How Romanowsky stains work and why they remain valuable—Including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme

School of Life Sciences, The University of Glasgow, University Avenue, Glasgow G12 8QQ, Scotland, UK.
Biotechnic & Histochemistry (Impact Factor: 1.44). 02/2011; 86(1):36-51. DOI: 10.3109/10520295.2010.515491
Source: PubMed


An introduction to the nomenclature and concept of "Romanowsky stains" is followed by a brief account of the dyes involved and especially the crucial role of azure B and of the impurity of most commercial dye lots. Technical features of standardized and traditional Romanowsky stains are outlined, e.g., number and ratio of the acidic and basic dyes used, solvent effects, staining times, and fixation effects. The peculiar advantages of Romanowsky staining are noted, namely, the polychromasia achieved in a technically simple manner with the potential for stain intensification of "the color purple." Accounts are provided of a variety of physicochemically relevant topics, namely, acidic and basic dyeing, peculiarities of acidic and basic dye mixtures, consequences of differential staining rates of different cell and tissue components and of different dyes, the chemical significance of "the color purple," the substrate selectivity for purple color formation and its intensification in situ due to a template effect, effects of resin embedding and prior fixation. Based on these physicochemical phenomena, mechanisms for the various Romanowsky staining applications are outlined including for blood, marrow and cytological smears; G-bands of chromosomes; microorganisms and other single-cell entities; and paraffin and resin tissue sections. The common factors involved in these specific mechanisms are pulled together to generate a "universal" generic mechanism for these stains. Certain generic problems of Romanowsky stains are discussed including the instability of solutions of acidic dye-basic dye mixtures, the inherent heterogeneity of polychrome methylene blue, and the resulting problems of standardization. Finally, a rational trouble-shooting scheme is appended.

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    • "Currently, application of RG stains is limited, especially for histopathology, owing to the erratic staining outcomes obtained by some workers. RG stains commonly are used in ways contrary to good practice so that tinctorial features and consistency are lost (Horobin 2011). Currently, there is no optimal protocol that ensures the technical and tinctorial benefi ts of RG staining as a counterstain for IHC procedures. "
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    ABSTRACT: We describe a detailed protocol for using Romanowsky-Giemsa (RG) counterstaining on formalin fixed, paraffin embedded tissue sections that are stained immunohistochemically (IHC) after antigen retrieval using hot acidic citrate buffer. RG staining is easy to perform and provides consistent results that are similar to hematoxylin and eosin (HE) staining. The counterstaining was applied after a variety of antibodies that used the DAB chromogen and the intensity of IHC stained structures was preserved. Moreover, RG counterstaining provided finer cell detail than HE, methyl green or nuclear fast red. A detailed troubleshooting guide is provided for the RG staining protocol.
    Full-text · Article · May 2013 · Biotechnic & Histochemistry
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    ABSTRACT: Protocols for immunocytochemical staining (ICC) and in situ hybridization (ISH) of air-dried Diff-Quick or May-Grünwald Giemsa (MGG)-stained smears have been difficult to establish. An increasing need to be able to use prestained slides for ICC and ISH in specific cases led to this study, aiming at finding a robust protocol for both methods. The material consisted of MGG- and Diff-Quick-stained smears. After diagnosis, one to two diagnostic smears were stored in the department. Any additional smear(s) containing diagnostic material were used for this study. The majority were fine needle aspirates (FNAC) from the breast, comprising materials from fibroadenomas, fibrocystic disease, and carcinomas. A few were metastatic lesions (carcinomas and malignant melanomas). There were 64 prestained smears. Ten smears were Diff-Quick stained, and 54 were MGG stained. The antibodies used for testing ICC were Ki-67, ER, and PgR, CK MNF116 (pancytokeratin) and E-cadherin. HER-2 Dual SISH was used to test ISH. Citrate, TRS, and TE buffers at pH6 and pH9 were tested, as well as, different heating times, microwave powers and antibody concentrations. The ICC was done on the Dako Autostainer (Dako(®), Glostrup, Denmark), and HER-2 Dual SISH was done on the Ventana XT-machine (Ventana / Roche(®) , Strasbourg, France). Optimal results were obtained with the TE buffer at pH 9, for both ICC and ISH. Antibody concentrations generally had to be higher than in the immunohistochemistry (IHC). The optimal microwave heat treatment included an initial high power boiling followed by low power boiling. No post fixation was necessary for ICC, whereas, 20 minutes post fixation in formalin (4%) was necessary for ISH. Microwave heat treatment, with initial boiling at high power followed by boiling at low power and TE buffer at pH 9 were the key steps in the procedure. Antibody concentrations has to be adapted for each ICC marker. Post fixation in formalin is necessary for ISH.
    Full-text · Article · Mar 2012 · CytoJournal

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