BEDSIDE URINARY MICROSCOPY
GIOVANNI BATTISTA FOGAZZI LECTURES SERIES
URINARY SEDIMENT: Part 5: Clinical practice II
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
Now what about the utility, the place of the examination of the urinary sediment in transplanted patients? The urinary sediment can tell us about the recurrence of a glomerular disease by showing the appearance of dysmoprhic red cells with or without red cell casts. It has been used in the diagnosis of acute cellular rejection by showing the presence of lymphocytes. Can be used in the diagnosis of urinary tract infection, which shows bacteria and white cells and especially in patients with transplanted kidney it’s easier to find white cell casts because these patients are more exposed to renal infection than in non-transplanted patients.
Finally, and it is the news of the day the urinary sediment is very important in showing decoy cells which are a typical marker showing the infection of polyoma V virus due to a BK virus, polyoma virus BK. I want to tell you about our experience with decoy cells in transplanted patients.
So what is first of all a short introduction to polyoma virus nephropathy. BK virus nephropathy, there is a mistake here, is a disease of the transplanted kidney due to the invasion of the renal parenchyma by polyoma virus BK. At renal biopsy there is acute tubular necrosis as you can see very well here, this is haematoxylin stain here the detachment of tubular cells from the basement membrane here, here also here. Interstitial cellular infiltrate as you can see very well here with or without tubulitis and typical tubular cytopathic changes as you can see here.
This is a typical tubular cell infected by polyoma virus BK.
This condition is characterised by a progressive deterioration of renal function in patients treated with new immunosuppressive agents such as tacrolimus and/or mycophenolate mofetil. It has a prevalence of about 5% and is associated with a graft loss of about 46% in a few months after diagnosis. And the golden standard for treatment is still the reduction of immunosuppression. Anti viral agents can be used but there are only anecdotal results in the literature.
The diagnosis is obtained by renal biopsy. The only way to have a diagnosis of the nephropathy caused by BK virus is renal biopsy but the examination of the urine sediment is very important in arising the suspicion that there maybe the infection caused by BK virus. As well as measurement of BKV DNA in blood and urine by real time PCR.
Urine cytology including practise is widely used for diagnosis of polyoma virus infection. It is based on the search of decoy cells. Decoy cells which are nothing but cells with typical nuclear changes induced by the BK virus.
From the literature we know that it is mostly, the method mostly used to look for decoy cells is the stain with Papanicolaou.
In the experience of Drachemberg who is one of the main actors in this field we know that there is the same prevalence of decoy cells in patients with acute serum creatinine, increase and in patients with stable serum creatinine however only the patients with an increase of serum creatinine have actually the nephropathy caused by BK virus.
Drachemberg has also found that there is a correlation between the number of decoy cells in the urine and the presence of the nephropathy. No nephropathy in patients with a small number of decoy cells. Drachemberg has also shown that there is a correlation between the number of decoy cells and the severity of the disease, of the nephropathy. With 3 or 4 plus decoy cells in the urine, you have always a severe nephropathy and she has also found that there is an agreement between decoy cells in the urine and the biopsy in 87% of cases. However, with a 10% false positives associated with decoy cells and again 3% false negatives associated with decoy cells. Thus to summarise the findings of Doctor Drachemberg we can say that if a patient, with a transplanted patient has many decoy cells in the urine and also has an increase of serum creatinine, the patient has a high probability of having BK virus nephropathy.
Another important actor in this field is Doctor Nickeleit who in a review paper published in 2000 in NDT showed that the presence of decoy cells in the urine when compared to the findings at biopsy has a very high sensitivity and specificity but a very low positive predictive value. Thus you may have many decoy cells in the urine but it is not said that the patient actually has a nephropathy caused by BK virus.
This data were confirmed by Hirsch who worked at the time with Doctor Nickeleit in Basel in this important paper and again he showed that Hirsch showed that decoy cells have a very low positive predictive value.
In our unit we go back to some questions raised before, we looked for decoy cells but we don’t use Papanicolaou stain, we only use phase contrast microscopy.
We use this because we are convinced that we have been able to demonstrate that both the nuclear and cytoplasmic changes caused by BK virus are easily seen and we use phase contrast because it is based on the same simple, quick and inexpensive method we use for the routine examination of urine sediment. In this way we cannot avoid using cytological techniques which need procedures, equipment and skills which may not be available in general labs.
This is the method, the same I have shown you at the beginning of the course, now we express the result, our results, decoy cells as number per 50 high power fields.
This is the list of the possible alteration changes which are observed by phase contrast microscopy in decoy cells which I show you here. The most frequent change is the enlargement of the nucleus which appears as a ground-glass. Another possibility is multiple inclusion bodies in the nucleus and also inclusion bodies and vesicles in the cytoplasm. Another type of change is inclusion bodies in the nucleus which are of a very peculiar shape, very regular. Another important rather frequent finding is a unique inclusion body which gave the cell the appearance of a hybrid cell which in this case was found within a cast which suggests that there is kidney disease and more rarely we can have vesicular nuclei and cells like this which can be easily confused with neoplastic cells.
Here the same pattern of things which you can see by Papanicolaou. You see here inclusion bodies within the nuclei.
Here another one.
We have compared in our lab the findings obtained by phase contrast microscopy and Papanicolaou stain in 18 patients and we found that there were the same cellular changes with both methods. In some cases Papanicolaou showed better details of nuclear inclusions however, the quality of the stain was not always consistent with Papanicolaou and in addition to this Papanicolaou was more complex and time consuming than phase contrast only, 40 minutes versus 15 minutes.
Then we also wanted to be sure that what we defined as decoy cells were actually decoy cells so we performed electron microscopy in 11 urinary samples and we found the typical nuclear changes in all cases and in 10/11 we were able to demonstrate that what we defined as decoy cells actually contained the virus particle.
You see here margination of chromatin with this material within the nucleus.
Again here you see nuclear inclusion bodies
and here see the nucleus which is gorged with virus particles. So phase contrast microscopy is also useful to identify decoy cells and without needing any stain.
We use this method to follow our patients with BKV nephropathy in our unit. We have 8 patients now and you see here that in some patients there is a good correlation between the findings in the urine and the findings in the blood with the measurement of DNA, for instance you have a patient here with a follow up of 47 patients. Here the 12 decoy cells in the urine at the beginning when there was a sharp increase of serum creatinine. At the last observation the DNA in the blood is negative and also decoy cells are negative.
We also found a correlation between the severity of BKV nephropathy and the number of decoy cells in 4 patients with mild BKV nephropathy the number of decoy cells, the mean number was 10 and here in patients with moderate to severe disease the number of decoy cells in the urine was much higher.
We also studied the features of the urine in these patients and we found that macrophages were frequent 4/5 patients.
We extended this observation to more sediments and we studied 13 sediments and we found macrophages in 12 sediments. Now we can say today that the presence of macrophages is another feature of the urine sediment of patients with infection due to BK virus.
What do we know today about urinary macrophages?
We know that they can be easily identified by phase contrast microscopy as granular macrophages
or vacuolar macrophage
or as phagocytic macrophages.
To be sure that they are actually macrophages for a period of time we stay in the urine with a monoclonal antibody which was CD68 (clone PGM1) and all the cells which had the appearance of macrophages were actually positive with this stain.
What is the clinical meaning of macrophages in the urine? In 1992 by conventional microscopy with staining it was stated that macrophages were a marker of infection and inflammation. 5 years ago in a very nice review paper Otta reported his results, his previous results and showed that when looked for with flow cytometry macrophages are typical elements of patients with proliferative glomerulonephritis in active phase while by using immuncyto staining he showed that macrophages appear as oval fat bodies, in patients with non-selective proteinuria. Last year by immunofluorescence and again immunoclonal antibody it was found that macrophages were a typical finding in patients with active IgA nephropathy. We with our study using simple phase contrast microscopy have shown and found that macrophages are typical of the infection due to BK virus.
The examination of the urine sediment is the most inexpensive and quick test in our hands. It is also one of the few tests which can be performed today at the bedside. We know that when performed with the correct methodology and knowledge, it can be useful in a wide range of renal diseases as we have seen glomerulonephritis, macroscopic hematuria, patients with a kidney transplant etc.
So great is the potentiality of the examination of the urine sediment that it should be carried out by the physician himself. The 2 to 5 minutes of additional time consumed with this test often will richly be rewarded.
With this statement I have a question. Why, then, is such a valuable test so frequently neglected by us nephrologists?
THE SIXTH PART WILL BE PUBLISHED ON MARCH 8TH, 2007