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Dr G.B Fogazzi
Research Laboratory on Urine, Unit Operativa di Nefrologia
Fondazione IRCCS, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena
Milan, Italy
Slide 1
Slide 2
Slide 3
Prof Fogazzi: This is the outline of the course. After a brief introduction, I will speak
about the main methodological aspects concerning the urinary sediment; the
particles of the urinary sediment of nephrological importance with their clinical
implications; the urinary sediment in the clinical practice and, finally, I will draw
some conclusions.
Slide 4
Slide 5
From the historical point of view, we know that the urinary sediment was introduced
into clinical practice in the late 1830s in Paris at La Charit hospital by Franois Rayer
and his pupil Eugne Napolon Vigla. By the end of the 19th century, all the main
particles had been identified and the main urine profiles had been described.
However, in the subsequent century, the 20th century, the urinary sediment
examination knew only a progressive decline with only very few exceptions. One was
the original and important work of Thomas Addis in the 1920s and the other was the
publication, in 1982, of a paper by Fairley and Birch on the utility of urinary
erythrocyte morphology evaluation by phase contrast microscopy in patients with
hematuria.
Slide 6
What is the situation today? In most instances, urinary sediments are examined in
central laboratories far from bedside and without the correct equipment and
knowledge, and with the dream to entrust the whole task to automated instruments.
These are already on the market, one being UF 100, which is based on flow
cytometry, and the other iQ200, which is based on images obtained by a video
camera. Last but not least, too often the urinary sediment examination is neglected
even by nephrologists.
Slide 7
Slide 9
Methodological aspects are very important for urine sediment. The main
methodological aspects include: a correct urine collection; a standardised method for
the handling of the urine; the use of a proper microscope and the use of a proper
report to describe the findings. All these aspects are described in detail in the
document published by the European Urinalysis Group 5 years ago as a supplement
of the Scandinavian Journal of Clinical and Laboratory Investigation (2000; Vol 60,
Suppl 231) as well as in our book (Fogazzi GB, Ponticelli C, Ritz E. The Urinary
Sediment. An Integrated View 2nd Edition. Oxford , Oxford University Press, 1999).
Slide 10
As to urine collection, its important to give the patient written and simple
instructions. According to the strategy of the single lab, we can ask the patient to
supply the first or the second urine of the morning. In our lab, we ask for the
second urine, since overnight urine, due to its prolonged permanence in the bladder
can favour the lysis of particles. We suggest the patient to avoid strenuous physical
effort in the hours preceding the test, since this may influence in various ways the
findings (for instance by causing haematuria and/or cylindruria). We advise the
patient to clean the external genitalia in an ordinary way. In this respect, we do not
suggest special procedures since the more the procedures suggested are
complicated, the less the patient is compliant. In order to avoid contamination, the
male has to uncover the glans and female to spread the labia of the vagina. For the
same reason, we always suggest to collect midstream urine. Its important to
remember that urine collection during menstruation must be avoided because of the
high probability of blood contamination. It is also important to use a proper urine
container (with a capacity of at least 50 to 100 mL, an opening of at least of 5 cm to
allow easy collection of urine for both men and women, a wide base to avoid
accidental spillage, a cap to avoid leakage, a label for patient identification). It is no
more time for the patient to collect the urine into jugs, bottles, cans, etc.
Slide 11
What about a standardized method for the handling of urine? Why is standardization
of the handling of the urine important? It is important because only with a
standardized method we can obtain quantitative reproducible results. The slide
shows the method that we use in our lab. We ask the patient to supply the second
urine of the morning produced over a period of 2 hours; then, we centrifuge a 10 mL
aliquot of urine for 10 minutes at 400 G , which correspond to 2,000rpm with our
centrifuge. Then, we remove with a pump a fixed volume of supernatant urine, which
is 9.5 mL. Then, with a Pasteur pipette, we gently but thoroughly re-suspend the
sediment in the remaining 0.5 mL of urine. Then, with a precision pipette, we
transfer 50 L of resuspended urine to a glass slide, which is covered with a coverslip
of a fixed surface, namely 32 x 24 mm . Then, we examine the samples at low and
high magnification (160x and 400x) within 3 hours from urine collection. For routine
practice, we express the particles as lowest/highest number seen by microscopic
field. When we want to produce scientific results, we count the number of the cells
found over 20 high power field, as we will see in the last part of my speech (see The
urinary sediment findings in proliferative and non proliferativer glomerular
diseases). With this method we were able to obtain reproducible results, which in
addition correlated significantly with the number of particles found by the counting
chamber.
Slide 12
And now the microscope. The microscope must be of good quality, must be equipped
with a low and high magnification, and must, and I want to stress must, be equipped
with phase contrast and polarized light.
Slide 13
Slide 14
Its very simple to explain. On the right you see bright-field, and on the left you see
phase contrast. You see that with phase contrast the particles are much better seen
against the background than with bright-field, and this without the use of stains! I
want to stress that the European Guidelines strongly recommends the use of phase
contrast microscopy.
Slide 15
Slide 16
And why polarized light? Polarized light is extremely useful to correctly recognize the
crystals. For example, you see uric acid crystals as seen by phase contrast
microscopy (slide 15), and what happens when we use polarized light (slide 16).
Under polarized light, uric acid crystals assume a typical polychromatic appearance,
which is useful to identify them.
Slide 17
which under polarized light appear as Maltese crosses which is, shining particles
containing a black cross whose arms are regular and symmetrical. This feature
allows the identification of lipid particles, especially when they come with an atypical
appearance.
Slide 19
Now, the urinary sediment report. This is the report we use in our lab. After the
patient details, we have pH, density (or specific gravity), haemoglobin and leukocyte
esterase as detected by dipstick. Then the particles: erythrocytes (with their
morphological classification; see below), leukocytes, tubular cells, transitional cells
(from the deep and superficial layers of the uroepithelium), squamous cells, casts,
lipids, crystals, bacteria, and yeasts. We also have a space for a brief conclusive
comment. I want to stress the importance of having in the report the findings
obtained by dipstick. Why this?
Slide 20
Let me give you an example. You see in this slide a sample with a pH of 6.0, a
density of 1.006, a +++ haemoglobin and +++ leukocyte esterase by dipstick.
However, by microscopy we find a low number of erythrocytes and leukocytes, which
is in contrast with +++ haemoglobin and +++ leukocytes esterase. Is there any
explanation for this discrepancy? Yes there is. The discrepancy is due to the fact that
low density causes the lysis of erythrocytes and leukocytes, which therefore cannot
be seen by microscopy. In contrast, in other instances we may have negative
haemoglobin and many erythrocytes by microscopy. This may be due, for example,
to the presence of large concentrations of Vitamin C in the urine, which reduces the
sensitivity of dipstick for haemoglobin. Therefore, it is always important to match the
findings obtained by dipstick with those obtained with microscopy and to try to
explain them. Thus, our comment for the sample shown in the slide is: Mild
erythrocyturia and leucocyturia. Please note the discrepancy between dipstick for
haemoglobin and leukocyte esterase and microscopy. This is probably due to cell
lysis caused by low density. The final message on this point is that examining the
urine only by dipsticks or only by microscopy exposes to the risk of false results. This
risk is reduced when both methods are used on the same sample.
PART II
Slide 21
Slide 22
And now the particles of the urinary sediment of nephrological importance with their
clinical meaning. In the urine sediment we can find cells, lipids, casts, crystals and
microorganisms.
Slide 23
What about cells? We have two groups of cells in the urinary sediment: the cells
deriving from blood and the cells of epithelial origin. The cells deriving from blood
include: erythrocytes, leukocytes, and macrophages. The epithelial cells include:
renal tubular cells, transitional cells, and squamous cells. For the lack of time, today
I will not speak about transitional and squamous cells.
Slide 24
What about erythrocytes? In our lab, they are a frequent finding, being observed in
53% of the samples. Since the early 1980s we know that in the urine we can find
two main types of erythrocytes: the so-called glomerular (or dysmorphic)
erythrocytes and the so-called non-glomerular (or isomorphic) erythrocytes.
Slide 25
While this slide shows a nice example of non glomerular or isomorphic erythrocytes
i.e., erythrocytes with a spherical shape and regular contours, containing (greenbluish cells) or not (colourless cells) haemoglobin.
Slide 27
In the same year, Fassett and co-workers, again from Australia, published a paper in
Lancet in which, besides confirming the results of Fairley and Birch, established that
a haematuria is of glomerular origin when it contains >80% dysmporhic
erythrocytes, while it is of non glomerular origin when >80% of erythrocytes are
isomorphic.
Slide 30
Using this criterion, they obtained a correct diagnosis in 115/120 patients with a
glomerular disease and in 100/105 patients with a urological disorder. The same
criterion was adopted by other investigators such as Dr De Santo from Naples and Dr
Rath from London, UK, and if we put all the results together we see that a correct
diagnosis could be obtained in 93-94% of cases.
Slide 31
Thus, put in this way the whole matter could seem a little complicated and not very
useful in clinical practice. However, some years ago Khler and co-workers published
an important paper, which overtook some of the problems mentioned above.
Slide 33
In their paper, Khler and co-workers showed that there is a subtype of dysmorphic
erythrocyte, which they called acanthocyte, which can be easily (and less
subjectively) identified due to its shape of a ring from which one or more blebs
protrude (slide 33 shows an acanthocyte as seen by scanning electron microscopy)
and which they found to be a marker of glomerular bleeding.
Slide 34
In this slide you can see how easily the acanthocytes can be identified by phase
contrast.
Slide 35
The paper of Khler and co-workers stimulated other groups to investigate the utility
of the search of acanthocytes in the urine. The main studies published so far have
shown that by using a cut-off for acanthocytes of 5% a glomerular bleeding could
be identified with a 52-100% sensitivity and a 96-100% specificity. In addition,
Khler and co-workers subsequently showed that if the patient supplies a second
urine sample, sensitivity goes up to 72%, and to 84% if supplies a fourth urine
sample. Thus, my advice is to start the evaluation of erythrocyte morphology by the
search of acanthocytes. If they are not present, we proceed with the search of the
other dysmorphic red cells.
Slide 37
What is the main indication for the evaluation of urinary erythrocyte morphology in
clinical practice? Its persistent isolated microscopic haematuria of unknown origin
(see below slides 109-117). In this condition, the evaluation of red cell morphology
helps in deciding whether the patient has to be submitted to a nephrological workup rather than to a urological one. This saves to the patient inappropriate
investigation such as cystoscopy for a patient with haematuria due to a glomerular
disease.
Slide 38
These are eosinophils, which can be identified only after staining (in this case MayGrnwald-Giemsa). Eosinophiluria has been considered as a marker of acute
interstitial nephritis.
Slide 41
After the publication in the New England Journal of Medicine in 1986 of this paper by
Nolan and colleagues we know that eosinophiluria can be found in a wide range of
conditions, not only in acute interstitial nephritis.
Slide 43
In fact, by using a specific stain for eosinophils which is, Hansel stain, it was found
that these cells were present not only in acute interstitial nephritis, but also in rapidly
progressive glomerulonephritis, and acute prostatitis. In addition, subsequent studies
demonstrated that eosinophiluria can also be found in acute renal failure caused by a
cholesterol embolism, urinary schistosomiasis, Schnlein-Henoch purpura nephritis,
etc. Therefore, I believe that eosinophiluria cannot be longer considered as a specific
marker of acute interstitial nephritis. For this reason in our lab we have abandoned
the search of urinary eosinophils.
Slide 44
The utility of the search of lymphocytes in the urine sediment has been largely
investigated in the 1980s and early 1990s. Most studies have demonstrated that
they are an early marker of acute cellular rejection of renal allograft, with a 80-90%
Now renal tubular cells. If you remember slide 23, among the cells deriving from
blood I also mentioned macrophages. I will speak about these cells in the last part of
the course (see slides 163-171).
Slide 47
As you can see in the slide, there are different morphological types of renal tubular
cells, which derive from different tubular segments, from the proximal convoluted
tubule to the collecting duct.
Slide 48
Slide 49
This is an example of a distal tubular cell, which is smaller than the previous one,
and has a smaller cytoplasm.
Slide 50
And this is an example of a collecting duct cell. Compared to the two previous ones,
it has a columnar aspect and a basal nucleus. Renal tubular cells can be recognised
by phase contrast microscopy without difficulty, even though some experience is
needed. They can be confused with transitional cells of the deep layers of the
uroepithelium. However, while tubular cells are seen in a nephrological context, the
transitional cells are seen in patients with urological disorders.
Slide 51
What is the clinical meaning of tubular cells in the urine? They always indicate a
renal tubular damage. Therefore, they are a marker of acute tubular necrosis.
Moreover, they can be found in acute interstitial nephritis, in acute cellular rejection
of renal allograft etc. However, they can also be found in glomerular diseases
especially of proliferative type, as we will see later on.
Slide 52
Now, lipids. Lipids can be found in the urine as: fatty droplets, in clusters or isolated;
oval fat bodies, a definition which goes back to the 19th century; fatty casts, and
cholesterol crystals. In most instances, they are the consequence of lipid
ultrafiltration due to an impairment of glomerular basement membrane (GBM)
permeability. Therefore, they are a marker of GBM damage, as it occurs in
glomerular diseases. However, rarely, lipiduria can be due to lipid storage diseases,
such as Fabry disease.
Slide 53
These are lipid particles within the cytoplasm of a proximal renal tubular cell after
they have been ultrafiltered at glomerular level and reabsorbed by the tubular cells
and organised into lysosomes.
Slide 55
This is a typical oval fat body. Today we know that they are nothing but
macrophages or renal tubular cells gorged with lipid particles.
Slide 56
And here, a typical fatty cast. As already mentioned above (see slides 17 and 18),
under polarized light lipid particles show the typical appearance of Maltese crosses.
Slide 57
And here a typical cholesterol crystal, which instead does not polarize light.
Slide 58
What is the clinical meaning of urinary lipids? They are a typical marker of heavy
proteinuria as it is found in nephrotic syndrome. However, this is only a general rule,
because lipiduria has been described also in patients with mild to moderate
proteinuria, and in patients with nonglomerular disorders such as polycystic kidney
disease (Kirk et al. Urinary lipid bodies in polycystic kidney disease. Am J Kidney Dis
1985; 5: 49-53). Another situation in which lipiduria can be found is Fabry disease.
Slide 59
What are the urinary sediment findings in Fabry disease? By phase contrast, we see
cells laden with lipids as well as free fatty particles. By polarising light, we see the
so-called "Maltese crosses", and by electron microscopy we find lysosomal inclusions,
which appear as myelin figures or myelin bodies.
Slide 61
To show some images of lipiduria as found in Fabry disease, I have to turn to the
work A Colour Atlas of Urine Microscopy written by Birch DF, Fairley KF, Becker GJ,
and Kincaid-Smith P, and published by Chapman & Hall (London) in 1984. Here there
is a fatty particle under phase contrast microscopy,
and here the same particle under polarized light. You can see very well the Maltese
cross.
Slide 63
PART III
Slide 64
Now casts. I will not speak about the whole spectrum of casts we can find but only
the most important ones. Casts are elements, which form in the distal tubules and
collecting ducts of the kidney. They have a matrix, which is Tamm-Horsfall
glycoprotein, which is produced by the thick ascending segment of the loop of Henle.
There are different types of casts with different clinical meanings.
Slide 65
What about the clinical meaning of casts? One cause that I think is important,
whatever particle is contained in the cast, this particle comes from the kidneys.
Slide 66
Thus if we have an erythrocyte cast, we must know, we must remember that this
means that the red cells come from the kidneys. With a sensitivity however, which
seems to be low.
Slide 67
Several investigators have looked for erythrocyte casts in patients with various types
of glomerulonephritis and you see here the percentage of patients which have been
found is rather low from 22 to 38% even though a more recent paper published by
the group of Doctor Khler has shown that if they are found in a very extensive way,
the percentage goes up to 86%. I will tell you later on what is our experience in 100
patients with glomerulonephritis.
Slide 68
Leukocyte casts. Again they tell us that the leukocytes come from the kidney which
may happen both in patients with glomerulonephritis and in patients with for
instance renal infection, pyelonephritis.
Slide 69
Epithelial casts, which contain tubular cells these casts are typically seen in patients
with acute tubular necrosis but they are also seen in patients with glomerular
nephritis and again Ill show you later on our findings.
Slide 70
Bacterial casts, extremely rare but again these tell us that the bacteria comes from
the kidney.
Slide 71
We can even have yeast casts but in this case this is a candial cast, again infection
comes from the kidneys.
Slide 72
Now the big chapter of urinary crystals. We can very arbitrarily distinguish 3 groups
of crystals, the so-called common crystals, the pathological crystals and crystals due
to drugs.
Slide 73
Common crystals first, uric acid crystals, which we always find in acidic urine.
Slide 74
What is the clinical meaning of these common crystals? In most instances, 99.99%
of cases, uric acid calcium oxalate and calcium phosphate crystals are simply due to
a transient super saturation of the urine which is caused by foods, dehydration or
changes of urine pH and/or temperature upon stenting. However, especially when
they are persistent in the same patient they may be associated with a metabolic
disorder such as hypercalcuria, hyperoxaluria or hyperuricosuria.
Slide 79
So they suggest that there is a metabolic disorder in these cases and very rarely
they can be associated with acute renal failure due to intrarenal precipitation of
crystals, which happens in 2 very well-known clinical situations which are acute urate
nephropathy which is associated with an intratubular precipitation of uric acid
crystals and the other situation is the poisoning entrained by ethylene glycol
ingestion which is associated with intratubular precipitation of calcium oxalate
crystals, which then appear in the urine.
Slide 80
Cysteine crystals they are a typical marker of patients with cysteinuria. The more the
urine is acidic, the higher is the possibility to find these crystals in the urine.
Slide 83
Now 2,8 dihydroxy adenine crystals. These crystals are similar, maybe similar to uric
acid crystals at least for their colour, which is an amber colour, brownish, yellowish
colour. They have spherical particles with irradiations which have origin in the centre
of the crystal and then spread towards the edge of the crystals.
Slide 84
These are crystals seen by phase, by bright-field microscopy and under polarised
light they appear as maltese crosses very similar to lipid particles.
Slide 85
Are these crystals important? The answer is yes. These crystals are due to the
hereditary deficiency of adenine phosphoribosyl transferase enzyme. APRT is an
enzyme which catalyses the transformation of adenine into adenosine
monophosphate. If there is a deficiency of this enzyme, adenine is transformed by
the xanthine oxidase into 2, 8 dihydroxy adenine, which is a molecule, which is
highly insoluble at any pH, which means that it goes, it precipitates always in the
urine present.
Slide 86
We know that there are 2 types of APRT deficiency, which is a condition, which is
inherited in an autosomal recessive manner. Type 1 which is seen in Caucasian
people is associated with a virtually absent enzyme activity while type 2 APRT
deficiency is seen in Japanese and is associated with a low but however, some
activity of the enzyme.
Slide 87
From the clinical point of view this condition has been well studied in Iceland where
the condition is endemic and you see here that any age can be affected by the
disease with a ratio male-female which is 1:1 and 65% of patients may have
recurrent radiolucent stone disease which is often confused with uric acid stone
disease. There will be 26% of patients with acute renal failure due to intratubular
precipitation of the substance, 17% of patients with chronic renal failure probably
due to chronic interstitial nephritis and 96% of patients may have crystalluria.
Slide 88
You see here these are the lumina of the tubules they are completely obstructed by
precipitated crystals while here crystals are probably within the cytoplasm of cells. It
is important to recognise this condition because after the renal biopsy the patient at
the time was on dialysis, after the identification of the crystals allopurinol had been
given and the patient was able to recover from acute renal failure.
Slide 90
The diagnosis of the deficiency of APRT. We can measure the level of the residual
enzyme activity in erythrocyte lisate. We can measure the urine to dehydroxy
adenine by high performance liquid chromatography, we can perform ultraviolet and
infrared spectrophotometry of stones and we can finally examine the urine sediment.
Slide 91
What is the role of urine sediment examination in this condition? It is well written
here, this statement of Doctor Alderperson an expert of this condition, skilful and let
me add motivated urinary microscopy is the single most important diagnostic
procedure because urinary 2 8-dehydroxy-adenine crystals are usually abundant in
untreated patients. So with a simple test we can do very important clinical things.
Slide 92
Now the main types of crystals due to drugs. A number of drugs can cause
crystalluria. For instance sulphadiazine and acyclovir, the antiretroviral agent
indinavir, piridoxylate which is used in some European countries as a coronary dilator.
Slide 93
the nature of which here was confirmed by spectroscopy which was performed in
Paris by our colleague and friend Professor Daudon in indinavir crystal and in
acyclovir crystals.
Slide 97
Slide 98
Slide 99
What are the factors, which favour the precipitation of drugs in kidneys? First of all is
a drug overdose, dehydration, hypoalbuminemia which causes the presence of a high
percentage, proportion of unbound drug in the blood and urine pH, some drugs
precipitate at acidic pH for instance, amoxycillin while neutral pH or alkaline pH is
necessary for the precipitation of ciprofloxacin crystals.
Slide 100
What are the clinical manifestations of drug crystalluria? One is isolated and
asymptomatic crystalluria, the most frequent one then they can cause hematuria,
microscopic or gross hematuria with or without leucocyturia. Crystals are very
frequently associated with stones, which can cause obstructive uropathy and they
can even cause acute renal failure due to intratubular precipitation of crystals with
the same mechanisms Ive just described for 2,8 dihdyroxiadenine.
Slide 101
General rules to follow when we think that there is a crystalluria due to drugs in the
urine. First think of a drug whenever you come across crystals with unusual
appearance. Second, if you have this feeling, this impression ask the patient if and
which drugs she/he is taking, then check the renal function because acute renal
failure may be a consequence of crystalluria and then hydrate the patient and reduce
and discontinuate the drug to prevent acute renal failure.
Slide 102
Microorganisms. We know here everybody knows that in the urine we can find
bacteria, yeast protozoa and parasites. Bacteria can be present in the urine as a
marker of infection or of contamination of the urine. Candida most of the time comes
from contamination from the genital secretion as well as Trichomonas vaginalis.
Enterobius vermicularis is very rare and it can be seen as a result of the
contamination of the urine from faeces especially in children. A true infection is
always caused by schistosoma haematobium. Urinary sediment plays an important
role in the diagnosis of schistosoma haematobium. Urinary schistosomiasis may not
be a problem in our countries, in Europe, in the United States but is a very important
clinical problem in a number of developing countries.
Slide 103
The urine sediment shows in patients with urinary schistosomiasis the presence of
the eggs of the schistosoma hematobium which are very easily recognised because
they are very big first of all 120-50 micrometers, a typical shape and this spine
which is a terminal spine which allows us to differentiate schistosoma haematobium
from schistosoma mansoni which has a spine which is lateral but which is not found
in the urine but is found in faeces.
Slide 104
Urinary schistosomiasis as I told you is endemic in many geographic areas, look here
in many areas of Africa going from areas North of the Sahara and in many sub
Saharan Africa, it is also present in the Middle East and here you see this is Iraq.
This is responsible for a recurrent bouts of gross hematuria, which are recurrent and
cyclic, and in many areas of sub-Saharan rural Africa it is also known as the agent
causative of "male menstruation". It can cause obstructive uropathy, bladder
carcinoma and glomerulonephritis.
Slide 105