J. clin. Path., 1974, 27, 741-750

Separation of abnormal cells in the peripheralblood by means of the zonal centrifugePATRICIA M. EGAN' AND J. V. GARRETT with an appendix by C. W. GILBERT

From the Department ofPathology, Christie Hospital and Holt Radium Institute, and Paterson Laboratories,Manchester

SYNOPSIS The basic principles of rate centrifugation in the large volume zonal centrifuge and itsapplication to the separation of abnormal or rarely occurring cells in the blood are described.Details of the method, gradient media, and collection and examination of the fractions obtained aregiven. A description of the cells found in normal human blood, in a subject undergoing knownimmunological stimulus, and in glandular fever and Hodgkin's disease is given and the implicationsare discussed.

In recent years it has become possible by sophisti-cated centrifugation techniques, particularly byusing suspending media with a density gradient, toseparate particles according to their size and density.This has obvious applications in haematology andin particular the method was attractive as a possiblemeans of separating for further examination theabnormal cells often seen in buffy coat prepara-tions in various conditions, especially Hodgkin'sdisease (Crowther, Fairley, and Sewell, 1969;Bouroncle, 1966; Halie, Eibergen, and Nieweg,1972).

Basic Principles and Development of Method

The equations governing the sedimentation ofparticles in a liquid are given in the appendix. Theseequations show how the sedimentation velocity andhence the separation of particles depends on theirdensity relative to the suspending medium and parti-cularly on their diameter, since this occurs as asquared term. Much work on density gradient separ-ation of cells has been done in gradients made upin tubes either as a continuous gradient (Shortman,1968) or as a discontinuous gradient composed ofsteps each of decreasing density layered above oneanother (Dicke, Tridente, and Van Bekkum, 1969).

In order to overcome the many theoretical and

'Present address: Mathilda and Terence Kennedy Institute of Rheu-matology, Bute Gardens, Hammersmith, London W6 7DW.

Reprint requests to: Dr J. V. Garrett, Pathology Department, ChristieHospital, Manchester, M20 9BX.

Received for publication 30 May 1974.

practical limitations of tube gradients the zonalcentrifuge was developed (Anderson, 1966). Forcell separation this has the advantage that walleffects are eliminated and the large capacity of therotor, over 1 litre of gradient, enables considerablenumbers of cells to be used in the initial suspension.Conversely it has the disadvantage that the differentcell fractions have to be recovered from a largevolume of liquid. In this paper we are only con-cerned with the large volume type A slow speed,zonal rotor (Measuring & Scientific EquipmentLtd, Crawley, Sussex) on the MSE Mistral 6Lcentrifuge.The separation of cells in this apparatus is by the

rate zonal principle, that is to say, it is effected bythe rate of sedimentation of the cells according tothe differential sedimentation equation and does notdepend on the cells reaching different density equili-brium positions (Boone, Harell, and Bond, 1968).A refinement of the density gradient was describedby Noll (1967) in which the increasing force exertedon the particles as the result of the increasing radialdistance during centrifugation is counterbalancedby increasing the viscosity of the medium towardsthe periphery. Such a gradient is termed isokineticand allows maximum separation of the cells by therate zonal method.

If the relevant values are known they may besubstituted in the sedimentation formula and a smallcomputer programme developed to predict sedi-mentation results in different media by cells ofdifferent density and size. Such programmes ofgradient sedimentation conditions have been de-veloped by Pretlow and his coworkers (Pretlow and

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Boone, 1969; Pretlow, 1971; Pretlow and Luberoff,1973) and applied to isokinetic tube gradients. Thistype of calculation has now been extended for usewith the zonal rotor and the details are given in theappendix. By this means it is possible to predictthe radial positions reached by cells of stated sizeand density after a run at a specified speed for aspecified time in a gradient of known viscosity anddensity.

Materials and Methods

BLOOD SAMPLESTwenty ml peripheral blood was collected into ahepatinized container without preservative.

FICOLLFicoll (Pharmacia) was used to make up the con-tinuous density gradient. A stock solution, 20% w/v indistilled water, was prepared and treated at 4°C withde-ionizing resin AG501-X8D (BioRad Labs)(Gorczynski, Miller, and Phillips, 1970).

PREPARATION OF THE CELL SUSPENSIONFor examination of lymphocytes and abnormal cellsit was found desirable to remove phagocytic cellsand this was done by mixing the blood samplewith approximately one third its volume of 1%methyl cellulose (Dow) and incubating this withcarbonyl iron powder (G.A.F. Chemical Division,Manchester) 100 mg/20 ml blood (Coulson andChalmers, 1967). The tube was shaken gently byrepeated inversion on a slow rotator for 30 minutesat 37°C. The mixture was then placed over a magnetfor 15 minutes and the clumps of iron particles andmost of the phagocytic cells allowed to sediment.The lymphocyte-enriched supernatant was layeredonto a preliminary gradient of Ficoll-Triosil andspun at 400 g for 20 minutes (Harris and Ukaejiofo,1970).The lymphocytes were collected from the gradient

interface, washed once in minimal essential medium(MEM, see below) and resuspended in a 30 mlvolume of 1% Ficoll in MEM and the total numberof cells noted. The final cell suspension containedbetween 5 x 106 and 5 x 107 nucleated cells.

GRADIENT MEDIA AND CENTRIFUGATIONMETHODA 10 x concentrated solution of minimal essentialmedium was prepared from stocks of amino acids,vitamins, and glutamine (Flow Labs) in an Earle'sbase without calcium. The stock solution of Ficollwas diluted with the appropriate quantities of con-centrated MEM to make 11% and 5.64% heavyand light solutions, having densities between 1-039

and 1017, and an osmolarity of 300 milliosmoles.The density values of the solutions were altered insome experiments as indicated by the predictionformula.The medium was buffered at pH 7 2 with Hepes

(N-2-hydroxy-ethyl piperazine-N'-2-ethansulphonicacid, BDH Chemicals), at a final concentration of10 mM.A gradient, linear by volume, was generated from

the periphery in the rotor, cooled to 4°C and spin-ning at 300 rpm using the MSE gradient former andHIFLO Pump, 500 ml of light and 490 ml of heavysolution being mixed to provide 990 ml of gradient.One hundred ml of 150% Ficoll (density 1 35) wasadded at the end of the gradient to act as a cushionand prevent further cell sedimentation. This solu-tion was coloured by the addition of lissamine greenso that the position of the cushion was visible, whichwas useful to know when unloading the rotor. Thespace remaining was filled with 250% sucrose solu-tion.A 30 ml cell suspension was applied to the gradient

at the centre of the rotor and finally a further 30 mlof MEM overlay was added.The speed and time for each run was that pre-

viously predicted by the computer programme togive the maximum cell separation. At the end of therun the rotor speed was reduced to 300 rpm and thegradient pumped out by introducing 250% sucroseat the periphery.Twenty-two-ml fractions were collected, a small

sample of each was taken for cell count by theCoulter counter, and the refractive index of everythird fraction was measured to check the density.The remaining cell suspension was spun to con-

centrate the cells which were then washed and re-suspended in 2 ml 199 medium.

PREPARATION OF SLIDESThis cell suspension was again centrifuged and re-suspended in a sufficient volume of 199 + 15 % BSAto give a final concentration of approximately 1 x105 cells/ml. Samples of this suspension, each of0 2 ml, were used in the Shandon cytocentrifuge toprepare slides which were then stained by Leishman'sstain.The density of the gradient at any point can be

obtained from the refractive index as a linear rela-tionship exists between the refractive index of Ficollsolutions and their density (Bach and Brashler,1970).Figure 1 shows refractive index measurements

obtained from recovered gradient samples demon-strating the linearity of the gradient it is possibleto obtain over the entire length, deviations onlyoccurring at the two ends where mixture with MEM

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Separation ofabnormal cells in the peripheral blood by means of the zonal centrifuge

REFRACTINDEX1.3500

.3490

1.3480

1.3470

1.3460

3450

1 3440

1 3430

1.3420

1.3410

I unn

*IVE

Z9: REFRACTIVE INDEX/TUBE NUMBER No. /

/7

7el

A-~e7,,e

U0-.0

700"Oox1~I?...............4 8 12 1 20 24 28 32 36 40 4446

TUBE NUMBER

Fig 1 Refractive index measurements on recoveredgradient samples.

overlay or higher concentration Ficoll cushionoccurred.

Results

The possibilities of the method and the agreement ofthe actual and predicted sedimentations were ex-plored by using readily available cell lines. These

51 1.048 17.3^1

46 1.068 17.31Lzz 34 I 1.068 7.6f

41

z3/ /.Z 1.048 7.6#L31

211/x 70

1617&~~~~~

0 8 16 24 32 40 48 56 64 72MINUTES

Fig 2 Predicted sedimentation of cells from two celllines of densities 1P048 and 1P068 and diameters 7-6 j.mand 17 3 zm at 1400 rpm. Gradient density 1P024-1 042.

were a cell line LS from a mouse lymphoma L 5178Y and a line YR from a rat Yoshida sarcoma. TheLS cells have an average diameter of 7-6 ,tm and adensity of 1 048 to 1-068. The YR cells had an aver-age diameter of 17-3 ,tm and a density of 1-043 to

FRACTION NUMBERFig 3 Actual separation of cells from the two cell linesas shown by the percentage of each per fraction.

Si

46

41

cc 36E

z 31z0F 26

U.21

16

I.Q 5, 1.07 5v L67 0, 1.07Q,

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iX111 /

X1 X40 OKX

II#/// 71I1.z*I

Lo) -P//

a

0 8 16 24 32 40 48 56 64 n7 8UMINUTES

Fig 4 Predicted sedimentation of cells of density 107and 112, and diameters 8, 10, 12, 15 pm at 1000 rmp.Gradient density 1020-1038.

1.064. The diameters were obtained by eye-piecemicrometer measurements, the densities by iso-pycnic centrifugation in bovine albumin.The curves in fig 2 show the expected sedimenta-

tion in the gradient of Ficoll employed, and fig 3

.. . A

. . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . .

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Patricia M. Egan and J. V. Garrett

CELL COUNT140+f1200

1000

800

600

400

200

0

e.a~~~A.0. _. I

_ JI wV'-\.-.---BLANK4 8 12 16 20 24 28 32 36 40 44 48 50

TUBE NUMBER

Fig 5 Numerical distribution of various blood cells inzonalfractions (phagocytic cells removed).

shows the percentage of each type of cell recoveredin the fractions. It will be seen that there is reasonableagreement between the two. Thus after 10 minutes'centrifugation most of the 17-3 -m cells would beexpected to be in fraction 18 and higher and mostof the 7-6 ,um cells in fraction 11 or below. Thespread of cells is accounted for by the spread ofdensities.

Similar agreement with predicted results wasexperienced when blood cells were used and thedensities of lymphocytes and monocytes in Ficoll asdetermined by Pretlow and Luberoff (1973) used inthe sedimentation formula. From the curve obtainedfor the gradient usually employed (fig 4) it will beseen that there is an overlap to be expected of cellswith the same or similar diameter at slightlydiffering density. This was frequently observed,especially between fractions 18-26 as expected, andthere also often appeared to be a population ofsmall lymphocytes of rather greater density than themain population which was observed appearing inthe lower fractions mixed with large cells (see fig 22).

Figure 5 shows the usual pattern of distribution ofcell numbers among the fractions when whitecells from normal blood are treated. The peakaround fraction 12 is composed of small lymphocytesand relatively few cells are found in the lower tubes,but there is usually a small peak of larger cellsbetween fractions 30 and 40.

In blood the progressive purification from themixture of cells seen in the buffy layer preparationsfollowed in general the pattern shown in figures 6-9.

Results in Particular Conditions

NORMAL BLOOD (FIVE CASES)The first fractions contained platelets, if they hadnot already been removed by using defibrinatedblood, and the first lymphocytes to be recognizedwere a few of the very small lymphocytes sometimes

Fig 6 Buffy layer preparation peripheral blood ( x 400).

.,_V A.

Fig 7 Fraction 13. Almost all small lymphocytes( x 400).

.~~~~.........~I.

744

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4040-Ask.,

IF.Awk

14 ML.la

ik 'l;-

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Separation ofabnormal cells in the peripheral blood by means of the zonal centrifuge

SI

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M~~~ ....

. _@IV

:-.,.. ........

*.t...._

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Fig 9 Fraction 19. Plasmacytoid cells (normal blood)( x 400).

I

p._

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Fig 10 Fraction 22. Mostly larger basophilic cells(Hodgkin's disease) ( x 400).

Fig 11 Fraction 23. Transformed type cell in normalblood ( x 1000).

745

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seen in blood films, containing very little cyto-plasm around the dark staining nucleus and havinga diameter of approximately 96-11 ,um when ex-amined stained on the slide (compared with a redcell diameter of 7-9 ,um on the same slide).Next the fractions with the greatest number of

cells appeared and these were small lymphocytes,diameter 10 5-12 5 ,um (fig 7), with lower down anincreasing number of larger lymphocytes (fig 8)and then more basophilic or plasmacytoid cells(figs 9 and 10), but these were few in normal subjects.Lower down there may appear a few lymphocytesresembling mitogen transformed stimulated cellswith obvious nucleoli (fig 11) and finally, if theyhave not been removed from the original suspension,monocytes and polymorphs.

SUBJECTS WITH INFECTIONTwo cases with postoperative chest infectionsshowed a larger number of plasmacytoid and trans-formed type cells than normal.

GLANDULAR FEVER (THREE CASES)These cases were examined to see if the abnormalcells could be concentrated. As fractions furtherdown the gradient were examined cells of abnormalmorphological appearance were found. The orderin which the cells appeared was usually as follows:lymphocytes, large basophilic cells corresponding tothe characteristic cells seen in the blood; smallnumbers of cells with various kinds of irregularlyshaped nucleus or nucleus with a 'chunky' arrange-ment of the chromatin (fig 12); or cells with blast-like nuclei (fig 13).

HODGKIN S DISEASE (SIX CASES)Some of these, especially in the later stages withdisseminated disease, showed abnormal cells. Thesewere found in the denser fractions below the lym-phocytes and were large cells with basophiliccytoplasm and abnormal, irregularly shaped nuclei,usually with an abnormal appearance of the chro-matin (fig 14). These were in addition to the cellsprobably corresponding to transformed or reactivelymphocytes seen higher up the gradient (fig 15).The cells with irregular nuclei were similar to

those seen in cases of glandular fever but were oftenmore bizarre (fig 16).

FOLLOWING KNOWN ANTIGENIC STIMULUSIN A NORMAL SUBJECTOne subject was examined 10 days after receivingsmallpox vaccination and TAB inoculation when amarked vesicular vaccinial lesion was present. Inthe fractions with the highest concentration about

Fig 12 Glandular fever. Fraction 19. Abnormalchromatin pattern ( x 1000).

Fig 13 Glandular fever. Fraction 33. Blast like cells( x 1000).

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Separation ofabnormal cells in the peripheral blood by means of the zonal centrifuge

.....

Fig 14-_-

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Fig 16

Fig 14 Hodgkin's disease. Fraction 31. Cell withabnormal chromatin pattern ( x 1000).

Fig 15 Hodgkin's disease. Fraction 22. Transformedor reactive lymphocytes ( x 1000).

Fig 16 Hodgkin's disease. Fraction 31. Bizarre cells( x 1000).

Fig 17 Normal subject 10 days after antigenic stimula-tion. Fraction 33. Transformed type cells ( x 400). Fig 17

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Fig 18 After antigenic stimulus. Fraction 39. Largetransformed cell ( x 1000).

Fig 19 After antigenic stimulus. Fraction 30. Cellswith irregularly shaped nucleus (x 1000).

Patricia M. Egan and J. V. Garrett

30% of the cells were of the transformed blast typeat this time (figs 17 and 18).There were also cells with irregularly shaped

nuclei rather similar to those seen in Hodgkin'sdisease but not in such large numbers or of quitesuch abnormal appearance (fig 19). When the ex-amination was repeated seven days after the secondTAB inoculation the preparation showed very manyfewer of the large transformed blast-like cells, andrather more of the plasmacytoid and other types ofcell with basophilic cytoplasm and slightly eccentricnucleus but without an obvious nucleolus.

Discussion

The method has enabled a concentration of ab-normal cells from human blood to be made even if ithas not so far proved possible to isolate these aspure fractions. Nevertheless, this concentration ofthe abnormal cells, even if only small numbersare obtained, should facilitate their study.

Total recovery of cells was variable but appearedto be between 25% and 70%. Attention was firstdirected to deciding if the cell abnormalities ob-served could be due to artefacts produced by thegradient medium and centrifugation process.

Ficoll itself is an inert, non-ionized sucrose-epichlorohydrin co-polymer and in the concentra-tion used here it does not appear to be toxic. Viabi-lity counts with trypan blue-performed on someof the fractions, and after incubation of cells withthe medium for two hours-gave satisfactoryresults, at least 80% of the cells excluding the dye.Other workers have confirmed that Ficoll does nothave a deleterious effect on cells, thus Gorczynskiet al (1970) reported that even 36% Ficoll for threehours did not affect the plaque-forming cell abilityof mouse spleen cells, and Cooper and Bain (1971),who used a very similar method to that employedhere with guinea pig lymph node cells, observed notoxic effects as judged by plaque-forming abilityand mediation of transfer reactions.The question of the osmotic effect of gradient

density media has exercised a number of observers(Dicke et al, 1969; Gorczynski etal, 1970; Williamset al, 1972). We carried out osmolarity measurementsof Ficoll, and found that there was no appreciablealteration in osmolarity due to the Ficoll, thusagreeing with the data of Williams et al (1972) whoused a vapour pressure method and found that therewas no osmotic effect of Ficoll in concentrations be-low about 12%. It was, therefore, decided not to useconcentrat:ons of Ficoll above about 11 % and thegradients were made up to a toxicity of 300 milli-osmoles with a suitable concentration of MEM.To see if any morphological changes could be

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Separation ofabnormal cells in the peripheral blood by means of the zonal centrifuge

p/

Fig 20 Glandular fever. Fraction 23. Autoradiograph Fig 21 Hodgkin's disease. Fraction 30. Abnormal cells,showing labelled cells (x 1000). one with disrupted nucleus ( x 1000).

produced resembling those observed in the separatedcells, leucocytes were incubated with Ficoll ofvarious concentrations for three hours. It was foundthat at ai concentration of 12% or above changes inthe outline and cytoplasmic appearance with theformation of vacuoles could be seen in the lymph-ocytes in some preparations, but no nuclear changesof the type seen in the separated abnormal lympho-cytes were ever seen.We were able to demonstrate the viability of the

separated cells by incubating some fractions fortwo hours with tritiated thymidine and preparingautoradiographs. Figure 20 shows labelling of thenuclear DNA in the large cells concentrated fromglandular fever blood which are known to be inDNA synthesis.By zonal centrifugation we were able to find inw normal blood a few of the large reactive cells (fig 11)

elsewhere reported as occurring in Hodgkin'sdisease (Bouroncle, 1966). The cases of disseminatedHodgkin's disease showed the greatest number ofabnormal cells however. Some of these resembledthe transformed type cells seen in the normal subject

Fig 22 After antigenic stimulus. Fraction 41. Large undergoing a known, immunological stimulus,cell and small dense cell ( x 1000). others appeared definitely abnormal with irregu-

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larly shaped nuclei, and there were a few with dis-rupted nuclei (fig 21) perhaps suggesting a virusinfection. We have not so far seen double nucleatedcells of the Steinberg-Reed type. The glandularfever cases showed some similar cells, but the ab-normalities were not so marked as in the Hodgkin'scases.These observations show that by this method it is

possible to demonstrate unusual and apparentlyabnormal cells in the blood in a number of condi-tions and it remains to be seen whether any specificchanges are observable in the different conditions.If so these are more likely to be detected by specificimmunological staining, ultrastructural, or bio-chemical methods than by general morphology. Itappears possible however, by means of the zonalcentrifuge, to make preparations where the abnormalcells are concentrated to a considerable extent.

This work was supported by a short-term MedicalResearch Council grant and the Endowment Fundof the Christie Hospital. The tehcnical assistance ofPeter Baker is gratefully acknowledged.

References

Anderson, N. G. (1966). Zonal centrifuges and other separation sys-tems. Science, 154, 103-112.

Bach, M. K., and Brashier, J. R. (1970). Isolation of subpopulationsof lymphocytic cells by the use of isotonically balanced solu-tions of Ficoll. I. Development of methods and demonstrationof the existence of a large finite number of subpopulations.Exp. cell. Res., 61, 387-396.

Boone, C. W., Harell, G. S., and Bond, H. E. (1968). The resolutionof mixtures of viable mammalian cells into homogeneousfractions by zonal centrifugation. J. Cell Biol., 36, 369-378.

APPENDIX

The equation of motion of a cell in a zonal centrifugeis (Boone et al, 1968; Pretlow and Boone, 1969):

dr a2(Dp- Dm)w2rdt =187;

where r is the radial position of the cell at time t,w the angular velocity, a the diameter of the cell, Dpthe cell density, Dm and -q the medium density andviscosity at position r. The time taken for the cell tomove from a radius ro to a radius r or from a zonalvolume V0 to a volume V is

= 18 1 .71.dr'rOa2 c02 Dp - Dm r

andv 18 1 .i7,dVvo a2 W2 Dp - Dm K

Patricia M. Egan and J. V. Garrett

Bouroncle, B. A. (1966). Stemnberg-Reed cells in the peripheral bloodof patients with Hodgkin's disease. Blood, 27, 544-556.

Cooper, A. J., and Bain. A. G. (1971). A demonstration of a sub-population of rosetting cells in lymph nodes from sensitizedguinea-pigs by zonal centrifugation. Immunology, 21, 781-794.

Coulson, A. S., and Chalmers, D. G. (1967). Response ofhuman bloodlymphocytes to tuberculin PPD in tissue culture. Immunology,12, 417-429.

Crowther, D., Fairley, G. H., and Sewell, R. L. (1969). Significance ofthe changes in the circulating lymphoid cells in Hodgkin'sdisease. Brit. med. J., 2, 473-477.

Dicke, K. A., Tridente, E., and Van Bekkum, D. W. (1969). Theselective elimination of immunologically competent cells frombone marrow and lymphocyte cell mixtures. III. In vitro testfor detection of immunocompetent cells in fractionated mousespleen cell suspensions and primate bone marrow suspensions.Transplantation, 8, 422-434.

Gorczynski, R. M., Miller, R. G., and Phillips, R. A. (1970). Homo-geneity of antibody producing cells as analysed by their buoy-ant-density in Ficoll. Immunology, 19, 817-829.

Halie, M. R., Eibergen, R., and Nieweg, H. 0. (1972). Observationson abnormal cells in the peripheral blood and spleen inHodgkin's disease. Brit. med. J., 2, 609-611.

Harris, R., and Ukaejiofo, E. 0. (1970). Tissue typing using a routineone-step lymphocyte separation procedure. Brit. J. Haemat.,18, 229-235.

Noll, H. (1967). Characterization of macromolecules by constantvelocity sedimentation. Nature (Lond.), 215, 360-363.

Pretlow, T. G., and Boone, C. W. (1969). Separation of mammaliancells using programmed gradient sedimentation. Exp. molec.Path., 11, 139-152.

Pretlow, T. G. (1971). Estimation of experimental conditions thatpermit cell separations by velocity sedimentation of isokineticgradients of Ficoll in tissue culture medium. Analyt. Biochem.,41, 248-255.

Pretlow, T. G., and Luberoff, E. E. (1973). A new method for separat-ing lymphocytes and granulocytes from human peripheralblood using programmed gradient sedimentation in an iso-kinetic gradient. Immunology, 24, 85-92.

Shortman, K. (1968). The separation of different cell classes fromlymphoid organs. II. The purification and analysis oflymphocyte populations by equilibrium density gradientcentrifugation. Aust. J. exp. Biol. med. Sci. (1968) 46, 375-396.

Williams, N., Kraft, N., and Shortman, K. (1972). The separation ofdifferent cell classes from lymphoid organs. VI. The effect ofosmolarity of gradient media on the density distribution ofcells. Immutnology, 22, 885-899.

where

dVK= r.ddr

It should be noted that a and Dp are properties ofthe cell only and that Dm and 't/r or 7q/K are inde-pendent of the cell but vary down the gradient. Thephysical dimensions of the rotor establish a rela-tion between the radius r and the zonal volume Vand then the parameters of the gradient can bespecified in terms of V instead of r. Each gradientcan be specified by a list of Dm and -qlr at equalintervals of r or by Dm and -qlK at equal intervals ofV. Numerical integration of the equations can thenbe carried out quite rapidly using the trapesoidalmethod and many programmable desk calculatorsare adequate for this purpose.

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