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A Simple Stain for Differentiating Semen Constituents
0022-5347 /81/1265-0609$02.00/0 Vol. 126, November
THE JOURNAL OF UROLOGY
Printed in
Copyright© 1981 by The Williams & Wilkins Co.
[!. S
A
A SIMPLE STAIN FOR DIFFERENTIATING SEMEN CONSTITUENTS LAUREEN M. ALFORD AND DONALD J. RIVARD From the Division of Urological Surgery, Albany Medical College, Albany, New York
ABSTRACT
Extensive use of Sedi-stain in an outpatient infertility office has demonstrated that this commercially available urinary sediment preparation is a valuable and practical stain in semen analysis. The stain is inexpensive, has a long shelf life, is easy to apply, and produces immediate and reproducible results. The accuracy of Sedi-stain has compared favorably to other staining compounds used. It should prove to be an asset to the office investigation of male infertility. As andrology and seminology have become domains of the modern urologist practical demands of office infertility practice necessitate a simple method to aid· in the determination of sperm morphology and cellular content. Existing staining procedures are sophisticated and complex for a routine office clinical laboratory. In a small facility staining time, availability and expense should be important considerations. Since 1976 we have used in the urology laboratory at our hospital Sedi-stain, * a well recognized commercially available preparation for urinary sediment. This staining method has produced high quality specimen material, which is readily reproducible and has facilitated differentiation of cells of germinal origin from additional cellular content. Sedi-stain has compared favorably to existing stains used and should prove to be an asset to the office investigation of male infertility. Determinations of sperm morphology and cellular content are important aspects of office semen analysis. Two accepted standards for classification of sperm morphology have been established previously by Freund 1 and Eliasson. 2 Morphologic assignment and identification of associated cellular elements have been facilitated by the development of precise semen staining techniques. Widely used methods include Papanicolaou's, 1 hematoxylin, 3 eosin-nigrosin4 and Bryan's sperm stains. 5 Results with these staining techniques have been satisfactory, yet they remain sophisticated and complex for the routine laboratory that services a clinical office practice. Practical considerations in the office infertility laboratory should include cost, availability and staining time.
Since 1976 we have used Sedi-stain in the urology laboratory at our hospital in the preparation of office semen analysis. Although the exact chemical composition of Sedi-stain is not available commercially it is known to be similar in content and reactivity to the Sternheimer-Malbin stain. We examined 600 specimens from 200 infertile men on an outpatient basis. Analysis included total volume ejaculated, total sperm count, sperm count per ml. ejaculate, qualitative fructose analysis, estimation of viscosity, pH of specimen, estimation of sperm motility with assignment of motility index, and determination of sperm morphology and additional cellular content. TECHNIQUE
From a liquified fresh semen specimen a small aliquot of l or 2 drops of semen was mixed thoroughly with equal parts of Sedi-stain. Mixing was performed by gentle stirring with an applicator stick on a microscopic slide or in a test tube. A coverslip was placed on the wet mount stained semen preparation. Care was taken in preparing the slide to prevent an overflow of the mixture around the coverslip. Microscopic examination was done with high dry power and finer details were obtained with oil immersion. Morphologic differentiation of spermatic and cellular components was facilitated with this staining technique (see figure). DISCUSSION
As andrology and seminology have become domains of the modern urologist the practical demands of an office
B
D Morphologic differentiation of spermatic and cellular components using Sedi-stain and routine light microscopy. A, spermiophage. B, pinhead form. C, 2 normal sperm. D, spermatid, immature spermatic cell. E, 2 duplicate tails. F, leukocyte, polymorphonucleocyte. Accepted for publication January 30, 1981. * Clay-Adams Corporation.
609
610
ALFORD AND RIVARD
practice necessitate a simple method to determine sperm morphology. Staining methodology should meet the following criteria: 1) the method should be inexpensive, 2) the staining reagent should have a long shelf life, 3) the method should be easy to use and 4) there should be immediate staining results that are readily reproducible. The use of Sedi-stain in the urology office has demonstrated that it fulfilled these criteria adequately. Spermatozoa and cellular content of the specimen have stained immediately. Of benefit was the fact that erythrocytes, glitter cells, yeast and Trichomonas did not stain with this preparation. In addition, the acrosome of the spermatozoon only faintly captured the stain. Spermatocytes demonstrated dense purple nuclei, with a paler purple cytoplasm. Spermatids were distinguished easily from spermatocytes by a generally smaller size, and a more uniformly round single nucleus and cytoplasm. Spermiophages stained dark purple with clear cytoplasmic vacuoles. The morphologic differentiation of leukocytes was facilitated with this staining technique. Polymorphonucleocytes had characteristic dark purple granules in the cytoplasm and visible
nuclear filaments that connected nuclear segments. Lymphocytes were characterized by a smaller size, bluish color to the cytoplasm, absence of granules and nuclear segments, and eccentrically located nucleus. In addition, the nuclear size of polymorphonuclear cells and lymphocytes was generally much smaller than that of the immature, precursor spermatic cells. REFERENCES 1. Freund, M.: Standards for the rating of human sperm morphology. A cooperative study. Int. J. Fertil., 11: 97, 1966. 2. Eliasson, R.: Standards for investigation of human semen. Andrologie, 3: 49, 1971. 3. Amelar, R. D., Dubin, L. and Schoenfeld, C.: Semen analysis. An office technique. Urology, 2: 605, 1973. 4. Emilson, L.B. V., Dougherty, K. A., Cockett, A. T. and Urry, R. L.: Simultaneous determination of human sperm morphology and viability: simple office technique. Urology, 11: 488, 1978. 5. Couture, M., Ulstein, M., Leonard, J. and Paulsen, C. A.: Improved staining method for differentiating immature germ cells from white blood cells in human seminal fluid. Andrologia, 8: 61, 1976.
THE JOURNAL OF UROLOGY
Printed in
Copyright© 1981 by The Williams & Wilkins Co.
[!. S
A
A SIMPLE STAIN FOR DIFFERENTIATING SEMEN CONSTITUENTS LAUREEN M. ALFORD AND DONALD J. RIVARD From the Division of Urological Surgery, Albany Medical College, Albany, New York
ABSTRACT
Extensive use of Sedi-stain in an outpatient infertility office has demonstrated that this commercially available urinary sediment preparation is a valuable and practical stain in semen analysis. The stain is inexpensive, has a long shelf life, is easy to apply, and produces immediate and reproducible results. The accuracy of Sedi-stain has compared favorably to other staining compounds used. It should prove to be an asset to the office investigation of male infertility. As andrology and seminology have become domains of the modern urologist practical demands of office infertility practice necessitate a simple method to aid· in the determination of sperm morphology and cellular content. Existing staining procedures are sophisticated and complex for a routine office clinical laboratory. In a small facility staining time, availability and expense should be important considerations. Since 1976 we have used in the urology laboratory at our hospital Sedi-stain, * a well recognized commercially available preparation for urinary sediment. This staining method has produced high quality specimen material, which is readily reproducible and has facilitated differentiation of cells of germinal origin from additional cellular content. Sedi-stain has compared favorably to existing stains used and should prove to be an asset to the office investigation of male infertility. Determinations of sperm morphology and cellular content are important aspects of office semen analysis. Two accepted standards for classification of sperm morphology have been established previously by Freund 1 and Eliasson. 2 Morphologic assignment and identification of associated cellular elements have been facilitated by the development of precise semen staining techniques. Widely used methods include Papanicolaou's, 1 hematoxylin, 3 eosin-nigrosin4 and Bryan's sperm stains. 5 Results with these staining techniques have been satisfactory, yet they remain sophisticated and complex for the routine laboratory that services a clinical office practice. Practical considerations in the office infertility laboratory should include cost, availability and staining time.
Since 1976 we have used Sedi-stain in the urology laboratory at our hospital in the preparation of office semen analysis. Although the exact chemical composition of Sedi-stain is not available commercially it is known to be similar in content and reactivity to the Sternheimer-Malbin stain. We examined 600 specimens from 200 infertile men on an outpatient basis. Analysis included total volume ejaculated, total sperm count, sperm count per ml. ejaculate, qualitative fructose analysis, estimation of viscosity, pH of specimen, estimation of sperm motility with assignment of motility index, and determination of sperm morphology and additional cellular content. TECHNIQUE
From a liquified fresh semen specimen a small aliquot of l or 2 drops of semen was mixed thoroughly with equal parts of Sedi-stain. Mixing was performed by gentle stirring with an applicator stick on a microscopic slide or in a test tube. A coverslip was placed on the wet mount stained semen preparation. Care was taken in preparing the slide to prevent an overflow of the mixture around the coverslip. Microscopic examination was done with high dry power and finer details were obtained with oil immersion. Morphologic differentiation of spermatic and cellular components was facilitated with this staining technique (see figure). DISCUSSION
As andrology and seminology have become domains of the modern urologist the practical demands of an office
B
D Morphologic differentiation of spermatic and cellular components using Sedi-stain and routine light microscopy. A, spermiophage. B, pinhead form. C, 2 normal sperm. D, spermatid, immature spermatic cell. E, 2 duplicate tails. F, leukocyte, polymorphonucleocyte. Accepted for publication January 30, 1981. * Clay-Adams Corporation.
609
610
ALFORD AND RIVARD
practice necessitate a simple method to determine sperm morphology. Staining methodology should meet the following criteria: 1) the method should be inexpensive, 2) the staining reagent should have a long shelf life, 3) the method should be easy to use and 4) there should be immediate staining results that are readily reproducible. The use of Sedi-stain in the urology office has demonstrated that it fulfilled these criteria adequately. Spermatozoa and cellular content of the specimen have stained immediately. Of benefit was the fact that erythrocytes, glitter cells, yeast and Trichomonas did not stain with this preparation. In addition, the acrosome of the spermatozoon only faintly captured the stain. Spermatocytes demonstrated dense purple nuclei, with a paler purple cytoplasm. Spermatids were distinguished easily from spermatocytes by a generally smaller size, and a more uniformly round single nucleus and cytoplasm. Spermiophages stained dark purple with clear cytoplasmic vacuoles. The morphologic differentiation of leukocytes was facilitated with this staining technique. Polymorphonucleocytes had characteristic dark purple granules in the cytoplasm and visible
nuclear filaments that connected nuclear segments. Lymphocytes were characterized by a smaller size, bluish color to the cytoplasm, absence of granules and nuclear segments, and eccentrically located nucleus. In addition, the nuclear size of polymorphonuclear cells and lymphocytes was generally much smaller than that of the immature, precursor spermatic cells. REFERENCES 1. Freund, M.: Standards for the rating of human sperm morphology. A cooperative study. Int. J. Fertil., 11: 97, 1966. 2. Eliasson, R.: Standards for investigation of human semen. Andrologie, 3: 49, 1971. 3. Amelar, R. D., Dubin, L. and Schoenfeld, C.: Semen analysis. An office technique. Urology, 2: 605, 1973. 4. Emilson, L.B. V., Dougherty, K. A., Cockett, A. T. and Urry, R. L.: Simultaneous determination of human sperm morphology and viability: simple office technique. Urology, 11: 488, 1978. 5. Couture, M., Ulstein, M., Leonard, J. and Paulsen, C. A.: Improved staining method for differentiating immature germ cells from white blood cells in human seminal fluid. Andrologia, 8: 61, 1976.