The Urinary Sediment An Integrated View [3 ed.]

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A.MENARINI diagnostics

Edizione con -l °""· ......

Guida alla lettura in italiano

"

Giovanni B. Fogazzi I

The Urinary Sediment An Integrated View THIRD EDITION with a Historical Introduction by

J. Stewart Cameron

'i,

MASSON

Contributors "

J. Stewart Cameron, CBE, MD, FRCP Pubblicazione realizzata grazie al contributo di A. Menarini Diagnostic~

Department of Nephrology and Transplantation Guy's campus, King's College London, United Kingdom

Books Publishing Director: Tiziano Strambini Development Editor: Paola Leschiera

Maria Daniela Croci, technician of biomedical laboratory

Operations Director: Antonio Boezio Books Production Manager: Ornella Ceresa Creative Director: Giorgio Gandolfo Cover Design: Gaetano Altamura

Research Laboratory on Urine Nephrology Unit Fondazione IRCCS Ospedale Maggiore Policlinico Mangiagalli e Regina Elena Milano, Italy

© Elsevier Sri - All rights reserved 1993 1999 2001 2002 2009 2010 2010 -

First edition (Masson Spa - Milano) Second edition (Masson Spa - Milano) First reprint (Masson Spa - Milano) Second reprint (Masson Spa - Milano) Third edition Reprint (March) Reprint (June)

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without prior written permission from the publisher. Reproduction requests rnust be addressed to AIDRO, via delle Erbe 2, 20121 Milano - Phone and Fax 0039 02809506, e-mail: [email protected] Every effort has been made to ensure that the drug dosage schedules within this text are accurate and conform to standards accepted at tirne of publication. However, as treatment recommendations vary in light of continuing research and clinical experience, the reader is advised to verify drug dosage schedules herein with information found on product information sheets. This is especially true in cases of new or infrequently used drugs.

Giovanni B. Fogazzi, MD Research Laboratory on Urine Nephrology Unit Fondazione IRCCS Ospedale Maggiore Policlinico Mangiagalli e Regina Elena Milano, Italy Giuseppe Garigali, ScD Research Laboratory on Urine Nephrology Unit Fondazione IRCCS Ospedale Maggiore Policlinico Mangiagalli e Regina Elena Milano, Italy

Barbara Pirovano, ScD Laboratory Medicine Unit Ospedale di Romano di Lombardia, Italy

Sandra Secchiero, ScD Biomedical Research Centre Castelfranco Veneta, Italy ELSEVIER Elsevier Sri Via Paleocapa 7, 20121 Milano Tel. 02.88.184.1 www.elsevier.it Printed in Italy Printed by "Printer Trento" S.r.l.,Trento - Italy, June 2010

Simona Verdesca, MD esearch Laboratory on Urine Nephrology Unit Fondazione IRCCS spedale Maggiore Policlinico Mangiagalli e Regina Elena Milano, Italy

I

Preface to the third edition "

I 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

Contents •'

I

Foreword to the third edition Preface to the third edition Historical introduction (J.S. Cameron) References and notes

Chapter t.

1

16

G.B. Fogazzi and G. Garigali

Collection, preparation and examination of the samples, and report of the urinary findings Urine collection Preparation of the samples Inspection Preservation of samples Centrifugation Resuspension Preparation of slides Microscopic examination Report of findings The microscope for the analysis of urinary sediments The phase contrast microscope The polarized light The bright field microscope Other microscopic techniques The stains for urinary sediments References

Chapter 2.

VI VII

19 19 21 21 21 22 24 24 24

26 32 32 35 35 35 37··· 38

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

The formed elements of the urinary sediment Cells Erythrocytes Leukocytes Macrophages

41 41 42

49 54

XII

Contents

Renal tubular epithelial cells Transitional epithelial cells Squamous epithelial cells Lipids Casts Hyaline casts Granular casts Waxy casts Cellular casts Fatty casts Casts containing crystals and amorphous salts Casts containing microorganisms Pigmented casts Mixed casts Cylindroids Pseudocasts Mucus Crystals Common crystals Uric acid Amorphous urates and amorphous phosphates Calcium oxalate Calcium phosphate Triple phosphate Pathologic crystals Cholesterol Cystine Leucine Tyrosine 2, 8-dihydroxyadenine Crystals due to drugs Other crystals Hippuric acid Calcium carbonate Ammonium biurate Organisms Bacteria Yeasts Trichomonas vaginalis Schistosoma haematobium (urinary schistosomiasis) Enterobius vermicularis Contaminants Contaminants originating from the patient Contaminants originating in the laboratory Contaminants originating in the environment Possible misidentifications

References

57 63 69 71

77 80 83 85 88 92 92 92 95 97 99 101

103 105 108 108 113 115

120 123 127 127 128 130 130 130 133 133 133 134 135 136 136 1:39 141 142 143 144 144 148 150 152 153

XIII

Contents

Chapter 3.

G.8. Fogazzi and S. Verdesca

,.

159 159 168 168 168 168

Changes of urinary sediment caused by drugs Drug-related crystalluria Other changes induced by drugs Diuretics Drugs influencing urinary pH

R eferences

I

Chapter 4. G.8. Fogazzi 173 176

The urinary sediment of the normal subject References

Chapter 5.

G.8. Fogazzi and S. Verdesca The urinary sediment in the main diseases of the kidney and of urinary tract Minimal change disease and focal segmental glomerulosclerosis Urinary findings Membranous nephropathy Urinary findings IgA nephropathy Urinary findings Membranoproliferative glomerulonephritis Urinary findings Acute post-streptococcal glomerulonephritis Urinary findings Extracapillary glomerulonephritis Urinary findings Lupus nephritis Urinary findings Schonlein-Henoch purpura nephritis Urinary findings Diabetic nephropathy Urinary findings Nephropathies due to plasma cell dyscrasias Urinary findings Acute interstitial nephritis Urinary findings Eosinophiluria - is it a specific marker of ain ? Erythrocytic casts - can they be found in the urine of patients with ain? Chronic interstitial nephritis Urinary findings Acute tubular necrosis Urinary findings Renal transplantation Acute cellular rejection Urinary findings Polyomavirus BK infection Urinary findings

.

177 177 178 178 180 180 183 183 184 184 185 185 185 186 186 187 189 189 189 190 191 191 192 193 194 ,, 194 195 195 197 197 197 198 198 200

Contents

XIV

De novo or recurrent glomerulopathy Urinary findings Urinary tract infection Urinary findings Urological disorders Urinary findings References

203 203 203 204 204 204 206

IN TRO D UC TI ON

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ •'

HISTORICAL INTRODUCTION J. Stewart Cameron I

Chapter 6. G.8. Fogazzi and S. Verdesca Interpretation of the urinary sediment findings The nephrotic sediment The nephritic sediment The nephrotic and nephritic sediment The urinary sediment containing many renal tubular epithelial cells The urinary sediment containing increased numbers of erythrocytes The urinary sediment containing bacteria and leukocytes Minor urinary abnormalities References

Chapter 7.

8. Pirovano and G.8. Fogazzi

Automated systems for urinary sediment analysis Automated intelligent microscopy Flow cytometry Main performances of automated analyzers Carry over Precision Accuracy Advantages, limits and role of automated instruments References

Chapter 8.

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).

233 233 234 234 238 244 244

G.8. Fogazzi

Adjustment of the microscope 1. Adjustment according to the Kohler principle 2. Centering of the annular diaphragm of the condenser with the phase ring of the objective Index

221 221 224 226 226 229 229 230 232

«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 [l]).

S. Secchiero and G.8. Fogazzi

Quality control programs for urinary sediment Internal Quality Control External Quality Control Features of the Italian EQA Program "Urinalysis Performance" Results of "Urinalysis Performance" Comments on "Urinalysis Performance" References

Appendix.

211 211 214 216 216 217 218 218 219

247 247 249 251

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 associations of the various appearances of the urine are fanciful [2]. (Bartolomeus Anglicus, De proprietatibus rerum, Cambridge, Fitzwilliam Museum, MS 254, Folio 549; from MacKinney, L. Medical Illustrations in Medieval Manuscripts, 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 Provern;al 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).

Historical introduction

3

Although bloody urine had been noted in scarlatina! nephritis throughout the 17th and 18th centuries [13-15] , most notably by the astute Swedish paediatrician Rosen 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 Eugene Napoleon Vigla (1813-1872) (Figure 3) for the introduction of regular urine microscopy to clinical practice, described in 183 7 [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 "Traite des Maladies des Reins" published in three volumes between 1839 and 1841 [21], he writes:

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 Claudii Fabricii de Peiresc, senatoris aquisextiensis vita, per Petrum Gassendum, philosophum et matheseos professorem parisiensem . Hagae Comitis, Adriani Vlach, 1651) .

«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 !'urine, en revanche,fut cree par Rayer lui-meme" [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

4

J. Stewart Cameron

Historical introduction

5

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 3 Left: Pierre Olive Fran~ois Rayer (1793-1867), founder of French Nephrology. Right: his pupil and colleague Eugene Napoleon 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 Donne (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 deformes 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.

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FIGURE 5 One of the earliest illustrations of a urinary cast, taken from Simon's paper of 1843 [29] and reproduced 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 ofnephrotic 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

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;::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

Changes of urinary sediment caused by drugs

163

failure due to intratubular precipitation of crystals after a course with ciproftoxacin 750 mg twice daily for some days [25) . Interestingly, the crystals found irt 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 ciproftoxacin 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 x 5 µm to 360 x 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 3.3 Marked amoxycillin crystalluria showing "needles" and "shocks of wheat" (phase contrast, x

FIGURE 3.4 Birefringent crystals of amoxycillin (polarized light, x 160).

160).

FIGURE 3.5 A "broom bush-like" crystal of amoxycillin (phase contrast, x 400).

FIGURE 3.6 The same crystal by polarized light (x 400) .

FIGURE 3.7 A "star-like" crystal and "needles" of ciprofloxacin (phase contrast, x 256).

FIGURE 3.8 The same.crystals by polarized light (x 256).

164

G.B. Fogazzi and S. Verdesca

FIGURE 3.9 Many "needles" of ciprofloxacin (phase contrast, x 160).

FIGURE 3.10 The same crystals by polarized light (x 160) .

FIGURE 3.11 A clump of ciprofloxacin crystals with different shapes (phase contrast, x 400).

FIGURE 3.12 The same crystals by polarized light (x 400) .

Changes of urinary sediment caused by drugs

165

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]. I 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 2:1.015 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] . c;onsequently, 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 ).

Changes of urinary sediment caused by drugs

167

"

FIGURE 3.13 A "star-like" crystal and plates of indinavir (phase contrast, x 400).

FIGURE 3.15 Several crystals of indinavir with different shapes and sizes (phase contrast, x 160).

FIGURE 3.14 The same crystals by polarized light (x 400).

FIGURE 3.16 The same crystals by polarized light (x 160).

FIGURE 3.17 lndinavir crystal with the shape of irregular plates (phase contrast, x 160).

FIGURE 3.18 The same crystals by polarized light (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 an 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 LennoxGastaut 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 µrn to > 1300 µm), which can be either isolated or clumped

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G.B. Fogazzi and S. Verdesca

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.

Changes of urinary sediment caused by drugs

169

[6] SIMON D.I., BRosrus III F.C., ROTHSTEIN D.M. Sulfadiazine crystalluria revisited. Arch Intern Med 1990; 150: 2379-84. •' [7] PORTOLES J., TORRALBO A., PRATS D. et al. Acute renal failure and sulfadiazine crystalluria in kidney transplant. Nephrol Dial Transplant 1994; 9: 180-1. [8] DE SEQUERA P., ALBALATE M., HERNANDEZ J. et al. Acute renal failure due to sulfadiazine crystalluria in AIDS patients. Postgrad Med J 1996; 72: 557-8 . [9] LEHR D., ANTOPOL W. Specific morphology of crystals appearing in the urine during administration of sulfanilamide derivatives. Am J Clin Pathol 1942; 12: 200-9. [10] S16VALL J, WESTERLUND D , ALVAN G. Renal excretion of intravenously infused amoxicillin anQI ampicillin. Br J Clin Pharmacol 1985; 19: 191-201. [11] MOESCH C., RAVASSE P., LEROYER R. et al. Differents types de crystallurie d' amoxicilline. Ann Biol Clin 1990; 48: 331-5.

OTHER CHANGES INDUCED BY DRUGS

FoGAZZI G.B., CANTU M., SAGLIMBENI L. et al. Amoxycillin, a rare but possible cause of crystalluria. Nephrol Dial Transplant 2003; 18: 212-4. [13] BRIGHT D.A., GAUPP F.B. et al. Amoxycillin overdose with gross hematuria. West J Med 1989; 150: 698-9. [14] JoNES P, GABER L, NILSSON GR et al. Acute renal failure following amoxycillin overdose. Clin Pediatr

DI URETICS

[ 15]

BOFFA J.J., DE PRENEUF H., BouADMA L. et al. Insuffisance renale aigue par cristallisation d' amoxycilline. Presse Med 2000; 29: 699-701.

[16]

LABRIOLA L., JADOUL M ., DAUDON M. et al. Massive amoxicillin crystalluria causing anuric acute renal failure. Clin Nephrol 2003; 59: 455-7. BOURSAS M ., BENHASSINE L., KEMPF J. et al. Insuffisance renale obstructive par cristallurie a l'amoxycilline. Ann Fr Reanim 1997; 16: 908-10 JoNES H.M., SCHRADER W.A. Ampicillin crystalluria. Am J Clin Pathol 1972; 58: 220-3. CLARK R.F. Crystalluria following cephalexin overdose. Pediatrics 1992; 89 : 672-4. SCHLUTER G. Ciprofloxacin: review of potential toxicologic effects. Am J Med 1987; 82 (suppl 4A): 91-93.

[12]

1993; 32: 735-9.

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].

[17] [18] [19] [20] [21]

DRUGS INFLUENCING URINARY pH [22]

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.

[23] [24] [25] [26]

SWANSON B .N., BoPPANA V.K,, VLASSES P.H. et al. Norftoxacin disposition after sequentially increasing oral doses. Antimicrob Agents Chemother 1983; 23: 284-8. [28] POTTER J.L. , KRILL C.E. Acyclovir crystalluria. Pediatr Infect Dis J 1986; 5: 710-2. [29] BLOSSOM A.P., CLEARY J.D., PALEY W.P. Acyclovir-induced crystalluria. Ann Pharmacother 2002; 36: 526. [30] LYON A.W., MANSOOR A., TROTTER M. J. Urinary gems: acyclovir crystalluria. Arch Pathol Lab Med 2002; 126: 753-4. [31] SAWYER M .H., WEBB D.E. et al. Acyclovir-induced renal failure. Am J Med 1988; 84: 1067-71. [32] BRIGDEN D., RosLING A.E., Wooos N.C. Renal function after acyclovir intravenous injection. Am J Med 1982; 73 (Suppl lA): 182-5. [33] BECKER B.N., FALL P., HALL C . et al. Rapidly progressive renal failure due to acyclovir: case report and review of the literature. Am J Kidney Dis 1993; 22: 611-5. [27]

REFEREN CES

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BUCHANAN N. Sulphamethoxazole, hypoalbuminaemia, and renal failure. Br Med J 1978; 2: 172. CLARK R.F. Crystalluria following cephalexin overdose. Pediatrics 1992; 89: 672-4. PERAZZELLA M.A. Drug-induced renal failure: update on new medications and unique mechanisms of nephrotoxicity. Am J Med Sci 2003; 325: 349-62. CARBONE L.G., BENDIXEN B ., APPEL G .B. Sulfadiazine-associated obstructive nephropathy occurring in a patient with acquired immunodeficiency syndrome. Am J Kidney Dis 1988; 12: 72-5. SAHA! J., HEIMBERGER T., COLLINS K. et al. Sulfadiazine-induced crystalluria in a patient with the acquired immunodeficiency syndrome: a reminder. Am J Med 1988; 84: 791-2.

THORSTEINSSON S., BERGANT., 0DDSDOTTIR S. et al. Crystalluria and ciproftoxacin, inftilence of urinary pH and hydration. Chemotherapy 1986; 32: 408-17. NIX D .E., SPIVEY J.M., NORMAN A. et al. Dose-ranging pharmacok:inetic study of ciprofloxacin after 200, 300, and 400 mg intravenous doses. Ann Pharmacother 1992; 26: 8-10. CHOPRA N., FINE P., PRICE B. et al. Bilateral hydronephrosis from ciprofoloxacin induced crystalluria and stone formation. J Urol 2000; 164: 438 . SEDLACEK M., SURIAWINATA A .A., SCHOOLWERTH A. et al. Ciprofloxacin crystal nephropathy - a new cause of acute renal failure . Nephrol Dial Transplant 2006; 21: 2339-40. STRATTA P., LAZZARICH E. et al. Ciproftoxacin crystal nephropathy. Am J Kidney Dis 2007; 50: 330-5. FoGAZZI G.B ., GARIGALI G., BRAMBILLA C. et al. Ciproftoxacin crystalluria. Nephrol Dial Transplant 2006; 21: 2982-3.

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DAUDON M., EsTEPA L., VIARD J.P. et al. Urinary stones in HIV-I -positive patients treated with indinavir. Lancet 1997; 349: 1294-5. TASHIMA K.T., HOROWITZ J.D., RosEN S. Indinavir nephropathy. N Engl J Med 1997; 336: 138-40. KOPP J.B., MILLER K.D. , M1CAN J.A.M. et al. Crystalluria and urinary tract abnormalities associated with indinavir. Ann Intern Med 1997; 127: 119-25. BERNS J.S., COHEN R.M., SILVERMAN M. et al. Acute renal failure due to indinavir crystalluria and nephrolithiasis: report of two cases. Am J Kidney Dis 1997; 30: 558-60. PERAZELLA M.A. , KAsHGARIAN M., COONEY E. Indinavir nephropathy in an AIDS patient with renal insufficiency and pyuria. Clin Nephrol 1998; 50: 194-6. MARTINEZ F., MoMMEJA-MARIN H. , EsTEPA-MAURICE L. et al. Indinavir crystal deposits associated with tubulo-interstitial nephropathy. Nephrol Dial Transplant 1998; 13: 750-3. TSAO J.W., KOGAN S.C. Indinavir crystalluria. N Engl J Med 1999; 340: 1329. GAGNON R.F., ALLI A.I., EDWARDES A.K. et al. Low urine pH is associated with reduced indinavir crystalluria in indinavir-treated HIV-infected individuals. Clin Nephrol 2006; 65: 13-21. KOPP J.B ., FALLOON J., FILIE A. et al. Indinavir-associated interstitial nephritis and urothelial inflammation: clinical and cytologic findings. Clin Inf Dis 2002; 34: 1122-8. DIELEMAN J.P., v AN ROSSUM A.M.C., STRICKER B.C.H. et al. Persistent leukocyturia and loss of renal function in a prospectively monitored cohort of HIV-infected patients treated with indinavir. J Acquir Immune Defic Syndr 2003; 32: 135-142. SPENCE J.D., WONG D.G., LINDASY R.M . Effects of triamterene and amiloride on urinary sediment in hypertensive patients taking hydrochlorothiazide. Lancet 1985; ii: 73-5. FAIRLEY K.F., Woo K.T., BIRCH D.F. et al. Triamterene-induced crystalluria and cylindruria: clinical and experimental studies. Clin Nephrol 1986; 26: 169-73. RoY L.F., VILLENEUVE J.P., DUMONT A. et al. Irreversible renal failure associated with triamterene. Am J Nephrol 1991; 11: 486-9. DAUDON M ., REvEILLAUD R.J. , NORMAND M. et al. Piridoxylate-induced calcium oxalate calculi: a new drug-induced metabolic nephrolithiasis. J Urol 1987; 138: 258-60. LEHMANN D.F. Primidone crystalluria following overdose. A report of a case and an analysis of the literature. Med Toxicol 1987; 2: 383-7. BIRCH D.F., FAIRLEY K.F., BECKER G.J. et al. Colour Atlas of Urine Microscopy. London: Chapmann and Hall, 1994; pp. 129-30. SIGG T., LEIKIN J.B. Massive crystalluria in a patient taking primidone. Ann Emerg Med 1999; 33: 726-7. MOESCH C ., CHARMES J.P., BouTHIER F. et al. Calcium oxalate crystalluria in elderly patients and treatment with naftidrofuryl oxalate. Age and Ageing 1995; 24: 464-7. MOESCH C., RINCE M ., DAVDON M. et al. Renal intratubular crystallization of calcium oxalate and naftidrofuryl oxalate. Lancet 1991; 338: 1219-20. LE MEUR Y. , MOESCH C., RINcE M. et al. Potential nephrotoxicity of intravenous infusions of naftidrofuryl oxalate. Nephrol Dial Transplant 1995; 10: 1751-5. CuvELIER C., GoFFIN E. , CosYNS J.P. et al. Acute renal failure due to naftidrofuryl oxalate Praxilene® overdose in a kidney transplant recipient. Nephrol Dial Transplant 1995; 10: 1756-8. AUER B.L., AUER R.D., RODGERS A.L. Relative hyperoxaluria, crystalluria and haematuria after megadose ingestion of vitamin C. Eur J Clin Invest 1998; 28: 695-700. McALLISTER C.J., ScowDEN E.B., DEWBERRY F.L. et al. Renal failure secondary to massive infusion of Vitamin C. J Am Med Assoc 1984; 252: 1684. LAWTON J.M., CONWAY L.T., CROSSON J.T. et al. Acute oxalate nephropathy after massive ascorbic acid administration. Arch Intern Med 1985; 145: 950-1. RAMASWAMY C.R. , WILLIAMS J.D., GRIFFITHS D.F.R. Reversible acute renal failure with calcium oxalate cast nephropathy - possible role of ascorbic acid. Nephrol Dial Transp lant 1993; 8: 1387-9.

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WONG K., THOMSON C. , BAILEY R.R. et al. Acute oxalate nephropathy after a massive intravenous dose of vitamin C. Aust NZ J Med 1994; 24: 410-1. ,• SINGH A., SARKAR S.R., GABER L.W. et al. Acute oxalate nephropathy associated with orlistat, a gastrointestinal lipase inhibitor. Am J Kidney Dis 2007; 49: 153-7. RENGSTORFF D.S. , MILSTONE A.P., SEGER D.L. et al. Felbamate overdose complicated by massive crystalluria and acute renal failure. Clin Toxicol 2000; 38: 667-9. MEIER K.H., OLSON KR., OLSON J.L. Acute felbamate overdose with crystalluria. Clin Toxicol 2005 ; 43: 189-92. PERAZZELLA M.A. Crystal-induced acute renal failure . Am J Med 1999; 106: 459-65. I DAUDON M., JUNGERS P., LACOUR B. Interet clinique de l'etude de la cristallurie. Ann Biol Clin 2004; 62: 379-93 . DEREBAIL V.K., McGREGOR J.G. , COLINDRES R.E. et al. Acute kidney injury in a patient with P. carinii pneumonia. Kidney Int 2009; 75: 865-6. IMHOF P.R., HusHAK J., SCHUMANN G. et al. Excretion of urinary casts after administration of diuretics. Br Med J 1972; 2: 199-202. BRADEN L., SANCHEZ P.O., FITZGIBBON J.P. et al. Urinary doubly refractile lipid bodies in non-glomerular renal diseases. Am J Kidney Dis 1988; 11: 332-7. SCHUETZE., SCHAEFER R .M ., HEIDBREDER E . et al. Effect of diuresis on urinary erythrocyte morphology in glomerulonephritis. Klin Wochenschr 1985; 63: 575-7. McQuEEN E.G., ENGEL G.B. Factors determining the aggregation of urinary mucoprotein. J Clin Pathol 1966; 19: 392-6. FRIEDMAN I.S ., ZUCKERMAN S., COHN T.D. The production of urinary casts during the use of cation exchange resins. Am J Med Sci 1951; 221: 672-7 .

CHAP TER

4

•'

THE URINARY SEDIMENT OF THE NORMAL SUBJECT G.B. Fogazzi

I

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. WBCs mean No. (range)

Casts mean No. (range)

Author [ref]

Subjects studied

Addis [1]

74 adults

65 ,750/12 h (0-425,000)

322,500/12 h (32,400-1 ,835,000)

1,040/12 h (0-4,270)

Lyttle [2]

74 children

15,181±400/12 h (0-129 ,000)

322 ,184 ± 25,500 (9,0002,822 ,000/12h)

1,085 ± 123/12 h (0-12,916)

Aas [3]

119 newborns

90,129/mm 3 (0-630,000)

1,865,529/mm 3 (42 ,000-13,500,000)

12,852/mm 3 (0-440 ,000)

I

G.B . Fogazzi

174

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 x 106 erythrocytes/L (i .e. < 14,000/mL) and< 4 x 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 x 106/L versus 1.2 x 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 4.1and4.2? 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. Author [ref]

Subjects studied

Normal erythrocyturia

Birch [5]

376 adults

< 8,000/ml

Pollock [6]

27*

< 1,000/ml

Loh [7]

419 children

< 14,000/ml

*Age and sex not described in the paper.

Normal leukocyturia

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). I 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 µL 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.

.-

Author [ref]

Tratchman [9]

Children

?

Schroder [1 OJ

Children

?

Piqueras [11]

Children

Uncentrifuged

> 5/ml

Topham [12]

Adults

Uncentrifuged

> 5 x 106/L

Mc Gregor [13]

Adults

Uncentrifuged

Chow [14]

Adults

Centrifuged

> 2/HPF

Kovacevic [15]

Young males

?

> 5/HPF

> 240,000/12 hours