AN ALBUM OF URINARY MICROSCOPY IMAGES IN A CLINICAL CONTEXT
G.B. Fogazzi, Milan, Italy
S. Verdesca, Milan, Italy
So, now a case concerning a renal transplant recipient with progressive decline of renal function.
In June 1999 a 31-year old man received a kidney transplant for end stage renal disease due to a congenital obstructive uropathy. Immunosuppressive therapy included tacrolimus, mycophenolate mofetil and prednisone. Fourteen months later serum creatinine had gone up gone up to 4.3 mg/dL in spite of treatment with intravenous methylprednisolone pulses.
The urine sediment of this patient contained many unusual and abnormal cells (slide 80 and 81), some of which were found within casts (arrows).
Based on these findings, we hypothesized the presence of a polyomavirus BK-induced nephropathy. This was shortly after confirmed by the renal biopsy findings, which I’m going to show you.
At low power magnification, a diffuse interstitial mononuclear cellular infiltrate could be seen, which in some areas more was more prominent (arrows) than in others.
In addition, there was a quite severe tubular damage, as demonstrated by the necrotic changes of the tubular epithelium (red arrows). However, this was not the only tubular abnormality, in fact, there also was this cell within a tubular lumen (indicated by the blue arrow), which had quite an enlarged and dark nucleus.
Other cells with an enlaged nucleus were found in other tubules, either within the tubular epithelium (slide 85 and 86, arrows)
or within the tubular lumen, either isolated (slide 87, arrow),
or within the matrix of casts (slide 88, arrow).
Thus, renal biopsy confirmed our hypothesis of a polyomavirus BK nephropathy. Polyomavirus BK (BKV) is a DNA virus, which is present in about 80% of the general population, where it lodges within the cells of the uroepithelium, which lines the urinary tract form the renal calyces to the bladder. In immunocompetent individuals, BKV is in state of latency, while in immunocompromised subjects, it may reactivate and cause clinical disturbances. In these last years, it has been found that in renal transplant recipients treated with tacrolimus, mycophenolate mofetil, or sirolimus, BKV can cause the so-called BKV nephropathy. This is found in 1% to 5% of such patients, and can lead to graft loss in about 50% of cases. In our unit we have seen so far 8 patients with this condition.
How can BKV reactivation be suspected? In several ways, one of these being the finding of the so-called “decoy cells” in the urinary sediment.
Decoy cells are epithelial cells (mostly of renal tubular origin) with typical nuclear changes (slide 91).
Usually, the search of decoy cells is carried out in specialized labs by the means of cytological techniques based on Papanicolaou stain. However, in our lab we use the same simple technique we use for all urine samples, which is based on well standardized procedures for centrifugation, removal of supernatant urine, and examination of the sample by phase contrast microscopy, without any stain.
By Papanicolaou stain, you see here (slide 93) the so-called “ground glass” appearance, which is due to very enlarged nuclei (arrows).
While here (slides 94 and 95) you see several inclusion bodies of different size within the nucleus of some cells (arrows).
By electron microscopy you can see very well another typical change caused by BKV which is, the “margination” of the chromatin (arrows). Besides this, you can see countless small gray “dots” within the nucleus, which are nothing but viral particles.
Viral particles, which can cause these peculiar structures (arrows).
You can see that in some instances the nucleus is completely filled with these viral particles, which have an average diameter of 45 Ä.
So far we have seen how decoy cells look like with Papanicolaou stain and with electron microscopy. Now, what about their appearance with phase contrast microscopy?
We’ll, let’s go back to the images I have shown you at the beginning of this case. These are cells with a “ground glass” appearance (Fig. A and C). This is a cell with the margination of the chromatin (Fig. B, top, even though the cell too small to be seen), while this is a cell with multiple inclusion bodies in the nucleus (Fig. B, bottom). In Fig. D, you can see cells with an enlarged nucleus, which were trapped within a cast, which undoubtedly indicates their renal origin
Here, you can see how phase contrast microscopy shows very easily and nicely the cells with the so-called “eye bird” appearance. Also these cells can be either free in the urine (Fig. E, top) or within a cast (Fig. F, arrow). Fig. G shows a cells a vacuolated nucleus, which is a rare finding, while Fig. H shows other rare variants of decoy cells, with an unusual cellular shape and coarse chromatin pattern (top), which mimic the cells which can be found in the urine of patients with uroepithelial malignancies.
Here, you can see other images of decoy cells as seen with phase contrast microscopy: again a “ground glass” appearance (right side, top and bottom), multiple inclusion bodies (center), and a decoy cell with multiple intracytoplasmic vacuoles (left side)
The various types of decoy cells I have shown you so far differ remarkably from the normal tubular cells, shown in slide 102, in which the nucleus is smaller and contains only nucleoli.
In our lab, we have compared the results obtained with phase contrast microscopy with those obtained Papanicolaou stain in 43 urine samples from 18 transplanted patients all with decoy cells in the urine. We found that the two techniques gave similar results, even though Papanicolaou obtained, at times, better details of nuclear changes. However, in our hands, the quality of the stain was not always consistent, and at times it did not allow a reliable examination of the sample. In addition, Papanicolaou was more complex and time consuming (about 40 minutes vs 15 minutes). In addition, while a nephrologist is usually not familiar with Papanicolaou stain, he/she is (or should be) familiar with urine sediment examination. Thus, with our approach, we can look for decoy cells at bedside ourselves, without the necessity of sending the samples to the cytology lab.
What is the relationship between decoy cells in the urine and BKV nephropathy? Studies published in 1999 and 2001 by Drachemberg and co-workers show that there is a good correlation between the number of decoy cells and the presence of the nephropathy and between the number of decoy cells and the histological grading of BKV nephropathy. In addition, BKV nephropathy is more likely when decoy cells are associated with an acute increase of serum creatinine. In Drachemberg experience, decoy cells give false positive a 9.6% false positivity and 2.9% false negativity.
Hirsch and co-workers investigated the value of decoy cells compared to other tests in diagnosing BKV nephropathy. In a cohort of 78 kidney transplant recipients treated with tacrolimus or mycophenolate, who were prospectively followed-up for 85 weeks, decoy cells were found in about 29% of patients after an average of 16 weeks from transplantation. BKV DNA was found in the blood in about 13% of patients, after an average of 23 weeks. However, when the patients were submitted to renal biopsy, BKV nephropathy was found only in about 6% of patients.
Based on these findings, decoy cells had a very high sensitivity but a very low (29%) positive predictive value. Therefore, the finding of decoy cells does not necessarily imply the presence of BKV nephropathy. However, it certainly heralds the reactivation of BKV in the urinary system, which should alert the clinician to check for both BKV viraemia and serum creatinine levels.
My final message for this case is that decoy cells can easily be looked for and seen at bedside by nephrologists by using phase contrast microscopy and a conventional preparation of the urine samples.
Again, we published our findings in this short paper some years ago in NDT.
Now, we have another discussion session (about case 3 and case 4).
Question. My first comment and question would be regarding the first case. A few years ago in a review paper on IgA nephropathy, in which more than 200 patients were followed for more than 25 years, it was shown that in the follow up the number of red blood cells was the most important risk factor for progression of disease and it was stated that a patient with more than 100,000 red blood cells/μl had a worst outcome. So my question regarding the first case is, in this woman she doesn’t have high blood pressure, no proteinuria, normal renal function but let’s say that she has 500.000 red blood cells, would you biopsy her? Regarding the second case Dan Brennan just wrote a paper on prevention of BK nephropathy arguing that if you detect decoy cells in the urine, in his case he uses PCR in the urine and in the blood, he suggests that you should decrease the immunosuppression. In your opinion if you find decoy cells in the urine would you advise us to start to decrease immunosuppression as well?
Fogazzi. Let me start from the second question. If we find decoy cells, especially in high numbers, let’s say, in our experience, at least 10-15 over 50 high microscopic fields at 400x magnification, in repeated samples we consider this as a sign of reactivation of BKV. Therefore, we ask for the measurement of BKV-DNA levels in the blood and of serum creatinine, and follow-up the patient closely. The reduction of immunosuppression is the first procedure to be done to prevent or cure BKV nephropathy. As for the first question, if I have a patient with normal renal function, no hypertension, no proteinuria at all and a severe microscopic haematuria, especially if she/he is a young patient, I may consider renal biopsy, even though the lesions which we most often find (thinning and/or splitting of glomerular basement membrane, mesangial proliferation, or other changes) are usually not treatable.
Question. The case of the 66year-old lady. If she had come to you from the start, would you have done a biopsy?
Fogazzi. I was keen to perform renal biopsy in this lady, because after 17 years of investigation she did want to know what she actually had. However, we didn’t perform renal biopsy because there was a large cyst in the lower pole of the left kidney. In patients with isolated microscopic haematuria we perform renal biopsy only in selected cases, as were the 16 patients I have described above (see slides 69-71).
Question. I have a question concerning the percentage of dysmorphic erythrocytes which is considered as the cut off to suspect or diagnose a glomerular bleeding
Fogazzi. Your question is a very good one. If you go through the literature, you’ll find that for one author the cut off is 80%, for another it is 40%, for another one 17%, and so on and so forth. In our study which is going to be published in Pediatric Nephrology, we found that a cut off of ³ 40% for dysmorphic erythrocytes and of ³5% for acanthocytes is reliable and useful to identify a haematuria of glomerular origin.
Question. Do you use phase contrast for all your patients?
Fogazzi. Yes, why not?. Phase contrast has an added value compared to bright field microscopy, and is very simple to use. It should also be remembered that international guidelines do recommend the use of phase contrast microscopy for all urinary sediments.
Question. I have two questions for you. One is: how many red blood cells do you need to use the percentage? When you have only 5 erythrocytes/high power field is that valid? The other question: my feeling is that we find red blood cell casts rarely. Is there something in the preparation to improve the yield of these casts?
Fogazzi. Thank you, good questions. The first one: if you have only 5 red blood cells/high power field what do you do? In our lab, for all cases with isolated microscopic haematuria of unknown origin, including those with few erythrocytes/high power field, we evaluate the morphology of 100 red blood cells. If there are 5 erythrocytes/microscopic field, this means that we examine 20 high power fields, a procedure which takes no more than 5-10 minutes. The results we obtained in our 16 patients with microscopic haematuria (see slides 69-71) were not at all influenced by the number of erythrocytes/microscopic field. The second question: in my opinion, red blood cell casts are rarely seen because they are rarely looked for.
Question. I think I look for them quite a lot.
Fogazzi. For patients with isolated microscopic haematuria of unknown origin, we always look for erythrocytic casts carefully and with a standardized method. We do this by examining 50 low power (160x) microscopic fields, and with this method we find erythrocytic casts in about 11% of samples (see again slides 69-71). Thus, you are correct, these casts are rare, but less rare than usually thought if you look for them systematically and intensively.
Question. And you spin for 5 minutes?
Fogazzi. For 10 minutes.
Question. Concerning the decoy cells have you done in parallel with both Papanicolaou and phase contrast. Phase contrast images didn’t convince me. We cannot apply so easily the term “ground glass” image to the phase contrast. This is typical for Papanicolaou or for blue sections.
Fogazzi. As I said, we did compare Papanicolaou with phase contrast in 43 urine samples from 18 patients, and the results were very comparable. Only occasionally we had better nuclear details with Papanicolaou. However, in our hands Papanicolaou did not give consistent results: with some samples the quality was excellent, while with others it was very poor. But above all, while we as nephrologists can easily prepare a sample for conventional urine microscopy, we are usually not able to prepare samples for Papanicolaou. Thus, with this latter method you have to send the urine to another lab, where it may happen that not all people know what the problem with decoy cells is. Thus, I think that with some experience and a good microscope we can try to manage the whole problem ourselves. This is my message in the end.
THE FIFTH PART WILL BE PUBLISHED ON JUNE 19TH, 2008