J Clin Pathol 1988;41:786-792

Laboratory techniquesUse of dark ground microscopy in haematologyA S TODD, W K BARNETSON Department ofPathology, University ofDundee, Scotland

SUMMARY The novel combination of vital staining, quick drying of thin films, and dark groundmicroscopy furnishes new information about organelles, granules, and precipitates in blood cells. Thepreparations are permanent and give optical signals of high contrast which depend on refraction ordichroism of the object. Mitochondria are readily shown and can be counted and sized. Pilotobservations suggest that mitochondria in red cells may give information about cell maturity andsplenic function; that the size oflymphocyte mitochondria may reflect the state of activation ofthosecells; and that the sensitivity of unstable haemoglobin detection may be greatly enhanced. Themethod is unsuitable for the study of granulocyte mitochondria.

Dark ground microscopy has been little used inhaematology and rarely on dried material. Its use forthe examination of reticulocyte preparations' gavesuch striking effects that we were led to explore otherapplications in haematology.Dark ground microscopy differs from the bright-

field method in that the refractile properties of theobject are exploited rather than suppressed (by"clearing"); contrast is higher; dyed objects show acolour complementary to that seen under brightfield.

In this paper we report the dark ground demonstra-tion of the following: (in unstained preparations)malarial parasites; red cell inclusions; neutrophil andeosinophil granulocytes; iron granules; melanin.

(In vitally stained preparations) mitochondria;Heinz bodies; unstable haemoglobin precipitates;platelet granules; basophil granulocytes; trypano-somes; reticulocytes.

(In fixed, stained material) punctate basophilism2;iron granules.

Material and methods

Blood was obtained from samples from normalvolunteers, or was taken from the residue of clinicalsamples submitted for "routine" blood counts. In allcases the anticoagulant was ethylenediaminetetra-acetic acid (edetic acid) dipotassium salt. Pilotexperiments showed that heparinised samples gavesimilar results.

Seminal fluid was from samples submitted forfertility testing.

Accepted for publication 9 February 1988

Dyes used were Janus green B, colour index (CI) No11050 from GT Gurr, specified "for mitochondrialstaining", as an aqueous solution of 01% w/v;acridine orange (basic orange 14, CI No 46005) as a0-1% w/v aqueous solution for vital staining, and as a0-3% solution in citrate-saline for stainingreticulocytes; brilliant cresyl blue CI No 51010, (BDHPoole, Dorset) 0 5% in ethanol (for reticulocytes); 1%in citrate saline, for unstable haemoglobin precipita-tion and staining.

Stains used were modified Wright's stain (MerciaDiagnostics); eosin BDH Poole Dorset. 1-0%aqueous; citrate-saline (trisodium citrate 3-8% inwater 1 part; sodium chloride 0-85% in water 4 parts).

IMMERSION AND MOUNTING MEDIAA set of Cargille Index of refraction liquids (RPCargille Laboratories, Cedar Grove, New Jersey,USA) was used to study refraction effects. These wereapplied under a No 1 coverslip to prevent mixing withthe immersion oil.To conserv- the expensive reference fluids, a

medium of approximately defined refractive index('n') was made by mixing dimethyl phthalate(n = 1-514 to 1-516) with dibutyl phthalate(n = 1 492 to 1-494), or with l-Bromo-naphthalene(n = 1-6583 (BDH, Poole, Dorset). The resultant 'n'can be calculated from the formula:

nm X vm = n, X V2 + n2 X V2where v,.2 and n,a2 are the volume and refractive index,respectively, of the components, and vm and nm of themixture.

For permanent preparations either BPS3 or EuperalVert (Difco) were used.

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Use ofdark ground microscopy in haematology

STAINING METHODSVital (non-lethal) staining was obtained with acridineorange and Janus green B: 5.pl of 0-1% solution ofdyeis added to 0 5 ml ofwhole blood, mixed, and allowedto react for 20 to 60 minutes, then prepared formicroscopy as films or buffy coat smears. Acridineorange may be combined with Janus green B as acounter stain, 5 p1 of each being added to 0 5 ml ofblood.

Post-vital (lethal) staining was obtained withbrilliant cresyl blue (for reticulocytes): 4 drops of a0 5% solution of the dye in ethanol are dried in a 10mm diameter plastic tube. Four drops of whole bloodare added, left to stain for 10 minutes, and thensmeared as thin films.

Brilliant cresyl blue was used for unstablehaemoglobin precipitation and staining4: 5 drops ofwhole blood are added to an equal volume of a 1%solution of the dye in citrate-saline and allowed tostain for either two hours at 37°C or overnight at roomtemperature. Films are then made in the usual way.

Acridine orange was used for reticulocytes: onedrop of 0-3% solution is mixed on a slide with onedrop ofblood and after 20 seconds smeared and dried.

Wright's stain for blood films was applied using theAmes Haematek staining machine. Perls's stain wasused for iron. The film was fixed in methanol for 30minutes, air dried, stained in: 4% hydrochloric acid (1volume) 4% potassium ferrocyanide (5 volumes) for10 minutes; rinsed in 1% lithium carbonate and indistilled water; counterstained in 1% aqueous eosin;washed and dried.Thin blood films were made in the usual way.

MICROSCOPYThe smears were either unmounted, using Leitzimmersion oil (n = 1-518) or mounted in one of avariety of media of known 'n' separated from theimmersion oil by a No 1 coverslip. Oil immersionobjectives (Zeiss x 40 planapochromat NumericalAperture (NA) 10 with iris diaphragm; Reichertplanapochromat x 100 NA 1-25 with iris diaphragm)were used, mounted on a Reichert Zetopan IImicroscope using Tungsten illumination and an oilimmersion dark ground condenser NA 1-42.A Leitz Ortholux II microscope with Ploem

illuminator equipped for Rhodamine fluorescence wasused.

APPLICATION OF IMMERSION/MOUNTING MEDIAAs dark ground microscopy is very sensitive todiscontinuities in refractive index it is important toavoid air bubbles in the medium. Usually this can beachieved by wetting the smear with xylene beforeapplying mountant or oil to the slide. The xylene seemsto cause no optical disturbance.

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Results

UNSTAINED MATERIALPlasmodiaThin blood films containing Plasmodium vivax,Plasmodium malariae, and Plasmodium falciparumwere examined. Parasites showed as a cluster of highlyrefractile slightly bluish granules (fig 1). Bright fieldillumination showed that the granules were malarialpigment. Plasmodium vivax gave the weakest signal,although this may depend on the maturity of thetrophozoites and hence the pigment content. Somedistinguishing features of the type of plasmodia are:pigment granules in trophozoites of P vivax are finerthan in other forms; the band form of trophozoite inP malariae may be recognised; applique forms ofPfalciparum are well shown.

In Giemsa stained "thick films" gametocytepigment shows as clumps of brightly refractilegranules which permits rapid screening, withverification under bright field illumination. Beck et alhave shown similar effects during immunofluorescencestudies on malaria.5Another pigment, melanin, has an identical

appearance under dark ground illumination (fig 4).Some of the leucocytes can be identified in the

unstained state. Neutrophil granulocytes are packedwith tiny refractile granules of a slightly greenish hue,the nuclear lobes showing as negative staining amongthe granules. Eosinophils have coarser, highlyrefractile, slightly yellow, hollow granules.Dark ground examination of unstained films can

show inclusions in red cells. In films from cases ofhyposplenism, of severe alcoholism, and frompremature infants a variety of droplets, granules, andincrustations with iron can be seen (fig 3). Irongranules are very refractile and have pronouncedgreenish-blue colour. This identification was

Fig 1 Lymphocytefrom a case ofglandularfever showingring-like structure ofmitochondria. (Janus Green B, darkground.)

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Fig 2 rig 3

Fig 4 Fig 5

Fig6 Fig7

Fig 2 Bandform ofPlasmodium malariae, (Unstained,dark ground.)

Fig 3 Red cellsfrom chronic alcoholic showing inclusionsand vacuoles. (Unstained, dark ground.)

Fig 4 Melanin granules in monocyte. Buffy coatfrom caseofmelanocarcinoma. (Wright's stain, dark ground.)Fig 5 Mitochondria in platelets. Non-stainingferruginousgranules in red cells. (Janus green B vital, dark ground.)

Fig 6 Monocytes (bottom left, top right and lymphocyte(centre (showing yellow mitochondria. Polymorphs (leftand centre) showing mostly non-staining granules. (Janusgreen B, dark ground.)

Fig 7 Neutropil showing rod-like mitochondria: red cellswith solitary and multiple mitochondria. (Janus Green B,dark ground.)

confirmed by subsequent staining of marked fields byPerls's stain and examination under brightfieldillumination. Under dark ground, Prussian bluegranules are bright red against the vivid green of theeosin stained red cells.

VITALLY STAINED BLOODJanus green BWith this dye, mitochrondria are seen as brightorange-yellow granules which, at the highest mag-

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Use ofdark ground microscopy in haematologynification, appear complex, consisting ofsignet-ring orbracelet-like groups of smaller components (fig 4).Granule-containing cells were identified by markingor charting fields under dark ground then staining thesmears with Wright's stain for brightfield examina-tion, or by mounting the smear in medium of slightlylower refractive index (n) to show the plasma andnuclear membranes of the cell. Euperal Vert(n = 1.47) is satisfactory and is a permanent moun-

tant.The granules are normally seen in a few red cells

(less than 1 0%), in most platelets (fig 5), in two thirdsofmonocytes, in nearly all lymphocytes, in about 10%of neutrophils (fig 7), and rarely in eosinophils. Theirsize varies from 0 3 pm to I 0 pm. They are largest inlymphocytes, especially activated forms, and smallestin monocytes and platelets. Monocytes contain thelargest number of Janus green staining bodies (up to100), lymphocytes up to 25, neutrophils up to seven,

platelets from two to 25, eosinophils one or two. Ingranulocytes the Janus green bodies are often"bacillary" in form (fig 7). Many more Janus green

bodies may be seen in neutrophils from cases showingneutrophil leucocytosis and toxic granulation.

In glandular fever, in samples showing many Turkcells, and in blood treated with phytohaemagglutininthe lymphocyte mitochondria seem larger and are

more obviously annular (fig 1). In blood from infantsJanus green staining oflymphocytes ranges from nil tothe intense concentration seen in Turk cells. In chroniclymphocytic leukaemia the granules are more

numerous (fig 8) and may be larger.Janus green staining granules may be seen in up to

1% of red cells in normal blood. The proportion ofsuch cells may be increased, both in cases showingreticulocytosis and when splenic function isdiminished or absent. By combining vital Janus greenstaining and post-vital acridine orange staining,mitochondria and reticulocytes can be studied in thesame preparation. Janus green is added to blood andafter 15 to 30 minutes at 20°C a drop of the mixture isstained for reticulocytes with acridine orange. Inaddition to the orange-yellow mitochondrial granules,such preparations show nuclei of a dark dull greencolour, and reticulocyte granules coloured a vividturquoise. Other highly refractile pale turquoisegranules are seen in all platelets and in some

lymphocytes, red cells, monocytes and polymorphs(fig 9). Using this combined method, up to one third ofreticulocytes show stained mitochondria, and nearlyall red cells with stained mitochondria containreticulocyte material, the exception being cases ofhyposplenism, severe alcoholism, and prematurity ininfants. In alcoholics as many as 17% of the red cellsmay have stained mitochondria unaccompanied byreticulocyte material-that is, in "mature" cells-

789refractile white or pale turquoise granules also seen inthe red cells in these cases look like defunct remnantsof mitochondria.When mouse blood containing trypanosomes

(T Brucei) was stained with Janus green in the usualway it showed intense staining of the kinetoplasts andless consistent, weaker staining ofother granules alongthe bodies of the parasites. These less intensely stainedgranules showed best when the preparation wasmounted in Euperal Vert (fig 10).The combination of vital staining with Janus green

B, quick drying, and dark ground microscopy can beapplied to cells cultured on coverslips. Fibroblasts,normal keratinocytes, and squamous carcinoma cells6were stained by adding Janus green B to thesupernatant culture medium to a final concentrationof 5 x 10-6 w/v. After 20 minutes the coverslipswere rinsed in balanced salt solution containing 2%bovine albumin (to minimise crystallisation of saltsduring drying). This method shows that mitochondriaare long beaded and thread-like in fibroblasts (fig 1 1),granular in keratinocytes, and apparently moreabundant in the malignant cells (fig 12). Theappearances are almost identical with those of cellsvitally stained with the fluorochrome laser dyeRhodamine 123, reported by Johnson et al.7When Janus green B is applied to seminal fluid in the

same way as to blood, about one third of thespermatozoa show granular staining of the mito-chondrial spiral. The association between staining andmotility has not yet been established.

Acridine orange (vital staining)This provides an excellent counterstain for mito-chondrial staining with Janus green B and can beapplied simultaneously. Cell granules show variousshades of bluish-green and the nuclei have a moreyellowish-green colour; basophil granules are brightlyrefractile and of an unsaturated turquoise colour.

Brilliant cresyl blue (post-vital)Blood stained for reticulocytes and viewed underdarkfield conditions shows the ribonuclease as brightapple green granules, with the usual reticular dis-tribution.' Heinz bodies formed "in vivo" have anorange tint; those made in vitro by exposure tophenylhydrazine have a strong red colour.

Brilliant cresyl blue is also used as the standard dyefor showing the presence of "unstable" haemoglobinin the red cells, by exposing the blood to the dye forabout two hours. The haemoglobin H (HbH) granulesfill the red cell and under dark ground are yellow to redin colour (fig 14). Even when scanty the affected cellsare easy to identify because of the high contrast of theimage. The staining of unstable haemoglobin fades ina few weeks unless mounted in BPS.

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Fig 8

Fig 10

Fig 14

Fig 8 Lymphocytes in chronic lymphocytic leukaemiawith densely packed mitochondria. (Janus green B, darkground.)

Fig 9 Red cells containing precipitated RNA (greenishblue) and mitochondria. (Janus green B, acridine orangepost-vital, dark ground.)

Fig 10 TBrucei in mouse blood showing kinetoplasts andmitochondrial granules. (Janus green B, dark ground.)

Fig. 11 Fibroblastfrom coverslip culture showing chainsofmitochondria. (Janus green B, dark ground.)

Fig 12 Squamous carcinoma cells in culture showingmany mitochondria. (Janus green B, dark ground.)

Fig 14 Red cells, ofwhich all but three are packed withprecipitated HbH. One (lower centre) also contains highlyrefractile reticulocyte granules. (Brilliant cresyl blue, post-vital, dark ground.)

Nizet used dyes other than cresyl blue for theidentification of reticulocytes under darkfield micro-scopy, and ofthese the best is acridine orange; stainingis almost instantaneous, and the colour (saturatedturquoise) is as vivid as when the same dye is used as afluorochrome.

Wright's stainUnder darkfield illumination Romanowsky stainedpreparations show enhanced detail of nuclear struc-

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Use ofdark ground microscopy in haematologyture and, by refraction, show cytoplasmic granulesthat are poorly visualised under brightfield (fig 2).Dark ground is especially useful for showing punctatebasophilism and was formerly used in screening forlead poisoning.2

Discussion

Dark ground microscopy has largely been neglectedin haematology, perhaps because it has the reputa-tion for being an exacting method, but the modern"research" microscope is usually equipped with amultipurpose substage system which avoids most ofthe difficulty in setting up. The staining techniques wedescribe are simple to apply, mostly requiring no morethan the addition of a few microlitres of dye to theblood sample.The colours displayed by the stained granules were

at first surprising, in view of the colour of the sameobjects seen in transmitted light. Reference to spectraldata for the dyes used8 shows that the absorptionpeaks for the dyes correspond to the colours seenunder darkfield conditions. This can be verified byplacing a calibrated graded interference filter in thenarrow light beam coming from the substage of themicroscope. By moving the filter to and fro, thewavelength of maximum granule brightness can beidentified and confirmed to be that of the spectralabsorption peak for the dye. It seems that the dyedgranule acts as a dichroic mirror, passing thosewavelengths we see in brightfield, reflecting those seenin the dark ground.The reflection colour is modified by admixture with

light transmitted through the granules and by reachingthe objective as a result of refraction. Thus the colourand contrast ofthe image can be modified by changingthe refractive index of the mounting medium whichchanges the amount of refracted light. Further varia-tion of the reflection signal seems to result from thenature of the surface to which the dye is attached. Forinstance, brilliant cresyl blue stains platelet granulesgreenish yellow and the released lipid dark red.

Until the introduction of electron microscopy forthe study of cells, the standard method of identifyingmitochondria was by vital staining with Janus green B9or Pinacyanol.'0 Since then the method has been littleused, probably because in the standard technique(using wet preparations under brightfield illumina-tion) contrast is low and the granules and cells are inconstant Brownian movement. The combination ofmethods described here gives images of high contrastfrom stable, permanent preparations. The ultra-structural studies of Tanaka" confirm that vitalstaining with Janus green B in low concentration isspecific for mitochondria. Electron micrographs oflymphocytes stained with Janus green B show

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Fig 13 Mitochondria in phytohaemagglutinin-stimulatedlymphocytes: (a) unstained. (b) after Janus green B. Notemyelinfigureformation and dye precipitation inmitochondria.

extensive damage to mitochondria (fig 13), suggestingthat the dye is more toxic than formerly supposed.'2Electron microscopy shows one major discrepancy;many more mitochondria are detected in granulocytesthan by the dye method. When the fluorochromeRhodamine 6G is used instead ofJanus green B, manymitochondria are seen in the granulocytes, whichagrees with the ultrastructural findings. For all otherblood cells, Janus green B, Rhodamine 6G, andelectron microscopy seem to give comparable results.The reason for the discrepancy may lie in the

variable plasma membrane permeability of thedifferent cell types for each dye used. Granulocytesfrom cases showing a toxic neutrophil leucocytosisand increased Janus green B staining, may haveplasma membranes that are more permeable by thedye.The other dye classically used for mitochondrial

staining is Pinacyanol. It gives vivid staining but has alow solubility in aqueous media and the colour fades.The use of dark ground illumination greatly

enhances the contrast of unstable haemoglobinprecipitated by brilliant cresyl blue. Experiments now

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in progress suggest that certain combinations of dyeconcentration, time, temperature and age of samplemay give false positive reactions. The increasedsensitivity of the dark ground method should permitbetter standardisation of this test.

We are grateful to Drs M J W Faed andW J Herbertfor samples and to Professor J S Beck and Dr H BGoodall for useful criticism. We thank Mr R Fawkesfor photographic help, Mr G Milne for electronmicroscopy and Mr D Clark for haemoglobinanalysis.

Referen

1 Nizet A. Recherches sur la physiopathologie des hematies. AciaMedScand 1944;117:1 19-215.

2 Albahary C. L'Hematie a granulations basophiles. Sang1953;24:539-46.

3 Kirkpatrick J, Lendrum AC. Further observations on the use ofsynthetic resins as a substitute for Canada balsam. J PatholBacteriol 1941;S3:441-3.

4 Lehmann H, Huntsman RG. Man's haemoglobins. Oxford: NorthHolland Publishing, 1974:391.

5 Beck JS, Logic AW, McGregor IA. Antigenic changes during thelife cycle of plasmodium falciparum. Experentia 1981 ;26:1365-6.

6 Kondo S, Aso K. Establishment of a cell line of human skinsquamous cell carcinoma in vitro. Br J Dermatol 1981;105:125-32.

7 Johnson LV, Walsh ML, Chen LB. Localisation of mitochondriain living cells with Rhodamine 123. Proc Natl Acad Sci USA1984;77:990-4.

8 Lillie RD. HJ Conn's biological stains. Baltimore: Wilkins andWilkins, 1969.

9 Michaelis L. Die vitale Farbung, eine Darstellungs methode derZellgranula. Arch Mikr Anat 1900;55:558-75.

10 Hetherington DC. Pinacyanol as a supra-vital microchondrialstain for blood. Stain Technol 1936;1 1 :153-4.

11 Tanaka Y. Deposition of Janus green B and pinacyanol inmitochondria of supravitally stained KB cells. Exp Cell Res1968;52:338.

12 Bessis M. Living blood cells and their ultrastructure. Berlin:Springer 1973:683.

Requests for reprints to: Dr A S Todd, ConsultantHaematologist, Ninewells Hospital and Medical School,PO Box 120, Dundee DDI 9SY, Scotland.

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