Urine Under Microscope

A basic and worth information for diagnostic is urine microscopy. ideally it should be by the physician at his clinic to add and correlate diagnosis promptly. this will make physician confident in dealing with patients. it also help in follow up the consequences in some important glomerulopathies.

1. Urine Under Microscope Dr. A brar A li Katpar Department of Nephrology King K halid Hospital-Hail Kingdom of Saudi Arabia.

2. Introduction Urinalysis is perhaps the most common investigation performed in clinical practice it is noninvasive inexpensive and easy to perform test, which provides wealth of useful information especially on patients with renal disease. It not only assists in the diagnosis of renal disease but can also provide information about the severity and outlook of the disease. In order to obtain maximum information from this elementary investigation, the physician looking after the patients should preferably do itself. Because he alone will ultimately be in better position to correlate the findings with the disease and fully grasp its implications .

3. Urine Collection For microscopy, urine should be collected in an open-mouth, clean but not necessarily a sterile container. Although the first morning specimen is most concentrated and acidic due to overnight fast, the second urine of the morning is preferable for microscopy, as it too is concentrated and acidic but without overnight stay in the bladder which causes lysis of the cells and cast .

4. For reliable urine microscopy and culture, carefully collected Midstream Urine without contamination from the genitalia is essential. For this, the patient must be instructed about the correct procedure. The patient should be explained in plain simple language that before collecting a specimen of his/her urine, hands and external genitalia should be gently washed with water or saline: disinfectants should not be used. Also that overzealous cleaning is harmful, because it can possibly cause abrasions of the periuretheral area and result in bleeding; thus contaminating the specimen. The patient should also be explained that it would be ideal for him/her to have a moderately full bladder. Furthermore to avoid contamination men should be advised to retract their foreskin (if Present) and women should hold their labia apart. Once they start voiding, the first 200ml of urine should be discarded, and the next 100-200 ml of urine should be collected in container by moving it in and out of free-flowing urine stream.

5. If a proper mid-stream specimen cannot be collected as in the case of infants, or young children, or aged persone because of there physical shortcoming or mental handicap, the requisite urine specimen can be obtained either by an open-ended catheter or suprapubic aspiration.

6. STORAGE OF URINE To avoid any possible alterations in physical or chemical feature, urine should ideally be analyzed with in an hour of Voiding. With passage of time the cells in the specimen tend to lyse and cast disintegrate especially in urine with low specific gravity and or alkaline pH. Moreover, with prolong stay at room temperature, bacteria have tendency to multiply obscuring the very cells and casts. Several means of preservations of cells with chemical reagents (0.5% Glutaryldehyde , Boric Acid, Formalin) and refrigeration at 4 degrees centigrade have been proposed. But these tends to interfere with chemical reaction. On the other hand refrigeration of urine causes crystals to precipitate, obscuring cells and cast .

7. Starting

8. URINE MICROSCOPY To obtain maximum possible information it is important to examine the urine using a phase-contrast microscope with the added facility of polarized light. Colorless, or unstained objects, have little effect on the amplitude of light waves; hance observing such objects under bright field is not very helpful. With phase-contrast microscopy, light passing through a relatively thick or dense part of a cell, such as the nucleus, is retarded compared with light passing through media only, or a thin part of the specimen. The degree of retardation, and the extent of the phase-shift that result, is proportional to the thickness of the object.

9. The the phase-contrast microscope thus exploit the interference effects that takes place when the two types of light recombine. If the waves complement each other, the light is brighter, while if they are 180 degrees out of phase, one cancels other, and the image becomes dark. To sum up phase-contrast microscopy enhances the ability to see and identify cells and cast, which are not visible with bright-light microscopy. Polarized light is particularly useful in identifying free fat, cholesterol and crystals of calcium oxalate, uric acid.

10. To quantify formed elements in the urine, most authors recomned centrifugation of a fixed volume of urine, transferring a drop of urine from the deposit to a glass slide and then counting the cells under high power. With this semi-quantitative method, cells are described as number or a range of cells observed per high power field(HPF).

11. This method has been found open to error at almost every step. The number of cells/HPF depend largely upon various factors namely: a- The quantity of urine centrifuged b- The type and speed of centrifuged c- The duration of centrifugation d- The method of discarding the supernatant and e- The amount of urine in which the sediment is re-suspended.

12. The volume of urine under the cover slip also varies and depends upon the size of the drop of urine, weight of the coverslip and viscosity of the urine. It is not easy to control so many variables; hence this method of semi-quantitative estimation of cells can be erroneous. Therefor, with a view to avoid such errors, counting of cells using uncentrifuged urine and a counting chamber is recommended. A Fuchs-Rosenthal counting chamber is particularly useful

13. FORMED ELEMENTS IN THE URINE

14. Red blood cells Red Blood Cells may be found in normal urine. With semi-quantitative methods, up to 1-2 RBC/HPF are considered normal. With phase-contrast microscopy, the upper limit of normal in un-centrifuged urine is 15000 cells/ml and all are of glomerular origin. About 30% of cells are lost either with centrifugation or resuspension . Therefore upper limit of normal in a centrifuged urine is 10,000 cells/ml. The normal limit with bright light is lower, as some of the cells seen with phase contrast are either not visible or misinterpreted as debris. RBC’s are likely to undergo several morphological changes in the urine. Some of these changes are non specific and due to physiochemical environment. FIG:6

15. Brich and fairley were the first to note that rbc’s cells in the urine of patients with glomerulonephritis have peculiar changes (fig:7-13). Other s subsequently confirmed these findings.

16. White blood cells Like RBC ’s , w hite blood cells (WBC’s) may be found in normal urine. With the semi-quantitative method of estimation, 1-2 cells/HPF is considered normal. Using a counting chamber and phase-contrast microscope 2000wbc/ hpf is considered to be the upper limit of normal in properly collected midstream urine specimen. Neutrophils are the m ost frequent WBC ’s in urine, with an average diameter of 10 micrometer they are slightly larger than a RBC . They have a granular cytoplasm and a lobulated nucleus. Increased numbers of neutrophils in the urine indicates urinary-tract infection.

17. When their number increases without bacteriuria or growth on culture, renal tuberculosis, renal calculi, renal papillary necrosis, polycystic kidney disease and infection with fastidious organism should be considered. Lymphocytes are smaller than neutrophils. Although they can be identified with phase-contrast microscopy, their identification usually requires special staining. They are frequently found in the urine of renal transplant recipient’s during episodes of acute cellular rejection. Eosinophils are slightly larger than neutrophils and are only identified by special stains. They are seen in acute interstitial nephritis and in patients with athero-embolic disease .

18. Mucus Mucus in the urine appears as ribbon-like threads of variable width and length. It is a normal constituent of urine and of no pathological significance.

19. Cylindroids These are similar to cast with one of their ends resembling a mucus thread. In the past there was some controversy over their origion . It is now clear that they are composed of tamm-horsfall mucoprotein and are therefore cast. They may contain particles like cast. Fig:

20. Cast Cast Are elongated cylindrical structure. They acquire shape from the tubular lumen (distal tubular and collecting ducts) in which they are formed. They are composed of Tamm- Horsfall glycoprotein. Cast formation is favored by factors which promote aggregation of Tamm- Horsefall proteins, which include increased urinary concentration of electrolytes, hydrogen ions, and ultra-filtered proteins. Iteraction between the protein and hemoglobin, myoglobin, Bence -Jones proteins or radio contrast media also favor formation. Different formed elements transported along the nephron are traped in them producing various types of Cast (Table). Igor Tamm & Frank Horsfall Glycoproteins Uromodulin Gene= UMOD Chromosome16

21. HYALINE CAST These consist of Tamm- Horsfall Mucoprotein only. As they have low refractive index they easily escape detection if a bright-field microscope is used. They are occasionally present in normal individuals, but are increased in renal diseases. They are also observed in patients with acute cardiac failure, fever, those receiving loop diuretics, and in normal people after strenuous activity.

22. GRANULAR CAST These contain granules that may be either fine or coarse. Granules consist of ultra-filtered proteins or degenerated cells. Their presence in the urine is always pathological. Large numbers of coarse granular casts are particularly seen in acute tubular necrosis.

23. WAXY CAST Waxy casts are highly retractile, usually large with clear cut edges. Their composition is different from other cast, and are resistant to dissolution to alkaline urine. They are typically found in patients with advanced renal failure.

24. ERYTHROCYTE CAST Erythrocyte cast show enormous Variation in morphology, and contains variable number of RB C’s . They indicate glomerular bleeding.

25. LEUKOCYTE CAST These contain variable number of neutrophils. They are commonly found in acute or chronic pyelonephritis. They are also seen in proliferative glomerular lesions like acute post streptococcal glomerulonephritis, active mesangiocapillary glomerulonephritis and lupus nephritis. It is at time, difficult to distinguish leukocytes within the cast from tubular epithelial cells.

26. FATTY CAST These contain variable amount of lipids and are usually seen in the urine of patients of with N ephrotic syndrome.

27. Lipid Droplets Lipid droplets can be seen in the urine either as free droplets, within the renal tubular epithelial cells Fig: or with in the Cast. Lipids are usually seen in the urine of patient with heavy proteinuria. How ever they are also described in patients with Low-Grade proteinuria.

28. Oval Fat Bodies Oval Fat bodies are renal tubular epithelial cells full of cholesterol esters. The fat is easily identified with polarized-light microscopy when a ‘Maltese Cross’ appearance is seen.

29. Squamous Epithelial Cells Thease are large (mean diameter 55 um) flat cells with abundant granular cytoplasm and a small central nucleus. They are exfoliated from the bladder and urethra. Un women with vaginal discharge, they contaminate the urine.

30. Renal Tubular Epithelial Cells Renal tubular epithelial cells, most commonly found in the urine are from the proximal tubules. They are round to ovioid cells with an average diameter of 13um and have a single nucleus. Normally they are not present in the urine. They may be found in increase in number after exercise and after ingestion of certain drugs. They are frequently present in case of acute tubular necrosis and acute renal allograft rejection.

31. Erthryophagocytes An erythrophagocyte is renal tubular epithelial cell whose cytoplasm contains erythrocytes that are phagocytosed during their passage along the nephron. It may be seen in all form of the glomerulonephritis and has the same significance as erythrocyte Cast.

32. Myelin Bodies In F abry’s disease, glycosphingolipid laden epithelial cell of Loop of H enle and distal convoluted tubules desquamate and are detectable in the urine sediment. Lipids in these bodies demonstrates “Maltese Cross” under polarized light. They can be differentiated from cholesterol drplets by their irregular shape. FIG:

33. Candida Candida is the most frequent yeast found in the urine. They appear as pale-green cells often nucleated, and with smooth well-defined walls. They are commonly seen in the urine because of contamination from genitalia. They can also grow in urinary tract of patient with: diabetes mellitus; having indwelling catheters; on prolong antibiotic therapy; or receiving immunosuppression.

34. Bacteria Bacteria are frequently seen in urine sediment. They do not necessarily indicate infection and may be the result of contamination of urine. Presence of leukocytes with bacteria increase the probability of the infection.

35. Crystals The urine can contain several types of crystals. Uric Acid, Amorphous U rates, Calcium oxalates, Cystine , Leucine, Tyrosine, and Cholesterol crystals are found in acid urine. Whereas crystals of C alcium phosphate, Tripple Phosphate and Amorphus Phosphate are found in alkaline urine.

36. Thank you