Technical methods

Technical methods

A standardized Romanowsky stainprepared from purified dyes

P. N. MARSHALL, S. A. BENTLEY, AND S. M. LEWISDepartment of Haematology, Royal PostgraduateMedical School, Hammersmith Hospital, Du CaneRoad, London W12 OHS

Stains consisting of combinations of various thiazinedyes with eosin, for example, those of Giemsa,Leishman, and Wright, are used routinely to studythe cellular morphology ofblood and bone marrow.The major problem in the use of these Romanowsky-type stains is batch-to-batch variations in theirstaining properties as a result of variations in theirdye composition and the presence of contaminatingmetal salts (Lillie, 1943 and 1944a; Lillie and Roe,1942; Marshall et al, 1975a; Saal, 1964; Scott andFrench, 1924).The extent of these variations in a large number

of commercially available Romanowsky-type stainswas demonstrated in a previous study (Marshallet al, 1975a). In this study it was found that thesimplest successful stain examined contained methy-lene blue, azure B, and eosin and was only mini-mally contaminated with metal salts. Accordingly,we have produced a stain from purified samples ofthese dyes.

Materials and Methods

PURIFICATION OF DYESMethylene blue and azure B were purified as des-cribed previously (Marshall and Lewis, 1975).Commercial samples of eosin (table) were freed ofcontaminating metal salts and volatile materialand converted to the free acid by the followingscheme:

(a) The commercial dye was dissolved at 10 g/lin 0-1 M aqueous ammonia. Any insoluble materialwas removed by filtration.

(b) 0-2 M aqueous hydrochloric acid was addedto this solution until precipitation ceased.

(c) The precipitate was removed by filtrationand washed several times with dilute aqueoushydrochloric acid (ca 0'0005 M).

Received for publication 19 May 1975,

Dye Supplier and Batch No. Sulphated Ash yielded %or Purchase Date By Dried After desalting

CommercialProduct

BASF 6698403 19-87 0-27BDH 145810 16 50 <0*05Eastman PE4 21-59 <0.05Edward Gurr Feb 1973 16-63 0-10R.A. Lamb0715 19-53 0-48R. A. Lamb 2621 18-18 <0 05

Table Sulphated ash analyses ofeosin samples

(d) Finally, the precipitate was dried at 135-140°Cfor approximately 4 hours.The purity of this free acid was confirmed by

sulphated ash analyses.

FORMULATION OF STAINPurified methylene blue, azure B, and eosin weremixed in varying proportions. In some mixtureseither methylene blue or azure B was omitted.The mixtures were dissolved in 1:1 v/v glycerol-methanol, in which the dyes are readily soluble andhighly stable (Lillie, 1944b). These solutions werediluted with varying proportions of Sorensen'sphosphate buffer, pH 6-8 or 7-2, 0-00132 M (availablefrom Mercia, Watford, Herts). Films prepared ongood quality microscope slides (Chance PropperLtd, Smethwick, Warley) were fixed in methanol,stained for varying times in these solutions, anddifferentiated in buffer pH 6-8 or 7-2. They were thenair-dried and mounted in a synthetic resin (Diatex,R. A. Lamb Ltd).The stained films were examined by an observer

who was unaware of the composition of the stainsused and they were compared with duplicate filmsstained with a traditional May-Grunwald-Giemsasequence as routinely used in this laboratory (Dacieand Lewis, 1968).

OPTIMAL STAINING PROCEDUREAfter testing a number of staining solutions andstaining times, the procedure presented below wasfound to give excellent results:

(a) The following quantities of the purified dyeswere mixed: methylene blue, 9 0 mg; azure B,17-0 mg; eosin, 13-0 mg. The mixture was dissolvedin 20 ml of 1:1 v/v glycerol-methanol by gentlestirring for a few minutes. (This stock solution

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Technical methods

4

.5

.'t. -U

Fig. 3

I.

Fig. S Fig. 6

Figs 1 to 6 Material stained using the procedure described in the text.

Figs 1 and 2 Photomicrographs ofa normal bloodfilm.Fig 3 Photomicrographs ofa bloodfilmfrom a case of fi-thalassaemia major showing anisochromasia, polychromasia,andpunctate basophil stippling.Fig 4 Photomicrograph ofa bloodfilmfrom a case ofacute myeloblastic leukaemia. The blast cellhasprominentnucleoliand contains an Auer rod.Figs 5 and 6 Photomicrographs of a bone marrow film from a case of megaloblastic anaemia showing erythroid andgranular precursors.

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Technical methods

may be stored for many months without alterationin its composition.)

(b) The solution was diluted with 100 ml ofSorensen's phosphate buffer, pH 6-8, 0-00132 M.

(c) Films of blood (collected into EDTA-K2anticoagulant, 15 mg/ml of blood) and aspiratedbone marrow were fixed for 5-10 minutes in meth-anol. They were stained by immersion in thediluted solution for 10 minutes.

(d) Stained films were differentiated for 1 minutein the pH 6-8 buffer, air-dried, and mounted.

Results and Discussion

Typical examples of the appearances of films stainedby the recommended technique are shown in figs 1 to6. These show the typical scheme of colourationproduced by successful Romanowsky-type stains.It should be noted, however, that the colour balanceand some of the more subtle features seen in thefilms, especially fine granulation, have been lostin thereproduction. Although the number ofcolouredcomponents in this stain is small (see figs 7 and 8)compared with that typically seen in commercialstains (Marshall et al, 1975a) the characteristic subtlevariation in colour was present.

Stains not containing methylene blue gave resultsapproaching a Romanowsky effect but lacking this

Zq SOLVENT FRONT

I Tribromofluores(ein

lEosin

Azure BMethylene blueORIGIN

Fig 7 Thin-layer chromatogram (prepared according toMarshallandLewis (1974)) ofthe Romanowsky staindescribed in the text. The major components are methy-lene blue (Rr 0059), azure B (Rf 0-11), and eosin (Rf 0 60).A trace oftribromofluorescein (Rf 0-72) is present.

) ci

(CH,),23(C,2

Br() BB(CH NH-CH

Brr

H- OOH

C.I. 52015

C.I. 52010

C.1.45380(free acid)

Fig 8 Structuralformulae ofthe dyes used to preparethe staining solution (a) methylene blue; (b) azure B;and (c) eosin (free acid).

subtle colour variation. The presence of methyleneblue enhanced nucleolar staining and red cellpolychromasia. In addition, it resulted in monocytecytoplasm being stained grey in contrast to the blue-grey colouration seen in its absence. Those stainsnot containing azure B stained chromatin blueinstead of purple.Replacement of the pH 6-8 buffer by one of pH

7*2 resulted in an orange-grey colouration of redcells. The staining of white cells was unaffected.Stains prepared from different batches of purifieddyes gave identical results. To date, about 200 filmsof normal and pathological blood and bone marrowhave been stained. On no occasion was any stainingvariation seen. This reproducibility obtained froma stain prepared from purified dyes represents adistinct advantage over that of currently availablecommercial stains (Marshall et al, 1975a). Anotheradvantage which makes the stain particularlysuitable for routine use is that the stock solution inglycerol-methanol is rapidly prepared and may beused immediately. It may be noted that the prep-aration of stock solutions of Giemsa, Jenner, andWright stains (Baker et al, 1966; Lillie, 1969)involves a lengthy solution and ageing process. Thestock solution of our new stain is extremely stable.Once diluted with buffer, the stain must be usedwithin a period of about 3 hours; diluted solutionsof the conventional Romanowsky-type stains showsimilar stability.The effectiveness of the procedure for desalting

eosin is illustrated in the table. Commercial samplesof eosin contain a small proportion of tribromo-

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fluorescein (Marshall et al, 1975b). This is notremoved by the desalting procedure, although itmay be removed chromatographically (Graichenand Molitor, 1959). However, we have found thatthis is unnecessary for the present staining procedure.Tribromofluorescein-free eosin and eosin deliber-ately contaminated with a much larger proportionof tribromofluorescein than is present in commercialsamples gave indistinguishable results.

This work was supported by a grant to S. M. Lewisfrom the Department of Health and Social Security.

References

Baker, F. J., Silverton, R. E., and Luckcock, E. D. (1966).An Introduction to Medical Laboratory Technology, 4thedition, p. 524. Butterworths, London.

Dacie, J. V. and Lewis, S. M. (1968). Practical Haematology,4th edition. Churchill, London.

Graichen, C. and Molitor, J. C. (1959). Studies on coal-tarcolors. XXII. 4,5-dibromofluorescein and related bromo-fluoresceins. J. Ass. off. agric. Chem., 42, 149-160.

Lillie, R. D. (1943). A Giemsa stain of quite constantcomposition and performance, made in the laboratory from

Modifications to improve thereliability of the Coulter 'S'P. F. J. NEWMAN, E. ROBERTSON, AND M. J. MUIRHaematology Department, Royal Infirmary,Edinburgh, Scotland

The Coulter Counter Model 'S' System (CoulterElectronics Ltd) measures automatically the haema-tological parameters haemoglobin concentration,white cell count, red cell count, and mean cellvolume and also computes from them the haemato-crit, mean cell haemoglobin, and mean cell haemo-globin concentration. The instrument can handleone sample every 20 seconds and can be both preciseand reliable (Barnard et al, 1969). However, in ourlaboratory the model 'S' is used to process up to 800samples per day, and we have found that theseconditions of virtually continuous operation exposelimitations in the design of the instrument with aconcomitant fall off in its reliability. We now de-scribe some of these limitations and suggest ways ofmodifying the machine to overcome them.

Received for publication 10 July 1975.

eosin and methylene blue. Publ. Hlth Rep. (Wash.), 58,449-452.

Lillie, R. D. (1944a). Factors influencing the Romanowskystaining of blood films and the role of methylene violet.J. Lab. clin. Med., 29, 1181-1197.

Lillie, R. D. (1944b). The deterioration of Romanowskystain solutions in various organic solvents. Publ. HlthRep. (Wash.), Suppl. 178, 1-16.

Lillie, R. D. (1969). H. J. Conn's Biological Stains, 8th edition,pp. 438-439. Williams and Wilkins, Baltimore.

Lillie, R. D. and Roe, M. A. (1942). Studies on polychromemethylene blue. I. Eosinates, their spectra and stainingcapacity. Stain Technol., 17, 57-63.

Marshall, P. N., Bentley, S. A., and Lewis, S. M. (1975a)An evaluation of some commercial Romanowsky stains.J. clin. Path., 28, 680-685.

Marshall, P. N., Bentley, S. A., and Lewis, S. M. (1975b).A procedure for assaying commercial samples of eosin.Stain Technol., 50, 107-113.

Marshall, P. N. and Lewis, S. M. (1974). A rapid thin-layerchromatographic system for Romanowsky blood stains.Stain Technol., 49, 235-240.

Marshall, P. N. and Lewis, S. M. (1975). The purification ofmethylene blue and azure B by solvent extraction andcrystallization. Stain Technol., 50, in press.

Saal, J. R. (1964). Giemsa stain for the diagnosis of bovinebabesiosis. I. Staining properties of commercial samplesand their component dyes. J. Protozool., 11, 573-582.

Scott, R. E. and French, R. W. (1924). Standardization ofbiological stains. Milit. Surg., 55, 229-243 and 337-352.

The Rinse and Drain Mechanisms

The most troublesome limitation exposed when theinstrument was run continuously was the failureof the aperture baths to drain completely. (Thishappened several times within a period of threemonths.) In the original version of the instrument,during each cycle waste fluid was drawn undervacuum from the aperture baths into the wastechamber and then discharged to waste under 0 35kgf/cm2 positive pressure. Owing to the low surfacetension of the effluent, caused by the lysing agent,and the turbulence created by the vacuum, there wasa tendency for excess foam to accumulate in thewaste chamber which then overflowed into the trapbottle and from there into the switching valve (SV)No. 6, which controls the vacuum in the pneumaticcard (L2). This caused the switching valve to becomeprogressively less efficient with the result that theaperture baths were sometimes only partiallydrained, and carry-over between successive sampleswas observed. The valve eventually failed com-pletely. The manufacturers then modified the instru-ment by fitting an anti-foam device (Coulter Altera-tion Number ECO-0367) which gave only partialimprovement; however the excess foam still over-

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