HANDS-ON COURSE

BEDSIDE URINARY MICROSCOPY

GIOVANNI BATTISTA FOGAZZI LECTURES SERIES

URINARY SEDIMENT: Part 1: Methods

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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Prof Fogazzi: This is the outline of the course. After a brief introduction, I will speak about the main methodological aspects concerning the urinary sediment; the particles of the urinary sediment of nephrological importance with their clinical implications; the urinary sediment in the clinical practice and, finally, I will draw some conclusions.

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From the historical point of view, we know that the urinary sediment was introduced into clinical practice in the late 1830s in Paris at La Charité hospital by François Rayer and his pupil Eugène Napoléon Vigla. By the end of the 19th century, all the main particles had been identified and the main urine profiles had been described. However, in the subsequent century, the 20th century, the urinary sediment examination knew only a progressive decline with only very few exceptions. One was the original and important work of Thomas Addis in the 1920s and the other was the publication, in 1982, of a paper by Fairley and Birch on the utility of urinary erythrocyte morphology evaluation by phase contrast microscopy in patients with hematuria.

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What is the situation today? In most instances, urinary sediments are examined in central laboratories far from bedside and without the correct equipment and knowledge, and with the dream to entrust the whole task to automated instruments. These are already on the market, one being UF 100, which is based on flow cytometry, and the other  iQ200, which is based on images obtained by a video camera. Last but not least, too often the urinary sediment examination is neglected even by nephrologists.

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In our unit at the Ospedale Maggiore of Milan, we examine urine sediments ourselves. We examine about 2,300 samples per year, most of which contain abnormalities. Most of our sediments come from our ward and clinic.

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Methodological aspects are very important for urine sediment. The main methodological aspects include: a correct urine collection; a standardised method for the handling of the urine; the use of a proper microscope and the use of a proper report to describe the findings. All these aspects are described in detail in the document published by the European Urinalysis Group 5 years ago as a supplement of the Scandinavian Journal of Clinical and Laboratory Investigation (2000; Vol 60, Suppl 231) as well as in our book (Fogazzi GB, Ponticelli C, Ritz E. The Urinary Sediment. An Integrated View 2^nd Edition. Oxford , Oxford University Press, 1999).

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As to urine collection, it’s important to give the patient written and simple instructions. According to the strategy of the single lab, we can ask the patient to supply the first or the second urine of the morning. In our lab, we ask  for the second urine, since overnight urine, due to its prolonged permanence in the bladder can favour the lysis of particles. We suggest the patient to avoid strenuous physical effort in the hours preceding the test, since this may influence in various ways the findings (for instance by causing haematuria and/or cylindruria). We advise the patient to clean the external genitalia in an ordinary way. In this respect, we do not suggest  special procedures since  the more the procedures suggested are complicated, the less the patient is compliant. In order to avoid contamination, the male has to uncover the glans and female to spread the labia of the vagina. For the same reason, we always suggest to collect midstream urine. It’s important to remember that urine collection during menstruation must be avoided because of the high probability of blood contamination. It is also important to use a proper urine container (with a capacity of at least 50 to 100 mL, an opening of at least of 5 cm to allow easy collection of urine for both men and women, a wide base to avoid accidental spillage, a cap to avoid leakage, a label for patient identification). It is no more time for the patient to collect the urine into jugs, bottles, cans, etc.

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What about a standardized method for the handling of urine? Why is standardization of the handling of the urine important? It is important because only with a standardized method we can obtain quantitative reproducible results. The slide shows the method that we use in our lab. We ask the patient to supply the second urine of the morning produced over a period of 2 hours; then, we centrifuge a 10 mL aliquot of urine for 10 minutes at 400 G , which correspond to 2,000rpm with our centrifuge. Then, we remove with a pump a fixed volume of supernatant urine, which is 9.5 mL. Then, with a Pasteur pipette, we gently but thoroughly re-suspend the sediment in the remaining 0.5 mL of urine. Then, with a precision pipette, we transfer 50 mL of resuspended urine to a glass slide, which is covered with a coverslip of a fixed surface, namely 32 x 24 mm . Then, we examine the samples at low and high magnification (160x and 400x) within 3 hours from urine collection. For routine practice, we express the particles as lowest/highest number seen by microscopic field. When we want to produce scientific results, we count the number of the cells found over 20 high power field, as we will see in the last part of my speech (see “The urinary sediment findings in proliferative and non proliferativer glomerular diseases”). With this method we were able to obtain reproducible results, which in addition correlated significantly with the number of particles found by the counting chamber.

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And now the microscope. The microscope must be of good quality, must be equipped with a low and high magnification, and must, and I want to stress must, be equipped with phase contrast and polarized light.

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This is the microscope we use in our lab. Why phase contrast?

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It’s very simple to explain. On the right you see bright-field, and on the left you see phase contrast. You see that with phase contrast  the particles are much better seen against the background than with bright-field, and this without the use of stains! I want to stress that the European Guidelines strongly recommends the use of phase contrast microscopy.

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And why polarized light? Polarized light is extremely useful to correctly recognize the crystals. For example, you see uric acid crystals as seen by phase contrast microscopy (slide 15), and  what happens when we use polarized light (slide 16). Under polarized light, uric acid crystals assume a typical polychromatic appearance, which is useful to identify them.

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Polarized light is also important to correctly identify lipid particles,

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which under polarized light appear as “Maltese crosses” which is, “shining” particles containing a “black cross” whose arms are regular and symmetrical. This feature allows the identification of lipid particles, especially when they come with an atypical appearance.

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Now, the urinary sediment report. This is the report we use in our lab. After the patient details, we have pH, density (or specific gravity), haemoglobin and leukocyte esterase as detected by dipstick. Then the particles: erythrocytes (with their morphological classification; see below), leukocytes, tubular cells, transitional cells (from the deep and superficial layers of the uroepithelium), squamous cells, casts, lipids, crystals, bacteria, and yeasts. We also have a space for a brief conclusive comment. I want to stress the importance of having in the report  the findings obtained by dipstick. Why this?

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Let me give you an example. You see in this slide a sample with a pH of 6.0, a density of 1.006,  a +++ haemoglobin and +++ leukocyte esterase by dipstick. However, by microscopy we find a low number of erythrocytes and leukocytes, which is in contrast with +++ haemoglobin and +++ leukocytes esterase. Is there any explanation for this discrepancy? Yes there is. The discrepancy is due to the fact that low density causes the lysis of erythrocytes and leukocytes, which therefore cannot be seen by microscopy. In contrast, in other instances we may have negative haemoglobin and many erythrocytes by microscopy. This may be due, for example, to the presence of large concentrations of Vitamin C in the urine, which reduces the sensitivity of dipstick for haemoglobin. Therefore, it is always important to match the findings obtained by dipstick with those obtained with microscopy and to try to explain them. Thus, our comment for the sample shown in the slide is: ” Mild erythrocyturia and leucocyturia. Please note the discrepancy between dipstick for haemoglobin and leukocyte esterase and microscopy. This is probably due to cell lysis caused by low density.”  The final message on this point is that examining the urine only by dipsticks or only by microscopy exposes to the risk of false results. This risk is reduced when both methods are used on the same sample.

 

THE SECOND PART WILL BE PUBLISHED ON SEPTEMBER 28TH