2.205 The same crystals shown in Figure
ance (phase contrast, original magnification x 400). 2.204 as seen by polarized light. Note that also the bi-
refringence features are atypical. These crystals could
correctly be identified only after infrared spectroscopy
analysis (original magnification, x 400).
APRT
Adenine Adenosine monophosphate
Xantine
oxidase
2,8-dihydroxyadenine (2,8-DHA)
(highly insoluble at any pH)
FIGURE 2.206 The metabolic pathway which leads to the production of 2,8-DHA.
crystals due to drugs

Several drugs can occasionally cause transient crystalluria, in isolation or in conjunction
with other urinary abnormalities and a wide range of clinical implications. These are described
in details in Chapter 3.
other crystals
hippuric acid
Hippuric acid appears as elongated hexagons (Figure 2.207).
Hippuric acid crystals are a rare finding. They usually do not have clinical significance.
FIGURE 2.207 A hippuric acid crystal (phase con-

trast, x 400).
calcium carbonate
Calcium carbonate is found in alkaline urine and appears mostly as “dumbels” (Figures
2.208 and 2.209). However, other appearances are possible [128]. The adding of acetic acid
to the urine sample containing calcium carbonate causes the production of carbon dioxide,
which is revealed by the appearance of urine effervescence.
Calcium carbonate is a rare finding in humans, while it is frequently seen in the urine of
herbivores, especially horses. Its clinical meaning in humans is not known, even though it
might be associated with the ingestion of large quantities of vegetables [128].
FIGURE 2.208 Different types of calcium carbonate FIGURE 2.209 The same crystals shown in Figure 2.208
crystals found in the urine of a horse, the nature of as seen by polarized light (original magnification x 400).
which was confirmed by infrared spectroscopy analysis
(phase contrast, original magnification x 400).
ammonium biurate
Ammonium biurate is found in alkaline or neutral urine. The typical appearance is that of
the so-called “thorn apples” which are yellow-brown spheres with spicules (Figure 2.210 and
2.211). Under polarized light, ammonium biurate shows a strong birefringence.
Ammonium biurate crystals are usually observed in urine rich of ammonia, a fact which is
caused by bacteria which split urea [109].
FIGURE 2.210 An ammonium biurate crystal (phase FIGURE 2.211 The same crystal shown in Figure 2.210
contrast, original magnification x 400). as seen by polarized light (original magnification, x 400).
organisms
Several organisms can be identified in urinary sediments (Table 2.9).
bacteria
By phase contrast microscopy, bacteria appear as dark grey or black particles. Rods may
be isolated, in pairs or in long chains. The same is true for cocci (Figures 2.212-2.214). Rods
and cocci are easily identifiable, but sometimes cocci may be confused with amorphous
urates or phosphates. The movement typical of cocci may be useful for this differentiation.
Bacteria may adhere to squamous epithelial cells (Figure 2.62), or clump into masses of
variable size (Figure 2.215).
Bacteria are not present in uninfected urine, but urinary samples may acquire bacteria due
to the fact that urine is usually collected, handled and analysed under non-sterile conditions.
Another important preanalytical aspect is that bacterial growth is favoured by a long delay
between urine collection and urine examination.
Urinary infection can reasonably be suspected if bacteria are present in freshly voided
midstream urine, particularly if numerous leukocytes are also present [129,130] (Figure
2.216). However, it should not be forgotten that, especially 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 (Figure 2.65) with or without Candida and/or Trichomonas vaginalis. In
urinary tract infections with involvement of the kidney, besides leukocytes and bacteria, also
leukocytic casts and even bacterial casts can be found [101].
TABLE 2.9 The microorganisms of the urinary sediment.

Category Types
Bacteria Rods and cocci
Yeasts Candida
Protozoa Trichomonas vaginalis
Schistosoma haematobium
Parasites
Enterobius vermicularis
FIGURE 2.212 Rods isolated and in pairs (phase con- FIGURE 2.213 Rods in chains (phase contrast, origi-
trast, original magnification x 400). nal magnification x 400).
FIGURE 2.214 A chain of cocci (phase contrast, origi-

nal magnification x 400).
FIGURE 2.215 A clump of rods (phase contrast, origi- FIGURE 2.216 Bacteria intermingled with polymor-
nal magnification x 400). phonuclear leukocytes (phase contrast, x 400).
yeasts
Yeasts are unicellular organisms which reproduce by budding and separation of daughter
cells. Candida are the most frequent yeasts found in the urine. There are more than 80
Candida species, but only a few of them are pathogenic for humans. Of these, Candida
albicans is the most commonly found species.
Candida appear as pale-green cells with smooth and well-defined walls. The nucleus
is at times visible, and the cytoplasm is homogenous without apparent organelles. The
shape of cells, ovoid, spherical or elongated, is characteristic of the particular Candida
species (Figures 2.217 and 2.218). The cells of Candida albicans are ovoid, while those of
Candida kruzei are elongated, but differentiation of Candida species based exclusively on
microscopy is unreliable. Round Candida cells may resemble erythrocytes and some types
of monohydrated calcium oxalate crystals, but Candida are often nucleated and, especially,
show budding. After the urine has been left standing, abundant pseudomycelia, i.e. chains of
elongated unseparated Candida or clumps, can be seen (Figures 2.219 and 2.220).
In our experience, 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 (Figure 2.65).
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 the
latter condition, candidal casts can be found in the urine [102,103].
FIGURE 2.217 Candida with buds (phase contrast, FIGURE 2.218 Elongated Candida (phase contrast, x 400).
FIGURE 2.219 Pseudomycelia (phase contrast, x 400). FIGURE 2.220 A large clump of Candida intermingled
with polymorphonuclear leukocytes (phase contrast,
trichomonas vaginalis
Trichomonas vaginalis (TV) is protozoon. It has an ovoid to round shape, and is barely
larger than a polymorphonuclear leukocyte. Its distinguishing morphological feature is the
presence of five flagella one of which, bent backwards, is linked to the body by an undulating
membrane (Figures 2.221). When alive, TV can readily be identified by the motility of the
flagella and its rapid and irregular movements through the slide (Figure 2.222). However,
dead TV is difficult to distinguish from polymorphonuclear leukocytes (Figure 2.223).
The finding of TV in the urine usually indicates contamination from genital secretions,
this protozoon being a frequent cause of vaginitis or urethritis. In typical cases, the urine con-
tains not only TV but also massive amounts of squamous epithelial cells, polymorphonuclear
leukocytes, bacteria and/or Candida albicans.
FIGURE 2.221 The structure of Trichomonas vaginalis.
FIGURE 2.222 A schematic representation of the mov- FIGURE 2.223 Trichomonas vaginalis. Note the flag-
ements of Trichomonas vaginalis through the slide. ella (phase contrast, original magnification x 400).
schistosoma haematobium (urinary schistosomiasis)

Infection due to Schistosoma haematobium (SH) is endemic in several geographic regions
(the Middle East, particularly in the Nile valley, West and South Africa, some areas of the
Arabic peninsula, etc.) and affects about 100 million people.
Infection is acquired through contact with contaminated waters. The life cycle of the
parasite begins with the shedding of the ova through infected urine into a freshwater basin
such as a pond, river or lake. In a few days, the ova hatch with the release of the miracidium,
which is the embryo of the parasite. Then, the miracidium enters an intermediate host, which
is represented by a freshwater snail of the genus Bulinus. After some time, the snails release
thousands of cercariae, which are able to penetrate through the skin of persons bathing in the
contaminated water. From the skin, through the venous system, cercariae reach the venous
plexus of the bladder and of the lower end of the ureters, where the adult female deposits
thousands of ova in the mucosa, submucosa or even in the muscular layer. The presence of
the ova stimulates the formation of vesical and ureteric eosinophilic granulomata, mucosal
hyperaemia and ulcers, and polypoid vegetations. This causes haematuria (which is the most
frequent clinical manifestation of SH infection) and obstructive uropathy [131].
Although serological procedures are available, in endemic areas the most common method
to diagnose SH infection is the search for ova in the urine sediment [132]. These can be
visualized and counted after filtration of urine through paper or Nuclepore filters or after
urine centrifugation.
The ova of SH measure 115-170 μm x 40-70 μm, and have an ovoid shape with a typical
terminal spike (Figure 2.224). They usually contain a well evident miracidium, even though
occasionally, eggshells empty of miracidium can be seen [133], or even free miracidia outside
eggshells [133,134].
The probability of finding the ova in the urine sediment increases if the urine is collected
between 10 a.m. and 2 p.m., when the excretion of the ova reaches its peak [135], and after
a physical effort (such as a run or a walk), which favours the detachment of the ova from the
bladder mucosa. Counting of ova in the urinary sediment yields an estimate of the severity
of the infection.
Besides ova, the urine of patients infected with SH usually shows other particles. In 50
samples containing variable numbers of ova, and collected in an endemic area of Benin
Republic (West Africa), we found: erythrocytes (100%), leukocytes (92%), superficial
transitional cells (28%), and bacteria (34%). However, leukocytes and bacteria in such cases
could also be due to contamination from genitalia, which is particularly frequent in developing
countries as a consequence of poor hygienic conditions, or to superimposed bacterial urinary
infections, whose prevalence is increased in patients with SH [136]. The use of Hansel’s stain
has shown that urinary leukocytes found in SH infection also include eosinophils, which
derive from the vesical and ureteric lesions caused by the parasite [50].
enterobius vermicularis
The parasite Enterobius vermicularis (EV) is occasionally found in the urine of children,
either as a contaminant – from the anus, genitalia or urethra – or as a parasite of the bladder.
Ova measure about 25 to 50 μm, have one flat side while the other is rounded, and have a
double-layered wall (Figure 2.225).
EV inhabits the caecum and the colon in which it lives, adhering to the intestinal mucosa.
When gravid, the females detach from the intestinal mucosa and migrate towards the rectum.
Once they reach the anus, they crawl onto the perianal and perineal skin, where they deposit
the eggs and die. The diagnosis is made by placing adhesive tape over the perianal skin and
looking for pinworms and their ova on the adhesive tape.
FIGURE 2.224 An egg of Schistosoma haematobium FIGURE 2.225 Eggs of Enterobius vermicularis (phase
(phase contrast, original magnification x 400). contrast, x 400).
contaminants
The term “contaminant” applies to all elements which enter the urine after it has left the
bladder. The possible contaminants are numerous and can derive from the patient, from the
laboratory or from the environment (Table 2.10). The contaminants are not important per se,
but must be identified correctly in order to avoid misinterpretation. Contamination can partly
be avoided by proper patient preparation, urine collection and clean working conditions.
contaminants originating from the patient

Blood contamination regularly occurs during menstruation.
Erythrocytes, leukocytes, or bacteria may contaminate the urine of women with
genital infections such as vaginitis. In men, these particles can be due to urethritis or
balanoposthitis.
Spermatozoa are often present in the urine for some hours after intercourse, also in the
urine of women. When coiled, they can resemble acanthocytes (Figure 2.226). Besides free
spermatozoa, also “sperm bodies” can be found in the urine sediment [137]. These consist in
phagocytes containing numerous spermatozoa, whose heads are within the cytoplasm of the
cell, while the tails are sticking out. Their clinical significance is unclear.
Subjects with poor hygiene may have pediculosis pubis, and the parasite may be found in
the urine.
Faeces and intestinal cells can be found in the urinary sediment as a consequence of
vesicointestinal fistulae of whatever origin or after ileal bladder reconstruction. Faeces
appear as particles of variable shape and size which may contain tissue strands, vegetable
and/or muscle fibres (Figure 2.227). Intestinal cells appear as mononucleated cells with a
large nucleus and a thin cytoplasmic rim [138].
Enterobius vermicularis has been mentioned above (Figure 2.225).
Cloth fibres derive from clothes. They can be either flat or cylindrical, can exhibit the
texture of the cloth, and are at times coloured (Figures 2.228-2.231). In the latter case, they
may be confused with casts (the so-called “pseudocasts”, page 101). Fibres made of synthetic
materials are often birefringent under polarized light (Figures 2.136 and 2.137).
Talcum can be found in the urine when it is applied to the genital area. Talcum particles
have a crystalline structure and their morphology varies from pin-like particles to large
irregular bodies. Under polarized light they are strongly birefringent (Figures 2.231-2.232).
Cream and detergent particles can contaminate the urine when used to clean external
genitalia. They may appear as masses or pseudocasts (Figure 2.233 and 2.234). Cream
particles may also derive from lubricants used in urological manoeuvres [139].
TABLE 2.10 Principal contaminants of the urine.

Patient Laboratory Environment
Erythrocytes* Starch powder Pollen granules

Leukocytes* Glass fragments Plant cells
Squamous epithelial cells Air bubbles Fungal spores:
Bacteria* Alternaria
Spermatozoa Helminthosporium
Trichomonas vaginalis Epicoccum
Pubic hair Cladosporium
Pediculosis pubis Fibres
Faeces
Intestinal cells
Enterobius vermicularis
Cloth and synthetic fibres
Talcum
Creams and detergents
Starch powder
* Contaminants when deriving from urethra or genital secretion.
FIGURE 2.226 Spermatozoa. Note the coiled sperma- FIGURE 2.227 A partially digested muscle fibre due
tozoon (arrow) which might be misidentified as acan- to contamination of the urine from faeces. Note that
thocytes or G1 cells (phase contrast, x 400). this particle might be misidentified as a cast (phase
contrast, x 400).
FIGURE 2.228 A flat cloth fibre with a clearly visible FIGURE 2.229 A cylindrical cloth fibre, which is easily
texture (phase contrast, x 160). identifiable as a contaminant due to its peculiar pink
colour (phase contrast, x 160).
FIGURE 2.230 A rolled-up cloth fibre with knots (pha-

se contrast, x 160).
FIGURE 2.231 Talcum particles with irregular shape FIGURE 2.232 The same particles shown in Figure
(phase contrast, x 400). 2.231 as seen by polarized light (x 400).
FIGURE 2.233 A cream particle (phase contrast, ori- FIGURE 2.234 A large cream particle which might be
ginal magnification x 400). misidentified as a waxy cast (phase contrast, 160).
contaminants originating in the laboratory

Starch appears as translucent roundish to polygonal particles, with a nucleus-like centre
(Figure 2.235). Under polarized light, starch particles resemble birefringent “Maltese crosses”,
from which they differ because they produce a black cross whose arms are asymmetrical
(Figure 2.236. Compare it with Figures 2.67 and 2.116). Starch mostly derives from the
powder contained in the gloves worn by the personnel of clinical laboratories. Occasionally,
however, it may also derive from patients who use starch powder to keep subpannicular
and inguinal skin folds dry [140,141] or “Merfen powder” to cure inflammation of external
genitalia [142].
Glass fragments are transparent particles of irregular shape with hard edges (Figure
2.237). At times, it may be difficult to differentiate glass from irregular uric acid crystals.
However, the amber colour of the latter and the lack of birefringence of glass allow the
correct identification. Glass slivers are due to microscopic fragments released from either
microscopic slides or coverslips.
Air bubbles are an artefact rather than a contaminant. Most often they result from air
bubbles caused by the resuspension with a pipette of the sediment after centrifugation or
from air trapped between the slide and the coverslip. Air bubbles have a round shape and dark
double contours (Figure 2.238).
FIGURE 2.235 Starch particles with different sizes FIGURE 2.236 The same starch particles shown in
and shapes (phase contrast, original magnification Figure 2.235 as seen by polarized light. Note the asymmet-
x 400). rical arms of the crosses (original magnification x 400).
FIGURE 2.237 A glass sliver (phase contrast, x 400). FIGURE 2.238 Air bubbles (phase contrast, x 400).
contaminants originating in the environment

Several elements from environmental sources, especially form the air, may contaminate
the urine while it is handled in the laboratory.
Pollen granules have a morphology which greatly varies according to their nature (Figures
2.239 and 2.240).
Plant cells have distinct double-layered walls surrounding a large nucleus.
Fungal spores include Alternaria (Figure 2.241), Helminthosporium (Figure 2.242),
Epicoccum (Figure 2.243) and Cladosporium (Figure 2.244).
In a study of 1,600 urinary sediments conducted over a 1-year period, we have found that
fungal spores, especially Alternaria, appeared in the urine throughout the year, with a peak
from June to October on rainy days. Since the spores are usually present in soil and plants,
our finding suggests that rain moves spores from their original location to the air, through
which they reach the laboratory.
FIGURE 2.239 Pollen (family Leguminosae) (phase con- FIGURE 2.240 Pollen (family Pinaceae) (phase con-
trast, x 400). trast, x 400).
FIGURE 2.241 Alternaria (phase contrast, x 400). FIGURE 2.242 Elminthosporium (phase contrast, x 400).
FIGURE 2.243 Epicoccum (interference contrast, x 500). FIGURE 2.244 Cladosporium (phase contrast, x 400).
possible misidentifications
Several elements of the urinary sediments may be misidentified because their appearance
is more or less similar to that of other elements. Misidentifications can be avoided only by
a sound knowledge of the morphology of all the elements of the urinary sediment coupled
with experience. The main possible misidentifications have already been discussed in various
parts of the text and are summarized in Table 2.11.
TABLE 2.11 Main possible misidentifications.

Possible misidentifications How to avoid it
The ring of acanthocytes/G1 cells is thicker

Acantocytes/G1 cells versus coiled than the coiled tail of spermatozoa.
spermatozoa Moreover, these are usually associated
with uncoiled spermatozoa
Isomorphic erythrocytes versus ovoid mono- Ovoid calcium oxalate crystals polarize light
hydrated calcium oxalate crystals while erythrocytes do not
Isomorphic erythrocytes versus round Round Candida without buds are usually
Candida without buds associated with Candida with buds
Trichomonas vaginalis shows flagella and
Polymorphonuclear leukocytes versus rapid movements. When dead it does not
Trichomonas vaginalis move, but lacks the typical lobated nucleus
and cytoplasmic granules of leukocytes
Deep transitional cells have a thinner
Ovoid renal tubular cells versus ovoid deep cytoplasm than tubular cells. Associated
transitional cells particles are helpful in differentiating the
two types of cells
Lipid droplets show “Maltese crosses” with
Lipid droplets versus starch particles symmetrical arms while starch particles
(under polarized light) show “Maltese crosses” with asymmetrical
arms
Pseudocasts usually have harder edges,
Casts versus pseudocasts more irregular contours, more variable size
or colours than casts
Uric acid crystals have a typical amber
Uric acid crystals versus glass slivers colour and polarize light while glass slivers
do not
Urates form in acid urine, phosphates
Amorphous urates versus amorphous
in alkaline urine. Urates polarize light,
phosphates
phosphates do not
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CHAPTER 3
CHANGES OF URINARY SEDIMENT
CAUSED BY DRUGS
G.B. Fogazzi and S. Verdesca
Several drugs can cause urinary sediment changes. In several instances, these changes do
not have clinical implications, while on other occasions they may indicate renal pathologies.
In the latter case, the analysis of the urinary sediment is often a clue to the correct diagnosis.
Therefore, the physician should be aware of the urinary changes which can be induced by
drugs.
drug-related crystalluria
A variety of drugs may cause transient crystalluria, in isolation or in conjunction with
other urinary abnormalities or with even acute renal failure (Table 3.1). The factors usually
favouring the formation of drug crystals are drug overdose, dehydration, hypoalbuminaemia
(which increases the fraction of unbound drug which is ultrafiltered by the glomerulus), high
or low urine pH [1,2]. An important risk factor for the development of acute kidney injury
is the presence of an underlying renal impairment, which probably causes the exposure of a
fewer number of functioning nephrons to the crystal-forming agent. Excessive drug dosing
for the underlying renal filtration rate is another contributing factor [3].
The most important drugs which can cause crystalluria are discussed below.
Sulfadiazine. This is the treatment of choice for Toxoplasma encephalitis in patients with
AIDS. It is a short-acting sulphonamide, which is excreted rapidly by the kidneys and has a
low solubility, especially at urine pH < 5.5. This feature is responsible for the precipitation
of sulfadiazine crystals and/or calculi within the urinary system. The spectrum of renal
manifestations is wide and includes asymptomatic crystalluria, haematuria and acute renal
failure secondary to obstructive uropathy or intratubular obstruction [4-8]. Crystals and
stones dissolve with hydration and urine alkalinization, and the renal manifestations usually
reverse in a few days.
Sulfadiazine crystals appear as strongly birefrigent “shocks of wheat” or “shells” with
an amber colour and radial striations (Figures 3.1 and 3.2). These features distinguish them
from other sulphonamide crystals [9]. The search for crystals in the urine is one of the
measures suggested to monitor patients undergoing sulfadiazine therapy [6]. Although their
presence alone may not indicate renal injury, the finding should prompt hydration and urine
alkalinization, if not even a reduction or discontinuation of the drug.
160 G.B. Fogazzi and S. Verdesca
TABLE 3.1 Main types of crystals and clinical manifestations caused by drugs.
Drug Crystal Clinical manifestations

Birefringent “shocks of wheat” or Asymptomatic crystalluria,
Sulfadiazine “shells” with radial striations and haematuria, acute renal
amber colour failure, obstructive uropathy
Birefringent needles, Isolated crystalluria,
Amoxicillin “shocks of wheat”, haematuria, acute renal
“broom bush-like” failure, obstructive uropathy
Birefringent needles, “stars”,
Isolated crystalluria, acute renal
Ciprofloxacin “sheaves”, “fans”, “butterflies”,
failure, obstructive uropathy
etc.
Asymptomatic crystalluria,
Birefringent needles with sharp or
Acyclovir acute renal failure, haematuria
blunt extremities
and leukocyturia
Birefringent irregular plates, acute renal failure, obstructive
Indinavir
“crosses”, “stars”, “fans”, etc. uropathy, sterile leucocyturia,
interstitial nephritis
Birefringent coloured spheres Asymptomatic crystalluria,

Triamterene
(brown, green, orange, red) ?acute renal failure
Piridoxylate Asymmetrical hexagons Urinary stones
Primidone Birefringent hexagons
transient haematuria
Naftidrofuryl Birefringent monohydrated Asymptomatic crystalluria,
oxalate calcium oxalate acute renal failure
Birefringent monohydrated Crystalluria, haematuria,
Vitamin C
calcium oxalate acute renal failure
Calcium oxalate
Orlistat Acute renal failure
(?mono- or ?bi-hydrated)
(?Birefringent) sharp needles
Felbamate Haematuria, acute renal failure
isolated or in clumps
Amoxycillin. This }-lactam antibiotic is absorbed in the gastrointestinal tract and

is excreted by the kidneys (about 90% of the drug being secreted by the proximal tubules
and the remaining 10% being excreted by glomerular filtration) [10]. Amoxycillin can
cause transient asymptomatic crystalluria without renal damage [10-12], crystalluria
with gross haematuria [13], or crystalluria with gross haematuria and acute renal failure,
either oliguric or non oliguric [14-16]. It is hypothesized that haematuria and acute renal
failure derive from the tubular damage and medullary congestion caused by intratubular
precipitation of crystals [14], however no renal biopsies have been performed so far. Another
possible but less frequent mechanism for renal function impairment is obstructive uropathy,
due to the massive precipitation of macroscopic crystals in the renal pelvis [17]. Once
FIGURE 3.1 A crystal of sulfadiazine with typical FIGURE 3.2 Strongly birefringent sulfadiazine crys-
shape (“shock of wheat”) and striations (phase con- tals (polarized light, x 256).
trast, × 400).
amoxycillin is discontinued, the clinical manifestations always resolve: crystalluria usually

within 24 hours, gross haematuria within three days, and acute renal failure in 3-17 days.
Factors that favour crystal precipitation are drug overdose, low diuresis, or acid urine [10,14].
Amoxycillin crystals appear as “needles”, “shocks of wheat”, or “broom bush-like”
structures, which are all strongly birefringent under polarized light (Figures 3.3-3.6)(for
other images see also reference 12). Therefore, amoxycillin crystals differ remarkably from
common crystals. However, they may be similar to other crystals caused by antibiotics, such
as ampicillin [18] or cephalexin [19].
Ciprofloxacin. This fluoroquinolone antibiotic can cause crystalluria in alkaline urine
(especially at pH >7.3) after oral or intravenous administration [20-22]. A few cases of acute
renal failure have been described in association with ciprofloxacin crystalluria. In one patient,
renal function impairment was due to obstructive uropathy caused by massive precipitation of
drug crystals in the distal ureters and bladder, after a 24-day treatment at a dose of 500 mg twice
a day [23]. In another patient, whose renal function was already impaired before treatment, an
oliguric acute renal failure was observed after a course with ciprofloxacin 750 twice a day for
eight days. Clue to the diagnosis was the finding of ciprofloxacin needle-shaped birefringent
crystals in round conglomerates in the urine [24]. Two other patients developed acute renal
FIGURE 3.3 Marked amoxycillin crystalluria showing FIGURE 3.4 Birefringent crystals of amoxycillin (po-
“needles” and “shocks of wheat” (phase contrast, x larized light, x 160).
160).
FIGURE 3.5 A “broom bush-like” crystal of amoxycil- FIGURE 3.6 The same crystal by polarized light (x 400).
lin (phase contrast, x 400).
failure due to intratubular precipitation of crystals after a course with ciprofloxacin 750 mg
twice daily for some days [25]. Interestingly, the crystals found in the kidneys were similar to
ciprofloxacin crystals described by other investigators in the urine [24,26].
In order to better know the morphology of ciprofloxacin crystals, we induced a transient
and isolated crystalluria in the alkalinized urine (pH = 8.5) of a healthy volunteer by the
oral administration of 250 mg of ciprofloxacin and sodium bicarbonate 500 mg four times in
24 hours [26]. Ciprofloxacin crystals, whose nature was confirmed by infrared spectroscopy,
appeared with a large variety of shapes (“needles”, “stars”, “sheaves”, “fans”, “butterflies”
and other unusual appearances) and sizes (from 30 × 5 μm to 360 × 237 μm). Common to all
crystals was a lamellar structure and a strong birefringence. While some crystals, especially
the largest ones, had a brownish hue, others were colourless (Figures 3.7-3.12) (For other
images see reference 26).
Norfloxacin also can cause crystalluria in alkaline urine (pH > 7.0), however at single
doses of 1,200 and 1,600 mg, which are by far higher than to dose used in clinical practice
[27]. Norfloxacin crystals have a spherical appearance with ragged edges and orange-green
highlights [27].
FIGURE 3.7 A “star-like” crystal and “needles” of cip- FIGURE 3.8 The same crystals by polarized light (x 256).
rofloxacin (phase contrast, x 256).
FIGURE 3.9 Many “needles” of ciprofloxacin (phase FIGURE 3.10 The same crystals by polarized light
contrast, x 160). (x 160).
FIGURE 3.11 A clump of ciprofloxacin crystals with FIGURE 3.12 The same crystals by polarized light
different shapes (phase contrast, x 400). (x 400).
Acyclovir. The antiviral drug acyclovir can cause crystalluria especially when it is
administered as a rapid intravenous bolus (= 500 mg/m2) and/or when the patient is dehydrated
[3]. Crystalluria may either be asymptomatic [28-30] or associated with acute renal failure,
which is usually reversible after discontinuation of the drug [31-33].
Acyclovir crystals are strongly birefringent and needle-shaped with either sharp or blunt
extremities [28-30]. When they are abundant, the urine acquires a silky and opalescent
macroscopic appearance [28]. Haematuria and leukocyturia are a frequent association of
acyclovir crystalluria [32].
Indinavir. The inhibitor of HIV-1 protease, indinavir, when given at the therapeutic dose
of 800 mg three times a day can cause asymptomatic crystalluria, acute renal failure due to
urolithiasis, or intratubular precipitation of crystals [34-40]. Crystal formation is strongly
influenced by urine pH, since indinavir is insoluble at pH >6.0, while its solubility increases
exponentially at lower pH values, with complete solubility at pH 3.0 [41]. Thus, it is not surprising
that in 579 urine samples from 54 HIV-infected patients the prevalence of indinavir crystalluria
was 60% at urine pH >7.5, while it was 12.7% at pH 5.0 [41]. In the same study also urine
specific gravity (SG) was found to be an influencing factor, the prevalence of crystalluria being
higher at SG Γ1.015 than SG 1.005 (64% vs 16.7%)[41]. Frequently, indinavir crystalluria is
associated with sterile leukocyturia with or without renal function impairment [42,43], which is
seen as a marker of a possible drug-induced interstitial nephritis and/or urothelial inflammation
[42]. Indinavir crystalluria is quite pleomorphic: crystals appear either as flat irregular plates
exhibiting an internal layering, or as “crosses”, “stars” or fan-shaped structures, and are always
birefringent under polarized light [3,35,37,40] (Figures 3.13-3.18).
Triamterene. The diuretic triamterene can cause transient and asymptomatic crystalluria
in acidic urine [44,45]. A case of irreversible acute renal failure with intratubular precipitation
of triamterene crystals (but without crystalluria) has been reported [46]. Consequently,
triamterene crystals must be regarded as a potential cause of severe renal tubular injury.
Triamterene crystals are spherical and predominantly brown in colour. Under polarized
light, they appear as “Maltese crosses” [45]. In most cases, these crystals are associated with
brown casts, which are also due to triamterene.
Piridoxylate. This is an equimolar combination of glyoxylic acid and pyridoxine used for
the treatment of coronary disease. It can cause a unique form of calcium oxalate trihydrate
crystalluria, which is usually associated with piridoxylate stones [47].
Piridoxylate crystals are asymmetrical hexagons, which disappear completely from the
urine after withdrawal of the drug [47].
Primidone. The barbiturate primidone can be a cause of crystalluria following overdose
[48,49] or even normal maintenance doses [50]. The urinary abnormalities include isolated
crystalluria or crystalluria associated with transient haematuria [48,50].
Primidone crystals are birefringent hexagons which appear singly or in conglomerates. In
the latter case, they can resemble crystals of cystine [49].
Naftidrofuryl oxalate. This vasodilator can cause either asymptomatic crystalluria, which
has been reported after oral administration in elderly patients [51], or crystalluria associated
with acute renal failure, which is observed after intravenous injection. Renal damage is due
to the intratubular precipitation of crystals [52-54]. Crystals caused by naftidrofuryl oxalate
are made of monohydrated calcium oxalate (see Figures 2.159-2.164).
FIGURE 3.13 A “star-like” crystal and plates of indi- FIGURE 3.14 The same crystals by polarized light
navir (phase contrast, x 400). (x 400).
FIGURE 3.15 Several crystals of indinavir with differ- FIGURE 3.16 The same crystals by polarized light
ent shapes and sizes (phase contrast, x 160). (x 160).
FIGURE 3.17 Indinavir crystal with the shape of ir- FIGURE 3.18 The same crystals by polarized light
regular plates (phase contrast, x 160). (x 160).
Vitamin C. When given in high doses, especially intravenously, vitamin C can cause
monohydrated calcium oxalate crystalluria (see Figures 2.159-2.164). This can be either
asymptomatic [55] or associated with acute renal failure due to the intratubular precipitation
of calcium oxalate crystals [56-59].
Orlistat. This drug is an oral inhibitor of gastrointestinal lipase used to obtain weight
reduction in obese patients. At intestinal level, orlistat acts by reducing fat absorption with a
potential increase of oxalate absorption, which may result in an increase of urinary oxalate
excretion. One patient, who has recently been described, developed reversible acute renal
failure associated with intrarenal precipitation of oxalate crystals, increased urinary oxalate
excretion, and numerous calcium oxalate crystals in the urine sediment (whether they were
mono- or bi-hydrated was not specified) [60]. The co-presence of stage 3 chronic kidney
disease and dehydration were favouring factors for the onset of acute kidney injury.
Felbamate. This is an antiepileptic drug used to treat seizure disorders and Lennox-
Gastaut syndrome. Felbamate has a low-protein binding (25-35%) and approximately 50%
of the drug is normally excreted unchanged in the urine. To date, 2 patients with felbamate
overdose complicated by massive crystalluria have been described. In one patient, felbamate
crystalluria was associated with acute renal failure, which reversed after the discontinuation
of the drug [61]. In the other patient, crystalluria was associated with microscopic haematuria,
while renal function was not impaired [62]. Felbamate crystals appear as sharp needle-like
structures of variable size (90 μm to >1300 μm), which can be either isolated or clumped
together in a cat-tail configuration [62]. It is stated that under polarization they are strongly
birefringent, however the published images do not seem to support this view [62].
A number of other drugs such as cephalexin, ampicillin, acetyl salicylic acid, xylitol, or
ceftriaxone can rarely cause crystalluria, usually with no clinical implications [63,64,64b].
As a general rule, the finding in a urine sample of numerous crystals with unusual and
pleomorphic appearances should always raise the suspicion of a drug crystalluria (without
forgetting, however, that some drugs cause “just” calcium oxalate crystalluria). The suspicion
should prompt the question if and which drug(s) the patient is taking. If a drug of those described
above is identified, renal function should immediately be checked and the drug should possibly
be reduced or withdrawn in order to prevent the development of acute renal failure.
In addition, risk factors such as dehydration, hypoalbuminaemia, or urine pH favouring
crystallization should be corrected.
other changes induced by drugs

diuretics
Drugs such as furosemide or ethacrynic acid can cause transient hyaline cylindruria [65].
Since this cylindruria peaks at 3-6 h and usually disappears by 24 h, it does not have any
clinical relevance [65].
Hydrochlorothiazide or furosemide can cause the appearance of pseudoanisotropic
material resembling free fat [66].
In haematuric patients with glomerulonephritis, loop diuretics cause a transient increase of
isomorphic erythrocytes with a consequent decrease in the percentage of dysmorphic cells [67].
drugs influencing urinary ph

All drugs which can cause alkaline urine, such as sodium bicarbonate, can reduce the
number of casts. This happens because aggregation of Tamm-Horfall glycoprotein, i.e. the
matrix of casts, is less at alkaline pH [68]. Conversely, large doses of ammonium exchange
resins (or drugs acidifying the urine) give rise to the appearance of a large number of granular
casts as a result of increased acidity and solute concentration in the urine [69]. These casts
usually do not have clinical significance.
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CHAPTER 4
THE URINARY SEDIMENT
OF THE NORMAL SUBJECT
G.B. Fogazzi
Since the time of Thomas Addis (see “Historical Introduction” for details), 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.
Some of the early studies, the results of which are summarized in Table 4.1, showed
that erythrocytes, leukocytes-renal tubular epithelial cells (which were grouped together
because it was difficult to distinguish the two types of cells) and casts ranged from zero to
variable numbers [1-3]. Casts were almost invariably of the hyaline type [1,2], even though
granular casts or even epithelial casts could be found, especially in newborns [3]. Those
studies showed that there were large differences in the numbers of cells and casts considered
as normal (Table 4.1).
Subsequently, with the use of stains, it became possible to distinguish leukocytes from
renal tubular epithelial cells [4]. It was shown that while the excretion rates of the latter are
relatively constant in a given individual from day to day and over periods of many months,
there were great inter- and intra-subject variations in the excretion of leukocytes. In addition,
while the excretion of renal tubular epithelial cells (and of erythrocytes) was significantly
higher in males than in females, the excretion of leukocytes was significantly higher in
females [4].
TABLE 4.1 Excretion of erythrocytes (RBCs), leukocytes (WBCs) and casts found in some early
studies.
RBCs WBCs Casts

Author Subjects
mean No. mean No. mean No.
[ref] studied
(range) (range) (range)
65,750/12h 322,500/12 h 1,040/12 h

Addis[1] 74adul ts
(0-425,000) (32,400-1,835,000) (0-4,270)
322,184 ± 25,500
15,181 ± 400/12 h 1,085 ± 123/12 h
Lyttle [2] 74 children (9,000-
(0-129,000) (0-12,916)
2,822,000/12h)
90,129/mm3 1,865,529/mm3 12,852/mm3
Aas[3] 119ne wborns
(0-630,000) (42,000-13,500,000) (0-440,000)
174 G.B.F ogazzi
In recent times, further data have been published concerning the normal excretion of
erythrocytes [5,6] and of erythrocytes and leukocytes [7] (Table 4.2).
Birch et al. studied the erythrocyturia of 376 healthy adult subjects (151 males and 225
females), aged 18-82 years (median age 23 years) [5]. 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 (whose sex
and age were not given in the paper) [6]. 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 (described
as 228 males and 160 females), aged 2-16 years [7]. Using uncentrifuged urine, a Neubauer
counting chamber and phase contrast microscopy, it was found that 95% of children excreted
< 14 × 106 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. [5] and of Loh et al. [7], the morphology of
erythrocytes was also evaluated. This was found to be consistently dysmorphic [5,7], i.e. of
glomerular origin. These results were confirmed partially by Fasset et al., who studied 50
healthy 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” [8].
Thus, the studies described give very different figures in the excretion of erythrocytes
and leukocytes in the urine of the normal subject, a fact which explains the very different
definitions of pathologic microscopic haematuria which can be found in the literature (Table
4.3) [9-15].
How can one explain the different results shown in Tables 4.1 and 4.2? A partial explanation
is that the methods used to collect, prepare and analyse the urine samples were not the same
in the different studies. For instance, Birch et al. [5] and Pollock et al. [6] used somewhat
TABLE 4.2 Excretion of erythrocytes and leukocytes found in some recent studies.
Subjects Normal Normal

Author [ref]
studied erythrocyturia leukocyturia
Birch [5] 376 adults < 8,000/mL --
Pollock [6] 27* < 1,000/mL --
Loh [7] 419 children < 14,000/mL < 4,000/mL
* Age and sex not described in the paper.

The urinary sediment of the normal subject 175
different centrifugation procedures, while Loh et al. used uncentrifuged samples [7]. In
addition, there were large differences in the number of subjects studied. Due to the large
variations in the excretion of cells and casts, this type of study should include adequate
numbers of individuals. However, the study of Pollock et al. [6] included only 27 subjects.
Further possible explanations are the difference in the male to female ratio, and the different
ages of the subjects studied. For instance, compared with adults, newborns seem to excrete
more leukocytes/renal tubular epithelial cells and casts (Table 4.1), and children seem to
excrete more erythrocytes (Table 4.2).
This lack of consistent and sound results is a major problem, and may explain why
several laboratories do not provide normal values for urinary erythrocytes and leukocytes,
as we found in two surveys performed among the renal and non renal laboratories in Italy
[16,17]. However, without these figures, it is impossible to define microscopic haematuria or
pathological leukocyturia correctly.
In the authors’ opinion, every laboratory should try to obtain the normal values for both
erythrocyte and leukocyte excretion. This may be done by a careful selection of the subjects to
be studied and by using a standardized method for urine collection, handling and analysis.
In our laboratory, we studied 70 adult subjects (22 female, 48 male) aged 16-53 years
[18]. They were defined as normal if they had: negative clinical history for kidney and
urinary tract diseases, normal blood pressure, creatinine clearance > 80 mL/min/1.73 meter2,
normal physicochemical urinalysis, negative urine culture, and normal kidney and bladder
ultrasonography. The second urine of the morning, produced over 2 hours, was collected.
Then, a 10 mL aliquot of urine was centrifuged at 400 g (which corresponds to 2,000 rotations
per minute with our centrifuge) for 10 minutes. After removal of 9.5 mL of supernatant urine
and resuspension of the sediment in the remaining 0.5 mL, 50 ΗL of resuspended urine was
TABLE 4.3 Different definitions of microscopic haematuria used by different investigators.

In some studies the information about the centrifugation procedures used was
missing.
Definition
Author [ref] Patients Urine sample of microscopic
haematuria
Tratchman [9] Children ? > 240,000/12 hours
Schröder [10] Children ? Γ 10/mm3
Piqueras [11] Children Uncentrifuged > 5/mL
Topham [12] Adults Uncentrifuged > 5 × 106/L
Mc Gregor [13] Adults Uncentrifuged Γ 10 × 106/L
Chow [14] Adults Centrifuged > 2/HPF
KovaĆeviĄ [15] Young males ? > 5/HPF

176 G.B. Fogazzi
transferred to a glass slide. After covering the samples with a 24 × 32 mm coverslip, the
samples were analysed by phase contrast microscopy at × 400. Erythrocytes and leukocytes
were reported as mean number ± SD observed over 20 microscopic fields (HPF).
Erythrocytes were 5.8 ± 5.7/20 HPF (range 0-26); leukocytes were 3.1 ± 3.5/20 HPF
(range 0-17). These figures were not influenced by patient gender, urine volume, specific
gravity or pH.
Due to these findings, in our laboratory we define as normal an erythrocyturia with Φ 1
erythrocyte/HPF and a leukocyturia with Φ 1 leukocyte/HPF (see table 1.5).
References
[1] ADDIS T. The number of formed elements in the urinary sediment of normal individuals. J Clin Invest
1926; 2: 409-21.
[2] LYTTLE J.D. The Addis sediment count in normal children. J Clin Invest 1933; 12: 87-93.
[3] AAS K. The cellular excretion in the urine of normal newborn infants. Acta Paediatr 1961; 50:
361-70.
[4] PRESCOTT L.F. The normal urinary excretion rates of renal tubular cells, leucocytes and red blood cells.
Clin Sci 1966; 31: 425-35.
[5] BIRCH D.F., FAIRLEY K.F., WHITWORTH J.A. et al. Urinary erythrocyte morphology in the diagnosis of
glomerular hematuria. Clin Nephrol 1983; 20: 78-84.
[6] POLLOCK C., PEI-LING L., GYÖRY A.Z. et al. Dysmorphism of urinary red blood cells – value in
diagnosis. Kidney Int 1989; 36: 1045-9.
[7] LOH E.H., KENG V.W., WARD P.B. Blood cells and red cell morphology in the urine of healthy children.
Clin Nephrol 1990; 34: 185-7.
[8] FASSETT R.G., HORGAN B.A., MATHEW T.H. Detection of glomerular bleeding by phase-contrast
microscopy. Lancet 1982; ii: 1432-4.
[9] TRACHTMAN H., WEISS R.A., BENNET B. et al. Isolated hematuria in children: indications for renal
biopsy. Kidney Int 1984; 25: 94-9.
[10] SCHRÖDER C.H., BOMPTEMPS C.M., ASSMANN K.J.M. et al. Renal biopsy and family studies in 65
children with isolated hematuria. Acta Pediatr Scand 1990; 79: 630-6.
[11] PIQUERAS A.L., WHITE R.H.R., RAAFAT F. et al. Renal biopsy diagnosis in children presenting with
haematuria. Clin Nephrol 1998; 12: 386-91.
[12] TOPHAM P., YOUNG S., HARPER S. et al. Isolated microscopic haematuria in the genitourinary clinic: the
value of renal biopsy. Int J STD & AIDS 1997; 8: 558-62.
[13] MC GREGOR D.O.M., LYNN K.L., BAILEY R.R. et al. Clinical audit of the use of renal biopsy in the
management of isolated microscopic hematuria. Clin Nephrol 1998; 49: 345-8.
[14] CHOW K.M., KWAN B.C., LI P.K. et al. Asymptomatic isolated microscopic haematuria: long-term
follow-up. Q J Med 2004; 97: 739-45.
[15] KOVACEVIC Z., JOVANOVIC D., RABRENOVIC V. et al. Asymptomatic microscopic haematuria in young
males. Int J Clin Pract 2008; 62: 406-12.
[16] FOGAZZI G.B., GRIGNANI S. Urine microscopic analysis – an art abandoned by nephrologists? Nephrol
[17] FOGAZZI G.B. Urinalysis performance. Programma di valutazione esterna di qualità sul sedimento
urinario. Anno 2001-2003. Pisa: Pacini; 5-10.
[18] FOGAZZI G.B., PASSERINI P., BAZZI M. et al. Use of high power field in the evaluation of formed elements
of urine. J Nephrol 1989; 2: 107-12.
CHAPTER 5
THE URINARY SEDIMENT IN THE MAIN DISEASES
OF THE KIDNEY AND OF URINARY TRACT
This chapter describes the urinary sediment findings in the main diseases of the kidney
and urinary tract. For most diseases, definition, aetiology and pathogenesis, pathology and
clinical course are briefly described, which allows a better understanding of the urinary
findings.
minimal change disease

and focal segmental glomerulosclerosis
Definition. Minimal change disease (MCD) is mainly a disease of children even though
it may also occur in adults. It is characterized by full-blown nephrotic syndrome i.e., severe
proteinuria without hypertension and renal failure. Slowly progressing impairment of renal
function and appearance of hypertension are exceptional.
Focal segmental glomerulosclerosis (FSGS) affects both children and adults and is
considered a variety of MCD with adverse renal prognosis. Most cases of FSGS are
“idiopathic”, but it may also occur in patients with AIDS, morbid obesity, reflux nephropathy,
heroin addiction, reduced renal mass, etc.
Aetiology and pathogenesis. For MCD, its possible association with lymphomas or
atopy suggests that it may be a condition due to a disregulation of cell mediated immunity.
Lymphocyte-derived cytokines altering the glomerular permeability were proposed as
possible pathogenetic mechanism many years ago, but this hypothesis remains elusive. A
circulating factor acting on podocytes has not yet been identified.
The aetiology of idiopathic or primary FSGS is by definition unknown. In some patients,
clinical data support similar aetiologic factors as those hypothesized for MCD. In other
patients, circulating permeability factors, which are only partially defined biochemically,
have been identified. Detachment of podocytes from the glomerular basement membrane
is also considered a possible initiating process. Various types of secondary FSGS exist:
familial, virus-associated (e.g., HIV), due to drugs (e.g., heroin, pamidronate), mediated
by adaptive structural-functional responses (e.g., reduced renal mass, reflux nephropathy,
surgical renal ablation), or initially normal renal mass (hypertension, obesity, sickle cell
disease). These forms have different pathogenetic mechanisms, which lead to the same
FSGS phenotype.
Pathology. In MCD, the glomeruli are normal by light and immunofluorescent microscopy.
By electron microscopy, effacement of foot processes of podocytes is seen. In FSGS, the
typical lesion is represented by focal and segmental scarring of the glomerular tufts (Figure
5.1, top left), which may be associated with increased endocapillary cellularity, collapse of
the glomerular tufts, the so-called tip lesion, etc.
Clinical course. In MCD, the renal prognosis is usually benign. Most patients are cured
eventually, although this may take many years.
In most patients remission of proteinuria is obtained with a course of high-dose prednisone,
but many patients relapse after the steroid is stopped. A stable remission may be obtained in
many cases with cytotoxic agents or cyclosporin.
Many patients with idiopathic FSGS progress to end-stage renal failure. Only a minority
of patients respond to steroids or other immunosuppressive agents.
urinary findings
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 (see Chapter 6).
In MCD, lipiduria is less frequent than in other glomerular diseases [1], and microscopic
haematuria is absent or mild. We found it only in 7/15 patients (47%), with the number of
erythrocytes ranging from 0 to 5/high power field (mean ± SD: 1 ± 1).
In primary FSGS, microscopic haematuria is more frequent and less mild than in MCD.
We found it in 16/20 patients (80%), in whom the number of erythrocytes ranged from 0 to
20/high power field (mean ± SD: 5 ± 5).
Any deviation from the above patterns is suspicious. For instance, the sudden appearance
(or sharp increase) 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
Definition. Primary membranous nephropathy (MN) is the most frequent cause of nephrotic
syndrome in adults. Less frequently, the disease is secondary to systemic lupus, hepatitis B or
C infection, malignancy, or drugs (e.g., non-steroidal anti-inflammatory agents).
Aetiology and pathogenesis. MN is an immunocomplex-mediated disease. Immunocom-
plexes, presumably formed locally, deposit on the external side of the glomerular basement
membrane, forming immunodeposits containing mainly IgG and complement.
Pathology. Diffuse thickening of the capillaries of the glomerular tuft of variable degree,
with no or only mild mesangial cell proliferation (Figure 5.1, top right). Immunohistology
FIGURE 5.1 Some non proliferative glomerular diseases described in this chapter. Top left: focal segmental glomerulo-
sclerosis (the arrow indicates the area with sclerosis) (AFOG stain, x 400); top right: membranous nephropathy with
its typical deposits (red dots) on the external side of the glomerular basement membrane (AFOG stain, x 630);
bottom left: light chain deposition disease with an acellular nodular lesion (bottom right) and diffuse thickening of
the glomerular basement membrane (AFOG stain, x 400); bottom right: diabetic nephropathy with diffuse mesangial
matrix increase, diffuse thickening of the glomerular basement membrane, and segmental mesangial hypercellularity
(haematoxylin & eosin, x 250).
shows IgG and C3 deposition on the external side of the glomerular basement membrane.
In advanced phases, immunodeposits are incorporated into the basement membrane and are
gradually reabsorbed.
Clinical course. This is highly variable, but 30-40% of patients progress to end-stage renal
failure while a few may go into spontaneous remission. In addition, some patients may suffer
from thrombotic complications caused by the nephrotic syndrome. The course of the disease
can be modified by immunotherapy.
urinary findings
Proteinuria in the nephrotic range is usually associated with microscopic haematuria, heavy
cylindruria, and lipiduria. Table 5.1 shows the urinary findings we observed in 40 patients
with IMN, 32 of whom had a proteinuria Γ 3.5 g/24 hours. A mild-to-moderate haematuria
was found in 80 % of cases, a mild leukocyturia in 17.9%, and few RTECS in 57.5%. Fatty
casts were the most frequent (100% of samples) and the most abundant, being followed by
RTEC casts (52.5%), erythrocytic casts (37.5%), and leukocytic casts (only 1 case, 2.5%).
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 extracapillary proliferation [2].
iga nephropathy
Definition. IgA nephropathy (IgAN) is the most common type of glomerulonephritis
worldwide. It may present with either recurrent episodes of gross haematuria (usually triggered
by an upper respiratory tract infection) or persisting urinary abnormalities i.e., microscopic
haematuria with or without proteinuria. Renal function can be either normal or impaired. On
rare occasions, there is rapidly progressive renal failure due to extracapillary proliferation.
Aetiology and pathogenesis. The precise aetiology of IgAN is unknown. It may result
from a glomerular (mesangial) response to a variety of foreign antigens, which have
however disappeared from the kidneys at the time of renal biopsy. Alternatively, it may be
an autoimmune disease directed against mesangial antigens, or it may develop from antigen-
independent mechanisms such as IgA glycosylation.
Pathology. Mesangial cell proliferation is the most typical lesion (Figure 5.2, top left).
However, in rare instances, the disease may be associated with other patterns such as
minimal glomerular abnormalities or extracapillary proliferation. The diagnostic lesion is
the presence, at immunofluorescence microscopy, of dominant or co-dominant mesangial
IgA deposits with or without IgG and C3.
Clinical course. The short-term renal prognosis is usually favourable, but in the long term
a sizeable proportion of patients will progress to end-stage renal failure. Poor prognosis is
predicted by proteinuria > 1.0 g per day, elevated serum creatinine at presentation, and severe
tubulointerstitial lesions at renal biopsy. Various types of treatment including corticosteroids
with or without cytotoxic drugs can improve the clinical course.
TABLE 5.1 Urinary sediment findings in idiopathic membranous nephropathy, IgA nephropa-
thy, and extracapillary/necrotizing pauci-immune glomerulonephritis (GN).
Idiopathic
IgA Extracapillary/
membranous
nephropathy Necrotizing GN
nephropathy
Number of patients 40 37 19
M/F 25/15 30/7 6/13
Age
56.6 ± 17.3 42.7 ± 14.2 53.1 ± 14.9
mean ± SD
16-78 18-75 31-81
range
S-Creatinine
1.2 ± 0.6 2.0 ± 2.0 3.7 ± 3.0
mean ± SD
0.5-4.1 0.8-11.5 1.0-12.9
range
N with proteinuria 40 (100%) 37 (100%) 19 (100%)
mean ± SD (g/24h) 6.7 ± 4.6 2.3 ± 3.0 3.6 ± 3.2
range 0.4-20.2 0.4-16.6 0.4-11.2
N with haematuria 32 (80%) 37 (100%) 19 (100%)
mean ± SD * 109.0 ± 89.5 658.8 ± 663.4 909.9 ± 696.7
range 21-356 23-2000 68-2000
N with leukocyturia 7 (17.9%) 18 (48.6%) 16 (84.2%)
mean ± SD * 50.0 ± 30.9 63.4 ± 59.7 88.1 ± 65.1
range 24-114 28-290 28-258
N with RTECs 23 (57.5%) 26 (70.3%) 16 (84.2%)
mean ± SD * 3.3 ± 2.7 4.8 ± 4.5 9.5 ± 10.5
range 1-8 1-17 1-40
N with RBC casts 15 (37.5%) 32 (86.5%) 17 (89.4%)
mean ± SD + 1.8 ± 1.3 6.0 ± 5.0 7.1 ± 5.7
range 1-5 1-18 1-22
N with WBC casts 1 (2.5%) 6 (16.2%) 6 (31.6%)
mean ± SD + - 2.0 ± 1.3 1.8 ±1.3
range - 1-4 1-4
N with RTEC casts 21 (52.5%) 28 (75.7%) 13 (68.4%)
mean ± SD + 1.8 ± 0.9 6.2 ± 5.8 8.2 ± 8.1
range 1-5 1-22 1-31
N with fatty casts 40 (100%) 32 (86.5%) 8 (42.1%)
mean ± SD + 27.9 ± 19.2 13.1 ± 12.4 12.4 ± 12.3
range 2-77 1-52 1-38
Haematuria and leukocyturia were defined as > 20 erythrocytes (RBCs) and > 20 leukocytes (WBCs)/20 high power
fields (HPFs) at x 400 respectively. * = RBCs, WBCs and renal tubular epithelial cells (RTECs) were expressed
as number counted over 20 HPFs. Since for RTECs no cut off for normality exists, all cells were considered and
calculated in the mean ± SD (while for RBCs and WBCs the mean ± SD was calculated considering only the samples
with > 20 cells/20 HPFs). For each sample, 100 casts were looked for, which were classified in 8 categories. In this
table only 4 types of casts were reported as number/100 (+). All samples were examined a few hours before renal
biopsy and were prepared according to the standardized method described in Table 1.6.
FIGURE 5.2 Some proliferative glomerular diseases described in this chapter. Top left: IgA nephropathy, with me-
sangial cell proliferation and increase of the mesangial matrix (haematoxylin & eosin, x 250); top right: extracapillary
glomerulonephritis with a large cellular “crescent” occupying Bowman’s space (trichrome stain, x 250); bottom left:
class IV lupus nephritis with several mesangial and subendothelial deposits (in red) (AFOG stain, x 400); bottom
right: necrotizing glomerulonephritis, with a large area of fibrinoid necrosis (red area) (AFOG stain, x 250).
urinary findings
Table 5.1 shows the urinary sediment findings we observed in 37 patients with IgAN, 7 of
whom had a proteinuria Γ 3.5 g/24 hours. A mild-to-very severe haematuria was present in
100% of cases, a mild-to-moderate leukocyturia in 48.6%, and few renal tubular epithelial
cells in 70.3%. Erythrocytic casts and fatty casts were the most frequent (86.5% each), being
followed by renal tubular epithelial cell casts (75.7%) and leukocytic casts, which were the
most rare (16.2%).
Our findings on erythrocytes and leukocytes are similar to those reported by Ibels et al.
[3], 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. [4], 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.
New techniques applied to urine sediment investigation (i.e., immunofluorescence with
monoclonal antibodies) have shown that patients with progressive IgAN have an increased
excretion of podocytes [5].
Among glomerular diseases, IgAN is also one of the most frequent causes of 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 [6,7], while others
found mainly dysmorphic erythrocytes [8] or Γ 5% acanthocytes [9,10].
membranoproliferative glomerulonephritis
Definition. Membranoproliferative glomerulonephritis (MPGN) may be a primary
(idiopathic) or a secondary disease, which may be associated with hepatitis B or C infection,
bacterial endocarditis, visceral abscesses, infected ventriculoatrial shunt, malaria, type II or
III cryoglubulinaemia, autoimmune diseases, etc.
The primary form may present after an infection of the upper respiratory tract or may be
discovered because of oedema or because of minor urinary abnormalities with or without
renal function impairment.
Aetiology and pathogenesis. For primary MPGN, type I and type III subtypes (see pathology)
are the result of glomerular immune complex deposition from the circulation. Type II MPGN
is linked to a continual overactivation of the alternative pathway of complement, which in
most cases is due to the presence of the autoantibody C3 nephritic factor. The pathogenesis
of the secondary forms varies according to the nature of the associated condition.
Pathology. An increase in mesangial cells and matrix, and a thickening of capillary walls
with the typical “double track” appearance are the typical lesions, common to all forms
of MPGN. By immunohistology and electron microscopy, three different types of MPGN
can be identified: type I MPGN (with granular subendothelial deposits of C3 and IgM);
type II MPGN or “dense-deposit disease” (with ribbon-like C3 and electron-dense substance
within the glomerular basement membrane) and type III MPGN (with granular C3 and both
subendothelial and subepithelial electron-dense deposits and marked dirsruption of the
glomerular basement membrane).
Clinical course. Most patients with the MPGN of whatever type develop slowly progressive
renal insufficiency. In some cases, however, there may be a rapid progression to renal failure
due to a superimposed extracapillary proliferation.
urinary findings
A wide spectrum of urinary changes is possible in MPGN of whatever type. These include
isolated dysmorphic microscopic haematuria, microscopic haematuria and proteinuria
(20-30% of cases), nephrotic syndrome (40-60% of cases) and macroscopic haematuria
(10-20% of cases). Thus, the finding of a nephritic or of a nephritic and nephrotic sediment
(see Chapter 6) is not uncommon in patients with MPGN. Interestingly, in a large study on
the prognostic factors of primary MPGN [11], 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

Definition. Acute post-streptococcal glomerulonephritis (PSGN) is the most common
glomerulonephritis in children, especially in developing countries. In industrialized countries,
PSGN affects more adults than in the past, especially debilitated elderly individuals.
The typical picture is represented by the appearance of gross haematuria with oedema,
hypertension and impaired renal function in a patient who, 2-3 weeks previously, had an
acute streptococcal throat infection or erysipelas. In a minority of cases, isolated microscopic
haematuria or microscopic haematuria and proteinuria are observed.
Aetiology and pathogenesis. PSGN is caused by glomerular deposition of immunocomplexes,
the formation of which is triggered by nephritogenic streptococcal antigens.
Pathology. In the acute phase, there is diffuse endocapillary proliferation with exudative
lesions (i.e., polymorphonuclear infiltration) associated with abundant parietal and mesangial
deposits of C3.
Clinical course. In children, the renal prognosis is usually favorable, renal function
spontaneously recovers, and hypertension disappears within a few days, while urine
abnormalities may disappear only after months or years. In adults, complete clearing up of
the urinary findings is less likely, and abnormalities persist in many patients. Even patients
who apparently recover from the acute episode may subsequently develop proteinuria,
hypertension and renal insufficiency, probably as a consequence of glomerular hypertension
and hyperfiltration in remnant nephrons.
urinary findings
In the acute phase, the urinary findings correspond to those of a full-blown nephritic
syndrome (see Chapter 6). However, as in lupus nephritis, diffuse and active glomerular
changes may occasionally be associated with normal – or only mildly altered urine [12-15].
In patients who go into remission, proteinuria and haematuria usually disappear by the end
of the first year, but in some 15% of patients, isolated microscopic haematuria may persist
for years.
extracapillary glomerulonephritis
Definition. Extracapillary glomerulonephritis is a condition characterized by rapid
deterioration of renal function over days or a few weeks due to the formation of “crescents”
(i.e., proliferation of cells within Bowman’s space) in the majority of glomeruli.
Aetiology and pathogenesis. Extracapillary glomerulonephritis may be associated with:
(i) anti-glomerular basement membrane antibodies (e.g., Goodpasture’s syndrome); (ii)
immunocomplex disease (e.g., systemic lupus erythematosus and IgG-IgM cryoglobulinae-
mia); and (iii) pauci-immune systemic vasculitis (e.g., Wegener’s granulomatosis or microsco-
pic polyarteritis). Whether an “idiopathic” variety of extracapillary glomerulonephritis exists
is still uncertain.
Pathology. Extracapillary proliferation (i.e., crescents in > 50% of glomeruli) with or
without glomerular fibrinoid necrosis is the distinguishing lesion (Figure 5.2, top right
and bottom right). While Goodpasture’s syndrome is characterized by linear deposition
of IgG along the glomerular basement membranes, by immunohistology immunocomplex
diseases are associated with granular deposition in the glomeruli of immunoglobulins and/
or complement. Pauci-immune systemic vasculitis, instead, shows negative or scanty and
unspecific immunohistological findings.
Clinical course. When left untreated, extracapillary glomerulonephritis of whatever
cause leads rapidly to irreversible renal failure. Early recognition is crucial, since prompt
therapeutic intervention with high-dose steroids and cytotoxic drugs, with or without plasma
exchange, reverses the renal prognosis and may even bring about healing.
urinary findings
In the active phase, extracapillary 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 [16]. Also in our experience,
haematuria was always present and very severe, and was associated with marked erythrocytic
cylindruria in > 89% of cases (Table 5.1). Mild to moderate leukocyturia and shedding of
RTECs were also frequent (84.2% each), as well as RTEC casts (68.4%) and fatty casts
(42.1%). Leukocytic casts were observed in > 31% of cases.
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 [17] and a rise in the number of urinary erythrocytes [18]. One must be aware
that these findings can be observed in whatever type of extracapillary 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 [19].
lupus nephritis
Definition. Lupus nephritis (LN) is a frequent manifestation of systemic lupus erythe-
matosus, and contributes substantively to its morbidity. LN can occur in either asymptomatic
patients or in patients suffering from extrarenal symptoms such as fever, arthritis, dermatitis,
pleuritis, etc.
Aetiology and pathogenesis. Systemic lupus is a disease due to a wide variety of
autoantibodies directed against nuclear components such as nucleosome, quaternary antigens
of the chromatin and small nuclear and cytoplasmic ribonucleoproteins. The renal disease is
caused either by in situ formation of immunocomplexes or by the trapping in the kidneys of
circulating immunocomplexes containing aggregates of immunoglobulins and complement
components.
Pathology. A wide spectrum of renal lesions is possible in LN, for which six different
immunohistologic classes are recognized: minimal mesangial nephropathy (class I),
mesangial proliferative disease (class II), focal proliferative glomerulonephritis (class III),
diffuse proliferative glomerulonephritis (class IV) (Figure 5.2, bottom left), membranous LN
(class V), and advanced sclerosing LN (class VI). In addition, class III and class IV can be
either active (proliferative) or inactive (sclerosing) or a combination of both lesions; class V
can be associated with proliferative aspects (the so-called mixed forms); transformation from
one class to another is possible; various extraglomerular lesions are possible such as acute
interstitial nephritis, vasculitis, vasculopathy, and thrombotic microangiopathy.
Immunohistology varies according to the histological class and proliferative/chronic lesions.
In proliferative active disease, there are usually abundant glomerular and extraglomerular
(tubular, interstitial and/or vascular) deposits of immunoglobulins and complement, which
are not seen in the sclerosing lesions.
Clinical course. LN is characterized by a remitting and relapsing course. Modern
therapeutic strategies neither cure lupus nor completely prevent relapses. Each major relapse
(either nephritic or proteinuric) can leave residual and cumulative irreversible renal damage,
which is often subclinical. The more episodes of relapses, especially of the nephritic type, the
greater the likelihood is of progression to chronic renal failure [20].
urinary findings
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 [21]. Early diagnosis of renal disease is of paramount
importance, and patients with known or suspected lupus should undergo urinalysis at regular
intervals [20].
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 [22].
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 [23], 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), as shown by the preliminary results of a study in progress at our renal unit on
patients with LN (Table 5.2). However, one must be aware that rare cases of active lupus
nephritis with minor urinary changes and inactive sediment can occur [24,25].
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 [26].
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 (Table 5.2).
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 [23].
In a lupus patient with a progressive increase in serum creatinine, it is extremely important
to interpret the 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

Definition. Schönlein-Henoch purpura (SHP) is among the most frequent systemic diseases
in children. The typical cases present with symmetrical skin purpura, arthralgia, abdominal
pain and bloody diarrhoea associated with mild urinary abnormalities or with nephritic or
nephrotic syndrome (see Chapter 6). Renal function can be either normal or impaired, with a
proportion of patients presenting with rapidly progressive renal failure.
Aetiology and pathogenesis. SHP is considered to be the systemic form of IgAN, with
similar pathogenetic aspects (see above). Some foods, drugs, infections or even insect stings
are possible triggering factors.
Pathology. A wide spectrum of glomerular lesions is possible, from minimal change
disease to extracapillary glomerulonephritis. Mesangial proliferative glomerulonephritis,
however, is the most frequent form. As in IgAN, the diagnostic change is the predominance
of IgA deposits in the mesangial areas.
TABLE 5.2 Urine sediment findings in lupus nephritis.
Class III Class IV Class V
Number of patients 6 15 5
M/F 0/6 1/14 1/4
Age
39.0 ± 14.2 32.5 ± 11.4 31.8 ± 5.8
mean ± SD
25-63 13-70 24-39
range
S-Creatinine
0.8 ± 0.3 1.5 ± 0.9 1.4 ± 1.7
mean ± SD
0.5-1.2 0.1-3.5 0.4-4.5
range
N with proteinuria 6 (100%) 15 (100%) 5 (100%)
mean ± SD (g/24h) 1.1 ± 0.5 4.3 ± 3.4 1.9 ± 1.7
range 0.7-2.0 0.9-10.6 0.2-4.6
N with haematuria 6 (100%) 6 (40%) 3 (60%)
mean ± SD * 478.8 ± 315.9 488.9 ± 547.8 26.3 ± 4.9
range 24-864 68-2000 23-32
N with leukocyturia 6 (100%) 13 (86.6%) 2 (40%)
mean ± SD * 307.7 ± 381.7 346.9 ± 412.2 51 ± 26.9
range 34-1048 37-1279 32-70
N with RTECs 6 (100%) 9 (60%) 2 (40%)
mean ± SD * 11.3 ± 15.2 12.4 ± 8.7 14.0 ± 18.4
range 2-42 1-29 1-27
N with RBC casts 5 (83.3%) 14 (93.3%)
1 (20%)
mean ± SD + 13.2 ± 45.0 5.9 ± 4.3
--
range 7-19 1-15
N with WBC casts 3 (50.0%) 5 (33.3%)
mean ± SD + 5.3 ± 3.2 5.4 ± 6.1 0
range 3-9 1-16
N with RTEC casts 4 (66.6%) 10 (66.6%) 2 (40%)
mean ± SD + 9.0 ± 5.9 5.8 ± 4.0 6.5 ± 3.5
range 1-14 1-12 4-9
N with fatty casts 6 (100%) 12 (80%) 2 (40%)
mean ± SD + 7.8 ± 4.2 13.1 ± 15.3 1.5 ± 0.7
range 4-15 3-62 1-2
Haematuria and leukocyturia were defined as > 20 erythrocytes (RBCs) and > 20 leukocytes (WBCs)/20 high power
fields (HPFs) at x 400 respectively. * = RBCs, WBCs and renal tubular epithelial cells (RTECs) were expressed
as number counted over 20 HPFs. Since, for RTECs, no cut off for normality exists, all cells were considered and
calculated in the mean ± SD (while for RBCs and WBCs the mean ± SD was calculated considering only the samples
with > 20 cells/20 HPFs). For each sample, 100 casts were looked for, which were classified in 8 categories. In this
table only 4 types of casts were reported as number/100 (+). All samples were examined a few hours before renal
biopsy and were prepared according to the standardized method described in Chapter 1, Table 1.6.
Clinical course. Patients presenting with only mild urinary changes usually have a favourable
long-term prognosis. Chronic renal failure, however, may develop in patients presenting with
nephritic or nephrotic syndrome.
urinary findings
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 [27].
diabetic nephropathy
Definition. Together with retinopathy and neuropathy, diabetic nephropathy (DN) is a
microvascular complication of both type 1 and type 2 diabetes mellitus. In type 1 diabetes,
DN occurs usually 10-15 years after the onset of the disease, while in type 2 diabetes (because
of uncertainty about the onset of the disease), it occurs at variable intervals after diagnosis.
Poor glycemic control and duration of diabetes are the major risk factors for DN, which
today is the leading cause of end stage renal failure in the developed world. Hyperfiltration
(i.e., increase in glomerular filtration rate) and microalbuminuria (i.e., excretion of albumin
in the range of 30-300 mg/24 hours) are the typical signs of early or incipient DN. Overt
DN is characterized by persistent detectable proteinuria, which over time tends to reach the
nephrotic range, with high blood pressure, and progressive renal insufficiency.
Aetiology and pathogenesis. The mechanisms by which hyperglycemia leads to DN are not
entirely elucidated. However, in various cell culture studies it has been shown that glucose
induces hypertrophy, extracellular matrix deposition, and TGF-} production, and that chronic
hyperglycemia can lead to nonenzymatic glycation of advanced glycated products. These are
increased in the serum of patients with DN, and have also been localized in diabetic glomeruli.
Pathology. The glomeruli show uniform and diffuse thickening of the glomerular basement
membrane, due to collagen IV accumulation, and mesangial expansion. This can be either diffuse
or nodular being, expanded by nodules of periodic acid-Schiff (PAS)-positive material (the so-
called nodular form of Kimmelstiel Wilson’s glomerulosclerosis) (Figure 5.1, bottom right).
Clinical course. Over the course of several years, progression to end-stage renal failure in
association with nephrotic range proteinuria and high blood pressure is virtually certain.
urinary findings
Initially, only microalbuminuria is found, the detection of which necessitates the use of
specific methods such as immunochemical assays utilizing antialbumin 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 12.5% to 35% of patients with
type 1 clinically diagnosed DN [28,29], and in 15% to 35% [30,31] of patients with
biopsy-proven DN associated with type 2 diabetes mellitus. More recently, microscopic
haematuria, defined as Γ 8 erythrocytes/μL, was 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 [32]. Interestingly, Γ 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.
All these findings support a previous study in which a discrete microscopic haematuria
with erythrocyte casts was found in 5 patients with biopsy-proven uncomplicated diabetic
nephropathy and in another 4/30 patients with clinically diagnosed DN [33].
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 [34].
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 [35]. 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

Definition. Plasma cell dyscrasias are neoplasms characterized by the production of
monoclonal immunoglobulins or their fragments by a clone of pathological B cells. Due
to the high renal plasma flow, the glomerulus is the first structure in the body in which
monoclonal proteins are deposited.
Aetiology and pathogenesis. The renal damage is the consequence of either glomerular
ultrafiltration of the monoclonal proteins (myeloma cast nephropathy) or their deposition
within the glomeruli, tubules and vessels (AL amyloidosis and light chain deposition
disease).
Pathology. Monoclonal gammopathies may cause different renal diseases, the main ones
being: (i) myeloma cast nephropathy, whose distinguishing feature at renal biopsy is the
presence of intratubular casts surrounded by multinucleated macrophagic cells; (ii) AL
amyloidosis, in which amyloid deposits involve glomerular and extraglomerular structures;
and (iii) light chain deposition disease (Figure 5.1, bottom left), in which the monoclonal
light chain deposits mainly along the tubular basement membranes and along the walls of
glomerular capillaries.
Clinical course. Progressive renal insufficiency with episodes of acute renal failure,
favored by dehydration or the use of non steroidal anti-inflammatory drugs, is typical of
myeloma cast nephropathy. Typical cases with amyloidosis or light chain deposition disease
are associated with nephrotic range proteinuria and progressive renal insufficiency.
urinary findings
In patients with myeloma cast nephropathy, there usually is a proteinuria which is due
mainly 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 light chains.
Conventional urine microscopy is usually unspecific in myeloma cast nephropathy.
However, in stained samples “myeloma cells” may occasionally be found in the urine
[36,37]. These are cells with oval to round eccentric nuclei with 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 [38].
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, while we have found it
in ~ 65% of patients with light chain deposition disease [39].
In both amyloidosis and light chain deposition disease, the appearance of a nephritic
sediment may herald the superimposition of extracapillary proliferation.
By immunofluorescence microscopy applied to urinary sediment, we found that patients
with monoclonal gammopathies excreted casts which contained predominantly the
monoclonal light chain responsible for the disease [40].
Several electron microscopy studies were devoted in the past to the search for amyloid
fibrils in the urine sediment. After the first apparently positive results, it was shown that this
type of investigation obtained uncertain results [41].
acute interstitial nephritis

Definition. The term acute interstitial nephritis (AIN) encompasses a heterogenous group
of disorders which have in common the rapid decline of renal function associated with
acute infiltration of the renal interstitium by different types of cells, while the glomeruli are
unchanged.
In antibiotic-related forms, the patient often presents with fever, skin rash, arthralgia and
sometimes gross haematuria. Symptoms are less defined and more variable in the other
forms.
Aetiology and pathogenesis. Most frequently, AIN results from hypersensitivity to a drug
(more often antibiotics, especially }-lactamic antibiotics, and non-steroidal anti-inflammatory
agents). However, AIN may also be due to immunological disorders (e.g., systemic lupus,
Sjögren’s syndrome) or complex bacterial or viral infection (e.g., Hantaviruses). For some
patients, no cause can be identified (e.g., AIN associated with uveitis).
Pathology. Interstitial cellular infiltrates, interstitial erythrocyte extravasation, interstitial
oedema, tubulitis and tubular necrosis are the distinguishing features of AIN (Figure
5.3, left). In some patients, granulomata can also be present. Interstitial eosinophils are a
frequent finding, especially in antibiotic-related AIN. In the forms caused by non-steroidal
anti-inflammatory drugs, a glomerular disease (minimal change disease or membranous
nephropathy) is also very frequently present.
Clinical course. In AIN secondary to drugs, if the drug is discontinued, renal function
usually improves, with the improvement being favoured by the use of corticosteroids.
Permanent renal damage is possible if diagnosis and treatment are delayed. In AIN due to
immunological disorders or to bacterial or viral infections, the renal disease heals with the
treatment of the underlying immunological or infectious disease. AIN associated with uveitis
usually reverses with corticosteroids.
urinary findings
In }-lactamic-related AIN, proteinuria is usually mild. Leukocyturia and haematuria may be
absent in the initial phase of the disease, while they are found in all patients in the full-blown
phase (gross haematuria is observed in 60% of cases). Eosinophiluria seems to be always
present [42]. Urinary changes clear up in days to weeks after the withdrawal of the drug [43].
The urinary changes caused by other antibiotics are less well defined. However, they seem
to be similar to those described for }-lactamic antibiotics.
In AIN due to non-steroidal anti-inflammatory drugs, due to the concomitant glomerular
disease, proteinuria is a constant finding, and frequently it is in the nephrotic range. Occasionally,
the urinary sediment is normal or mildly changed, but most frequently it is characterized
by leukocyturia, haematuria or casts (hyaline, granular, waxy, containing leukocytes but not
erythrocytes). Renal tubular epithelial cells are observed in ~ 15% of patients, and lipiduria in
~ 20%. Eosinophiluria seems to be rare, but from the review of the literature it is unclear how
extensively it has been studied in patients with this variety of AIN [43].
In AIN associated with bacterial infections, proteinuria is frequent but is usually < 1.5
g/24 hours. Haematuria is seen in 80% of cases, and is almost invariably microscopic.
Surprisingly, leukocyturia is rare [43].
Of the forms of AIN associated with viral infections, that caused by Hantaviruses (which
are transmitted to man by rodents, and are present especially in Korea, Japan, China, Eastern
Europe and Scandinavia) is the best studied. Proteinuria is seen in almost all patients, and
is frequently in the nephrotic range. Microscopic haematuria is observed in ~ 75% of cases,
but it is frequently very mild and of short duration. Occasionaly, however, there may even
be gross haematuria. Leukocyturia seems to be more frequent than microscopic haematuria.
With the recovery of renal function, the urinary changes also reverse [43].
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 both, but it may even
be normal. In only a few patients has eosinophiluria been found [43].
Two aspects of urinary sediment in AIN deserve a separate comment.
FIGURE 5.3 Left: acute interstitial nephritis associated with uveitis. Note the two intratubular casts (arrows). This
finding explains the abundant cylindruria which can be observed in this condition (AFOG stain, × 100). Right: chronic
interstitial nephritis with typical interstitial fibrosis (PAS stain, × 250).
eosinophiluria – is it a specific marker of ain?

Eosinophiluria is usually considered as a marker of AIN. Old studies support this view
[42] as well as several case reports in which AIN was caused by a wide spectrum of drugs
including ciprofloxacin, omeprazole, vancomycin, fluindione, or linezolid [44-48]. 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 [43]. 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. [49-53]. In a study by Ruffing et al. [54], eosinophiluria was found in
only in 6/15 patients with clinically diagnosed AIN, and in 10/36 patients with other renal
disease. Thus, eosinophiluria as a marker of AIN had a 40% sensitivity, a 72% specificity,
and only a 38% positive predictive value.
For all these reasons, it is our opinion that eosinophiluria can no longer be seen as a
specific marker of AIN.
erythrocytic casts – can they be found in the urine of patients with ain?
It is commonly thought that erythrocytic casts are so rare in AIN that their presence
should suggest the diagnosis of a glomerular disease [55, 56]. However, erythrocytic casts
were described in a patient with AIN by Sigala et al. [57], and we have recently found many
erythrocytic casts associated with a severe isomorphic microscopic haematuria in the urine
sediment of a patient with AIN due to amoxycillin + clavulanic acid. 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. [9].
Thus, the possibility that erythrocytic casts can be found in the urine of patients with AIN
should be considered and further observation and investigation are needed.
chronic interstitial nephritis

Definition. The term chronic interstitial nephritis (CRIN) encompasses a heterogeneous
group of disorders which have in common the progressive scarring of the renal
tubulointerstitium, a process which is characterized by tubular atrophy, mononuclear cellular
infiltration, and interstitial fibrosis.
Aetiology and pathogenesis. Drugs (e.g., analgesics, cisplatin, lithium, cyclosporin), heavy
metals (e.g., lead, cadmium), metabolic disorders (e.g., hyperuricaemia, hypercalcaemia,
hyperoxaluria), urinary tract infections, chronic urinary tract obstruction, radiation, immunological
disorders (e.g., systemic lupus erythematosus, Sjögren’s syndrome), haematopoietic disorders
(e.g., sickle cell disease, multiple myeloma), vascular diseases (e.g., nephrosclerosis), hereditary
diseases (e.g., medullary cystic disease, hereditary nephritis), granulomatous diseases (e.g.,
sarcoidosis) and endemic diseases (e.g., Balkan nephropathy, nephropatia epidemica) can all
cause CRIN. In addition, idiopathic forms are possible.
Tubular injury of whatever cause results in the release of chemotactic substances and the
expression of leukocyte adhesion molecules which attract macrophages and T-cells into the
interstitium. This leads to the release of growth factors which stimulate fibroblast proliferation
and activation, which results in interstitial matrix accumulation.
Pathology. The typical renal lesions of CRIN are represented by: tubular atrophy with
flattened epithelium, tubular dilatation, and tubular basement membrane thickening;
interstitial fibrosis, due to the accumulation of collagen, and patchy or diffuse mononuclear
cell infiltrate, due to monocyte-macrophages and, especially, T-lymphocytes (Figure 5.3,
right). Glomeruli are normal or only mildly changed.
Clinical course. It is typically indolent. The early manifestations, which are those of tubular
dysfunction, go often undetected. A slowly progressive renal insufficiency is typical.
urinary findings
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 } 2-
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, and proteinuria (40-50% of cases),
which is usually < 1 g/24 h. Microscopic haematuria is seen in 30-40% of patients, 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 [58].
A worsening of haematuria with or without atypical urothelial cells (see below urological
disorders) 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

Definition. In hospitalized patients, acute tubular necrosis (ATN) and prerenal uremia are
the two most frequent causes of acute renal failure (ARF) (i.e., rapid decline of renal function
over hours to weeks). The term ATN should be applied to cases of ARF in which the renal
biopsy shows the typical changes of tubular cell damage. In clinical practice, however, the
diagnosis of ATN is often made on clinical grounds only.
Aetiology and pathogenesis. ATN is usually due to ischemic or nephrotoxic injury. Thus
the main causes of ATN include: shock/hypovolemia, nephrotoxic drugs (e.g., gentamicin,
acyclovir, foscarnet, paracetamol, herbal remedies) or nephrotoxic substances (e.g., cadmium,
mercury, ethylene glycol, radiographic contrast, myoglobin, haemoglobin).
The pathogenesis of ATN is complex, being due not only to tubular factors, but also to
impaired haemodynamic factors, endothelial cell injury, and inflammatory factors.
Pathology. Dilatation of the tubular lumen with flattening and degenerative/necrotic
changes of tubular epithelium, with consequent sloughing of tubular cells from the tubular
basement membrane, is the diagnostic lesion of ATN (Figure 5.4). In addition, intraluminal
cellular debris and casts (granular and renal tubular epithelial cell [RTEC] casts), and
interstitial oedema are usually seen.
Clinical course. ATN is a potentially reversible condition. In uncomplicated cases recovery
occurs over 2 to 3 weeks. The patient must be sustained through the phase of renal failure by
conservative treatment or appropriate dialysis methods.
FIGURE 5.4 Acute tubular necrosis. Left: greatly damaged tubular cells (arrowheads) and partial loss of the tubular
epithelium (arrows) (trichrome stain, x 400); right: an intratubular finely granular cast (arrowheads) and an intratu-
bular cast containing renal tubular epithelial cells (arrows) (trichrome stain, x 400).
TABLE 5.3 Main urinary parameters to distinguish between prerenal ARF and ARF due to acute
tubular necrosis (modified from ref. 59).
Parameter Prerenal Renal
Specific gravity >1.020 ~1.010
Osmolality (mmol/kg) >500 >300
Sodium (mmol/L) <20 >40
Fractional excretion of Na (%) <1 >2

urinary findings
Some urinary parameters are very important for differentiating the patient with prerenal
ARF from the patient with ATN (Table 5.3) [59]. In addition to them, the examination of the
urinary sediment, when performed by a skilled practitioner, provides valuable and unique
information about the events occurring within the kidney [60,61].
In ATN, urine sediment shows variable numbers of RTECs, both necrotic and viable [62-64],
at times even fragments of the tubular epithelium, and abundant cylindruria (granular casts
and RTEC casts) [64b]. In addition, depending on the cause of the tubular damage, other
particles can be seen [65,66]. 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 [67] or haemoglobinuria;
• the presence of a massive crystalluria can suggest ethylene glycol poisoning (which
causes atypical spindle-like monohydrate calcium oxalate crystals) [68], acute uric
acid nephropathy (which causes uric acid crystals) [69], or ATN due to intrarenal
precipitation of a drug. In this last case, 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), as described in detail in Chapter 3;
• the presence of severe haematuria associated with erythrocytic casts strongly suggests
a primary or secondary proliferative glomerulonephritis in an active phase (see above
extracapillary glomerulonephritis) [70].
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-}-glucosaminidase, and alanine aminopeptidase [59].
renal transplantation
Renal transplantation represents the treatment of choice for many uraemic patients. With
the available immune suppressive drugs, the graft and patient survivals are excellent for
recipients of either living-related or cadaveric kidneys. However, several complications can
occur after transplantation, for some of which the examination of the urinary sediment can
be of valuable help.
Acute cellular rejection

It is a condition mainly due to cytotoxic T-lymphocytes specifically directed against donor
antigens.
In the typical acute “cellular” rejection, renal biopsy shows interstitial cellular infiltrates due
to activated lymphocytes, monocytes/macrophages, plasma cells and polymorphonuclear cells.
Cellular infiltrates can involve the tubules, which can cause the so-called “tubulitis” and even
patchy acute tubular necrosis. In the most severe cases, there also are vascular lesions with
intimal proliferation, fibrinoid necrosis of the media and polymorphonuclear cell infiltration.
An abrupt increase of serum creatinine is the typical clinical sign.
urinary findings
In early studies, several investigators demonstrated that acute cellular rejection was
frequently associated with the occurrence of lymphocyturia or with a sharp increase in pre-
existing lymphocyte excretion. Various types of stains as well as very different criteria for
defining lymphocyturia were used with variable results. Other investigators found that the
appearance of a large number of renal tubular cells was a more reliable index of rejection
[71], especially when it was associated with oxalate crystals, a dirty background, increased
erythrocyturia, mixed cell clusters, lymphocytes and cellular mitoses [72]. More recent
approaches include the identification of lymphocytes and tubular cells by monoclonal
antibodies [73], the search of both T lymphocytes and of HLA-DR expression on tubular
cells [74], the use of flow cytometry to demonstrate increased excretion of HLA-DR- and
CD3-positive cells [75], or the use of enzyme-linked antibodies directed to lymphocytes
subpopulations and monocyte/macrophages [76].
Clearly, this type of investigation can be performed in cytopathology or research
laboratories only, without forgetting that the final diagnosis of acute rejection is based on
renal biopsy findings.
Polyomavirus BK infection
In recent years, this viral infection has become an important clinical problem in kidney
transplant recipients due to the use of powerful immune suppressive agents such as tacrolimus,
mycophenolate mofetil, or sirolimus.
Polyomavirus BK (BKV) is a DNA virus which belongs to the family of Papovavirus.
Primary infection occurs during infancy through the respiratory or gastroenteric tract without
clinical signs. About 80% to 90% of the general population have antibodies against BKV,
which in the latent state resides within the transitional epithelium of the uropethelium and
within the tubular cells of the renal medulla.
In renal transplant recipients under the effect of pharmacological immune suppression,
BKV can reactivate and cause a renal disease known as BKV nephropathy (BKVN). This has
a 2% to 9% prevalence, occurs early after transplantation, and is heralded by a progressive
increase of serum creatinine in the absence of symptoms. Irreversible loss of the kidney
occurs in about one fourth of the affected patients.
At renal biopsy, the diagnostic lesion is represented by cytopathic changes in the tubular cells,
whose nuclei are heavily altered by the virus (Figure 5.5). In most cases, these cells can easily
be identified by traditional histological techniques, but sensitivity is increased by the use of
FIGURE 5.5 BKV nephropathy, with typical tubular BKV-infected cells. Top left: a tubular cell with an enlarged nu-
cleus which has a ground glass appearance (arrow) (haematoxylin & eosin, x 400); top right: a tubular cell with an
enlarged nucleus surrounded by a clear halo (arrow) (AFOG stain, x 400); bottom left: a tubular cell free in tubular
lumen with a peripheral clumping of nuclear chromatin (arrow) (PAS stain, x 400); bottom right: an intrabutubular
granulo-cellular cast which contains a BKV-infected cell with an enlarged nucleus and an evident dark inclusion body
(arrow) (PAS stain, x 250).
monoclonal antibodies against the SV 40 T-antigen of the virus. These changes are often associated
with interstitial cellular infiltrates with or without tubulitis and patchy tubular necrosis.
Treatment is based primarily on the reduction of immunosuppressive treatment, to which
cidofovir or leflunomide may be added.
urinary findings
Urine sediment examination plays an important role in recognizing the reactivation of
BKV and in the diagnosis of BKVN. In fact, these two conditions are associated with the
shedding in the urine of virus-infected cells, the so-called decoy cells.
These cells are usually identified by Papanicolaou stain on cytocentrifuged urine samples
(Figure 5.6) and show a number of changes at nuclear level, which consist in:
• nuclear enlargement, which confers a “ground glass” appearance and displacement of

the nucleus toward the periphery of the cell as if the nucleus were “escaping” from the
cell (the most common change);
• chromatin margination, which is chromatin clumping along the nuclear membrane
(common);
• abnormal chromatin patterns (e.g., coarse granules of variable size and shape, with
irregular arrangement) (common);
• single nuclear inclusion body surrounded by a peripheral halo, which confer a bird’s
eye appearance to the cells (rare);
• cytoplasmic vescicles (common).
All the changes described above are confirmed by transmission electron microscopy,
which in addition shows the presence, within the nucleus, of variable amounts of viral
particles with a diameter of about 45 Å (Figure 5.7).
For all these changes, decoy cells may at times mimic atypical uroepithelial cells as can
be found in inflammatory diseases and, especially, in malignant neoplasms of the excretory
urinary tract (see below urological disorders). Decoy cells can also mimic cells infected
by cytomegalovirus, especially when they have the so-called “bird’s eye” appearance (77).
The clinical setting and the measurement of appropriate biomarkers are mandatory in such
cases.
The value of decoy cells in diagnosing the reactivation of BKV and BKVN has extensively
been investigated. Dranchemberg et al. found a significant correlation between the number of
decoy cells in the urine and the occurrence of BKVN [78] and between the number of decoy
cells and the histological severity of BKNV [79]. Nickeleit et al., who were among the first in
describing BKVN in renal transplant recipients [80], found that decoy cells diagnosed BKVN
with a 100% sensitivity, a 95% specificity and a 100% negative predictive value. However the
positive predictive value was only 27% [81].
These latter findings were confirmed by Hirsch et al., who performed a prospective study
on 78 renal transplant recipients treated with either tacrolimus (37 patients) or mycophenolate
mofetil (41 patients) followed for 85 weeks [82]. In addition, by comparing decoy cells with the
quantitation of BKV DNA in the blood by real-time quantitative PCR, they found that the latter
increased on average later than decoy cells (28 weeks post-transplant vs 23 weeks) and that it
FIGURE 5.6 Decoy cells as seen by Papanicolaou stain.

All cells but one (bottom right) show an increase of the
nuclear size; all cells have abnormal chromatin patterns
and nuclear inclusion bodies of different size and shape.
The cells at top left and at bottom right also show cyto-
plasmic vescicles of different size (original magnification
x 400).
FIGURE 5.7 Decoy cells as seen by transmission electron microscopy. Left: a damaged tubular cell with enlarged
nucleus, chromatin (black granules) margination towards the nuclear membrane and intranuclear virus particles
(gray dots)(original magnification x 7,000); right: BK virus particles at high magnification (x 60,000)(arrows).
FIGURE 5.8 Decoy cells as seen by phase contrast mi- FIGURE 5.9 Other types of decoy cells as seen by
croscopy. Top, left and right: cells with an enlarged nu- phase contrast microscopy. Top and middle: cells with an
cleus and a typical ground glass appearance; middle: a “eye bird” appearance. This is due to the presence of one
cell showing an enlarged nucleus with several inclusion round, large nuclear inclusion body surrounded by a clear
bodies; bottom: a cell with a nucleus of normal size but halo; bottom: a cell with a nuclear inclusion body with a
with several inclusion bodies. Note the cytoplasmic vesi- “horseshoe” shape (original magnification, x 400).
cles (original magnification, x 400).
FIGURE 5.10 A urinary cast containing several decoy

cells with enlarged nucleus (arrows) as seen by phase
contrast microscopy (original magnification, x 400).
had a higher positive predictive value than decoy cells (50% vs 29%). More recently, Thamboo
et al. confirmed the utility of the search for decoy cells in the urine [83]. Furthermore, they
demonstrated that a significant number of decoy cells could already be found as early as the
second month after transplant, a finding which reinforces the value of decoy cells in monitoring
the patients at risk of reactivation of BKV.
From all this, it appears that the finding of decoy cells in the urine is a reliable indicator of
the reactivation of BKV, while BKVN can be suspected only if, besides decoy cells, increased
blood levels of BKV-DNA and increased serum creatinine are also found. Clearly, only the
renal biopsy can demonstrate with certainty the presence of BKVN.
In our laboratory, we currently perform the search for decoy cells in the urine of renal transplant
recipients. However, we do not use Papanicolaou staining on cytocentrifuged samples. We just
prepare the samples according to the usual method adopted in our laboratory for all urine
samples (see Table 1.6), search for decoy cells by phase contrast microscopy only without
any stain, and express the result as number of decoy cells counted over 50 HPFs at x 400.
In our experience, this method is simple and quick, avoids the need for sending the samples
to a specialized cytology laboratory, and is adequate for the identification of decoy cells
(84-86) (Figure 5.8-5.10).
We compared our method with Papanicolaou stain applied to cytocentrifuged smears on
48 urine samples, all containing decoy cells from 18 kidney transplant patients (unpublished
data). No major differences were seen in the detection of decoy cells, while Papanicolaou
technique in our hands was more time consuming (on average 40 minutes vs 15 minutes/
sample). We also compared our method with electron microscopy, and the latter confirmed
the presence of various quantities of intranuclear viral particles in 11/11 samples.
De novo or recurrent glomerulopathy

Glomerular diseases such as focal segmental glomerulosclerosis, IgA nephropathy,
membranous nephropathy, extracapillary glomerulonephritis and many others, can develop
in renal transplant recipients, either as a recurrent or a de novo disorder.
urinary findings
Urinalysis is very important in heralding the appearance of such disorders, either by
showing previously absent proteinuria or urine sediment changes, such as isolated microscopic
haematuria [87] or haematuria in combination with other changes as described above for the
glomerular diseases occurring in native kidneys.
urinary tract infection

Urinary tract infections (UTI), especially acute uncomplicated cystitis, are among the
most common medical disorders all over the world. The definitive diagnosis is based on
the presence of Γ 105 bacteria/ml of voided midstream urine, even though up to one third of
patients with cystitis may have lower colony counts.
urinary findings
Bacteriuria and leukocyturia are the hallmarks of urinary sediment in patients with UTI.
Superficial transitional epithelial cells, isolated or in aggregates, are also frequent. In acute
pyelonephritis, in which the infection involves the renal parenchyma, leukocyte casts and/or
bacterial casts can be found [88].
Usually there is a good correlation between the urine sediment findings and the results
obtained by urine culture [89-92]. However, urine microscopy can be limited by some false-
positive and false-negative results.
False-positive results occur especially due to:
• urine contamination from genital secretions, mostly in women with vaginitis. Such
cases, however, can usually be differentiated from true UTI by the presence of large
numbers of squamous epithelial cells of vaginal origin [93]. In our experience,
this condition frequently is recognized also by the co-existence of Candida or, less
commonly, Trichomonas vaginalis;
• the bacterial overgrowth which can be observed when urine is not handled and analysed
immediately after micturition, a fact which happens in most laboratories all over the world.
False-negative results, on the other hand, can be caused by:
• misintrepretation of bacteria (cocci are less easily identifiable than rods, and some
cocci can be confused with amorphous crystals);
• lysis of leukocytes, which is frequently seen when samples are left standing [90],
especially if urine pH is alkaline and/or specific gravity is low (e.g., Φ 1.010), and/or
the sample is analysed several hours after micturion.
urological disorders
Urological disorders such as cancer, urolithiasis, urinary tract obstruction, or hydronephrosis
are very frequently associated with urinary sediment changes.
urinary findings
Proteinuria is usually absent in urological disorders. However, in patients with renal
carcinoma, proteinuria can appear due to the invasion of the renal vein or the inferior vena cava
by a malignant thrombus [94], or a superimposed glomerular disease triggered by neoplastic
antigens. Heavy proteinuria can also be found during episodes of gross haematuria, which
is due to the fact that with bleeding, plasma proteins also enter the urinary tract. Proteinuria
may also be caused by the lysis of erythrocytes [95].
Urinary sediment abnormalities, in spite of the heterogeneous nature of urological disorders,
are restricted to microscopic or gross haematuria, leukocyturia, and/or the exfoliation of
transitional cells from the uroepithelium.
Microscopic haematuria is typically isomorphic and, in contrast to what is usually observed
in glomerulonephritis, it often shows a poor quantitative correlation with the severity of the
urological disease. This is especially true for bladder cancer, for which it has repeatedly been
demonstrated that large bladder tumours can cause mild haematuria (i.e., a few erythrocytes
per high-power field), and vice versa [96,97].
Transitional uroepithelial cells derive, in most instances, from the superficial layers of the
uroepithelium, while on some occasions they derive from its deeper layers. In our experience,
when the cells of the deep layers are present in large numbers (i.e., Γ 1/high-power field), the
presence of severe urological disorders is very likely [98] (see also Chapter 2).
FIGURE 5.11 Urothelial malignant cells as found by phase contrast microscopy in the urine of two patients with
bladder carcinoma confirmed by cystoscopy and histological examination. Patient 1.Top left: a cell with a very unusual
spindle-like shape and size (61 x 23 μm) and some isomorphic erythrocytes; top right: a mass of cells with unusual
shape. Patient 2. Bottom left: a bi-nucleated cell with unusual shape and increased size (88 x 23 μm); bottom right:
a very large (78 x 54 μm) and atypical cell with four nuclei (original magnification, x 400).
In clinical practice, the search for atypical “malignant” transitional uroepithelial cells in the
urine is widely used to diagnose and follow up the neoplasms of the urinary excretory system.
These cells are characterised by one or more of the following morphological changes [99]:
• increase of the diameter of the nucleus (> 25 μm)
• increase of the nuclear/cytoplasmic ratio
• multinucleation
• irregularity of nuclear shape
• increase in chromatin (hyperchromasia) with various chromatin patterns but especially
the reticulated and granulated ones
• increase in the size (> 5 μm) and/or number of nucleoli (> 3)
• unusual cellular shapes (snake, tadpole, etc.)
• appearance of cells in mass.
Usually, these changes are identified on fixed samples which have been stained by
Papanicolaou method. However, in our everyday work we have been able to identify atypical
transitional cells in several unfixed and unstained samples examined with phase contrast
microscopy (Figure 5.11), a finding which has been confirmed in a recent study [100].
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[98] FOGAZZI G.B., CARBONI N., PRUNERI G., et al. The cells of the deep layers of the urothelium in the
urine sediment: an overlooked marker of severe diseases of the excretory urinary system. Nephrol
[99] ITO K. et al. Color atlas of urinary cytology. St Louis: Ishyaku EuroAmerica Inc, 1992; 10-7.
[100] FERNÁNDEZ-ACENERO M.J., LORENCE D., CRIADO L. et al. Atypical cells in the urinary sediment: a
protocol for cytological analysis of the urinary sediment. Cytopathology 2008; 19: 381-4.
CHAPTER 6
INTERPRETATION OF THE URINARY
SEDIMENT FINDINGS
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 sediment; the nephritic sediment; the nephrotic
and nephritic sediment; the sediment with many renal tubular epithelial cells; the sediment
with increased numbers of erythrocytes; the sediment containing bacteria and leukocytes;
and the so-called minor urinary abnormalities.
This chapter integrates the information contained in Chapter 5.
the nephrotic sediment

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

• focal segmental glomerulosclerosis
• idiopathic membranous nephropathy
• diabetic nephropathy
• membranous lupus nephritis
• amyloidosis
• 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 (Figures 6.1 and 6.2 and Table 6.1).
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
FIGURE 6.1 The nephrotic sediment with massive li-

piduria (i.e. fatty casts and fatty droplets both isolated
and in aggregates) (phase contrast, × 160).
FIGURE 6.2 Left. An example of massive lipiduria (several oval fat bodies intermingled with lipid droplets of vari-
ous size) observed in a patient with nephrotic syndrome (phase contrast, original magnification x 160). Right. The
same image as seen by polarized light. Note the Maltese crosses.
Interpretation of the urinary sediment findings 213
TABLE 6.1 Urinary sediment findings in 52 patients with nephrotic syndrome due to minimal
change nephropathy (5 patients), focal and segmental glomerulosclerosis (9 pa-
tients), idiopathic membranous nephropathy (32 patients), and amyloidosis (6 pa-
tients).
Number of patients 52
M/F 30/22
Age
57.3 ± 16.5
mean ± SD
16-78
range
S-Creatinine
1.2 ± 0.6
mean ± SD
0.6-4.1
range
Proteinuria
7.5 ± 3.8
mean ± SD (g/24h)
3.5-20.2
range
N with haematuria 43 (82.7%)

mean ± SD * 124.4 ± 92.9
range 21-356
N with leukocyturia 12 (23.1%)

mean ± SD * 55.3 ± 38.4
range 21-143
N with RTECs 34 (65.4%)

mean ± SD * 4.0 ± 3.2
range 1-15
N with RBC casts 21 (40.4%)

mean ± SD + 1.8 ± 1.0
range 1-5
N with WBC casts 4 (7.7%)

mean ± SD + 1.5 ± 1.0
range 1-3
N with RTEC casts 32 (61.5%)

mean ± SD + 2.7 ± 2.8
range 1-16
N with fatty casts 49 (94.2%)

mean ± SD + 26.8 ± 17.4
range 2-66
Haematuria and leukocyturia were defined as > 20 erythrocytes (RBCs) and > 20 leukocytes (WBCs)/20 high power fields
(HPFs) at × 400 respectively. * = RBCs, WBCs and renal tubular epithelial cells (RTECs) were expressed as number counted
over 20 HPFs. Since for RTECs no cut off for normality exists, all cells were considered and calculated in the mean ± SD
(while for RBCs and WBCs the mean ± SD was calculated considering only the samples with > 20 cells/20 HPFs). For each
sample, 100 casts were looked for, which were classified in 8 categories. In this table only 4 types of casts were reported
as number/100 (+). All samples were examined a few hours before renal biopsy and were prepared according to the
standardized method described in Table 1.6.
glomerulosclerosis and idiopathic membranous nephropathy, and it is variable in diabetic

nephropathy. In these conditions, leukocyturia is uncommon and, if present, mild (Table 6.1).
Although lipiduria is the distinguishing feature of the nephrotic sediment, this is not invari-
ably present [1]. Lipids enter the urine because of impaired glomerular basement membrane per-
meability, 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 par-
tially reabsorbed by proximal tubular cells and transported for hydrolysis into lysosomes [2-5].
Then, they re-enter the tubular urine via regurgitation [2] or 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

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 glomerulonephtis
• extracapillary/necrotizing glomerulonephritis
• active class III and IV lupus nephritis
• Schönlein-Henoch purpura nephritis
• cryoglobulinaemic glomerulonephritis.
The hallmarks of the nephritic sediment are: erythrocyturia, leukocyturia, shedding of

RTECs and erythrocytic cylindruria (Figure 6.3 and 6.4).
This is confirmed by the findings we observed in 19 patients with extracapillary/necrotizing
glomerulonephritis (see Chapter 5, Table 5.1) and by the results we found in a study in which
we compared 52 patients with proliferative glomerular diseases with 48 patients with non
proliferative glomerular disorders [6]. In fact, the cohort of patients with a proliferative disorder
had significantly higher serum creatinine levels (2.1 ± 1.7 vs 1.3 ± 0.8 mg/dL), and a higher
prevalence of: microscopic haematuria (98.0 % vs 66.7%), leukocyturia (73.1% vs 18.8%),
FIGURE 6.3 The nephritic sediment with severe erythro- FIGURE 6.4 Marked erythrocytic cylindruria as can be
cyturia and cylindruria (granular and cellular casts) observed in nephritic sediment (phase contrast, x 160).
(phase contrast, × 160).
shedding of RTECS (82.7% vs 64.6%), erythrocytic cylindruria (84.6 % vs 39.6%), and RTEC
casts (94.2% vs 79.2%). These patients also had significantly higher numbers of: erythrocytes
(764.2 ± 720.4 vs 95.0 ± 137.1), leukocytes (64.2 ± 71.6 vs 13.1 ± 21.5), RTECs (8.4 ± 9.0 vs
2.6 ± 2.9) counted over 20 high power fields (x 400), and of erythrocytic casts (6.4 ± 7.9 vs 0.7
± 1.1) and RTEC casts (9.4 ± 10.5 vs 2.3 ± 3.0) expressed as number out of 100 casts/patient.
Proteinuria/24 hours, instead, was significantly higher in patients with non proliferative
glomerulopathies (5.0 ± 3.1 vs 2.9 ± 2.1). Based on the above urinary sediment findings, the
two groups of glomerulopathies could be correctly classified with 80.8% sensitivity and 79.2%
specificity. 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 [7-9], while they were not
found in non-proliferative glomerular disorders [10]. 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: (i) the
formation of casts within the tubular lumen, with entrapment into their matrix of erythrocytes
and leukocytes deriving from damaged glomeruli; and (ii) the co-existence of tubular
damage, which is observed in the most active forms of glomerulonephritis [11]. 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 [6,12,13]. 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 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 [14,15] or pauci-
immune renal vasculitis [16]. However, it is important to remember that 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 [17-19] and proliferative lupus nephritis [20,21].
In the nephritic sediment, haematuria is expected to be dysmorphic. However, in some
instances, it may be isomorphic in spite of the glomerular origin, which may be due to the
co-existence of renal insufficiency [22], necrotizing glomerulonephritis [23] or the use of
Henle’s loop diuretics [24].
the nephrotic and nephritic sediment

A urine profile with both nephrotic and nephritic features may be found in virtually
all proliferative glomerulonephritis. For example, we found it in 4/37 patients with IgA
nephropathy (10.8%), in 5/19 patients with extracapillary/necrotizing glomerulonephritis
(26.3%), and in 6/19 patients with class IV lupus nephritis (31.5%).
the urinary sediment containing many

renal tubular epithelial cells
This sediment 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. This pattern suggests the presence of acute tubular
necrosis as can be caused by hypotension or hypovolaemia [25-27].
• RTECs with brownish pigmented casts, which suggest myoglobinuria from
rhabdomyolysis [28,29], or haemoglobinuria.
• RTECs with a large number of erythrocytes, some leukocytes and erythrocytic casts,
which suggest the presence of a proliferative glomerulonephritis in active phase [11].
• RTECs with lipids, e.g., fatty droplets, oval fat bodies, fatty casts which 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), which can suggest acute interstitial nephritis.
• RTECs with crystals (e.g., uric acid, monohydrated calcium oxalate, 2,8 di-
hydroxyadenine crystals or crystals due to drugs), which suggest acute tubular damage
caused by intratubular precipitation of crystals [30,31].
the urinary sediment containing increased numbers

of erythrocytes
While it is accepted that there is some excretion of red blood cells (RBCs) in normal subjects,
there is no agreement at all on the number of RBCs which defines the pathological condition
known as microscopic haematuria (MH). Thus, while in our laboratory we define as MH the
finding of >1 RBC/high power field (x 400) (using the method described in Chapter 1, Table
1.6), for others the cut-off is established at very different levels (see Chapter 4, Table 4.3).
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 the
analysis of RBC morphology (see Chapter 2, page 42) it is possible to orientate the diagnosis,
at the very beginning of the workup, towards a glomerular (GH) or a non glomerular (NGH)
(= urological) origin of the haematuria [32,33]. The reliability of this approach has been
proven by different investigators.
Schramek et al. investigated 316 patients with IMH of unknown origin [34]. 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 [35]. 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.
We investigated 16 patients (10 children and 6 adults) with IMH, which was considered
of glomerular origin when there were Γ 40% dysmorphic RBCs and/or Γ 5% acanthocytes
[36]. 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%).
In another patient, with no glomerular changes at biopsy, a cluster of RBCs was found within
the renal tubules, which was an unequivocal sign of the renal origin of the haematuria. In the
same study, we also investigated the value of erythrocytic casts, and found that while they
had 100% specificity they had only 35.7% sensitivity. In spite of the different criteria used
to define glomerular or non glomerular haematuria, the results described above confirm that
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.
the urinary sediment containing bacteria and leukocytes

It is common knowledge that bacteriuria and leukocyturia are typical of urinary tract
infection. However, one must always remember that 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

This definition 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 described above is more frequently caused by
glomerular diseases when they are either mild or in the healing phase or evolving towards
chronicity.
One should also not forget that hyaline cylindruria with a few erythrocytes and leukocytes
may even be seen in normal subjects. To avoid misinterpretation, each laboratory shoud have
cut-offs for erythrocyturia and leukocyturia, a fact which is too often ignored.
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), which in our experience is
suggested by the finding of the same type of crystalluria in repeated samples.
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Nephrol 1988; 20: 429-38.
[5] STREATHER C.P., VARGHESE Z., MOORHEAD J.F. et al. Lipiduria in renal disease. Am J Hypertens 1993;
6: 353S-7S.
proliferative glomerular diseases. J Nephrol 2005; 18: 703-10.
[7] BURKOLDER P.M. Ultrastructural demonstration of injury and perforation of glomerular capillary
basement membrane in acute proliferative glomerulonephritis. Am J Pathol 1969; 56: 251-65.
[8] BONSIB S.M. GBM discontinuities. Scanning electron microscopic study of acellular glomeruli. Am J
Pathol 1985; 119: 357-60.
[9] BONSIB S.M. GBM necrosis and crescent organization. Kidney Int 1988; 33: 966-74.
[10] BONSIB S.M. Scanning electron microscopy of acellular glomeruli in nephrotic sindrome. Kidney Int
1985; 27: 678-84.
[11] VERDESCA S., BRAMBILLA C., GARIGALI G. et al. How a skilful and motivated urinary sediment
examination can save the kidneys. Nephrol Dial Transplant 2007; 22: 1778-81.
[12] FAIRLEY K.F. Urinalysis. In: SCHRIER R.W, GOTTSCHALCK C.W., eds. Diseases of the Kidney, 5th edn.
Boston: Little Brown, pp. 335-59.
[13] NAKAYAMA K., OHSAWA I., MAEDA-OHTANI A. et al. Prediction of diagnosis of immunoglobulin A
nephropathy prior to renal biopsy and correlation with urinary sediment findings and prognostic
grading. J Clin Lab Anal 2008; 22:114-8.
[14] HEBERT L., DILLON J.J., MIDDENDORF D.F. et al. Relationship between appearance of urinary red blood
cell/white blood cell casts and the onset of renal relapse in systemic lupus erythematosus. Am J Kidney
Dis 1995; 26: 432-8.
[15] FOGAZZI G.B., PASSERINI P. Der nephritische Sedimentbefund. Ther Umsch 1994; 51: 797-800.
[16] FUJITA T., OHI H., ENDO M. et al. Level of red blood cells in the urinary sediment reflects the degree of
renal activity in Wegener’s granulomatosis. Clin Nephrol 1998; 50: 284-8.
[17] COHEN J.A., LEVITT M.F. Acute glomerulonephritis with few urinary abnormalities. N Engl J Med
1963; 268: 749-53.
[18] ALBERT M.S., LEEMING J.M., SCAGLIONE P.R. Acute glomerulonephritis without abnormality of the
urine. J Pediatr 1966; 68: 325-9.
[19] GOORNO W., ASHWORTH C.T., CARTER N.W. Acute glomerulonephritis with absence of abnormal urinary
findings. Ann Intern Med 1967; 66: 345-53.
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[21] WOOLF A., CROKER B., OSOFSKY S.G. et al. Nephritis in children and young adults with systemic lupus
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[22] FOGAZZI G.B., MORONI G. Ematuria glomerulare e non glomerulare: studio della morfologia delle
emazie urinarie in pazienti portatori di malattie di vario tipo e con diverso grado della funzione renale.
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[23] SERRA A., TORGUET P., ROMERO R.R. et al. Normal urinary red blood cell morphology in segmental
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[28] GOLDSMITH B.M., HICKS J.M. Rhabdomyolysis: two pediatric case reports. Clin Chem 1985; 31: 314-7.
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management of isolated microscopic hematuria. Clin Nephrol 1998; 49: 345-8.
CHAPTER 7
AUTOMATED SYSTEMS
FOR URINARY SEDIMENT ANALYSIS
B. Pirovano and G.B. Fogazzi
Recently, instruments for the automatic examination of urinary sediments have been
introduced into the market. These instruments do not require any preparation of the specimens,
they are able to give high throughputs (= the examination of several tens of samples per hour),
and in most instances do not need trained personnel, and so have gained widespread diffusion.
This has happened especially in large laboratories, where high numbers of specimens (quite
often several hundreds every day) are asked to be examined, mostly for screening purposes.
In such situations the use of automated instruments offers a significant saving of time and
personnel resources, with a reduction in costs.
At present, three types of automated instruments are available on the market. Two
instruments are based on automated intelligent microscopy, the other on flow cytometry.
automated intelligent microscopy

Automated intelligent microscopy (AIM) encompasses fluids, electro-optics, and image
processing.
The fluid subsystem is based on the principle of slideless microscopy [1]. According to
this principle, the flowing particles can be observed microscopically when they are presented
within the focal depth of an image-gathering lens. Thus, in this system, the specimen flows as
a sheet in a flow chamber, being sandwiched between two layers of an enveloping fluid. By
careful control of the orthogonal distribution of shear forces in the flow chamber, asymmetric
elements are held in a stable oriented position in the flow, in order to expose their maximal
cross-sectional area normal to the direction of view. Furthermore, the particles assemble in the
plane of zero shear forced by fluid dynamics. This plane serves as the focal plane, it bisects all
particles irrespective of their size and contains the loci of the maximal edge intensities.
The electro-optical subsystem includes magnifying lenses, collimating lenses and a
stroboscopic lamp. The lamp fires in microsecond bursts to stop the motion of the moving
specimens. The stopped-motion view is observed through magnifying lenses, and the images
are collected by a video camera (Figure 7.1). AIM technology has been incorporated in the
first commercial automated analyzer of urinary sediments, the Yellow IRIS™ Urinalysis
Workstation [2], which has now been overtaken by a newer instrument, Iris iQ200 Urine
Microscopy Analyzer (Iris Diagnostic, Chatsworth, California, USA) [3,4].
222 B. Pirovano and G.B. Fogazzi
Urine sample
Flow of hydrodynamic focusing
Microscope CCD
objective camera
Lenses Ocular
Stroboscopic Flow cell
light Computer
Waste
FIGURE 7.1 Components of iQ200 analyzer (with modification from: Analisi Urine nuovi protagonisti. Iris iQ200
Automazione in microscopia. Instrumentation Laboratory S.p.A, 2005. Reproduced with permission).
iQ200 is based on capturing images from planar flow with a CCD (charge coupling device)
camera (Figure 7.1), and is equipped with an advanced auto particle recognition (APR)
software, which uses size, shape, contrast and texture to identify 12 particle categories. These
are: erythrocytes, leukocytes, leukocyte clumps, hyaline casts, pathological casts, squamous
epithelial cells, non squamous epithelial cells, bacteria, yeasts, crystals, mucus, and sperm. An
additional category is unclassified particles, which includes all individual images which cannot
be recognized very confidently by the APR software, and need to be reclassified by the operator.
The particles are quantitated as number/μL. However, quantitation as number/high or low
power field, or in classes, is also possible. The particles found can be displayed as black
and white images obtained by bright field microscopy on a screen by category (Figure 7.2)
for visual confirmation or reclassification by an operator. In this respect, the manufacturer
recommends that the images of all abnormal results are visually reviewed [4]. For doubtful
results, the specimen should be reanalyzed by manual microscopy.
The minimum urine specimen volume for iQ200 is 3 mL: 1 mL is aspirated into the
instrument and 2 μL are used for analysis. The throughput is of 60 samples/hour with good
walk-away capabilities (= level of automation, which is proportional to the interaction needed
between the instrument and the operator)[3,4].
sediMAX is another and the most recent automatic instrument (A. Menarini Diagnostics,
Florence, Italy) based on AIM. It is equipped with a software for the identification of the
following particles: erythrocytes, leukocytes, leukocyte clumps, squamous epithelial cells,
non squamous epithelial cells, crystals (uric acid, monohydrated and bihydrated calcium
oxalate, and triple phosphate), hyalin casts, pathological casts, bacteria, yeasts, mucus, and
spermatozoa. Quantitation of particles is either as number/μL or /high power field. The found
particles can be seen on a screen through black and white images, obtained by bright field
microscopy. At variance with iQ200, sediMAX supplies high power field like images (250x),
which show all the particles present in the microscopic field as with manual microscopy
(Figures 7.3 and 7.4). All particles can automatically be labelled on the screen, which
facilitates the visual identification and confirmation by the operator. However, single particles
FIGURE 7.2 Uric acid crystals as shown by iQ200 analyzer (from: iQ200 Analizzatore Automatico in Microscopia.
Raccolta di immagini. Instrumentation Laboratory S.p.A, 2005. Reproduced with permission).
can also be visualized, together with image zooming, which helps in the identification of
doubtful or unusual particles. The instrument automatically flags the “crowded samples”
requiring manual microscopy examination.
sediMAX requires 2mL urine aliquots. After aspiration, 200 μL of urine is automatically
loaded into a special cuvette, which is quickly centrifuged by the instrument to form a
sediment layer. The cuvette is then placed above the microscope, which takes 15 different
images for each sample, without the need for any solution. The maximal throughput is 80
samples/hour. sediMAX is predisposed to be connected with an instrument for the automatic
chemical analysis of urine [Bayer G. In: Workshop. The urinary sediment: its utility and a
new approach. Euromedlab, Amsterdam, 4 June 2007].
Only preliminary results are available at present for the performances of sediMAX. These
results were obtained by comparing the instrument with manual phase contrast microscopy
on 513 non centrifuged samples (457 pathological, 56 normal) for 6 particles (erythrocytes,
leukocytes, squamous epithelial cells, non squamous epithelial cells, pathological casts,
and yeasts) [Zaman Z. In: Workshop. The urinary sediment: its utility and a new approach.
Euromedlab, Amsterdam, 4 June 2007]. The findings obtained with software 5 are good
FIGURE 7.3 A microscopic field containing many hyaline-granular casts and a few renal tubular epithelial cell casts
as shown by sediMAX (original magnification x 250).
and promising (sensitivity Γ 80% for all particles but pathological casts [66.7%] and yeasts
[68.8%]; specificity Γ 80% for all particles but pathological casts [76.5%]; positive predictive
value Γ 90% for erythrocytes and leukocytes, from 38.4% to 57.1% for the other particles;
negative predictive value Γ 90% for all particles but erythrocytes [68%] and leukocytes
[75.6%]). In addition, no carry-over (i.e., contamination of a sample with particles contained
in an adjacent sample) was observed, and the rate of samples flagged as “crowded”,
considering that most of these were pathological, was only 9%. These results have recently
been confirmed by other investigators [5]. Definitive published studies are needed to confirm
these preliminary data.
flow cytometry
Flow cytometry incorporates the technology which for many years has been used for the
identification and count of blood cells. Thus, flow cytometers for urine are based on the
transformation of the sample into a laminar flow, which is obtained by passing a sheath liquid
around the sample itself (Figure 7.5). After automatic staining with a phenanthridine dye for
nucleic acid and a carbocyanine dye for cell membranes, the flow is irradiated with a laser
FIGURE 7.4 An example of the high power field image shown by sediMAX, in which squamous epithelial cells, su-
perficial transitional epithelial cells, and bihydrated calcium oxalate crystals can easily be identified (from Bayer G. A
new approach to urinary sediment. In: Workshop. The urinary sediment: its utility and a new approach. Euromedlab,
Amsterdam, 4 June 2007. Reproduced with permission) (x 250).
beam that can be focused with high coherency on a very small area in order to achieve the
highest irradiation efficiency (Figure 7.5). Both scattered light and fluorescence are detected,
which are then converted by a signal analyzer into four parameters: forward scattered light
intensity, fluorescence intensity, forward scattered light pulse width and fluorescence pulse
width. These data, along with impedance measurements, are converted from analogue signals
into digital information that allows the classification of the formed elements.
Flow cytometry technology has been used to develop the UF-100™ analyzer (Toa-
Sysmex, Kobe, Japan), a fully automated instrument which recognizes and quantitates urine
particles automatically without human interaction. The identified particles are: erythrocytes,
leukocytes, crystals, bacteria, yeasts, spermatozoa, squamous epithelial cells, small round
epithelial cells (a category which encompass renal tubular epithelial cells and transitional
epithelial cells), hyaline casts, and casts with inclusions.
While the first six particles are identified according to fluorescence intensity and forward
scattered light intensity (Figure 7.6 top), the other particles are identified according to forward
scattered light pulse width and fluorescence pulse width (Figure 7.6 bottom).
Figures 7.7 and 7.8 are examples of scattergrams and counts as shown by UF-100™.
Sheath fluid Sheath fluid
Dichroic mirror
Argon laser
Corrector Photomultiplier
lens tube
Laminar flow
of urine particles
Photo diode
FIGURE 7.5 Principle of flow cytometry incorporated in the UF-100TM analyzer.
The analyzer also gives a volumetric analysis of erythrocytes, which may be used to localize
the source of haematuria, “microcytic” erythrocytes being considered of glomerular origin
and “normocytic” erythrocytes of non glomerular origin. In this field, however, the published
results are still conflicting [6,7].
UF 100 requires 0.8 mL aliquots of uncentrifuged urine (9 μL of which are used for analysis),
it has a high throughput (100 samples/h), and automatically flags the more complex samples
which require the examination by manual microscopy.
Recently a new version of the instrument has been developed (UF-1000i-Toa-Sysmex, Kobe,
Japan), which has the same general features of the UF-100™ but is equipped with a special channel
for bacteria detection. At the moment little information is available about this new analyzer.
main performances of automated analyzers*

carry over
No significant carry-over (i.e., contamination of a sample with particles contained in an
adjacent sample) has been demonstrated by either iQ200 and UF-100™ analyzers. This even
after filling the samples with fresh erythrocytes, which tend to stick to surfaces and are the
best indicator of carryover problems [3,8,9].
* For the performances of sediMAX see p. 3.

FSC XT
Yeasts
Leukocytes
RBC
Sperms
Bacteria
FI
Flw
Casts with inclusions

Epithelial
cells
Hyaline casts
Fscw
FIGURE 7.6 Schematic representation of the distribution of the urine figured elements using the UF-100™ analyzer.
(top) Scattergram of fluorescence intensity (FI) and forward scattered light intensity (FSC). (bottom) Scattergram of
forward scattered light pulse width (Fscw) and fluorescence pulse width (Flw) (XT = crystals; RBC = red blood cells).
FIGURE 7.7 Example of leukocyte distribution in a region of high intensity scattered light. In this case, two popula-
tions of leukocytes (WBC) are seen (see text for explanation). RBC = red blood cells; BACT = bacteria; EC = epithelial
cells. The panel on the right shows the counts of the elements, expressed as number/μl and as number/microscopic
field (HPF = high-power field; LPF = low-power field).
FIGURE 7.8 Example of leukocyte distribution in a region of low intensity scattered light. In this case, only one
population of leukocytes is observed (see text for explanation).
precision
The precision profile (i.e., intra-assay imprecision measured in urine pools on different
counts of particles) of the flow citometry analyzer is better than that of iQ200 and of manual
microscopy at all particle concentrations. In fact, with UF-100™ the coefficient of variation
for erythrocytes, leukocytes and epithelial cells at concentrations of <10 cells/μL is <20%,
while it is 30% with iQ200, and 50% with manual microscopy [4,9].
accuracy
The accuracy (i.e., the difference from true value) of iQ200, using chamber counts for
comparison has been investigated in four independent studies [3,4,8,10]. All the authors
conclude that the analyzer performs well in recognizing and counting erythrocytes, leukocytes,
and squamous epithelial cells, with improvements after the reclassification by a trained
operator. For instance, in the study of Linko and co-workers [4], iQ200 detects erythrocytes,
leucocytes, and squamous epithelial cells with a coefficient correlation (r) of 0.894, 0.885
and 0.905 respectively, which improves to 0.948, 0.978 and 0.927 after reclassification.
However, the identification and counting of casts and non squamous epithelial cells is far
below manual microscopy (r = 0.000 and 0.342 respectively), and is unsatisfactory even after
reclassification (r = 0.732 and 0.499) [4]. Some improvement is also needed to improve the
specificity for yeast and crystal detection (r = 0.66 and 0.89 respectively) [4]. Another limit is
that on several occasions, the images provided by the instrument are of difficult interpretation
even for well trained operators, so that they cannot be properly classified [4].
The evaluation of the accuracy of the UF-100™ analyzer is more problematic because
some elements may exhibit different scatter and fluorescence patterns due to changes in their
morphology caused by factors such as urinary pH, osmolality, or the prolonged permanence
in the bladder. Figures 7.7 and 7.8 show, for instance, two scattergrams that document a
different leukocyte distribution. In the first case, two different populations of leukocytes can
be distinguished, while in the second case only one population is seen. The distribution of
leukocytes shown in Figure 7.7 identifies a population of swollen or damaged white blood
cells which reduced the forward scattered light, but not the fluorescence intensity. These
observations document the pre-eminent limit of flow cytometry: all the factors that can
induce a variation in size, shape, volume and fluorescence of elements, if they are outside the
pre-defined limits of the instrument, reduce the accuracy of the measurements.
The evaluation of the accuracy of the UF-100™, using manual microscopy for comparison,
was performed in two independent studies [9,11].
For erythrocytes, the concordance between the two methods was fairly good (r = 0.833).
The highest rates of disagreement ranged between 3.3% and 6.6%, and were related to the
overestimation of erythrocyte count by the UF-100™. The potential interferences were
caused by high concentrations of crystals, bacteria, and yeasts, whose distribution area is
close to that of erythrocytes (Figure 7.6 top).
For leukocytes, the correlation between the two methods was excellent (r = 0.933), and
no major disagreement was recorded. Excellent results were also obtained with squamous
epithelial cells and spermatozoa, while a 6.9% rate of false-positive results were observed for
yeasts [9]. For casts, however, the performance of the UF-100™ was unsatisfactory. Apart
from a poor positive correlation (r = 0.40), there were frequent false-negative results, which
ranged from 13.7% to 43%.
The reliability of the UF-100™ analyzer in detecting bacteria is particularly difficult
for several reasons. However, in one study in which a cut-off value for bacterial counts
of 3000/μL in association with leukocyturia defined as Γ 25/mL was selected, positive
cultures with a sensitivity of 94% and a specificity of 93% were found [12]. These data
disclose new perspectives on the use of the analyzer in the screening of urinary tract
infections.
A first preliminary evaluation of UF-1000i showed good results in comparison studies for
leukocytes, squamous epithelial cells and erythrocytes, with correlation coefficients of 0.990,
0.901 and 0.991 respectively [Pirovano B. In: Sismex European Urinalysis Symposium,
Wolfang, Austria, 19 October 2006]. In addition, elevated bacterial counts did not interfere
with erythrocyte detection, which was probably due to the special stain used for bacteria. For
the identification of casts and small round cells further studies are needed.
advantages, limits and role

of automated instruments
All automated analyzers allow the examination of high numbers of non centrifuged sam-
ples in short times. In addition, they are able to identify with acceptable accuracy some
urine particles, and supply quantitative results with small variation coefficients. Thus, these
instruments, compared with manual microscopy, offer several advantages (Table 7.1).
However, these instruments also have limitations (Table 7.1). In fact, they:
• do not distinguish renal tubular epithelial cells from superficial and deep transitional
epithelial cells, which are simply recognized as non squamous epithelial cells (iQ200
and sediMAX) or small round epithelial cells (UF-100™);
• underestimate casts, of which they can distinguish only the hyaline from the non
hyaline category;
• recognize only a few types of crystals such as uric acid and calcium oxalate;
• miss lipids completely.
Compared to UF-100™, iQ200 and sediMAX offer the possibility to review the images
on the screen, which may result in the partial identification of particles of importance, which
were not properly identified by the instrument. However, this result can be achieved by a well
trained and motivated operator and is time consuming.
For all these limitations, automated systems can replace manual microscopy only partly,
namely for samples which are normal or contain mild abnormalities. On the contrary, for
more complex samples which contain various combinations of blood-derived cells, renal
tubular epithelial cells, different types of casts, lipids, or crystals, these instruments can be
inadequate, as can often be the case for renal patients [13].
Thus, today the role of these instruments is to improve the workflow in large laboratories,
where several hundreds of samples are analyzed everyday, mostly for screening purposes.
After the majority of urine specimens has been analyzed automatically by the instruments
with acceptable accuracy, the most pathological samples can be left for manual microscopy
[14]. In the experience of one of the authors of this chapter, the introduction of flow cytometry
has reduced the need for manual microscopy by about 86% [9], which is in agreement with
the experience reported by other investigators [15].
TABLE 7.1 Main features of automated instruments compared with manual microscopy.
Automated Manual
Feature
instruments microscopy
Uncentrifuged urine
0.8 mL (UF-100™) Centrifuged urine
Urine sample
3.0 mL (iQ200) (10 mL)
2.0 mL (sediMAX)
Throughput High Low
Precision High Low
Accuracy for
The gold standard
erythrocytes, leukocytes, Good
method (*)
squamous epithelial cells
Accuracy for casts, non

The gold standard
squamous epithelial cells, Low
method (*)
crystals
Identification of fatty particles No Yes
Identification of glomerular and The gold standard

Conflicting results (UF-100™)
non glomerular erythrocytes method (*)
The gold standard

Accuracy for renal diseases Low
method (*)
No (UF-100™)
Possibility to see and review Yes (iQ200 and sediMAX)
No
the particles for confirmation or
reclassification
Costs Low High (**)
(*) Strongly operator dependent. (**) When considering that several trained operators are needed for the examination of
several hundreds of samples per day.
References
[1] BOLZ G., DEFOREST S.E. Flow analyzer and system for analysis of fluid with particles. US Patent no. 4,
338, 024, July 6, 1982.
[2] DEINDOERFER F.H., GANGWER J.R., LAIRD C.W. et al. The Yellow IRIS™” Urinalysis Workstation – the first
commercial application of “automated intelligent microscopy”. Clin Chem 1985; 31: 1491-9.
[3] ALVES L., BALLESTER F., CAMPS J. et al. Preliminary evaluation of the Iris IQTM 200 automated urine
analyzer Clin Chem Lab Med 2005; 43: 967-70.
[4] LINKO S., KOURI T.T., TOIVONEN E. et al. Analytical performance of the Iris iQ200 automated urine
microscopy analyzer. Chim Clin Acta 2006; 372: 54-64.
[5] GARIGALI G., BAYER G., CROCI M.D. et al Valutazione di sediMAX, un nuovo analizzatore automatico dei
sedimenti urinari. Biochimica clin 2008; 32: 496 (abstract).
[6] APELAND T., MESTAD O., HETLAND O. Assessment of haematuria: automated urine flowmetry vs microscopy.
[7] SCHARNHORST V., GERLAG P.G.G., NANLHOY MANUHUTU M.L. et al. Urine flow cytometry and detection of
glomerular hematuria. Clin Chem Lab Med 2006; 44: 1330-4.
[8] WAH D.T., WISES P.K., BUTH A. Analytical performance of the iQ200 automated urine microscopy analyzer
and comparison with manual counts using Fuchs-Rosenthal cell chambers. Am J Clin Pathol 2005; 123:
290-6.
[9] FENILI D., PIROVANO B. The automation of sediment urinalysis using a new urine flow cytometer (UF-100™).
Clin Chem Lab Med 1998; 36: 909-17.
[10] LAMCHIAGDHASE P., PREECHABORISUTKUL K., LOMSOMBOON P. et al. Urine sediment examination: a comparison
between the manual method and the iQ200 automated microscopy analyzer. Chim Clin Acta 2005; 358:
167-74.
[11] BEN-EZRA J., BORK L., MCPHERSON L.A. Evaluation of the Sysmex UF-100 automated urinalysis analyzer.
Clin Chem 1998; 44: 92-5.
[12] MANONI F., VALVERDE S., ANTICO F. et al. Field evaluation of a second-generation cytometer UF-100 in
diagnosis of acute urinary tract infections in adults patients. Clin Microbiol Infec 2002; 8: 662-8.
[13] KOURI T.T., KÄHKÖNEN U., MALMINIEMI K. et al. Evaluation of Sysmex UF-100 urine flow cytometer vs
chamber counting of supravitally stained specimens and conventional bacterial cultures. Am J Clin Pathol
1999; 112: 25-35.
[14] OTTIGER C., HUBER A.R. Quantitative urine particle analysis: integrative approach for the optimal
combination of automation with UF-100 and microscopic review with Kova cell chamber. Clin Chem
2003; 49: 617-23.
[15] DELANGHE J.R., KOURI T.T., HUBER A.R. et al. The role of automated urine particle flow cytometry in
clinical practice. Chim Clin Acta 2000; 301: 1-18.
CHAPTER 8
QUALITY CONTROL PROGRAMS
FOR URINARY SEDIMENT
S. Secchiero and G.B. Fogazzi
This chapter describes the Quality Control programs which can be used for urinary
sediment. The purpose of these programs is to obtain an examination of the urinary sediment
of good and reliable quality [1,2]. Internal Quality Control (IQC) and External Quality
Assessment (EQA) Programs integrate each other.
internal quality control

An Internal Quality Control (IQC) for urine microscopy should be done each day the test
is performed and should adhere to the following recommendations [2]:
• all personnel should follow the same documented procedures using the same
equipment, use the same terminology and report results in the same standard
format;
• duplicate urine sample examination should be used as a precision check for the
identification of the particles. Alternatively, control solutions containing erythrocytes
or leukocytes, which are commercially available, could be used;
• in case of disagreement on the presence or quantity of a microscopic element, the
examination should be repeated and a shared conclusion should be reached;
• unexpected control results should be identified, and appropriate corrective action
should be taken;
• recent reference texts, atlases, papers or online documents should always be available
for consultation, and experts’ opinions should be asked for in case of difficult and/or
doubtful findings.
In order to fulfil these recommendations, at the laboratory of the renal unit of Ospedale
Maggiore-Policlinico, Milano, where one of the authors of this chapter (G.B.F.) works:
• all the procedures and terminology used are standardized and written in detail in a
document which is kept on a shelf above the workbench;
• the microscope is adjusted according to Khöler principle (see Appendix) and phase
contrast is centred every time the examination of the urinary samples is started. In
234 S. Secchiero and G.B. Fogazzi
addition, a regular servicing of the microscope is done once a year by a specialized

technician;
• the exchange of opinions on difficult or doubtful findings is encouraged and dis-
cussion regularly takes place among the four persons who rotate on urine sediment
examination;
• once or twice a week, some samples, chosen among the most pathologic ones, are
reviewed for a check by the most expert microscopist of the group;
• all special and interesting findings are documented using a digital camera permanently
mounted on the microscope and filed on a computer program for this purpose;
• a specialized library containing several hundreds of scientific papers on various
aspects of urinary sediment examination and 16 atlases in different languages on the
same subject, is kept on shelves close to the microscope for consultation.
external quality control

Medical laboratories have a long tradition in the organisation of external EQA programs,
which started in 1947, when Belk and Sunderman published the results of a clinical chemistry
survey in the US [3].
Today, EQA programs are a key instrument for the improvement of laboratory quality,
and for some disciplines, they are an integral part of laboratories’ overall quality assurance
systems [4]. However, in spite of numerous documents and papers which stress the
importance of designing appropriate EQA schemes [4-10], several laboratory fields still lack
EQA programs.
EQA surveys on urinalysis are rare [11-13]. Of the few existing programs, some deal with
test strips and quantitative clinical chemistry analytes [12], while others also cover urinary
sediment.
The latter topic is included in the program run by Labquality, a Finnish non-profit
EQA scheme organisation which provides surveys also for Norway, the Baltic states and
Poland.
Urinary sediment is also included in the program run in Italy by the Centre of Biomedical
Research (CRB), which is an EQA scheme organisation with many programs in different
fields of laboratory medicine (www.centroricercabiomedica.it).
Interestingly, the College of American Pathologists (CAP) has recently introduced inter-
laboratory schemes focused on the new aspects of urinary sediment examination, which are
associated with the use of automated analyzers (see Chapter 7).
features of the italian eqa program “urinalysis performance”

The Italian EQA program, called “Urinalysis Performance” was set up in 2001 by a
promoting committee which included the representatives of the three Italian societies of
laboratory medicine and of the Italian Society of Nephrology [14].
This program is the first, and to date the only, Italian project for the standardization of
urine analysis. It is meant for use by Italian central laboratories, both public and private, and
by renal laboratories.
The aims of the program are: the evaluation of the laboratories’ performances; the training
support for the participants; the improvement of the efficiency and efficacy of urinary
sediment examination.
“Urinalysis Performance” includes two parts: one on test strips (which is not dealt with in
this chapter), the other on urinary sediment.
The part on urinary sediment is under the guidance and responsibility of one of us (G.B.F.),
who prepares and selects the images and also evaluates the answers of the participants of
each survey.
Today, the program consists in 4 surveys/year.
- Surveys 1 and 3. Each of these surveys shows two urine sediment particles. Each particle
is shown by both bright field and phase contrast microscopy and, when indicated (e.g.,
crystals or lipids), also by polarized light (Figure 8.1). The choice of showing the particles by
the three types of microscopy has a twofold motivation: (i) bright field microscopy was, and
still is, the method most widely used in routine practice and (ii) phase contrast microscopy
and polarized light are the methods recommended by international guidelines for everyday
work (1,2).
For each survey, the participants are asked to identify the particles shown. Moreover, for
one of the two particles (selected by the person in charge of the program), they are also asked
to indicate one clinical association, chosen among 4 or 5 possible options.
Over the years, in order to verify whether the program was able to achieve an improvement
in the identification capability of the participants, some particles were presented twice, by the
means of similar, but not identical images.
- Surveys 2 and 4. Each of these surveys presents a clinical case. These cases were
introduced because laboratory medicine is moving towards a clinical support service, and
guidelines and standards emphasise the importance of adding appropriate comments and
interpretation of results to medical reports and their assessment (15-21).
Clinical cases consist of a brief clinical history, which also include some key laboratory
data and four phase contrast microscopy images of particles found in the urine sediment
of the case presented (Figure 8.2). For clinical cases, the participants are also asked to
identify the particles shown, and to choose one possible clinical diagnosis among 4 to 5
proposed.
For each survey, the answers obtained are then evaluated as correct, incorrect, partially
correct, and no answer, and scored accordingly (5, 3, 0, and -2 respectively). For clinical
association, the answer is considered and scored only if the indicated particle (for surveys 1
and 3) or all four particles (for surveys 2 and 4), are correctly identified.
For each survey, the CRB edits a report for each laboratory, containing the judgement and
the scores obtained. Moreover, a summary of all participants’ answers is supplied, together
with a comment by the person in charge of the program on the images shown, their main
clinical correlates, and the answers supplied by participants.
At the end of each annual cycle, CRB prepares a report summarising the laboratory’s
performances and annual score, together with an overview of the results obtained by all
laboratories.
Today, the images of each survey are presented on the program’s website (www.urinalysis.
net) and the participants give their answers directly through it.
400x (pH 5.4)
400x
FIGURE 8.1 Survey 2-2003 for the identification of particles. Top: spindle-like uric acid crystals. Bottom: an oval fat
body. For both particles, left, bright field microscopy and, in the inset, polarized light; right, phase contrast micros-
copy. Note that for each particle the magnification was indicated, and for crystals, also the urinary pH.
20-30/high power field (x 400) 3-5/high power field (x 400)
2-4/over 20 low power fields (x 160) 1-3/over 20 lower power fields (x 160)
FIGURE 8.2 Survey 2-2007 showing the particles associated with clinical case 1.
Top, left: dysmorphic erythrocytes; right: renal tubular epithelial cells. Bottom, left: an erythrocytic cast; right: a
waxy cast.
The clinical case was presented as follows: a 45-year-old man hospitalised for rapidly progressive renal failure
(S-creatinine 1.2 mg/dL three months before hospitalisation) associated with the appearance of high blood pres-
sure (160/95 mmHg) and urinary abnormalities. Ultrasounds of the urinary system: normal.
Laboratory findings at hospitalisation: S-creatinine: 2.5 mg/dL (n.v. 0.5-1.0). U-protein/24 hours: 1.5 g (n.v. ≤0.14).
BUN: 95 mg/dL (n.v. 15-50). Urinary output/24 hours: 1,700 mL.
Possible clinical diagnosis (only one is correct):
acute nephritic syndrome nephrotic syndrome hypovolemic acute renal failure acute pyelonephritis
unilateral hydronephrosis due to ureteric stone.
results of “urinalysis performance”

The identification of particles. From 2001 to 2007, 84 images were sent, which showed
50 elements of urinary sediment (Table 8.1). The correct identification was highest for
bihydrated calcium oxalate crystals (100%) and triple phosphate crystals (99.6%), while it
was lowest for the leukocytic cast (9.2%) and the macrophage (10.9%).
Considering particle categories, a very high correct identification rate (obtained for
each particle from the sum of correct + partially correct answers) was obtained for micro-
organisms and crystals, followed in decreasing order by cells, lipids, casts and contaminants
(Table 8.2).
This part of the program also showed that, quite often, participants used inappropriate
terminology to define some particles. This happened especially with renal tubular epithelial
cells, transitional epithelial cells, and squamous epithelial cells, which were often defined as
“cells from the high, intermediate, or low urinary tract” respectively. For other particles, such
as erythrocytes and calcium oxalate crystals, the terminology used was often incomplete,
without specification as to whether the erythrocytes were isomorphic or dysmorphic, nor
whether calcium oxalate was mono- or bihydrated.
The particles presented twice. Twenty-four particles were presented twice. For 6 particles
(25.0%) there was a 4.6% to 27.7% (14.6 ± 8.5) decrease in the correct identification rate
when the particle was presented for the second time; for 4 other particles (16.6%) there were
no substantial differences between the first and second surveys (0 to + 0.2%); for the majority
of particles (14 out of 24, 58.3%), the identification rate increased by 2.6% to 77.2% (24.7 ±
19.7). For 11 out of 14 such particles (78.5%), the improvement between the first and second
survey was statistically significant (Table 8.3).
The clinical association. In the cycles from 2001 to 2003, when participants were free to
indicate one association of their choice, a very wide spectrum of answers was supplied, and
the answers were often of difficult interpretation, mostly because of the arbitrary and vague
terminology used. Moreover, there was a high rate of “no answer” (11.8 ± 5.5%, 5-28% per
survey).
Subsequently, with the introduction of multiple-choice answers, the correct clinical
association was indicated by more than 80% of participants for all but one particle (i.e.,
cholesterol crystals). Moreover, there was a substantial decrease in the rate of “no answer”
(2.5 ± 1.6%, 0.0 to 5.6% per survey) (Table 8.4).
The clinical cases. For the first case presented (Figure 8.2), among 168 laboratories out
of 325 which correctly identified all four elements presented (51.7%), the correct diagnosis
(acute nephritic syndrome) was given by 86.9% of participants. For the second clinical case,
among 125 laboratories out of 310 which correctly identified all the four elements shown
(40.3%), the correct diagnosis (ureteric stone) was given by 95.2% of participants.
TABLE 8.1 The particles sent to participants for identification in the period 2001-2007 and the
answers received.
Answers (%)
Number of
Urinary sediment particle Partially No
Correct Incorrect participants
correct answer
CELLS (N = 9)
Isomorphic erythrocytes 89.3 2.4 7.9 0.4 291
Dysmorphic erythrocytes 45.2 41.6 13.2 0.0 250
Acanthocytes 52.0 20.8 25.6 1.6 250
Leukocytes 96.9 1.4 1.4 0.3 291
Macrophage 10.6 0.3 83.4 5.7 309
Renal tubular epithelial cells 51.9 1.0 44.0 3.1 291
Deep transitional epithelial cells 45.2 41.6 12.4 0.8 250
Superficial transitional epithelial 41.9 14.8 42.3 1.0 291
cells
Squamous epithelial cells 88.1 0.0 11.9 0.0 361
LIPIDS (N = 4)
Aggregates of lipid droplets 61.2 29.8 6.1 2.9 245
Oval fat body 55.9 2.4 39.6 2.1 245
Fatty cast 74.7 0.9 24.0 0.4 229
Cholesterol crystals 53.9 1.6 42.9 1.6 245
CASTS (N = 15)
Hyaline 78.6 0.4 19.7 1.3 234
Hyaline-granular 74.3 0.0 24.8 0.9 234
Finely granular 64.1 1.7 33.8 0.4 234
Coarsely granular 59.9 0.6 38.9 0.6 321
Waxy 88.5 1.3 9.8 0.4 234
Granular-waxy 45.8 22.0 31.4 0.8 361
Erythrocytic 61.1 5.7 33.2 0.0 229
Leukocytic 5.5 3.7 90.8 0.0 327
Containing renal tubular epithelial 38.9 12.7 48.4 0.0 229
cells (RTECs)
Erythrocytic + RTECs 66.4 16.6 16.6 0.4 263
Leukocytic + RTECs 83.2 10.1 6.7 0.0 356
Haemoglobinic 91.0 2.5 6.5 0.0 355
Bilirubinic 43.2 31.5 25.0 0.3 324
Hyaline-granular cylindroid 68.0 15.8 16.2 0.0 291
Cylindroid containing erythrocytes 48.5 4.1 47.4 0.0 365
Continued
TABLE 8.1 Continued

Answers (%)
Number of
Urinary sediment particle Partially No
Correct Incorrect participants
correct answer
CRYSTALS (N = 13)
Uric acid 99.2 0.0 0.4 0.4 243
Calcium oxalate monohydrated 66.3 26.7 6.2 0.8 243
Calcium oxalate bihydrated 58.4 41.6 0.0 0.0 243
Triple-phosphate 99.6 0.0 0.4 0.0 243
Calcium phosphate 91.7 0.0 8.3 0.0 265
Calcium phosphate plate 71.0 0.0 27.4 1.6 263
Amorphous urates 86.4 1.1 12.5 0.0 265
Amorphous phosphates 80.4 3.4 16.2 0.0 291
Ammonium biurate 90.1 8.2 0.0 1.7 365
Cystine 94.7 0.0 5.3 0.0 265
Amoxycillin 12.1 50.4 36.0 1.5 355
Indinavir 63.4 0.6 26.1 1.5 344
Ciprofloxacin 25.4 42.8 31.5 0.3 327
MICRO-ORGANISMS (N = 4)
Bacteria 97.3 0.9 1.8 0.0 223
Candida 99.1 0.0 0.9 0.0 223
Trichomonas vaginalis 93.3 1.4 5.3 0.0 223
Eggs of Schistosoma
87.0 3.2 9.4 0.4 223
haematobium
CONTAMINANTS (N = 5)
Starch 50.6 0.4 47.8 1.2 245
Glass fragment 79.5 0.0 17.8 2.7 263
Fibre 91.1 0.7 8.2 0.0 291
Fungal spore (Alternaria) 61.1 29.3 8.3 1.3 324
Pseudocast 22.3 0.4 73.8 3.5 229
TABLE 8.2 Correct identification rates observed for each of the 6 categories of urinary particles
presented during the period 2001-2007.
Number
Particle Mean ± sd Median Range
presented
Micro-organisms 4 95.5 ± 4.0 96.4 90.2-99.1
Crystals 13 85.6 ± 14.3 91.7 62.5-100
Cells 9 71.6 ± 27.6 86.8 10.9-98.3
Lipids 4 70.1 ± 16.5 66.9 55.5-91.0
Casts 15 69.7 ± 21.4 74.3 9.2-93.5
Contaminants 5 67.0 ± 29.7 79.5 22.7-91.8
TABLE 8.3 First and second answers concerning the identification of the particles which were
presented twice.
Correct + partially correct
Urinary sediment particle
identifications (%)
Change
I II p-value
(%)
CORRECT IDENTIFICATION: DECREASE
Waxy cast 89.8 85.2 –4.6 0.123

Deep transitional cells 86.8 80.9 –5.9 0.054
Bilirubinic cast 74.7 60.1 –14.6 <0.001
Uric acid crystals 99.2 82.3 –16.9 <0.001
Hyaline cast 79.0 61.0 –18.0 <0.001
Isomorphic erythrocytes 91.7 64.5 –27.7 <0.001
CORRECT IDENTIFICATION: UNCHANGED
Calcium oxalate bihydrated crystals 100 100 0 -

Leukocytes 98.3 98.4 +0.1 0.924
Candida 99.1 99.3 +0.2 0.762
Triple-phosphate crystals 99.6 99.4 +0.2 0.807
CORRECT IDENTIFICATIONS: INCREASE
Dysmorphic erythrocytes 86.8 89.4 +2.6 0.359

Egg of Schistosoma haematobium 90.2 93.6 +3.4 0.129
Calcium oxalate monohydrated crystals 93.0 96.6 +3.6 0.066
Fatty cast 75.6 86.4 +10.8 0.001
Finely granular cast 65.8 82.4 +16.6 <0.001
RTECs 52.9 69.6 +16.7 <0.001
Starch 51.0 70.2 +19.2 <0.001
Oval fat body 58.3 83.2 +24.9 <0.001
Erythrocytic cast 66.8 96.3 +29.5 <0.001
RTECs cast 51.6 83.5 +31.9 <0.001
Superficial transitional cells 56.7 89.1 +32.4 <0.001
Macrophage 10.9 44.4 +33.5 <0.001
Cholesterol crystals 55.5 99.7 +44.2 <0.001
Leukocytic cast 9.2 86.4 +77.2 <0.001
TABLE 8.4 Answers concerning the clinical association in the period 2004-2007.
N with Correct clinical Answers (%)
Urinary
access to association
sediment
clinical (chosen among 4 to No
particle Correct Incorrect
association 5 options) Answer
Dysmorphic Glomerular
248 97.6 2.0 0.4
erythrocytes haematuria
Deep Damage to the
transitional 201 deep layers of the 99.5 0.5 0.0
cells uroepithelium
Active
Macrophage 158 86.7 12.0 1.3
glomerulonephritis
Renal disease with
Granular-waxy
165 deterioration of 90.3 7.3 2.4
cast
renal function
Acute renal failure
Cast containing associated with
269 89.2 10.4 0.4
RTECs acute tubular
necrosis
Active proliferative
Leukocytic cast 276 84.0 12.0 4.0
glomerulonephritis
Haemoglobinic Haematuria of renal
323 83.9 10.5 5.6
cast origin (glomerular)
Jaundice associated
Bilirubinic cast 140 with increased 94.3 3.6 2.1
conjugated bilirubin
Erythrocytic Haematuria of
345 89.9 7.5 2.6
cylindroid glomerular origin
Severe proteinuria/
Cholesterol
317 Nephrotic 74.8 21.4 3.8
crystal
syndrome
Crystalluria due to
Calcium oxalate
drugs (e.g., vitamin
monohydrated 212 91.9 5.7 2.4
C, naftidrofuryl
crystals
oxalate)
Urolithiasis from
Indinavir inhibitors of HIV-1
218 95.9 1.8 2.3
crystals protease (e.g.,
indinavir)
Egg of Infection of the
Schistosoma 300 urinary system due 91.0 5.3 3.7
haematobium to a parasite
Urine contamination
Starch 225 92.9 2.7 4.4
from environment
comments on “urinalysis performance”

The EQA programs which also include the examination of the urinary sediment are few.
However, the results obtained by “Urinalysis Performance” show that there is a great need
for such programs.
In fact, our program demonstrates that only some particles, such as micro-organisms and
the most common types of crystals, are known to participants. On the contrary, the knowledge
on particles such as renal tubular epithelial cells, and lipids is unsatisfactory, especially if one
considers the clinical implications they have.
Our results also show that EQA programs can improve the skills of the participants, as
shown by the results obtained for particles which were presented twice. In this respect, it is
worth noting that the highest and most significant improvements were obtained for particles
of clinical importance, such as the erythrocytic and leukocytic casts, which are a marker of
active glomerular disease.
EQA programs may also be a valuable tool for expanding the knowledge on particles
which are known only to specialists. In our program, this is clearly demonstrated by the
results obtained with the macrophage, which was almost totally misidentified when it was
presented for the first time, but whose correct identification increased by 33.5% when it was
presented for the second time.
EQA programs may also be used as a tool to improve the knowledge of the clinical
implications of the laboratory tests. The results obtained by our program with the first two
clinical cases presented, confirm the validity of this statement.
For all these reasons, the participation in EQA programs on urinary sediment should be
encouraged and sustained, especially by the Scientific Society of Laboratory Medicine.
References
[1] KOURI T., FOGAZZI G.B., GANT V. et al. European Urinalysis Guidelines. Scand J Clin Lab Invest 2000;
60 (Suppl 231): 39-47.
[2] CLSI (Clinical and Laboratory Standards Institute, ex NCCLS). Document GP 16-A3 – Urinalysis;
Approved guideline. Third edition (GP 16-A3), 2009; Vol. 29 N. 4.
[3] BELK W.P., SUNDERMAN P.W. A survey of the accuracy of chemical analyses in clinical laboratories.
Am J Clin Pathol 1947; 17: 853-96.
[4] LIBEER J.C. Role of external quality assurance schemes in assessing and improving quality in medical
laboratories. Clin Chim Acta 2001; 309: 173-7.
[5] HILL P., ULDALL A., WILDING P. Fundamentals in External Quality Assessment (EQA). Guidelines
on improving analytical quality by establishing and managing EQA schemes. Examples from basic
clinical chemistry using limited resources. IFCC 1996.
[6] LIBEER J.C., BAADENHUIJSEN H., FRASER G.C. et al. Characterization and classification of External
Quality Assessment Schemes (EQA) according to objectives such as evaluation of method and
participant bias and standard deviation. Eur J Clin Chem Clin Biochem 1996; 34: 665-78.
[7] CLSI (Clinical and Laboratory Standars Institute, ex NCCLS). Document GP 27-A2 – Using
Proficiency Testing (PT) to improve the clinical laboratory. Approved guideline. Second edition (GP
27-A2), 2007; Vol. 27 N. 8.
[8] ILAC. Guidelines for the requirements for the competence of providers of proficiency schemes. ILAC;
ILAC-G13; 2000.
[9] MAZIOTTA D., HAREL D., SCHUMANN G. et al. Guidelines for the requirements for the competence of
EQAP organizers in medical laboratories. IFCC/EMD/C-AQ, version 3/2002.
[10] SCIACOVELLI L., SECCHIERO S., ZARDO L. et al. External quality assessment schemes: need for recognised
requirements. Clin Chim Acta 2001; 309:183-99.
[11] TAKUBO T., TATSUMI N. Quality control in urinalysis. Southeast Asian J Trop Med Public Health 1999;
30 (Suppl 3): 136-48.
[12] BOISSON R.C., EYNARD J.C., CROZIER M. et al. French experience about quality assessment of
quantitative urinary analysis. Chim Clin Acta 2000; 297: 285-95.
[13] GUDER W.G., BOISSON R.C., FOGAZZI G.B. et al. External quality assessment of urine analysis in
Europe. Results of a round table discussion during the Symposium “From uroscopy to molecular
analysis”, Seeon, Germany, September 18-20, 1999. Clin Chim Acta 2000; 297: 275-84.
[14] FOGAZZI G.B. “Urinalysis Performance” Programma di Valutazione Esterna di Qualità sul sedimento
urinario. Anno 2001-2003. Pisa: Pacini, 2006.
[15] THE ROYAL COLLEGE OF PATHOLOGISTS. Guidelines for the provision of interpretative comments on
biomediacal reports. Bull R Coll Pathol 1998; 104: 25.
[16] VASIKARAN S.D., PENBERTHY L., GILL J. et al. Review of a pilot quality-assessment program for
interpretative comments. Ann Clin Biochem 2002; 39: 250-60.
[17] SCIACOVELLI L., ZARDO L., SECCHIERO S. et al. Interpretative comments and reference ranges in EQA
programs as a tool for improving laboratory appropriateness and effectiveness. Clin Chim Acta 2003;
333: 209-19.
[18] LIM E.M., SIKARIS K.A., GILL J. et al. Quality assessment of interpretative commenting in clinical
chemistry. Clin Chem 2004; 50: 632-7.
[19] CLINICAL PATHOLOGY ACCREDITATION (UK) Ltd. Standards for the Medical Laboratory. Scheffield, UK:
CPA, Version 2.01; 2009.
[20] ISO 15189:2007. Medical laboratories – Particular requirements for quality and competence. Ginevra:
ISO 2007.
[21] SCIACOVELLI L., SECCHIERO S., ZARDO L. et al. Risk management in laboratory medicine: quality
assurance programs and professional competence. Clin Chem Lab Med 2007; 45: 756-65.
appendix
ADJUSTMENT OF THE MICROSCOPE

G.B. Fogazzi
To obtain images of good quality (for both everyday work and photographs), the microscope
must be well adjusted.
With either bright Þeld or phase contrast, the microscope must be periodically adjusted
according to the Kšhler principle. This allows the correct centering of the light beam, which
provides a homogeneous illumination of the microscopic Þeld.
Phase contrast needs an additional procedure, which is the centering of the annular
diaphragm of the condenser with the phase ring of the objective (see Chapter 1, Figure 1.6).
The procedures described below are meant for microscopes of average to top quality,
equipped with a rotating universal condenser (Figure 1). This holds different lenses, one
for bright Þeld microscopy, another for dark Þeld illumination, and two or three for phase
contrast. The latter lenses are marked by the acronym PH, where PH1 matches with a × 10 or
× 20 objective, PH 2 with a × 40 objective, and PH3 for a × 100 objective.
1. adjustment according to the köhler principle

Bright Þeld microscope:
1. Focus on a sample using a low power objective (× 100, × 160 or × 200).

2. Lift the condenser up to the highest level, and close the Þeld diaphragm to the
maximum (Figure 1).
3. Center the beam of light through the two knobs of the condenser.
4. Lower the condenser until the edges of the Þeld diaphragm appear sharp.
5. Open the Þeld diaphragm until the edges disappear from the microscopic Þeld. To
avoid light dispersion, the opening should be only slightly larger than the microscopic
Þeld.
After these procedures, the condenser diaphragm (Figure 1) is partially closed to improve
the image deÞnition. Most people achieve this aim by moving the condenser downward.
However, this maneuver should be avoided, since it alters Kšhler adjustment.
248 G.B. Fogazzi
Pinion for the

adjustment
of the condenser
Universal
condenser
Condenser diaphragm
Field diaphragm
FIGURE 1 The microscope used in our laboratory (with which we obtained the images contained in this book).
Phase contrast microscope (see Chapter 1, page 32)
1. Focus on a sample using a low power objective (× 100, × 160, or × 200).

2. Shift the condenser lens from PH1 position to the bright Þeld condenser lens
position.
3. Lift the condenser up to the highest level, and close the Þeld diaphragm to the
maximum (Figure 1).
4. Center the beam of light through the two knobs of the condenser.
Adjustment of the microscope 249
5. Lower the condenser until the edges of the Þeld diaphragm appear sharp.
6. Open the Þeld diaphragm until the edges disappear from the microscopic Þeld. To
avoid light dispersion, the opening should be only slightly larger than the microscopic
Þeld.
7. Turn to the PH1 condenser lens.
With phase contrast, the image deÞnition depends only on the correct centering between
the annular phase of the condenser and the phase ring of the objective. Thus, there is no need
to act on the condenser diaphragm.
2. centering of the annular diaphragm of the condenser

with the phase ring of the objective
1. Replace one of the two eyepieces with the centering eyepiece supplied with phase
contrast microscope, the so-called phase telescope or auxiliary microscope.
2. Focus and then center the phase ring of the objective onto the ring (= annular
diaphragm) of the phase contrast condenser (see Chapter 1, Figure 1.6). This is done
by using two small knobs placed on the condenser (which differ from the small knobs
used for the adjustment according to the Kšhler principle).
3. Replace the phase telescope with the conventional eyepiece.
In the newest and best microscopes, the centering of the annular diaphragm with the phase
ring is more easily done by rotating two wheels placed in the condenser itself, without the
need for phase telescope.
It must be remembered that when changing to an objective with a different magniÞcation,
the annular condenser has to be changed in parallel, since the ring of the annular diaphragm
and the ring in the objective must match (i.e., condenser lens PH1 with × 20 objective, PH2
condenser lens with × 40 objective).
INDEX
A Cellular 88
Accuracy (for automated systems) 229-230 Complex 77
Acute cellular rejection 50, 57, 197-198 Containing crystals and amorphous salts 92
Acute interstitial nephritis 49, 57, 191-194 Containing microorganisms 92
Acute nephritic syndrome (see Urinary findings in) Epithelial (see Renal tubular epithelial cells)
Acute post-streptococcal glomerulonephritis 184-185 Erythrocytic 88, 89, 194, 197, 215
Acute tubular necrosis 57, 195-197, 216 Excretion rate 173
Acute uric acid nephropathy 108, 197 Fatty 8, 71, 75, 92, 93
Acyclovir 165 Fungal 92, 139
Addis Thomas 1, 13, 173 Granular 83-84, 168, 197
Air bubbles (see Contaminants) Haemoglobin 88, 95
Alternaria (see Contaminants) Hyaline 77, 80-82, 168
Ammonium exchange resins 168 Hyaline-granular 97, 98
Amoxycillin 160-161 Leukocytic 88, 90, 216
Amyloidosis 190, 191 Mixed 97
Annular diaphragm (of the phase contrast Myoglobin 95
microscope) 33, 249 Pigmented 95-96, 197
Automated intelligent microscopy 221-224 Renal tubular cell 57, 88, 90-91, 173, 216-217
Automated systems (for urinary sediment analysis) Waxy 85-87
221-231 CellFIX (for urine preservation) 21
Auxiliary microscope (see Phase telescope) Cells (of the urinary sediment) 41-70
Chronic interstitial nephritis 194-195
B Ciprofloxacin 161, 163-164
Bacteria (see Organisms) Cladosporium (see Contaminants)
Bacteriuria 204 Cloth fibres (see Contaminants)
Beale Lionel 8 Colour microphotography 14
Becquerel Alfred 4 Condenser (of the microscope) 32-33, 35
Bird Golding 2, 5-6, 8 Containers (for urine) 19
Bilharziosis (see Organisms, Schistosoma Contaminants 101, 144-151
haematobium) Air bubbles 148
Birefringence 35, 106 Alternaria 150
Bladder catheterization 20 Cladosporium 150
Bowman William 3 Cloth fibres 144
Brownian movement 49 Cream and detergent particles 144
Epicoccum 150
C Faeces 144
Candida (see Organisms) Glass fragments 148
Carryover (for automated systems) 226 Helminthosporium 150
Casts 5, 8, 77-98 Intestinal cells 144
Bacterial 92 Pediculosis pubis 144
Bilirubin 95 Plant cells 150
252 Index
Pollen 150 Dysmorphic 4, 43-44, 45, 46, 48

Spermatozoa 144 Excretion rate 173-176
Starch 71, 148 G1 cells 43
Talcum powder 144 Isomorphic 42, 168
Counting chambers 28 Sickle cells 44
Coverslip 24 Ethylene glycol 115, 197
Cream and detergent particles (see Contaminants) Extracapillary glomerulonephritis 185
Crystalluria 197, 219 (see also Crystals)
Crystals 2-4, 105-135 F
Acyclovir 165 Fabry’s disease 71-72
Ammonium biurate 135 Faeces (see Contaminants)
Amorphous phosphates 92, 113-114 Flow cytometry 224-226
Amorphous urates 113-114 Focal segmental glomerulosclerosis 177-178
Amoxycillin 160-161 Formaldehyde (for urine preservation) 21
Bihydrated calcium oxalate (Wedellite) 115 Frerichs Theodor 5
Calcium carbonate 134 Fuchs-Rosenthal counting chamber 28, 174
Calcium oxalate 92, 115-119
Calcium phosphate 120-122 G
Cholesterol 71, 127 Glass fragments (see Contaminants)
Ciprofloxacin 161, 163-164 Glutaraldehyde (for urine preservation) 21
Cystine 108, 128-129 H
2-8 Dihydroxyadenine 130-132 Haematuria 19
Drug-related 133, 159-168 Glomerular 43-44
Felbamate 167-168 Microscopic (see Microscopic haematuria)
Hippuric acid 133 Non glomerular 30
Identification 106 Helminthosporium (see Contaminants)
Indinavir 165 Henle Jacob 5
Leucine 130, 131 Hofmann Karl Berthold 8, 10
Monohydrated calcium oxalate (Whewellite) 115
Piridoxylate 165 I
Primidone 165 IgA nephropathy 180-183
Solubility features 106 Indinavir 165
Triamterene 165 Intestinal cells (see Contaminants)
Triple phosphate 123-126 Iris iQ200 Urine Microscopy Analyzer 221-222,
Tyrosine 130 226, 229, 230
Uric acid 108-112, 197
Cylindroids 99-100 J
Cylindruria 13, 19, 168, 214-215, 218-219 Johnson George 6
Cytomegalovirus 200
K
D Kark Robert 14
Decoy cells 200-203 Köhler principle (for microscope adjustment) 233,
De novo glomerulopathy 203 247, 249
Diabetic nephropathy 189-190
Dipstick L
Haemoglobin 25 Leukocytes 49-53
Leukocyte esterase 25 Eosinophils 49-50, 53, 193-194
Diuretics 168 Excretion rate 173-176
Donné Alfred 4, 13 Lymphocytes 50, 53, 198
Neutrophils 49
E Leukocyturia 204, 214, 218 (see also Leukocytes)
Eosinophiluria (see Leukocytes) Light chain deposition disease 190-191
Epicoccum (see Contaminants) Lipids 71-76, 211-214
Erythrocytes 42-48, 144, 214 Cholesterol 71, 127
Acanthocytes 43 Fatty casts 71, 92
Index 253
Free lipid droplets 71 Rods 136

“Maltese” crosses 8, 35, 71, 92 Schistosoma haematobium 142
Oval fat bodies 71 Trichomonas vaginalis 69, 136, 141, 204
Lipiduria 211-214 (see also Lipids) Yeasts 139
Lippman Richard W. 14 Orlistat 115, 167
Lupus nephritis 186-187, 216
Lymphocytes (see Leukocytes) P
Lymphocyturia (see Leukocytes) Pedersen Ib 14
Pediculosis pubis (see Contaminants)
M Peiresc (de) Nicolas Fabricius 2
Macrophages 54-56 Phase ring (of the phase contrast microscope) 33, 249
“Maltese” crosses (see Lipids) Phase telescope (of the phase contrast microscope)
Membranoproliferative glomerulonephritis 33, 249
183-184 Pigmenturia (see Urine, Colour)
Membranous nephropathy 178-180 Piridoxylate 165
Menstruation 20 Plant cells (see Contaminants)
Merfen powder 148 Plasma cell dyscrasias 190-191
Microscope (for urinary sediment) 32-38 Plastic bags (for urine collection) 20
Adjustment 233-234, 247-249 Polarization filters 35
Bright field 35, 234, 247 Pollen (see Contaminants)
Confocal scanning laser 36 Polycystic kidney disease 71
Fourier transform infrared 106 Polyomavirus BK infection 198-200
Immunofluorescence 36 Precision (for automated systems) 229
Interference contrast 35 Preservatives (of urine) 21-22
Phase contrast 14, 32-35, 174, 233, 235, 248-249 Primary hyperoxaluria 115
Polarized light 8, 35, 71, 106, 235 Primidone 165
Scanning electron 25 Proliferative glomerulonephritis 57, 197, 215, 216
Transmission electron 36 Pseudocasts 101-102, 144
Microscopic examination 24-26 Pseudo-Maltese crosses 130
Microscopic haematuria 4, 43
Midstream technique 20 Q
Minimal change disease 177-178 Quality Control Programs for urinary sediment
Minor urinary abnormalities 218-219 233-234
Mucus 103-104 Internal 233-234
Munk Fritz 8 External 234-244
Myeloma cast 191 Quantitation (of figured elements of urine) 28
Myeloma cast nephropathy 190, 191
Myeloma cells 191 R
Rayer Pierre 3, 4
N Recurrent glomerulopathy 203
Naftidrofuryl oxalate 115, 165 Relative centrifugal force (RCF) 23
Nasse Hermann 5 Renal papillary necrosis 195
Nephritic sediment (see Urinary sediment) Renal transplantation 197-203
Nephrotic and nephritic sediment (see Urinary Renal tubular epithelial cells 57, 88, 90-91, 195,
sediment) 198, 216-217
Nephrotic sediment (see Urinary sediment) Report (of urinary findings) 26-32
Nephrotic syndrome 57, 71-72, 211, 217 Rhabdomyolysis 95, 216
Neubauer counting chamber 174 Rieder Hermann 11
Rovida Carlo Leopoldo 13
O
Organisms 136-143 S
Bacteria 136-138, 204 Scherer Johann Joseph 5
Candida 69, 139-140, 204 Schistosoma haematobium (see Organisms)
Cocci 136 Schönlein-Henoch purpura 187, 189
Enterobius vermicularis 143 sediMAX 222-224, 230
254 Index
Simon Johann Franz 5 Lupus nephritis 186

Slide (preparation) 24 Membranoproliferative glomerulonephritis 184
Sodium bicarbonate 168 Membranous nephropathy 180
Spencer Edwin 14 Minimal change disease 178
Spermatozoa (see Contaminants) Nephrotic syndrome 211-214
Squamous epithelial cells 69-70 Normal subject 173-176
Stains 37-38 Pauciimmune systemic vasculitis
Hansel 37, 49-50 (see Extracapillary glomerulonephritis)
May-Grünwald-Giemsa 37, 49-50 Plasma cells dyscrasias 190
Methylene blue 37 Polyomavirus BK infection 198-203
Methylene green pyronin 37 Recurrent glomerulopathy 203
Monoclonal antibodies 38 Renal transplantation 197-203
Oil-red O 38, 54, 71 Schönlein-Henoch purpura nephritis 189
Papanicolaou 37 Urinary tract infection 49, 203-204
Prussian blue 38 Urological disorders 204-206
Sternheimer 37 Urinary sediment
Sudan III 38 Changes caused by drugs 159-168
Supravital 37 Containing bacteria and leukocytes 218
Wright 37, 49-50 Containing increased numbers of erythrocytes
Starch 71, 148 217-218
Sulfadiazine 159 Containing many tubular epithelial cells 216-217
Suprapubic puncture 20 Containing minor abnormalities 218-219
Nephritic 214-216
T Nephrotic 211-214
Talcum powder (see Contaminants) Nephrotic and nephritic 216
Tamm-Horsfall protein 14, 77, 85, 99, 168 Normal Subject 173-176
Thimerosal (for urine preservation) 21 Quality Control Programs 233-244
Transitional epithelial cells 57, 63-68, 204-206 Urinary tract infection 203-204
Deep 57, 63, 205 Urine
Malignant 204-206 Alkaline 19, 168
Superficial 57, 63, 205
Centrifugation 22-24
Triamterene 165
Collection 19-20
Trichomonas vaginalis (see Organisms)
Colour 21
Tyson James 8
Contamination 21, 136
U Diluted 19
UF-100TM analyzer 225-226, 227, 229, 230 Inspection 21
Ultzmann Robert 8, 10 pH 24-25
“Urinalysis Performance” Programs 234-244 Preparation (of slides) 24
Urinary findings in Preservation 21-22
Acute cellular rejection 197-198 Resuspension 24
Acute interstitial nephritis 192 Specific gravity 24
Acute nephritic syndrome 214-216 Turbidity 21
Acute post-streptococcal glomerulonephritis 185 Uroepithelium (see Transitional epithelial cells)
Acute tubular necrosis 198, 216 Urological disorders 204-206
Amyloidosis 191
Analgesic nephropathy 195
V
Vigla Eugène Napoléon 4
Chronic interstitial nephritis 195
Vitamin C 115, 167
De novo glomerulopathy 203
Vogel Julius 5
Diabetic nephropathy 189-190
Extracapillary glomerulonephritis 185-186 Y
Focal segmental glomerulosclerosis 178 Yeasts 139-140
Goodpasture’s syndrome Yellow IrisTM Urinalysis Workstation 221
(see Extracapillary glomerulonephritis)
IgA nephropathy 183 Z
Light chain deposition disease 190-191 Zernike Frits 32

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The Urinary

Espero que este libro le ayude en su formación profesional, en él

QFB. Daniel Arias López.

Mapastepec, Chiapas; México. Julio del 2017.

with a Historical Introduction by

Books Publishing Director: Tiziano Strambini

Operations Director: Antonio Boezio

© Elsevier Srl - All rights reserved

J. Stewart Cameron, CBE, MD, FRCP

Foreword to the third edition VI

Historical introduction (J.S. Cameron) 1

Chapter 1. G.B. Fogazzi and G. Garigali

Chapter 2. G.B. Fogazzi, G. Garigali, M.D. Croci and S. Verdesca

Renal tubular epithelial cells 57

Chapter 3. G.B. Fogazzi and S. Verdesca

De novo or recurrent glomerulopathy 203

Chapter 6. G.B. Fogazzi and S. Verdesca

Chapter 7. B. Pirovano and G.B. Fogazzi

Chapter 8. S. Secchiero and G.B. Fogazzi

Appendix. G.B. Fogazzi

FIGURE 1 Most medieval illustrations of physicians show

FIGURE 2 Nicolas Fabricius de Peiresc (1580-1637) who

«It is to be regretted that another method of investigation, microscopical examination, is

FIGURE 5 One of the earliest illustrations of a urinary

FIGURE 9 The urinary sediment as illustrated in John-

Medical Chirurgical Society in London in 1845, he was tartly reprimanded by an anonymous

Year Author(s) Country Title

Illustration of the constituents of urine,

Practical treatise on urinary and renal

The clinical examination of urine with

Year Author(s) Country Title

Robert Ultzmann und Atlas der physiologischen und

1896* Albert Daiber Switzerland Mikroskopie der Harnsedimente

Atlas der klinischen Mikroskopie

Year Author(s) Country Title

1880* Giulio Bizzozero Italy Manuale di microscopia clinica

Atlas der Mikroskopie

Mikroskopie und chemie

1914 Ch. Lesieur, M. Favre France Précis de microscopie clinique

FIGURE 13 Rieder’s illlustration of red cells in

FIGURE 14 Thomas Addis (1881-1949), who through-

FIGURE 15 The front cover of Richard

Acknowledgement. I am very grateful to Dr G.B. Fogazzi for much valuable help in

References and Notes

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

FIGURE 1.1 Suprapubic bladder puncture. The patient

preparation of the samples

TABLE 1.1 The main causes of abnormal urine colour.

Endogenous causes Exogenous causes

Haemoglobin Vegetable-derived substances

Pink Massive uric acid crystalluria in morbidly obese patients after

Brown Bilirubin, haemoglobin, myoglobin, homogentisic acid,

Black Haemoglobin, melanogen, homogentisic acid

Darkening upon standing Porphyrin, melanogen, homogentisic acid

has recently been investigated by comparing different preservation procedures on different

3 (2.8-8.4) FIGURE 1.2 Changes in cellular size with variations of the

Dipstick versus Haemoglobin Leukocyte esterase

Overall concordance 1755 (76.1%) 1913 (82.9%)

Disagreement 550 (23.9%) 392 (17.1%)

SURNAME ....................................................................... NAME ................................................................... DATE OF BIRTH ...................................................

ISOMORPHIC (%): ............................................ DYSMORPHIC (%): .................................................. ACANTHOCYTES(%): .................................................

RENAL TUBULAR EPITHELIAL CELLS: ........................................................................................................................................................................................

TRANSITIONAL CELLS. SUPERFICIAL: .................................................................................... DEEP: ......................................................................................