Summary
The need for the standardization of reagents and methods used in the histology laboratory is demonstrated. After definitions of dyes, stains, and chromogenic reagents, existing standards and standards organizations are discussed. This is followed by practical instructions on how to standardize dyes and stains through the preparation of reference materials and the development of chromatographic methods. An overview is presented of the problems concerned with standardization of the Romanowsky-Giemsa stain for cytological and histological application. Finally, the problem of how to convince routine dye and stain users of the need for standardization in their histology laboratories is discussed.
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References
Allison, R. T. (1982) The Biological Stain Commission. A model for the UK? Gazette IMLS 1, 3–4.
Allison, R. T. (1984) The need to standardise biological dyes. Gazette IMLS 28, 487–8.
Andersen, A. P., Lyon, H. & Jakobsen, P. (1991) Reagents. In Theory and Strategy in Histochemistry (edited by Lyon, H.), pp. 33–66. Berlin: Springer.
Baeten, W., Claereboudt, J., Colaeys, M., VanLiedekerke, B. M. & DeLeenheer, A. P. (1989) Application of fast atom bombardment and tandem mass spectrometry for the analysis of azure dyes. Spectrosc. Int. J. 7, 113–22.
Bins, M., Huiges, W. & Halie, M. R. (1985) Stability of azure B-eosin Y staining solutions. Br. J. Haematol. 59, 73–8.
Chayen, J. & Bitensky, L. (1991) Practical Histochemistry, 2nd edn. Chichester: Wiley.
Clark, G. (1974) Staining Procedures Used by the Biological Stain Commission, 3rd edn. Baltimore: Williams and Wilkins.
Clark, G. & Kasten, F. (1983) History of Staining, 3rd edn. Baltimore: Williams and Wilkins.
Conn, H. J. (1980a) The history of the Stain Commission. I. Preliminaries. Stain Technol. 55, 327–52.
Conn, H. J. (1980b) The history of the Stain Commission. III. Birth of the Stain Commission. Stain Technol. 56, 1–17.
Daruwalla, E. H. (1974) Physical chemistry of dyeing: state of dye in dye bath and in substrate. In The Chemistry of Synthetic Dyes (edited by Venkataraman, K.). New York: Academic Press.
Dean, W. W., Stastny, M. & Lubrano, G. J. (1977) The degradation of Romanowsky-type blood stains in methanol. Stain Technol. 52, 35–46.
Din, Deutsches Institut Für Normung (1987) DIN-Taschenbuch Medizinische Mikrobiologie. Berlin: Beuth.
Elias, J. M., Gown, A. M., Nakamura, R. M., Wilbur, D. C., Herman, G. E., Jaffe, E. S., Battifora, H. & Brigati, D. J. (1989) Special report: quality control in immunohistochemistry. Report of a workshop sponsored by the Biological Stain Commission. Amer. J. Clin. Pathol. 92, 836–43.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992a) Dye standards, Part I: terminology and general principles. Histochem. J. 24, 217–9.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992b) Dye standards, Part II, 1: Pyronin Y (CI45005). Histochem. J. 24, 220–3.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992c) Dye standards, Part II, 2: Methyl Green (CI 42585) and Ethyl Green (CI 42590). Histochem. J. 24, 224–7.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992d) Dye standards, Part II, 3: Thionin (CI 52000). Histochem. J. 24, 228–9.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992e) Dye standards, Part II, 4: Victoria Blue B (CI 45045). Histochem. J. 24, 230–2.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992f) Dye standards, Part II, 5: Pararosaniline (CI 42500). Histochem. J. 24, 233–5.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992g) Dye standards, Part II, 6: Rosaniline (CI 42510). Histochem. J. 24, 236–7.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992h) Dye standards, Part II, 7: Magenta II (no CI number). Histochem. J. 24, 238–9.
European Committee For Clinical Laboratory Standards, Subcommittee On Reference Materials For Tissue Stains, Eccls-Srmts (1992i) Dye standards, Part II, 8: New Fuchsin (CI 42520). Histochem. J. 24, 240–2.
Friedrich, K., Seiffert, W. & Zimmermann, H. W. (1990) Romanowsky dyes and Romanowsky-Giemsa Effect. 5. Structural investigations of the purple DNA-AB-EY dye complexes of Romanowsky Giemsa staining. Histochemistry 93, 246–56.
Giemsa, G. (1902) Färbemethoden für Malariaparasiten. Zbl. Bakt. 31, 429–30.
Giemsa, G. (1904) Eine Vereinfachung und Vervollkommnung meiner Methylenazur-Methylenblau-Eosinfärbung zur Erzielung der Romanowsky-Nochtschen Chromatinfärbung. Zbl. Bakt. 37, 308–11.
Giemsa, G. (1923) Das Wesen der Giemsa-Färbung. Centr. Bakt. 19, 99–105.
Green, I. E., Weintraub, H. A., Donnelly, B. S. & Mordecai, B. G. (1979) Sample preparation variation and its effect on automated blood cell differential analysis. Anal. Quant. Cytol. 1, 187–201.
Gupta, V. K. (1991) Synthetic dyes. In Handbook of Thin-Layer Chromatography (edited by Sherma, J. & Fried, B.) pp. 939–69. New York: Marcel Dekker, Inc.
Holmes, W. C. (1928) The spectrophotometric evaluation of mixtures of methylene blue and trimethylthionin. Stain Technol. 3, 45–8.
Horobin, R. W. (1980) The Use of Pure Dyes for Biological Staining. Colnbrook: Koch-Light.
Horobin, R. W. (1982) Histochemistry: an Explanatory Outline of Histochemistry and Biophysical Staining. p. 82; pp. 229–34. Stuttgart, London: Fischer Butterworths.
Horobin, R. W., Payne, J. N. & Jakobsen, P. (1987) Histochemical implications of the chemical and biological properties of SITS and some related compounds. J. Microsc. 146, 87–96.
Hoyer, P. E., Lyon, H., Jakobsen, P. & Andersen, A. P. (1986) Standardized Methyl Green-Pyronin Y procedures using pure dyes. Histochem. J. 18, 90–4.
Hüglin, D., Seiffert, W. & Zimmermann, H. W. (1986) Spektroskopische und thermodynamische Untersuchungen zur Bindung von Azur B an Chondroitinsulfat und zur Bindungsgeometrie des metachromatischen Farbstoff-komplexes. Histochemistry 86, 71–82.
ICSH (1984) ICSH reference method for staining blood and bone marrow films by azure B and eosin Y (Romanowsky stain). Br. J. Haematol. 57, 707–10.
Jefree, G. M. (1970) The histochemical differentiation of various phosphatases in a population of osteoclasts by a simultaneous coupling method using different diazonium salts, with observations on the presence of inhibitors in stable diazonium salts. Histochem. J. 2, 231–42.
Koji, T. & Nakane, P. K. (1990) Use of nucleotides as an alternative to formamide in non-radioactive in situ hybridization. Acta Histochem. Cytochem. 23, 327–34.
Lauder, R. M. & Beynon, A. D. (1989) Evidence against the existence of acetylated Sudan Black B. Histochemistry 93, 213–6.
Lillie, R. D. (1944) Spectroscopic data on paired mixtures of purified thiazins and of commercial thiazins. Publ. Health Rep. (suppl.) 178, 21–4.
Lyon, H. (ed.) (1991) Theory and Strategy in Histochemistry. Berlin: Springer.
Lyon, H., Jakobsen, P. & Andersen, A. P. (1983) Methyl Green-Pyronin staining with pure Pyronin Y. Histochem. J. 15, 605–6.
Lyon, H., Wittekind, D. & Schulte, E. (1991) Standardization of staining methods. In Theory and Strategy in Histochemistry (edited by Lyon, H.), pp. 509–17. Berlin: Springer.
Marshall, P. N. (1978) Romanowsky-type stains in haematology. Histochem. J. 10, 1–29.
Marshall, P. N. & Lewis, S. M. (1974) Batch variations in commercial dyes employed for Romanowsky-type staining: a thin-layer chromatographic study. Stain Technol. 49, 531–8.
Marshall, P. N. & Lewis, S. M. (1975) Metal contaminants in commercial thiazine dyes. Stain Technol. 50, 143–7.
Mowry, R. W. & Kasten, F. H. (1975) The importance of dye purification and standardization in biomedicine. Stain Technol. 50, 65–81.
Mowry, R. W., Longley, J. B. & Emmel, V. M. (1980) Only aldehyde fuchsin made from pararosanilin stains pancreatic B cell granules and elastic fibers in unoxidized microsections: problems caused by mislabelling of certain basic fuchsins. Stain Technol. 55, 99–103.
Müller-Walz, E. & Zimmermann, H. W. (1987) Über Romanowsky Farbstoffe und den Romanowsky-Giemsa Effekt. 4. Mitteilung: Bindung von Azur B an DNA. Histochemistry 87, 157–72.
Nelis, H. J. C. F. & DeLeenheer, A. P. (1986) Quality control of azure B preparations by liquid chromatography and standardization with azure B tetrafluoroborate. Clin. Chem. Acta 156, 247–58.
Pappenheim, A. (1911) ‘Panchrom’, eine Verbesserung der panoptischen Universalfarblösung für Blutpräparate jeder Art nebst Ausführungen aber metachromatische Farbstoffe und die metachromatische Potenz des Methylenblau. Fol. Haematol. 11, 194–218.
Schulte, E. K. W. (1991) Standardization of biological dyes and stains: pitfalls and possibilities. Histochemistry 95, 319–28.
Schulte, E. K. W., Lyon, H. O. & Hoyer, P. E. (1992) Simultaneous quantification of DNA and RNA in tissue sections. A comparative analysis of the Methyl Green-Pyronin technique with the Gallocyanin chromalum and Feulgen Procedures using image cytometry. Histochem. J. 24, 305–10.
Scott, B. E. & French, R. W. (1924) Standardization of biological stains. Milit. Surgeon 5, 229–43.
Scott, J. E. (1972) Lies, damned lies - and biological stains. Histochem. J. 4, 387–9.
Scott, J. E. & Mowry, R. W. (1970) Alcian Blue - a consumer's guide. J. Histochem. Cytochem. 18, 842.
Slifkin, M. A. (1971) Charge Transfer Interactions of Biomolecules. London: Academic Press.
Van DerPloeg, M. & VanDuijn, P. (1964) 5,6-dihydroxy indole as a substrate in a histochemical peroxidase reaction. J. R. Microsc. Soc. 83, 415–23.
VanDuijn, P. (1976) Prospects for microscopical cytochemistry. Histochem. J. 8, 653–76.
VanDuijn, P. & Van DerPloeg, M. (1980) Microscopic cytochemistry as matrix chemistry. In Ciba Foundation Symposium 73, pp. 209–29. Amsterdam: Excerpta Medica.
VanLiedekerke, B. M. & DeLeenheer, A. P. (1990) Analysis of xanthene dyes by reversed-phase high-performance liquid chromatography on a polymeric column followed by characterization with a diode array detector. J. Chromatogr. 528, 155–62.
VanLiedekerke, B. M., Nelis, H. J., Lambert, W. E. & DeLeenheer, A. P. (1989) High-performance liquid chromatography of quaternary ammonium compounds on a polystyrene-divinylbenzene column. Anal. Chim. 61, 728–32.
VanLiedekerke, B. M., DeSpiegeleer, B. M. & DeLeenheer, A. P. (1991a) High-performance thin-layer chromatographic analysis of thiazine dyes on silica-, cyano-, and reversed-phase C18 layers. J. Chromatogr. Sci. 29, 49–53.
VanLiedekerke, B. M., Nelis, H. J., Wittekind, D. H. & DeLeenheer, A. P., (1991b) Stability study of Azure B, Eosin Y and commercial Romanowsky Giemsa stock solutions using high performance liquid chromatography. Histochem. J. 23, 189–95.
Wied, G. L., Bartels, P. H., Bibbo, M. & Sychra, J. J. (1980) Cytophotometric markers for uterine cancer in intermediate cells. Anal. Quant. Cytol. 2, 257–63.
Wittekind, D. (1985) Standardization of dyes and stains for automated cell pattern recognition. Anal. Quant. Cytol. Histol. 7, 6–30.
Wittekind, D. (1986) On the nature of the Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. In Standardization and Quantitation of Diagnostic Staining in Cytology (edited by Boon, M. E. & Kok, L. P.). Leiden: Coulomb Press Leyden.
Wittekind, D. The Romanowsky-Giemsa stain. A treatise of an interdisciplinary subject. Progress in Histochem. Cytochem. (in preparation).
Wittekind, D. & Gehring, T. (1985) On the nature of Romanowsky-Giemsa staining and the Romanowsky-Giemsa effect. I. Model experiments on the specificity of Azure B-Eosin Y stain as compared with other Thiazine dye-Eosin Y combinations. Histochem. J. 17, 263–89.
Wittekind, D. & Grèzes, J. R. Sequential fluorescein derivative → thiazine dye stains (in preparation).
Wittekind, D., Kretschmer, V. & Löhr, W. (1976) Kann Azur B-Eosin Y die May-Grünwald-Giemsa Farbung ersetzen? Blut 32, 71–8.
Wittekind, D. H., Kretschmer, V. & Sohmer, I. (1982) Azure B-Eosin Y stain as the standard Romanowsky-Giemsa stain. Brit. J. Haematol. 51, 391–3.
Zanker, V. (1981) Definition der Farbstoffe. Acta Histochem. (suppl.) 24, 151–68.
Zimmermann, H. W. (1983) Physikalisch-chemische Grundlagen der Färbung für manuelle und apparative Zytodiagnostik. In Manual and Automated Diagnosis in Cytology (edited by Wittekind, D.). Microsc. Acta (suppl.) 6, 45–58.
Zipfel, E., Grèzes, J. R., Seiffert, W. & Zimmermann, H. W. (1981) Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa Effekt. 1. Mitteilung: Azur B, Reinheit und Gehalt von Farbstoffproben, Assoziation. Histochemistry 72, 279–90.
Zipfel, E., Grèzes, J. R., Seiffert, W. & Zimmermann, H. W. (1982) Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa Effekt. 2. Mitteilung: Eosin Y, Erythrosin B, Tetrachlorofluorescein. Spektrosckopische Charakterisierung der reinen Farbstoffe. Histochemistry 79, 539–55.
Zipfel, E., Grèzes, J. R., Naujok, A., Seiffert, W., Wittekind, D. & Zimmermann, H. W. (1984) Über Romanowsky-Farbstoffe und den Romanowsky-Giemsa Effekt. 3. Mitteilung: Mikrospektral-photometrische Untersuchung der Romanowsky-Giemsa Färbung. Spektroskopischer Nachweis eines DNA-Azur B-Eosin Y-Komplexes, der den Romanowsky-Giemsa-Effekt verursacht. Histochemistry 81, 337–51.
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Lyon, H.O., De Leenheer, A.P., Horobin, R.W. et al. Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents. Histochem J 26, 533–544 (1994). https://doi.org/10.1007/BF00158587
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DOI: https://doi.org/10.1007/BF00158587