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Staining Properties and Stability Ofa Standardised Romanowsky Stain

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Staining Properties and Stability Ofa Standardised Romanowsky Stain J Clin Pathol: first published as 10.1136/jcp.31.3.280 on 1 March 1978. Downloaded from Journal of Clinical Pathology, 1978, 31, 280-282 Staining properties and stability of a standardised Romanowsky stain P. N. MARSHALL', S. A. BENTLEY2, AND S. M. LEWIS From the Department of Haematology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS, UK SUMMARY An evaluation of the standardized Romanowsky stain of Marshall et al. has been made in a routine haematology laboratory. It was noted that this stain had several advantages over the May-GrUnwald Giemsa stain used in most British laboratories. These advantages include ease and speed of preparation, a shorter staining time, and reproducibility of results. These results are described in detail. The stability of the stock stain solution and of the 'working' stain (stock + buffer) has been studied by, respectively, thin-layer chromatography and visible spectroscopy. No change was detected in the composition of the stock solution at ambient temperature over a period of six months. Stability was unaffected by the composition of the container (polyethylene, PyrexTM, or soda-glass) or by daylight. The 'working' solution was stable for 3 hours. Thereafter a precipitate is formed, consisting of thiazine dyes and eosin in a molar ratio of -2: 1. copyright. The major problem in the use of Romanowsky Material and methods stains is batch-to-batch variation in their staining performance. This is a potential source of diagnostic PREPARATION OF STAIN AND STAINING error. Adoption of a reliable, standardized stain TECHNIQUE would not only eliminate such error but would also Dyes were purified, as described previously (Marshall ensure comparability of stained films from different et al., 1975c; Marshall and Lewis, 1975b). The stock laboratories and facilitate reference to illustrative solution of stain was prepared and used as described http://jcp.bmj.com/ atlases. Moreover, such a stain is essential for use by Marshall et al. (1975c). with automatic cell recognition systems. Variability Stained preparations were made from most of the in performance is usually due to variation in stain blood and bone-marrow samples arriving in our composition (Scott and French, 1924; Lillie and Roe, laboratory during one working week, approximately 1942; Lillie, 1943a, 1944a; Saal, 1964; Clemens and 1000 specimens in all. Duplicate preparations were Toepfer, 1968; Toepfer, 1972; Marshall and Lewis, made by our routine May-Grunwald Giemsa (MGG) 1974b, 1975a; Lohr et al., 1975; Marshall et al., technique (Dacie and Lewis, 1975). on September 27, 2021 by guest. Protected 1975a,b,c; Wittekind and Lohr, 1975; Wittekind et al., 1976). We have therefore developed a stain pre- STABILITY OF STAIN pared from pure dyes, which is of constant composi- The stock solution was stored at ambient tempera- tion and performance (Marshall et al., 1975c). Since ture (- 21QC) in polyethylene, PyrexT., and soda- our original report, in which the staining properties glass containers, in darkness and in daylight. All of different cell types were illustrated, we have con- containers were tightly stoppered to prevent evapora- tinued to study the stain's properties. In this paper tion but were only partially filled to allow contact of we describe its performance in routine use, list its the stain with atmospheric oxygen. Aliquots of staining properties, and provide data on its stability. stain were withdrawn at intervals over a period of six months, diluted with methanol, and analysed by 'Present address and address for correspondence: Coulter (Marshall and Lewis, Biomedical Research, 40 Bear Hill Road, Waltham, thin-layer chromatography MA 02154, USA. 1974a). The stability of the 'working' stain (stock + 2Present address: Room 4D22, Building 36, National buffer) was investigated as follows: samples were Institutes of Health, Bethesda, MD 20014, USA. prepared and aliquots withdrawn at intervals of up to eight hours. Where necessary, these were centri- Received for publication 25 July 1977 fuged to remove precipitate, the supernatant was 280 J Clin Pathol: first published as 10.1136/jcp.31.3.280 on 1 March 1978. Downloaded from Staining properties and stability ofa standardised Romanowsky stain 281 diluted with excess methanol, and visible absorption thiazine dyes underwent no oxidative demethylation spectra were made using a Unicam SP 800 Recording even though solutions were in contact with atmos- Spectrophotometer. pheric oxygen. There are few comparable data on other Romanowsky stains. Lillie (1944b) measured Results and discussion the changes in the visible absorption spectra of methylene blue-eosinate and Wright's stain with During the test period, peripheral blood and bone- time. The stains were found to be highly stable in marrow films were examined from patients with a glycerol-methanol (1:1 v/v), and this solvent mixture wide variety of disorders. The staining appearances was therefore selected for our stain. Stock solutions, of the vast majority of normal and pathological cell which are totally methanol-based, are much less types were thus seen. With the standardised stain red stable (Lillie, 1944b; Dean etaL., 1977). These include cells stained a clear pink, in contrast to a brownish the stains of Jenner, Leishman, May-Grunwald, and hue in our MGG preparations. The transition from the more recent stain of Wittekind et al. (1976). basophilic through polychromatic to orthochromatic Our 'working' solution proved stable in use for erythroblast was clearly demonstrated, as was the three hours. Thereafter, the formation of a precipi- presence of red cell inclusions such as Howell-Jolly tate made it necessary to prepare a fresh solution. bodies and siderotic granules. Malarial parasites Stain precipitation was investigated quantitatively by showed characteristic Romanowsky staining visible spectroscopy. Solutions of the 'working' reactions. stain diluted with methanol showed two absorption In white cells, the specific granules of neutrophils, maxima, one at 642 nm due to the thiazine dyes eosinophils, and basophils were well stained, as were (methylene blue and azure B), and another at 525 the azurophil granules of lymphocytes and mono- nm due to eosin. Thus, by measuring the reduction cytes, the primary granules of promyelocytes, and in the absorbances at these wavelengths, it was platelet granules. Toxic granulation of neutrophils possible to follow the amount and composition of was shown clearly. The stain gave an excellent the precipitate formed. It was found that the solution range of colouration in the mononuclear cells; cyto- was stable for three hours (Figure). After this, signi- copyright. plasm was grey in monocytes, a clear pale blue in ficant amounts of precipitate were formed, the rate lymphocytes, a deeper blue in blast cells and atypical of formation being greatest three to four hours after mononuclear cells, and a rich dark blue in plasma preparation. Precipitation ceased at eight hours, cells and proerythroblasts. Nuclear chromatin pat- when the concentration of eosin was effectively zero. terns were well stained and nucleoli were very clearly The composition of this precipitate was deduced as demonstrated. follows: the 'working' solution contains thiazine The appearances observed were thus in all respects dyes and eosin in the ratio of 2:1 by weight. Taking http://jcp.bmj.com/ those of an excellent Romanowsky stain, and the the molecular weight of the thiazine dyes as 350 (the results were comparable with the best MGG pre- molecular weight of the predominant dye, azure B parations. The latter will, of course, vary with the hydrobromide-that of methylene blue hydro- batches of stain used (see, for example, Marshall et chloride is not significantly different) and that of al. (1975a) and Toepfer (1972)) whereas our stain is of eosin (colour acid) as 648 (ignoring the small pro- constant composition and therefore gives consistent portion of 2',4',5'-tribromofluorescein), we see that results. in the freshly prepared 'working' solution the cationic on September 27, 2021 by guest. Protected Analysis of the stock solution of our stain showed and anionic dyes are present in the molar ratio of that its composition remained unchanged in the six- about 3f7:1. By eight hours the concentration of the month period under all conditions of storage. The cationic dyes in solution has been halved, whereas the 'U U100--o 0-0 c \0-F 100 0- -0 2 80- Figure Stability of 'working' ax solution of the stain: (left) 70\ change of absorbance at 642 _ o o O nm (thiazine dye absorption 6J o 0 40- 0_0 maximum) with time; (right) 5-s 20I change ofabsorbance at 525 0 0 nm (eosin absorption s040- >3 4 5 6 7 8 maximum) with time. TIME J Clin Pathol: first published as 10.1136/jcp.31.3.280 on 1 March 1978. Downloaded from 282 P. N. Marshall, S. A. Bentley, and S. M. Lewis concentration of eosin has fallen to virtually zero. violet. Journal ofLaboratory and Clinical Medicine, 29, Therefore, the molar ratio of thiazine dyes:eosin in 1181-1197. the precipitate is about 2:1. Similar studies on the Lillie, R. D. (1944b). The deterioration of Romanowsky 'working' solutions of commercially available stains stain solutions in various organic solvents. Public indicate the formation of similar precipitates, the Health Reports (Washington), Supplement, 178, 1-16. Lillie, R. D., and Roe, M. A. (1942). Studies on poly- rate of formation again being greatest three to four chrome methylene blue I. Eosinates, their spectra and hours after dilution of the stock. staining capacity. Stain Technology, 17, 57-63. In practical terms, the standardised stain has Lohr, W., Grubhofer, N., Sohmer, I., and Wittekind, D. several advantages in the routine laboratory. In (1975). The azure dyes: their purification and physico- addition to its stability, no lengthy process of dis- chemical properties. II. Purification of azure B. Stain solution of dyes and ageing of solutions is necessary. Technology, 50, 149-156.
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