Skip to main content
SpringerLink
Log in

Standardization of reagents and methods used in cytological and histological practice with emphasis on dyes, stains and chromogenic reagents

  • Review
  • Published:
The Histochemical Journal Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Allison, R. T. (1982) The Biological Stain Commission. A model for the UK? Gazette IMLS 1, 3–4.

    Google Scholar 

  • Allison, R. T. (1984) The need to standardise biological dyes. Gazette IMLS 28, 487–8.

    Google Scholar 

  • Andersen, A. P., Lyon, H. & Jakobsen, P. (1991) Reagents. In Theory and Strategy in Histochemistry (edited by Lyon, H.), pp. 33–66. Berlin: Springer.

    Google Scholar 

  • 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.

    Google Scholar 

  • Bins, M., Huiges, W. & Halie, M. R. (1985) Stability of azure B-eosin Y staining solutions. Br. J. Haematol. 59, 73–8.

    Google Scholar 

  • Chayen, J. & Bitensky, L. (1991) Practical Histochemistry, 2nd edn. Chichester: Wiley.

    Google Scholar 

  • Clark, G. (1974) Staining Procedures Used by the Biological Stain Commission, 3rd edn. Baltimore: Williams and Wilkins.

    Google Scholar 

  • Clark, G. & Kasten, F. (1983) History of Staining, 3rd edn. Baltimore: Williams and Wilkins.

    Google Scholar 

  • Conn, H. J. (1980a) The history of the Stain Commission. I. Preliminaries. Stain Technol. 55, 327–52.

    Google Scholar 

  • Conn, H. J. (1980b) The history of the Stain Commission. III. Birth of the Stain Commission. Stain Technol. 56, 1–17.

    Google Scholar 

  • 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.

    Google Scholar 

  • Dean, W. W., Stastny, M. & Lubrano, G. J. (1977) The degradation of Romanowsky-type blood stains in methanol. Stain Technol. 52, 35–46.

    Google Scholar 

  • Din, Deutsches Institut Für Normung (1987) DIN-Taschenbuch Medizinische Mikrobiologie. Berlin: Beuth.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Giemsa, G. (1902) Färbemethoden für Malariaparasiten. Zbl. Bakt. 31, 429–30.

    Google Scholar 

  • Giemsa, G. (1904) Eine Vereinfachung und Vervollkommnung meiner Methylenazur-Methylenblau-Eosinfärbung zur Erzielung der Romanowsky-Nochtschen Chromatinfärbung. Zbl. Bakt. 37, 308–11.

    Google Scholar 

  • Giemsa, G. (1923) Das Wesen der Giemsa-Färbung. Centr. Bakt. 19, 99–105.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Holmes, W. C. (1928) The spectrophotometric evaluation of mixtures of methylene blue and trimethylthionin. Stain Technol. 3, 45–8.

    Google Scholar 

  • Horobin, R. W. (1980) The Use of Pure Dyes for Biological Staining. Colnbrook: Koch-Light.

    Google Scholar 

  • Horobin, R. W. (1982) Histochemistry: an Explanatory Outline of Histochemistry and Biophysical Staining. p. 82; pp. 229–34. Stuttgart, London: Fischer Butterworths.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Lauder, R. M. & Beynon, A. D. (1989) Evidence against the existence of acetylated Sudan Black B. Histochemistry 93, 213–6.

    Google Scholar 

  • Lillie, R. D. (1944) Spectroscopic data on paired mixtures of purified thiazins and of commercial thiazins. Publ. Health Rep. (suppl.) 178, 21–4.

    Google Scholar 

  • Lyon, H. (ed.) (1991) Theory and Strategy in Histochemistry. Berlin: Springer.

    Google Scholar 

  • Lyon, H., Jakobsen, P. & Andersen, A. P. (1983) Methyl Green-Pyronin staining with pure Pyronin Y. Histochem. J. 15, 605–6.

    Google Scholar 

  • 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.

    Google Scholar 

  • Marshall, P. N. (1978) Romanowsky-type stains in haematology. Histochem. J. 10, 1–29.

    Google Scholar 

  • 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.

    Google Scholar 

  • Marshall, P. N. & Lewis, S. M. (1975) Metal contaminants in commercial thiazine dyes. Stain Technol. 50, 143–7.

    Google Scholar 

  • Mowry, R. W. & Kasten, F. H. (1975) The importance of dye purification and standardization in biomedicine. Stain Technol. 50, 65–81.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Schulte, E. K. W. (1991) Standardization of biological dyes and stains: pitfalls and possibilities. Histochemistry 95, 319–28.

    Google Scholar 

  • 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.

    Google Scholar 

  • Scott, B. E. & French, R. W. (1924) Standardization of biological stains. Milit. Surgeon 5, 229–43.

    Google Scholar 

  • Scott, J. E. (1972) Lies, damned lies - and biological stains. Histochem. J. 4, 387–9.

    Google Scholar 

  • Scott, J. E. & Mowry, R. W. (1970) Alcian Blue - a consumer's guide. J. Histochem. Cytochem. 18, 842.

    Google Scholar 

  • Slifkin, M. A. (1971) Charge Transfer Interactions of Biomolecules. London: Academic Press.

    Google Scholar 

  • 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.

    Google Scholar 

  • VanDuijn, P. (1976) Prospects for microscopical cytochemistry. Histochem. J. 8, 653–76.

    Google Scholar 

  • VanDuijn, P. & Van DerPloeg, M. (1980) Microscopic cytochemistry as matrix chemistry. In Ciba Foundation Symposium 73, pp. 209–29. Amsterdam: Excerpta Medica.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Wittekind, D. (1985) Standardization of dyes and stains for automated cell pattern recognition. Anal. Quant. Cytol. Histol. 7, 6–30.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • Zanker, V. (1981) Definition der Farbstoffe. Acta Histochem. (suppl.) 24, 151–68.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

  • 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.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pathology 134, Hvidovre Hospital, University of Copenhagen, DK-2650, Hvidovre, Denmark

    H. O. Lyon

  2. Laboratoria voor Medische Biochemie en voor Klinische Analyse, Universiteit Gent, B-9000, Gent, Belgium

    A. P. De Leenheer, W. E. Lambert & B. Van Liedekerke

  3. Department of Biomedical Science, University of Sheffield, S10 2TN, Sheffield, UK

    R. W. Horobin

  4. Anatomisches Institut, Johannes Gutenberg-Universität Mainz, D-55122, Mainz, Germany

    E. K. W. Schulte

  5. Anatomisches Institut II, Universität Freiburg, D-79001, Freiburg im Breisgau, Germany

    D. H. Wittekind

Authors
  1. H. O. Lyon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. P. De Leenheer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. R. W. Horobin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. W. E. Lambert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. K. W. Schulte
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. B. Van Liedekerke
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D. H. Wittekind
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00158587

Keywords

Search

Navigation