HANDS-ON COURSE |
BEDSIDE URINARY MICROSCOPY |
GIOVANNI BATTISTA FOGAZZI LECTURES SERIES |
URINARY SEDIMENT: Part 2: Particles I |
G.B. Fogazzi, Milan, Italy
|
Dr G.B Fogazzi
Research Laboratory on Urine, Unità Operativa di Nefrologia Fondazione IRCCS, Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena Milan, Italy
|
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
The
slide shows a good example of glomerular or dysmorphic erythrocytes. These are
cells with irregular shape, size, and cell membrane, which differ remarkably
from the image of erythrocytes we have stored in our mind.
Slide 26
While
this slide shows a nice example of non glomerular or isomorphic erythrocytes i.e., erythrocytes with a
spherical shape and regular contours, containing (green-bluish cells) or not (colourless cells) haemoglobin.
Slide 27
What
is the clinical implication of distinguishing glomerular from non glomerular
erythrocytes? In 1982 Fairley and Birch from
Slide 28
That
paper showed that glomerular or dysmorphic erythrocytes were found in patients
with haematuria caused by a glomerular disease, while non glomerular or
isomorphic erythrocytes were found only in patients with hematuria of
urological origin. Thus, it was concluded that the evaluation of urinary
erythrocyte morphology could be used to identify the source of hematuria.
Slide 29
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
Slide 31
However,
the evaluation of the urinary erythrocyte morphology is associated with some
drawbacks. First of all it requires experience. Then, it is exposed to the risk
of a low inter-observer reproducibility. Finally, even after more than 20 years
from the publication of the paper in Kidney International by Fairley and Birch
we still do not have univocal criteria for the classification of the
haematuria. In fact, there are investigators who consider a haematuria as
glomerular when 2 erythrocyte subtypes are present, others who say that there
must be at least 3 subtypes of erythrocytes, while others as we have just seen
use a >80% cut-off, others use other cut-offs, etc.
Slide 32
Thus, put in this way the whole matter could seem a little complicated and not very useful in clinical practice. However, some years ago Köhler and co-workers published an important paper, which overtook some of the problems mentioned above.
Slide 33
In
their paper, Köhler 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
And
here you see a diagram of the main types of acanthocytes.
Slide 36
The
paper of Köhler 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 ³5% a glomerular bleeding could be
identified with a 52-100% sensitivity and a 96-100% specificity. In addition,
Köhler 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? It’s
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 work-up 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
Now
leukocytes. In most instances, leukocyturia is due to polymorphonuclear
leukocytes, much less frequently to eosinophils or lymphocytes.
Polymorphonuclear leukocytes may derive from any segment of the urinary tract,
without forgetting genital contamination, which occurs especially in women with
vaginitis or leukorrhoea of whatever cause. The clinical meaning is of
leukocyturia is inflammation of whatever cause, including immunological
disorders such as glomerular diseases.
Slide 39
This
is an example of polymorphonuclear leukocytes as seen by phase contrast
microscopy. You can easily see their lobulated nucleus and their granular
cytoplasm.
Slide 40
These
are eosinophils, which can be identified only after staining (in this case
May-Grünwald-Giemsa). Eosinophiluria has been considered as a marker of acute
interstitial nephritis.
Slide 41
Is
this belief still true today? I don’t think so. Why?
Slide 42
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, Schönlein-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
What
about urinary lymphocytes?
Slide 45
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%
sensitivity. However, stains and cytological techniques are needed to identify
lymphocytes, and these techniques are usually available only in specialized
labs.
Slide 46
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
This
is an example of a proximal tubular cell, with a large nucleus surrounded by a
large granular cytoplasm.
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
This
is an example of lipid droplets, both in clusters and isolated.
Slide 54
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
Fabry
disease is due to the hereditary deficiency of the enzyme α-galactosidase A. It
is characterised by the accumulation of globotriaosylceramide (GL-3) in several
organs such as the heart, the brain, the skin and the kidneys. In the kidneys,
GL-3 accumulates in glomerular visceral epithelial cells, distal convoluted tubular
cells, and the cells of Henle’s loop.
Slide 60
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
Here,
you see the lysosomal accumulation of GL-3 by transmission electron microscopy,
with its typical “myelin body ”
structure. All this is interesting and useful, because it is possible to diagnose
and follow the course of the disease also by examining the urinary sediment. Of
course not necessarily with the electron microscope, but just with phase
contrast and filters for polarized light.
THE THIRD PART WILL BE PUBLISHED ON OCTOBER 26TH