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

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PART II

Link to Part I

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 Australia published this important and seminal paper in Kidney International.

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 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

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 19^th 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