J Clin Pathol: first published as 10.1136/jcp.28.8.680 on 1 August 1975. Downloaded from J. clin. Path., 1975, 28, 680-685 An evaluation of some commercial Romanowsky stains P. N. MARSHALL, S. A. BENTLEY, AND S. M. LEWIS From the Department ofHaematology, Royal Postgraduate Medical School, Hammersmith Hospital, Du Cane Road, London W12 OHS SYNOPSIS The staining properties of 43 commercial Romanowsky-type stains have been studied. Considerable differences in the appearance of stained blood films were observed with different batches of these stains, the staining of red cells being particularly variable. Attempts have been made to correlate staining patterns with stain composition as revealed by thin-layer chromato- graphy and sulphated ash analyses. In this way it has been possible to define some essential require- ments for satisfactory staining. Romanowsky-type stain variants such as those of were made by the National Physical Laboratory, Giemsa, Jenner, Leishman, and Wright are routinely Teddington, Middlesex. employed for the coloration of blood and bone- copyright. marrow films. All these stains contain a mixture of STAINING METHODS methylene blue and other closely related thiazine Specimens of venous blood from three subjects dyes and eosin. The variations in the staining proper- were collected into EDTA-K2 anticoagulant (1-5 ties of these different stain variants are well docu- mg/ml of blood). One of the subjects was haema- mented (Baker, 1970; Baker et al, 1966; Dacie and tologically normal, one had iron deficiency anaemia, Lewis, 1968; Lillie, 1969) as are the variations and the third chronic granulocytic leukaemia. Thin between different batches of stain which are nomin- films were made shortly after blood collection. They http://jcp.bmj.com/ ally of the same type (Cramer et al, 1973; Lillie, were dried in air and were fixed in methanol in 1944; Lillie and Roe, 1942; Price, 1968; Scott and accordance with standard practice (Dacie and Lewis, French, 1924). Staining variability is particularly 1968). Throughout this work only microscope slides troublesome when the needs of a routine depart- from a single manufacturer (Chance Propper Ltd, ment and the use of automatic staining equipment Smethwick, Warley) were employed since the call for a reliable, standardized procedure. staining of films may be affected by variations in In this study we describe the variation observed the quality of the glass on which they are spread on September 25, 2021 by guest. Protected in the coloration of blood films which have been (Scott and French, 1924). Films were stained with stained with a selection of commercially available the batches of Diff-Quik, Giemsa, Jenner, Leishman, Romanowsky-type stains. By correlating staining May-Grunwald, Romanowsky, and Wright stains performance with the chemical composition of the indicated in table I. Jenner and May-Grunwald stains, it has proved possible to define the character- stains were used in combination with Giemsa stain. istics of some successful commercial stains. In these combination stains, a single batch of Giemsa stain (R. A. Lamb, 3025). was used. All Materials and Methods staining was performed 'on slide' by the techniques described below. In all cases the aqueous buffer INVESTIGATION OF STAIN COMPOSITION employed was 0-066 M S6rensen's buffer, pH 6-8 The dye components of Romanowsky-type stains diluted before use 1: 20 with water (Dacie and Lewis, were separated by thin-layer chromatography. The 1968). method of Marshall and Lewis (1974a)wasemployed. Sulphated ash determinations on certain of the stains Diff-Quik Stain which are commercially available in powder form Films were fixed in methanol, stained for 15 sec in Received for publication 10 February 1975. solution I, followed by 15 sec in solution II. 680 J Clin Pathol: first published as 10.1136/jcp.28.8.680 on 1 August 1975. Downloaded from An evaluation ofsome commercial Romanowsky stains 681 Giemsa Stain One of the objectives of this study was to assess The commercial solution or a stock solution of the suitability of the Romanowsky-type stains 5 g/l stain powder in a mixture of glycerol (2 volumes) which are commercially available at the present time and methanol (3 volumes) was diluted 10 times with for the routine staining of blood films. In this buffer. Methanol-fixed films were stained in this assessment, the generally adopted staining pro- solution for 15 min. cedures were carefully standardized, but no attempt was made to adjust staining conditions to yield op- Jenner-Giemsa Stain timum results, since this is not practicable in busy, Methanol-fixed films were stained for 5 min in a routine laboratories. There is a generally accepted solution comprising the commercial solution or a scheme ofstaining which is expected ofRomanowsky 3 g/l stock methanolic solution of Jenner stain stained preparations, viz purple chromatin, blue powder (1 volume )and buffer (1 volume). Films were leucocyte cytoplasms, purple-black basophil then stained with Giemsa stain as already described. granules, red-pink eosinophil granules, purple neutrophil granules, purple platelet granules, and Leishman Stain pink red-cells. Based on this scheme, stained films Films were stained for 2 min with the commercial have been examined and the stains assessed as use- solution or a 15 g/l methanolic solution of the less, very poor, poor, fair, good or excellent (table I). commercial powder. This solution was then diluted These subjective ratings indicate the suitability of with twice its volume of buffer and allowed to act the stains for routine diagnostic purposes. Lowly for a further 10 min. rated stains are those which stain some cell compon- May-Grunwald-Giemsa Stain ents adequately but leave others unstained or stained Methanol-fixed films were stained for 5 min in the colours differing considerably from the accepted commercial solution or in a 3 g/l methanolic solution ones. It can be seen that staining properties varied of the May-Grunwald stain powder. Films were considerably in stains obtained from different suppliers. Indeed, this variability cannot be reduced then stained with Giemsa stain as already described. copyright. by dealing with a single supplier. It is unfortunate Romanowsky Stain that supply houses have done little to improve the Methanol-fixed films were stained for 5 min in the situation which has long been a cause of concern commercial solution diluted with an equal volume (Lillie, 1944; Lillie and Roe, 1942; Scott and of buffer. French, 1924). An attempt has been made to correlate staining properties with stain composition. In this section Wright Stain http://jcp.bmj.com/ Methanol-fixed films were stained for 5 min in a only the single stain procedures are discussed since solution comprising a 3 g/l methanolic solution of the results obtained with combination stains are the stain powder or the commercial solution (1 considered to be too complex for interpretation. volume), and buffer (2 volumes). Using those staining procedures described above, it was noted that the simplest stain of those yielding After staining, slides were differentiated for 4 min good or excellent results (Giemsa, BDH 685704/ in buffer, drained, air-dried, and mounted in Diatex 560113) contained dyes of Rfs 0 059 (methylene (R. A. Lamb Ltd). blue), 0-11 (azure B) and 0-60 (eosin), all as major on September 25, 2021 by guest. Protected components. Other relatively simple stains of this Results group (Giemsa, G. T. Gurr 06707; Leishman, E. Gurr, no batch No.; Romanowsky, R. A. Lamb The staining properties of the commercial stains, 2627) contained, in addition, a dye of Rf 0-72 together with their dye components, are presented in (tribromofluorescein) and in two instances one of table I. Sulphated ash analyses are given in table II. Rf 0 79 (fluorescein). However, the majority of successful stains contained all the following dyes, at Discussion least some of which were in quantities greater than traces: Rf 0-059 (methylene blue), 0-11 (azure B), A standardized Romanowsky-type stain is highly 0-15 (a dye chromatographically indistinguishable desirable not only to ensure consistently good from toluidine blue but whose identity is unknown), staining, which is the essence of morphological 0-19 (azure A), 0-24 (sym.-dimethylthionine), 0-60 diagnosis, but also to facilitate the exchange of (eosin), and 0-72 (tribromofluorescein). It thus material between laboratories. The advent of auto- appears that the components of Rfs 0-15, 0-19, matic cell recognition systems makes such a stain 0-24, and 0-72 are unessential for, although not essential. detrimental to, successful staining. In this study, we J Clin Pathol: first published as 10.1136/jcp.28.8.680 on 1 August 1975. Downloaded from 682 P. N. Marshall, S. A. Bentley, and S. M. Lewis Stain and Batch No. Components Present' 0-059 0-11 015 0-19 0-24 0-25 0-36 047 0-59 0-60 0-72 0-79 'Diff-Quik' I Harleco 3058 P Solution I 0 0 2 Solution It O 0 0 0 0 Giemsa I BDH 685704/560113 * *. 2 Difco 1169 O O J 0 3 Gurr 1324 O O 0 0 0 0 0 4 E. Gurr-no batch No. *0 0O 0 5 G. T. Gurr 06707 * * S 0 6 G. T. Gurr 17869 O 0 0 0 0 0 0 7 Hopkin and Williams 013954 * O O O O 0 O 0 8 R. A. Lamb 2670 * @0 0 0 0 9 R. A. Lamb 3025 0 0 0 0 0 01: 10 R. A. Lamb 3328 0 0 0 0 0 0 0 0 11 Merck 416168 * 0 0 0 0 * Jenner3 1 Difco 0402 0 0 0 0 0 0 2 E. Gurr-no batch No. * 0 0 0 3 G.