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A SIMPLE DIFFERENTIAL STAIN FOR URINARY SEDIMENT
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A SIMPLE DIFFERENTIAL STAIN FOR
URINARY SEDIMENT L. F. PRESCOTT M.A., M.B. Cantab. D. E. BRODIE M.D. Jefferson From the
Department of Medicine (Division of Clinical Pharmacology), Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. EXAMINATION of the urinary sediment is a traditional but frequently neglected procedure. It is often difficult to distinguish readily between polymorph leucocytes and small renal tubular cells, especially if degenerate forms are present (Addis 1949, Montgomerie et al. 1963). Collecthese cells are often referred to inaccurately as tively " white cells ", non-squamous white cells ", or leucocytes ". When large numbers of such cells are present, polymorph leucocytes (which we shall refer to as leucocytes) usually predominate, but if renal tubular cells are "
mistaken for
"
et al. 1963) diagnostic and arise. therapeutic The stain we describe here is based on the familiar peroxidase reaction, and permits rapid and specific identification of leucocytes. A modification of the copperbenzidine method of Sato and Shoji (1928) was described by Kaye (1958). This method has proved unreliable, since leucocytes in freshly passed normal urine often fail to react to the reagents, although urine which has stood for a time or is infected may stain satisfactorily. Freshly voided normal urine was found to have a substantial inhibitory effect on both leucocyte peroxidase and peroxidase obtained from crude horseradish extracts. This could have been due to the presence of unstable reducing substances which are inactivated by exposure to air or by bacterial action. To obtain consistently reliable staining, pH and hydrogen-peroxide concentration must be controlled. Leucocytes in urine are stained best at pH 5-0-6-5 and with 0-005-0-10% hydrogen peroxide. Higher concentrations of the latter tend to inhibit the leucocyte reaction, and with concentrations exceeding 0-02% erythrocytes stain densely. A hydrogen donor yielding a highly coloured oxidation product must also be present in adequate concentration. Benzidine is used in most peroxidase staining methods, but, because of its poor solubility in water, it does not stain leucocytes in the urinary sediment satisfactorily. A number of phenols, indo-phenols, aromatic amines, and leuco-dyes were investigated as possible alternatives, and of these 2:7 K. Laboratories Inc. 121, diaminofluorene (K. & Express Street, Engineer’s Hill, Plainview, New York, 11803) was chosen as an extremely sensitive indicator, stable enough to be used in a routine staining method. The observation that leucocytes slowly stain deep blue with 2:7 diaminofluorene even in the absence of added hydrogen peroxide demonstrates this sensitivity. Traces of higher peroxides are probably responsible for this effect, since it is abolished by the addition of catalase.
leucocytes (Scott
errors can
Method 300 mg. of 2:7 diaminofluorene and 130 mg. of phloxine B are dissolved in 70 ml. of warm 95% ethanol. To this is added 11 g. of sodium acetate (CH3COONa° 3H2O) dissolved in 20 ml. of 0-5% acetic acid and 1 ml. of 3% hydrogen peroxide. The mixture is allowed to stand for 48 hours and is ready for use after filtering. The shelf life in a dark bottle is many months, but occasional filtering may be required. Urine containing phosphate crystals should be cleared before centrifuging by
the addition of a few drops of glacial acetic acid, but excess should be avoided as very acid urines cause precipitation of dye. To 0-5 ml. of sediment 3 drops of stain are added and mixed thoroughly. Leucocytes are stained deep blue-black, renal tubular cells and squamous epithelial cells appear pink, while erythrocytes are stained red. The blue oxidation product of 2:7 diaminofluorene is soluble in leucocyte cytoplasm, since discrete pigmented granules are not observed as they are with the benzidine methods. Initially the cytoplasm is clear, or only slightly bluish, whereas the nuclei stain progressively darker blue. The cytoplasm then rapidly darkens, and the whole cell appears uniformly blue-black. This process is usually complete within 2 minutes. Some cellular distortion is occasionally seen with characteristic pseudopod-like extrusions and ballooning of the cytoplasm. Casts containing leucocytes are visible macroscopically as delicate blue-black threads and should be removed with a Pasteur pipette and examined separately. Striking preparations of mixed casts are obtainable by this method. Suspensions of cells prepared from scrapings of bladder, ureter, renal pelvis, and renal parenchyma obtained at necropsy were stained pink. These cells are known to contain little or no peroxidase. In contrast, leucocytes from blood and pus stain blue-black. Differential cell-excretion rates are measured with the following modification of the method of Houghton and Pears (1957). 10 ml. of well-mixed urine is spun at 1500 r.p.m. for 10 minutes in a graduated centrifuge tube. The supernatant is removed by careful aspiration, and 3 drops of stain are added to the sediment. Distilled water is added to the 0-5 ml. mark, and the whole is mixed thoroughly with a Pasteur pipette. Leucocytes, renal tubular cells, and erythrocytes are counted in ten large squares of a Neubauer counting chamber. The excretion-rate of each cell type is given by the following formula: For example, if the volume of urine collected over 3 hours is 400 ml., and 60 leucocytes are counted in ten large squares, the excretion-rate is:
Specimens containing large numbers of cells should be or counted uncentrifuged.
concentrated less,
Conclusion This staining method is simple, rapid, and reliable. The cells of urinary sediment can be easily identified and counted even under low-power magnification. Although its use so far has been restricted to urine, it should also be applicable to pleural, ascitic, and cerebrospinal fluids. This work was by a training grant (1 Tl HE-5615-01) from the National Heart Institute. REFERENCES
Addis, T. (1949) Glomerular Nephritis; p. 12. New York. Houghton, B. J., Pears, M. A. (1957) Brit. med. J. i, 622. Kaye, M. (1958) New Engl. J. Med. 258, 1301. Montgomerie, J. Z., North, J. D. K. (1963) Lancet, i, 690. Sato, A., Shoji, K. (1928) J. Lab. clin. Med. 13, 1058. Scott, J. T., Denman, A. M., Dorling, J. (1963) Lancet, i, 344.
"... It is nonetheless one of the blandest of 20th-century complacencies that education is a ’good thing ’. Whatever its quirks and defects, that the machine is benign ... We should call
schools and universities to account for the enormous of time and energy which they now consume. My own guess is that the first 15 years of an Englishman’s education does little for him of intellectual value beyond instilling certain interests, attitudes and habits of mind.... It is possible that, today, Einstein might be given a research grant; but with Darwin, who had five years of wasted university education behind him, the case is altogether different. What don, after all, can honestly claim that he would detect Darwin’s genius if he turned up among this year’s candidates for research ? He was just a nice young man with a good collection of beetles." - LIAM HUDSON, New Societv, Oct. 22, 1964, p. 13. our
amounts
A SIMPLE DIFFERENTIAL STAIN FOR
URINARY SEDIMENT L. F. PRESCOTT M.A., M.B. Cantab. D. E. BRODIE M.D. Jefferson From the
Department of Medicine (Division of Clinical Pharmacology), Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. EXAMINATION of the urinary sediment is a traditional but frequently neglected procedure. It is often difficult to distinguish readily between polymorph leucocytes and small renal tubular cells, especially if degenerate forms are present (Addis 1949, Montgomerie et al. 1963). Collecthese cells are often referred to inaccurately as tively " white cells ", non-squamous white cells ", or leucocytes ". When large numbers of such cells are present, polymorph leucocytes (which we shall refer to as leucocytes) usually predominate, but if renal tubular cells are "
mistaken for
"
et al. 1963) diagnostic and arise. therapeutic The stain we describe here is based on the familiar peroxidase reaction, and permits rapid and specific identification of leucocytes. A modification of the copperbenzidine method of Sato and Shoji (1928) was described by Kaye (1958). This method has proved unreliable, since leucocytes in freshly passed normal urine often fail to react to the reagents, although urine which has stood for a time or is infected may stain satisfactorily. Freshly voided normal urine was found to have a substantial inhibitory effect on both leucocyte peroxidase and peroxidase obtained from crude horseradish extracts. This could have been due to the presence of unstable reducing substances which are inactivated by exposure to air or by bacterial action. To obtain consistently reliable staining, pH and hydrogen-peroxide concentration must be controlled. Leucocytes in urine are stained best at pH 5-0-6-5 and with 0-005-0-10% hydrogen peroxide. Higher concentrations of the latter tend to inhibit the leucocyte reaction, and with concentrations exceeding 0-02% erythrocytes stain densely. A hydrogen donor yielding a highly coloured oxidation product must also be present in adequate concentration. Benzidine is used in most peroxidase staining methods, but, because of its poor solubility in water, it does not stain leucocytes in the urinary sediment satisfactorily. A number of phenols, indo-phenols, aromatic amines, and leuco-dyes were investigated as possible alternatives, and of these 2:7 K. Laboratories Inc. 121, diaminofluorene (K. & Express Street, Engineer’s Hill, Plainview, New York, 11803) was chosen as an extremely sensitive indicator, stable enough to be used in a routine staining method. The observation that leucocytes slowly stain deep blue with 2:7 diaminofluorene even in the absence of added hydrogen peroxide demonstrates this sensitivity. Traces of higher peroxides are probably responsible for this effect, since it is abolished by the addition of catalase.
leucocytes (Scott
errors can
Method 300 mg. of 2:7 diaminofluorene and 130 mg. of phloxine B are dissolved in 70 ml. of warm 95% ethanol. To this is added 11 g. of sodium acetate (CH3COONa° 3H2O) dissolved in 20 ml. of 0-5% acetic acid and 1 ml. of 3% hydrogen peroxide. The mixture is allowed to stand for 48 hours and is ready for use after filtering. The shelf life in a dark bottle is many months, but occasional filtering may be required. Urine containing phosphate crystals should be cleared before centrifuging by
the addition of a few drops of glacial acetic acid, but excess should be avoided as very acid urines cause precipitation of dye. To 0-5 ml. of sediment 3 drops of stain are added and mixed thoroughly. Leucocytes are stained deep blue-black, renal tubular cells and squamous epithelial cells appear pink, while erythrocytes are stained red. The blue oxidation product of 2:7 diaminofluorene is soluble in leucocyte cytoplasm, since discrete pigmented granules are not observed as they are with the benzidine methods. Initially the cytoplasm is clear, or only slightly bluish, whereas the nuclei stain progressively darker blue. The cytoplasm then rapidly darkens, and the whole cell appears uniformly blue-black. This process is usually complete within 2 minutes. Some cellular distortion is occasionally seen with characteristic pseudopod-like extrusions and ballooning of the cytoplasm. Casts containing leucocytes are visible macroscopically as delicate blue-black threads and should be removed with a Pasteur pipette and examined separately. Striking preparations of mixed casts are obtainable by this method. Suspensions of cells prepared from scrapings of bladder, ureter, renal pelvis, and renal parenchyma obtained at necropsy were stained pink. These cells are known to contain little or no peroxidase. In contrast, leucocytes from blood and pus stain blue-black. Differential cell-excretion rates are measured with the following modification of the method of Houghton and Pears (1957). 10 ml. of well-mixed urine is spun at 1500 r.p.m. for 10 minutes in a graduated centrifuge tube. The supernatant is removed by careful aspiration, and 3 drops of stain are added to the sediment. Distilled water is added to the 0-5 ml. mark, and the whole is mixed thoroughly with a Pasteur pipette. Leucocytes, renal tubular cells, and erythrocytes are counted in ten large squares of a Neubauer counting chamber. The excretion-rate of each cell type is given by the following formula: For example, if the volume of urine collected over 3 hours is 400 ml., and 60 leucocytes are counted in ten large squares, the excretion-rate is:
Specimens containing large numbers of cells should be or counted uncentrifuged.
concentrated less,
Conclusion This staining method is simple, rapid, and reliable. The cells of urinary sediment can be easily identified and counted even under low-power magnification. Although its use so far has been restricted to urine, it should also be applicable to pleural, ascitic, and cerebrospinal fluids. This work was by a training grant (1 Tl HE-5615-01) from the National Heart Institute. REFERENCES
Addis, T. (1949) Glomerular Nephritis; p. 12. New York. Houghton, B. J., Pears, M. A. (1957) Brit. med. J. i, 622. Kaye, M. (1958) New Engl. J. Med. 258, 1301. Montgomerie, J. Z., North, J. D. K. (1963) Lancet, i, 690. Sato, A., Shoji, K. (1928) J. Lab. clin. Med. 13, 1058. Scott, J. T., Denman, A. M., Dorling, J. (1963) Lancet, i, 344.
"... It is nonetheless one of the blandest of 20th-century complacencies that education is a ’good thing ’. Whatever its quirks and defects, that the machine is benign ... We should call
schools and universities to account for the enormous of time and energy which they now consume. My own guess is that the first 15 years of an Englishman’s education does little for him of intellectual value beyond instilling certain interests, attitudes and habits of mind.... It is possible that, today, Einstein might be given a research grant; but with Darwin, who had five years of wasted university education behind him, the case is altogether different. What don, after all, can honestly claim that he would detect Darwin’s genius if he turned up among this year’s candidates for research ? He was just a nice young man with a good collection of beetles." - LIAM HUDSON, New Societv, Oct. 22, 1964, p. 13. our
amounts