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
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.
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.
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.
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.
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.
is the clinical implication of distinguishing glomerular from non glomerular
erythrocytes? In 1982 Fairley and Birch from
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.
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.
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
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.
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.
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.
In this slide you can see how easily the acanthocytes can be identified by phase contrast.
And here you see a diagram of the main types of acanthocytes.
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.
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.
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.
This is an example of polymorphonuclear leukocytes as seen by phase contrast microscopy. You can easily see their lobulated nucleus and their granular cytoplasm.
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.
Is this belief still true today? I don’t think so. Why?
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.
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.
What about urinary lymphocytes?
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.
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).
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.
This is an example of a proximal tubular cell, with a large nucleus surrounded by a large granular cytoplasm.
This is an example of a distal tubular cell, which is smaller than the previous one, and has a smaller cytoplasm.
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.
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.
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.
This is an example of lipid droplets, both in clusters and isolated.
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.
This is a typical “oval fat body”. Today we know that they are nothing but macrophages or renal tubular cells gorged with lipid particles.
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”.
And here a typical cholesterol crystal, which instead does not polarize light.
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.
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.
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”.
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”.
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