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Microscopic Urinalysis with Phase Contrast Microscopy
THE JOURNAL OF UROLOGY
Copyright© 1972 by The Williams & Wilkins Co.
MICROSCOPIC URINALYSIS WITH PHASE CONTRAST MICROSCOPY MICHAEL A. RUSSO
AND
A. T. K. COCKETT
From the Division of Urology, University of Rochester School of Medicine and Dentistry, Strong Memorial Hospital, Rochester, New York
The science of histochemistry, coupled with a study of the naked cell employing light microscopy, leaves much to be desired. Unstained cells and organelles have refractive indices similar to that of the background medium; therefore, light waves passing through the cell and the adjacent background medium will have differences in phase but few differences in amplitude. Since the wavelength amplitudes of light passing through the background medium and cells are similar, the image of the cell and background medium are almost indistinguishable. Staining techniques provide sufficient differential changes in wavelength amplitude to produce marked differences in the images of the cell, cellular components and the background medium. 1 Phase contrast microscopes enhance the cellular and background differences. Phase microscopy changes the light passing through unstained cells, which the eye is unable to appreciate, into visible changes in wave amplitude, that is it separates the light directly transmitted by transparent cells from the light diffracted by these same cells and retards the phase of the diffracted light about a quarter of a wavelength with respect to the directly transmitted light wave. The 2 waves then will interfere with one another to produce visible changes in amplitude. Zernike is credited with the original mathematical and experimental description of this phenomenon. 2 It is our contention that phase contrast microscopy is readily and inexpensively applicable to microscopic urinalysis and that it is invaluable in examining unstained urinary sediment. MATERIALS AND METHODS
Fresh human urine samples were randomly obtained from the urology and nephrology clinics. These were centrifuged for approximately 2 to 5 minutes. The supernatant was discarded, the sediment was placed on a glass slide and a coverslip was applied. The A-0 Micro-Star binocular microscope was converted to a phase contrast microscope by substitution of 1) a base illuminator (fig. 1, A), 2) a 45x phase objective, dark contrast (fig. 1, B), 3) phase turret condenser mount with annular diaphragm and condenser centering wrenches, iris Accepted for publication June 11, 1971. Read at annual meeting of Northeastern Section, American Urological Association, Hershey, Pennsylvania, September 20-23, 1970. 1 Ross, K. A.: Phase Contrast and Interference Microscopy for Cell Biologists. New York: St. Martin's Press, Inc., 1967. 2 Zernike, F.: How I discovered phase contrast. Science, 121: 345, 1955.
diaphragm (fig. 1, C) and 4) phase aperture unit (fig. 1, D). Only a high dry objective (X 45) was used. Kodak Color Snap 35 Camera Model 2 and Kodak Panatomic (FX135-36) film was used to the same field as viewed with light and phase trast microscopy. Maximum definition and contrast of the light microscopic field were achieved decreasing the light source with the condenser. RESULTS
The hyaline cast and mucous threads are and clearly seen with the phase contrast (fig. 2, B and D). The same fields are visualized light microscopy and incorporated in the other of the plates (fig. 2, A and C). Coarsely granular casts with oval fat bodies and mass of cellular debris are visualized with light microscopy (fig. 3, A). All elements are seen clearly in the same field with (fig. 3, B). The sediment casts are seen clearly in the objective. Red blood cell elements in the ,·mu·~,~,'-" granulated cast are evident (fig. 4, B and D). finding is much less conspicuous under light micros~ copy (fig. 4, A and G). The hyaline casts are more discernible under phase than under light microscopy (fig. 5, A and G). The cellular elements are well distinguished. Mononuclear cells of renal tubular origin are able under phase from bilobed or trilobed white blood cells and the mononuclear Renal tubular cells engorged with fat are also found without difficulty in the phase contrast fields (fig. 5, B and D). Bacteria which are quite elusive in the unstained sediment examined with light microscopy (fig. 6, are seen clearly with phase contrast (fig. 6, B), as are the urethral epithelial cells. DISCUSSION
Examination of the urinary sediment is an tremely important diagnostic procedure in routine urologic practice. Proper examination and terpretation are imperative in screening a with primary renal disease, infection or uvvv'-'"''"· Heretofore, urinary sediment examinations have been attempted with light microscopy and variom, special sediment stains. Since the refractive indices of hyaline casts (Tamm Horsfall cellular elements and the background media of urine are similar, the amplitudes of light waves passing through these objects are not very different. 843
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RUSSO AND COCKETT
Fm. l. A, in base illuminator. B, 45X phase objective, dark contrast. C, phase turret condenser mount with annular diaphragm and condenser centering wrenches, iris diaphragm. D, phase aperture viewing unit.
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MICROSCOPIC URINALYSIS WITH PHASE CONTRAST MICROSCOPY
845
'
Fm. 2. A, light microscopy-hyaline cast. B, same cast under phase contrast microscopy. Reduced from X45. C, hyaline cast-light microscopy. D, same cast under phase contrast microscopy. Reduced from X45.
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RUSSO AND COCKETT
FIG. 3. A, coarsely granular cast and cellular debris-light microscopy. B, same cast with oval fat bodies and cellular debris-phase contrast microscopy. Reduced from X45.
IVIICROSCOPIC URINALYSIS WITH PHASE CONTRAST MICROSCOPY
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Fm. 4. A, coarsely granular cast with barely visible RBC component-light microscopy. B, same field under phase contrast microscopy. Note clearly visible RBC components. Reduced from X45. C, light view of REC in cast formation. D, same field with clear definition under phase contrast. Reduced from
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RUSSO AND COCKETT
Frn. 5. A, poorly defined cellular cast under light microscopy. B, same cast well defined under phase contrast. Note definition of cells and cellular components. Reduced from X45. C, cellular cast-barely visible antler light microscopy. D, same field under phase contrast. Note clarity of cellular components and hyaline components; also note different cell types. Reduced from X 45.
MICROSCOPIC URINALYSIS WITH PHASE CONTRAST MICROSCOPY
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Fm. 6. A, light microscopic view of urethral epithelial cells. Arrows point to bacteria (not clear under light microscopy). B, same specimen under phase contrast. Note easily detected bacteria; also note visibility of large nuclei of urethral cells. Reduced from X 45. Thus, the resultant visual images, as viewed through the light microscope, are invisible or at best difficult to recognize. 3 Phase contrast microscopes, by converting the phase changes of wavelengths into amplitude changes, can produce visible qualitative changes. From a practical standpoint this has been shown in our data and in another report. 4 Hyaline and granular casts which are barely detectable with light microscopy are detectable quickly and easily with the phase contrast microscope. Although cellular elements are visible with light microscopy, their inclusion in casts and their cellular types are not discernible. The opposite is true when phase contrast microscopy is used. Detection of bacteria in a high dry field is of great 3 McQueen, E. G. and Sidney, M. B.: Composition of urinary casts. Lancet, 1: 397, 1966. 4 Brody, L., Webster, M. C. and Kark, R. M.: Identification of elements of urinary sediment with phase-contrast microscopy. A simple method. J.A.M.A., 206: 1777, 1968.
diagnostic importance. Presently this can be done quickly and easily with an unstained urinary sediment by using a phase contrast microscope. We believe that phase contrast microscopy adds an important diagnostic technique to the urologist's armamentarium. We also believe that its application to a routine urologic practice can be accomplished easily and immediately by conversion of light microscopes to phase contrast microscopes. A maximum cost of $450, through application of the component parts shown in figure 1, is reasonable. 5 SUMMARY
Random human urine samples were collected and the urine sediment was visualized and under light and phase contrast microscopy. components, casts and bacteria were detected with greater ease and detail under phase contrast than under light microscopy. 5 American Optical Company. Price list for series 10 phases tar.
Copyright© 1972 by The Williams & Wilkins Co.
MICROSCOPIC URINALYSIS WITH PHASE CONTRAST MICROSCOPY MICHAEL A. RUSSO
AND
A. T. K. COCKETT
From the Division of Urology, University of Rochester School of Medicine and Dentistry, Strong Memorial Hospital, Rochester, New York
The science of histochemistry, coupled with a study of the naked cell employing light microscopy, leaves much to be desired. Unstained cells and organelles have refractive indices similar to that of the background medium; therefore, light waves passing through the cell and the adjacent background medium will have differences in phase but few differences in amplitude. Since the wavelength amplitudes of light passing through the background medium and cells are similar, the image of the cell and background medium are almost indistinguishable. Staining techniques provide sufficient differential changes in wavelength amplitude to produce marked differences in the images of the cell, cellular components and the background medium. 1 Phase contrast microscopes enhance the cellular and background differences. Phase microscopy changes the light passing through unstained cells, which the eye is unable to appreciate, into visible changes in wave amplitude, that is it separates the light directly transmitted by transparent cells from the light diffracted by these same cells and retards the phase of the diffracted light about a quarter of a wavelength with respect to the directly transmitted light wave. The 2 waves then will interfere with one another to produce visible changes in amplitude. Zernike is credited with the original mathematical and experimental description of this phenomenon. 2 It is our contention that phase contrast microscopy is readily and inexpensively applicable to microscopic urinalysis and that it is invaluable in examining unstained urinary sediment. MATERIALS AND METHODS
Fresh human urine samples were randomly obtained from the urology and nephrology clinics. These were centrifuged for approximately 2 to 5 minutes. The supernatant was discarded, the sediment was placed on a glass slide and a coverslip was applied. The A-0 Micro-Star binocular microscope was converted to a phase contrast microscope by substitution of 1) a base illuminator (fig. 1, A), 2) a 45x phase objective, dark contrast (fig. 1, B), 3) phase turret condenser mount with annular diaphragm and condenser centering wrenches, iris Accepted for publication June 11, 1971. Read at annual meeting of Northeastern Section, American Urological Association, Hershey, Pennsylvania, September 20-23, 1970. 1 Ross, K. A.: Phase Contrast and Interference Microscopy for Cell Biologists. New York: St. Martin's Press, Inc., 1967. 2 Zernike, F.: How I discovered phase contrast. Science, 121: 345, 1955.
diaphragm (fig. 1, C) and 4) phase aperture unit (fig. 1, D). Only a high dry objective (X 45) was used. Kodak Color Snap 35 Camera Model 2 and Kodak Panatomic (FX135-36) film was used to the same field as viewed with light and phase trast microscopy. Maximum definition and contrast of the light microscopic field were achieved decreasing the light source with the condenser. RESULTS
The hyaline cast and mucous threads are and clearly seen with the phase contrast (fig. 2, B and D). The same fields are visualized light microscopy and incorporated in the other of the plates (fig. 2, A and C). Coarsely granular casts with oval fat bodies and mass of cellular debris are visualized with light microscopy (fig. 3, A). All elements are seen clearly in the same field with (fig. 3, B). The sediment casts are seen clearly in the objective. Red blood cell elements in the ,·mu·~,~,'-" granulated cast are evident (fig. 4, B and D). finding is much less conspicuous under light micros~ copy (fig. 4, A and G). The hyaline casts are more discernible under phase than under light microscopy (fig. 5, A and G). The cellular elements are well distinguished. Mononuclear cells of renal tubular origin are able under phase from bilobed or trilobed white blood cells and the mononuclear Renal tubular cells engorged with fat are also found without difficulty in the phase contrast fields (fig. 5, B and D). Bacteria which are quite elusive in the unstained sediment examined with light microscopy (fig. 6, are seen clearly with phase contrast (fig. 6, B), as are the urethral epithelial cells. DISCUSSION
Examination of the urinary sediment is an tremely important diagnostic procedure in routine urologic practice. Proper examination and terpretation are imperative in screening a with primary renal disease, infection or uvvv'-'"''"· Heretofore, urinary sediment examinations have been attempted with light microscopy and variom, special sediment stains. Since the refractive indices of hyaline casts (Tamm Horsfall cellular elements and the background media of urine are similar, the amplitudes of light waves passing through these objects are not very different. 843
844
RUSSO AND COCKETT
Fm. l. A, in base illuminator. B, 45X phase objective, dark contrast. C, phase turret condenser mount with annular diaphragm and condenser centering wrenches, iris diaphragm. D, phase aperture viewing unit.
l,I
MICROSCOPIC URINALYSIS WITH PHASE CONTRAST MICROSCOPY
845
'
Fm. 2. A, light microscopy-hyaline cast. B, same cast under phase contrast microscopy. Reduced from X45. C, hyaline cast-light microscopy. D, same cast under phase contrast microscopy. Reduced from X45.
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RUSSO AND COCKETT
FIG. 3. A, coarsely granular cast and cellular debris-light microscopy. B, same cast with oval fat bodies and cellular debris-phase contrast microscopy. Reduced from X45.
IVIICROSCOPIC URINALYSIS WITH PHASE CONTRAST MICROSCOPY
847
Fm. 4. A, coarsely granular cast with barely visible RBC component-light microscopy. B, same field under phase contrast microscopy. Note clearly visible RBC components. Reduced from X45. C, light view of REC in cast formation. D, same field with clear definition under phase contrast. Reduced from
848
RUSSO AND COCKETT
Frn. 5. A, poorly defined cellular cast under light microscopy. B, same cast well defined under phase contrast. Note definition of cells and cellular components. Reduced from X45. C, cellular cast-barely visible antler light microscopy. D, same field under phase contrast. Note clarity of cellular components and hyaline components; also note different cell types. Reduced from X 45.
MICROSCOPIC URINALYSIS WITH PHASE CONTRAST MICROSCOPY
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Fm. 6. A, light microscopic view of urethral epithelial cells. Arrows point to bacteria (not clear under light microscopy). B, same specimen under phase contrast. Note easily detected bacteria; also note visibility of large nuclei of urethral cells. Reduced from X 45. Thus, the resultant visual images, as viewed through the light microscope, are invisible or at best difficult to recognize. 3 Phase contrast microscopes, by converting the phase changes of wavelengths into amplitude changes, can produce visible qualitative changes. From a practical standpoint this has been shown in our data and in another report. 4 Hyaline and granular casts which are barely detectable with light microscopy are detectable quickly and easily with the phase contrast microscope. Although cellular elements are visible with light microscopy, their inclusion in casts and their cellular types are not discernible. The opposite is true when phase contrast microscopy is used. Detection of bacteria in a high dry field is of great 3 McQueen, E. G. and Sidney, M. B.: Composition of urinary casts. Lancet, 1: 397, 1966. 4 Brody, L., Webster, M. C. and Kark, R. M.: Identification of elements of urinary sediment with phase-contrast microscopy. A simple method. J.A.M.A., 206: 1777, 1968.
diagnostic importance. Presently this can be done quickly and easily with an unstained urinary sediment by using a phase contrast microscope. We believe that phase contrast microscopy adds an important diagnostic technique to the urologist's armamentarium. We also believe that its application to a routine urologic practice can be accomplished easily and immediately by conversion of light microscopes to phase contrast microscopes. A maximum cost of $450, through application of the component parts shown in figure 1, is reasonable. 5 SUMMARY
Random human urine samples were collected and the urine sediment was visualized and under light and phase contrast microscopy. components, casts and bacteria were detected with greater ease and detail under phase contrast than under light microscopy. 5 American Optical Company. Price list for series 10 phases tar.