The rapid isolation from human blood of concentrated, white-cell-free preparations of Plasmodium falciparum

78 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE. Vol. 69. No. 1. 1975.

THE RAPID

ISOLATION FROM HUMAN BLOOD OF CONCENTRATED, FREE PREPARATIONS OF PLASMODIUM FALClPARUM

WHITE-CELL-

J. WILLIAMSONt AND B. COVER* tNationa1 Institute for Medical Research, London NW7 1AA. Investigation of the possible use of the erythrocytic forms of human malaria parasites to prepare an antimalarial vaccine, ideally requires a method which separates infected red cells from all other blood cells and constituents, and then liberates the parasites in such a form that they may be recovered free of host red cell elements. Although large-scale in vitro culture, or infection of simian hosts may ultimately yield practicable amounts of human malaria parasites, the richest source of mature forms currently remains the placenta infected with Plasmodium falciparum (CLARK, 1915; MCGREGOR et al., 1966); mature forms also appear to be more antigenic than peripheral blood ring forms (WILSON et al., 1973). Removal of white cells from a preparation of human blood intended for vaccination is desirable, not only to facilitate separation and purification of antigenic components, but to eliminate possible toxins (WILLIAMS and GRANGER, 1968; FRIMMER and LUTZ, 1969). This possibility should not be minimized, as in the common centrifugal methods for infected cell separation, the layer of parasitized cells is in the region of the buffy coat. Practicable methods for white cell removal are now widely available (Guns, 1970) and at least three have been successfully applied to human malarial blood: dextran sedimentation (ZUCKERMAN et al., 1967), differential sucrose gradient centrifugation (WILLIAMSON and COVER, 1966, 1971$) and cellulose powder filtration (FULTON and GRANT, 1956; PHILLIPS and TRIGG, personal communication). Techniques to free the parasite from its host red cell are numerous and ingenious, but all have disadvantages, although some of these may be less important for vaccine preparation than for other purposes. A major difficulty, graphically described by COOK et al., (1969) in their account of P. knowlesi isolation, is to ensure efficient removal of red cell membrane from the parasites. In the case of monkey red cells infected with schizonts of P. KnowZest the difficulty is obvious (Fig. l), as the parasitized red cell membrane is shrunk down over the irregular projections of the internal schizont. The problem should be much less acute with red cells infected with P. falciparum schizonts which are swollen and spherical (Fig. 2), and likely to be much more susceptible to rupture than cells infected with P. knowlesi schizonts. (see below), There have been at least 2 early attempts to induce protective immunity in man with extracts of human malaria parasites. KONSTANSOFF (1930) used P. vivax-infected human blood, lysed in aqueous phenol, with some success, stressing that the antigen was in a solution of the “Plasmodieneiweisz”. A much more ambitious attempt was made by HEIDELBERGER and his colleagues (1946a, b, c d), in which 500 ml. quantities of blood infected with P. vivax: were lysed in 51 volumes of cold formolized water saturated

with CO,. Very extensive washing to remove stromata and white cells probably also removed all soluble antigens, as the resultant deposit, the vaccine, although non-toxic, had no immumzing activity, and was considered “. . . a total failure”. The subsequent development of cell separation techniques as outlined above, offered a renewed opportunity to test their efficiency in the purification of human plasmodial preparations which would retain full antigenic activity. At the invitation of Dr. I. A. McGregor, Director, Medical Research Council Laboratories, Fajara, The Gambia, we undertook an investigation there of the applicability of differential sucrose gradient centrifugation to the separation and purification of P. falciparum erythrocytic stages and gametoqtes, and of nitrogen cavitation for host-cell-free parasite preparation. Some observations on the nature and antigenicity of the preparations are also recorded. *Present address : Biological Laboratories, University of Kent, Canterbury. We are indebted for unfailing assistance and hospitality to Dr. I. A. McGregor and his staff, Drs. M. E. Wilson, G. H. RCe, H. A. Wilkins and R. J. M. Wilson, Messrs. K. Fraser, P. J. Hall, K. Williams, R. K Bartholomew, C. E. J. Pyne, H. A. Garling and A. Rumgey. The nitrogen cavitation bomb was constructed by Mr. R. Bower and Mr.J. McClorry in the Engineering Division of the Institute at Mill Hill. We are also indebted to Dr. P. R. Stuart and Mr. J. S. Osborn of the National Physical Laboratory, for the scanning electron micrographs, and to Mr. M. A. Young for fluorescence emission studies. $Error in lines 10 and 11 of this account, ccGametocyte removal” should read ccGranulocyte removal”.

J; WILLIhElSOU

79

AND 3. COVER

I

FIG. I. Scanning electron micrograph of monkey erythrccytes with infected P. knowlesi schizonts. x 13000. (Samples from heavily-infected blood; the distortion of the red cells is due to infection and not to osmotic or other factors).

FIG. 2. Scanning electron micrograph of human erythrocytes infected with P. falciparum schizonts. x 14000. (Sampled from a sucrose gradient concentrate of heavily infected placental blood. There are sucrose crystals between the cells, which are covered with papillae; these appear to be part of the red cell membrane, and may correspond to the excrescences noted in electron micrographic sections by TRAGER, RUDZINSKA BURY, 1966).

and

BRAD-

Materials

and methods

P. falciparum-infected blood : Samples of heparinized peripheral blood containing ring forms or gametocytes were obtained by venepuncture of Gambian children, by courtesy of Dr. G. H. Rec. Trophozoitecontaining blood was provided by Mr. R. K. Bartholomew from large scale in vitro cultures (PHILLIPS, et al., 1972), and blood, heavily infected with schizonts, was obtained from the placentae of malarious Gambian women, as described by MCGREGOR et al., (1966). Differential parasite counts in thin films of human blood samples were kindly performed by Dr. M. E. Wilson; quantitative estimates of recovery, and concentration of parasites during fractionation, were made with a combination of counts on unfixed thick films stained with Field’s stain, FIELD, (1940) fixed thin films stained with Giemsa, and haemocytometry; platelets were counted according to WHITBY and BRITTON (1947). Samples for scanning electron microscopy were fized for 30 mins. in 05% phosphate-buffered glutaraldehyde (pH 7.4) washed in water and spread on cover slips. sucrose gradient centrifugatioz : The low-speed sucrose gradient separation technique and COVER, 1966; WILLIAMSON, 1967) has been in constant and satisfactory use in this laboratory for over 7 years in experiments on P. knowlesi; adaptation to large scale separations in the type Differential

(WILLIAMSON

80

ISOLATION OF CONCENTRATED, WHITE-CELL-FREE

PREPARATIONS

A zonal rotor (GUTTERIDGE et al., 1971) has given even sharper separation. The earlier technique was used in the present experiments with P. falciparum-infected blood samples, and as some practical details of it have not been previously reported in extenso, they are given here. Stock solutions (1) Krebs glucose saline (KGS) O@%aq. NaCl 1.15% aq. KC1 3.84% aq. MgS0,.7H,O Glucose 0.1 M phosphate* buffer pH 7.4 *Na,HPO,.l2H,O, 17.9 g.

100 ml. 4 ml. 1 ml. 3

0.27 g. 30 ml.

in 500 ml. (aq) N HCl (2)

10 ml.

I-

1.0 M sucrosein KGS 34.2 g. sucrose to 100 ml. KGS.

The sucrose gradients were prepared in KGS so as to provide an ionic environment conducive to cellular integrity. Linear gradients, O-25 M (8.55%) - 0.77 M (23.94%) sucrose, covered a density range of l-055 to 1.096, which spans the density of the various cells to be separated; an approximate check on cell densities, using calibrated o-dichlorbenzene/petroleum ether (S&lOO”) mixtures, gave the following figures : 1.03 Platelets (rat) 1.036 Leucocytes (rat) P. knowlesi-infected monkey red cells 1.061 1~08-1*10 Red cells (rat) The gradients can be prepared by layering and diffusion, by use of a simple 2-vessel gravity-feed mixer (cf. BRITTFN and ROBERTS, 1960; SAMIS, 1966), or more conveniently, with a peristaltic pump and a mixing vessel (BOZARTH et al., 1965; cf. AYAD et al., 1967) as described earlier (WILLIAMSON, 1967); the advantage of a multichaMe1 pump is that several gradients may be prepared simultaneously, and gradient profiles can be varied. Fifty ml. gradients in 100 ml. round-bottom centrifuge tubes (2.7 cm i.d. x 17.0 cm.) were loaded with blood samples according to the recommendations made earlier (WILLIAMSON and COVER 1966) (sample volume not more than l/3 of the gradient volume, and containing not more than 20% packed cells). Instead of centrifuging the gradients at 1,200 G-min, as carried out originally, continued use has shown that a more efficient separation may be made at 1,680 G-min. (e.g. 1,000 r.p.m., r 21.6 cm., 7 mins.); G = 1.12 x lo+ x (r.p.m.)2 x r (in cm.). The latter gradient centrifugation conditions were used here. Where only small blood samples were available, or continued sub-fractionation was performed, appropriately smaller volume gradients were used. After gradient centrifugation, cell layers were cleanly removed with a Pasteur pipette, the tip of which had a right-angle bend to prevent aspiration of underlying cell layers. The following counting system was applied to all blood samples before application to the gradient, and to all samples taken from the gradient; the latter, as appropriate, were washed and resuspended in KGS. Infected red blood cells (IRBC) from thick films Leucocytes (WBC) IRBC

(4

Uninfected red blood cells (RBC)

(4

Total cells

(4

from thin films by haemocytometry

J. WILLIAMSON

81

AND B. COVER

From counts (a) to (e), the following values were calculated: Total WBC (calculated for thin films)

b = a x c

IRBC (% total cells) Total IRBC WBC per lo8 IRBC IRBC yield

Enrichment factor

=

(f 1

c x 100 c+d$-f

k>

exg -iE

(h)

b x lo3 a h (gradient fraction x 100 = (original sample) --

= g (gradient fraction) (original sample)

Leucocyte numbers were estimated indirectly, as rapid fractionation of the samples was desirable and extensive additional haemocytometry would have been required for direct white cell counts. Indices of leucocyte contamination in samples are therefore expressed as a proportion of the number of IRBC present. Nitrogen cavitation : A cylindrical steel pressure vessel, of the type described by HUNTER and COMMERFORD (1941), was constructed in the Institute’s Engineering Division workshops to contain a 100 ml. glass

centrifuge tube. Removeable head, inlet and outlet needle valves and nylon tube connections were pressuretight up to 2,500 psi. Cells were suspended (10% v/v) in KGS containing 30 times the normal concentration of Mg+2, and 0.2 mM CaCI,, to preserve nuclear integrity and to prevent cell clumping (AVIS, 1969). To avoid sample loss on high-pressure ejection, the outlet nylon tube was led into a 2 litre separating funnel and held in place firmly when pressure was released at the outlet valve. Immunodaflusion: Where appropriate, separated samples were tested by Dr. R. J. M. Wilson for precipitinogenic activity, by double diffusion in agar gels, (WILSON et al., 1973) using antisera from immune Gambian adults.

Results Preliminary experiments were made in London to improve monocyte removal in the sucrose gradient separations, and to circumvent possible red cell agglutination, often encountered in acute P. fulcipurum malaria, which would complicate processing of infected blood. As granulocytes take up sucrose freely, they shrink in the hypertonic regions of the gradient, become more dense and are rapidly centrifuged to the bottom; monocytes, not being differentiated for endocytosis, are distributed more randomly. Two successive gradient separations of the bufIYy-coat-infected red cell layer of a P. knowlesi-infected blood sample, reduced lymphocyte and monocyte contamination of the resultant granulocyte-free infected red cell (IRBC) layer from approximately 05% to O*22o/oof the total IRBC. High lymphocyte and monocyte levels were encountered in infected Gambian blood samples and are noted below. In the event, red cell agglutination was not found to be a serious inconvenience in processing Gambian P. fulcipurum-infected blood. A more serious difficulty transpired in early attempts at gradient separations of placental blood to which sodium azide had been added as a preservative. In these samples, the abundant white cells, as a result of poisoning by azide, had thrown out fibrous processes by which they were entangled with each other and with adjacent red cells; clean separations on the sucrose gradient were thus prevented. One infected blood sample (C2; see Table II) was separated on a 60 ml. sucrose gradient which was then sampled sequentially in approximately 3 ml. volumes; the gradient samples were counted to give a quantitative analysis of cell distribution. Schizont-infected red cells are localized in the upper layers of the gradient, with a high concentration of granulocytes at the bottom; platelets are sequestered at the top,

82

ISOLATION

OF CONCENTRATED,

WHITE-CELL-FREE

PREPARATIONS

above the schizont layer. Only lymphocytes were present in all layers above the bottom granulocyte “button”. The following table (Table I) illustrates the calculations required (see Methods and Materials) to determine the extent of white cell removal, yield of infected red cells (at different levels of white cell contamination), and the enrichment of infected red cells in the final, as compared with the original, sample. A 3 ml. sample from the final stage of placenta (P2) processing was divided to give the pigmented schizont layer (P2A) and the residue (P2B). Each was diluted and applied to a 60 ml. sucrose gradient. After centrifugation, the schizont layer (P2Al and P2Bl) and the white cell “button” (P2A2 and P2B2) were removed from each gradient, washed and resuspended in KGS. P2A2 was put on a second gradient, centrifuged, and the schizont layer (P2A21) removed and examined. This placental sample contained azide; its effects are apparent from Table I, which shows the high proportion of WBC remaining in layer P2Al and the entrapment of half the IRBC in the granulocyte “button” P2A2. A second gradient separation of P2A2 produced a layer (P2A21) in which WBC were reduced from 592% to 0.18% of the IRBC, and the IRBC proportion was enriched 1.43 times; this purification was obtained at the expense of yield. Table I also shows that the residual placental blood layer P2B, even after gradient separation, carried only 4-5% IRBC compared with 90-95% IRBC in the P2A samples. For this reason, only the pigmented “schizont layer” was harvested from infected placental blood. TABLE I. An example of the application of the enumeration system devised for analysis of blood cell fractions from gradient separation. The sample prefix (P2) refers to the source (placenta P2); samples A and B are the pigmented “schizont” and residual layers respectively, of centrifuged placental blood. Sample P2A

P2Al

l(a) IRBC

2340

(b) WBC

112

Gnmt

Thick film

P2A2

P2A21

P2B

1229

810

1124

689

662

664

41

48

2

27

29

26

P2Bl

P2B2

(c) IRBC

537

91

343

534

27

25

38

(d) RBC

253

285

16

33

1000

600

655

25.7

3.04

20.3

1-O

0.11

o-11

1.5

(e) Total Cells ( x 10-7)

1504

270

531

86

916

245

123

(g) IRBC (% total)

65.5

24.0

90.5

94.0

2.63

4.00

5.47

985

64-8

478

80.8

23.8

9.80

6-75

3.34

5.92

0.18

3.92

4.38

3.92

6.58

48.5

8.20

41.2

28.1

1.37

1.43

1.52

2-08

Thin film

(f) WBC (talc)

(h) Total IRBC ( x lo-‘) WBC (% IRBC) IRBC yield (% original) Enrichment

factor

4.79

Using the above separation, counting and sampling techniques, the following results (Table II) were obtained for recovery of ring, trophozoite, schizont and gametocyte forms from P. fah>arum-infected blood. The first two placentae sampled contained sodium azide; its toxic effect on white cells is reflected in the low efficiency of white cell removal in Pl and the low yields of white-cell-free infected cells in P2. White cell removal from azide-free samples was consistently high (90-100%) except for placenta P4, which was only lightly infected, and culture C4, in which some clotting was evident. White cell numbers appear to rise proportionately with the infection, reaching values more than 5 times the Gambian normal, so that the need for efficient white cell removal from highly infected sources, such as placentae, is obvious. The average yield of purified infected red cells at 3 levels of white cell contamination (< l%, l-2% and 2-5%) is respectively 24.3%, 34.0% and 47.0% of the infected cells in the original sample. Considerable concentration of infected cells occurs during gradient separation, varying from 1.17- to 9.94-fold (average

J. WILLIAMSON

AND

B.

83

COVER

TABLE II. Recovery of P. fulciparum-infected red cells from placental, cultured peripheral, and peripheral human blood samples (representing schizont, trophozoite, ring and gametocyte stages). Samples were generally taken from the pigmented layer of centrifuged blood. Where this layer and the underlying layer were analysed, they are specified as U and L respectively; 0 refers to the original uncentrifuged blood sample. Sample

LayerPaasite

Initial infection

schizont

26.5

Early

65.5

(% Total Pl*

u P2+

-Trophozoite L +

<

1%

WBC

(%

orig.)

l-2%

WBC

3.15

8.20

6.58

0.01

69.3 100

50.1

1.75

100

14.8

ca 3.0x

1.60

51.3

P5

5.56

0.39

98.2

9.0

57.7

P6$

Pre-Schizont

54 5

2.45

96.7

15.9

27.4

P7

Late

x

30.3

Trophozoite

2

27 7

3.17

94.6

20.6

Cl

schizont

1.00

0.11

93.5

36

c2

schizont

1.70

0.03

96.9

29.2

35.4

1.08

0.10

U -~ L

Trophozoite

0 u

c5 U

0.29

20.2

100

0.80

33.7

77.4

7.49

64.0

2.45

0.22

95.0

Riig

8.90

0.18

93.8

L

7.50 Rim

Gl

Gametocyte

G2

GametOCYtC

from

16.0

2

39.9

1.17x

2

43.1

2.89

x

2

2.10x

1

67.8

6.50x

1

34.4

1.63x

2

67.2

2.20x

2

78.8

4.60

schimnt

R2

*Containing azide. tIAw infection. *Abnormal placenta

100

1.53 0.39

9.94x

100

2.73

4.85 Troohozoite

L

-

1.52x

0.28

9.56

PI *.-

1.43x

5.02

Schizont

01

WBC

No. of Gradients

1

96.2

Schizont

c3

2-5%

IRBC Enrichment (M=)

52.3

p4t

Culture

containins

2

0

Peripheral Blood

(Max)

yield

Schimnt 6.06

Placenta

Cells1

IRBC

% WBC Removal

2 2.63 50.2

P3

Initial WBC

33.2 73

75.5 4.51

91.6

0.31

0.60

95.9

0.34

0.67

99.8

5.70x

1

1.24x

2

47.8 46.1

1 0

1

12.8x 11.3

22.0

1 x

1

stillbirth.

In blood from the gametocyte-carriers (Gl and G2), the enrichment effect was much more pronounced, and gametocyte numbers were increased 12+3-fold and 22-fold respectively. Their localization in the gradient was less diffuse than that of the other parasite forms; in the case of Gl, a 2.0 ml. sample centrifuged on a 6.5 ml. gradient gave the following sequential distribution (from top to bottom of the centrifkge tube): Gametocytes per lo6 RBC WBC per lo6 RBC Sample vol. (ml.)

2.92fold).

3.0 0.5 o-9 1.0 o-9 0.9 0.9 0.7 0.2 Original blood

5,900

40,250 4,400 200 100

75 160 160 240 3,155

33.5 23.5 4,478 975 740 1,270 2,650 2,175 2,375 12,200

Attempts to prepare erythrocyte-free parasites by nitrogen cavitation: Before leaving London, the nitrogen bomb was tested with a 20 ml. sample of normal adult human blood, the packed cells of which were resuspended to 20% (v/v) in Krebs saline. Pressure was built up

84

ISOLATION

OF CONCENTRATED,

WHITE-CELL-FREE

PREPARATIONS

over 5 minutes to 1,000 psi with nitrogen, and the sample was kept at this pressure for 30 minutes. RBC breakage was 958% after pressure release; a second identical run on the same sample raised the breakage to 99.7%. These conditions were initially used on 3 samples of P. falciparum-infected Gambian blood which had been concentrated and freed from white cells by sucrose gradient centrifugation. The first sample comprised a pool of the 3 most heavily infected layers, containing 52.4% of the total IRBC, from the separation of an in vitro culture (C2) described earlier, the second comprised the pooled IRBC layers (P2A1, P2Bl) from the sucrose gradient separation of schizont-containing blood from placenta P2 (cf. Table I), and the third was similarly derived from placenta P3. After nitrogen cavitation, the “bombed” suspension was centrifuged, and in the case of the C2 and P3 preparations, the deposit was separated further by centrifugation in a 10 ml. sucrose gradient. The C2 gradient gave 2 very fine upper layers and a bottom deposit, and microscopic examination showed that the second layer consisted of a heterogeneous mixture of chromatin granules and masses, with amorphous yellow particles, and that the deposit was of similar composition but containing many distorted red cells and large crvstalloid yellow masses. As these crystalloid masses fluoresced a deep brown to purple colour when excited by long ultraviolet illumination (max 365 mu.; Chance filter OX1 with 250 w. mercury pressure lamp, Zeiss cardioid immersion darkground condenser, x 100 fluorite objective), they were considered to be haem; the possibility of their being lipofuscin in nature was discounted, as these pigments tend to fluoresce in the range blue to yellowgolden. The P3 gradient showed only one layer, which contained a large amount of debris, a few intact IRBC and numerous unstained refractile vesicles, about the size of a merozoite, which may have been pigment vacuoles. The placental (P2) blood preparation was not separated further; the deposit from centrifugation showed that, in addition to cell debris and pigment clumps, about one-third of the trophozoite-infected red cells had ruptured to release free parasites. As a pressure of 1,000 psi seemed to be too high for uniform recovery of intact free parasites, another small sample of pooled IRBC gradient layers from placenta P3 was subjected to nitrogen cavitation at 500 psi for 30 minutes. The product, which contained large numbers of fme particles, numerous “pigment vacuoles” and a few free schizonts, was put on a 10 ml. sucrose gradient and centrifuged. Only one faint upper band was apparent, which consisted exclusively of “pigment vacuoles” of uniform size. Pressure and exposure time were further reduced with two samples of in vitro culture trophozoitecontaining blood, and a sample of peripheral blood containing ring forms. Four IRBC layers from sucrose gradient separations of culture blood sample C3 were pooled and “bombed” at 250 psi for 15 minutes. After centrifugation of the product, the residue was found to contain large numbers of unbroken cells, “pigment vacuoles” , yellow haem agglomerates and many free trophozoites; it was applied to a 10 ml. sucrose gradient and centrifuged. Three layers were discernible; the top showed large numbers of “pigment vacuoles”, a few red cells and some pigment, the middle, very large numbers of free parasites and a few red cells, and the lower, large numbers of red cells, a few of which were infected. The sediment at the bottom of the gradient consisted of cell debris, large numbers of red cells and many very large agglomerates of yellow haem. Another 4 IRBC samples from sucrose gradient separations of culture blood (C4), were pooled and “bombed” at a slightly lower pressure (200 psi for 15 minutes). Centrifugation of the product gave a residue which consisted largely of unbroken uninfected red cells, together with many fine particles and haem agglomerates. A final experiment was carried out with 3 IRBC fractions from a sucrose gradient separation of peripheral blood (R2). The pooled fractions were resistant to a pressure of 200 psi for 15 minutes, but further exposure to 800 psi for 15 minutes, broke the cells effectively. The deposit from the centrifuged product contained no recognizable free ring forms and consisted of ceil debris, many intact red cells and masses of yellow haem agglomerates. Discussion The only other well documented attempt to harvest P. fakiparum from infected human blood is that of ZUCKERMANet al., (1967) who used dextran sedimentation to remove white cells from samples of peripheral blood collected in the Gambia and in Thailand. As their objective, to provide purified parasite material with antigenic activity, was similar to our own, the following points of comparison may be made. So far, the dextran sedimentation method has been applied only to peripheral blood carrying the early ring forms, which appear not to be so immunogenic as the later trophozoite and schizont forms (WILSON

J. WILLIAhUON

AND B. COVER

85

et al., 1973). As the accelerated sedimentation of red cells in the presence of dextran is due to its haemagglutinating properties, we decided in the early development of our method, to avoid its use (WILLIAMSON and COVER, 1966); Ficoll, which is often preferable to sucrose for density gradient preparation (cj. ALI and FLETCHER, 1971) because it produces less osmotic damage, is not entirely free of haemagglutination drawbacks (~~ATHIAS et al., 1969), and is also expensive for large scale use. As red cells infected with the later trophozoite and schizont stages are known to become “sticky”, haemagglutinating agents such as dextran would seem to be undesirable; in support of this assumption, Professor Zuckerman has told us that the dextran sedimentation technique was not effective with heavily infected placental blood. Although ZUCKERMAN et al. (1967) did not report in detail on their yields of parasite material and on the extent and variability of white cell removal, the relevant data were recorded and retained in the MRC Laboratories in Fajara. By courtesy of Dr. I. A. McGregor and Mr. K. Williams, and with Professor Zuckerman’s kind permission, we have been able to use some of these data for comparison with our own. From 23 samples purified by the dextran technique, with original parasitaemias ranging from 0.2 to 7.4% (mean 3+64%), the efficiency of WBC removal ranged from 67 to 116% (mean, 852%). As there was no selective removal or concentration of IRBC, calculation of yield was restricted to the recovery of free parasites liberated by saponin lysis and is not relevant to the estimates presented here. ZUCKERMAN et al., (1967) found that, on high-speed centrifugation, residual saponin-affected white cells released gummy nucleic acid aggregates in which numerous free parasites were trapped. This experience is further evidence for the desirability of effective removal of white cells, especially as saponin lysis is likely to be more acceptable for parasite antigen preparation than immune lysis. The overall efficiency of white cell removal on sucrose gradients (90.2 -+ 13.6%) was comparable to that of the dextran method (85.2 f 15*4%), but a considerable factor in this comparison is that the dextran separation technique took 10 hours to perform; sucrose gradient separations take only 7 minutes centrifugation time, so that production of leucocyte-free and concentrated infected red cell preparations is considerably accelerated. The cellulose filtration method of FULTON and GRANT (1956) may be a useful alternative for white cell removal from malarious human blood; using this method FITCH (1969) claimed more than 99% white cell removal from P. berg/&infected mouse blood, and ROCK et al., (1971) showed that white cells were removed from P. Knowlesi-infected monkey blood to a level of “less than 1 lymphocyte per lo4 parasites”. The method, dependent on the adhesive properties of leucocytes, has illustrious origins. ALMROTH WRIGHT (1926) found that simple filtration through filter paper removed leucocytes from defibrinated blood, and FLEMING (1926) showed that compressed plugs of teased filter paper were also very efficient, and that the few unretained cells were lymphocytes. More recently WILLIAMS and RICHARDS(1973) and RICHARDS and WILLIAMS (1973) have described their application of the technique to the requirements of in citro culture of P. berghei from rat blood. Blood has been filtered undiluted (ROCK et al., 1971), with plasma replaced by citrate-saline (FULTON and GRANT, 1956), diluted 1 : 1 (FITCH, 1969; BAGGALEY and ATKINSON, 1972), and diluted 1 : 6 (WILLIAMS and RICHARDS, 1973); the efficiency of white cell removal appears to have been high in all cases,but the last authors indicate that after passageof a volume of blood suspension equivalent to approximately twice the column volume, an increasing number of damaged lymphocytes began to appear. No extensive details of recovery, of column capacity, or of the efficiency of platelet removal, are given in any of these accounts; the report of ROCK et al., (1971) implies that platelets must be removed separately. The method appears likely to be practicable with P. f&z$zrum-infected human blood, but it will require assessment in at least as much detail as is given here in the present method or as was given to the dextran sedimentation method by ZUCKERMAN et al., (1967). All the methods seem likely to give infected red cell preparations which are viable and capable of use for metabolic analysis. In the sucrose gradient separation, infected cells are only briefly in contact with sucrose solution at a level where the concentration is not far above isotonicity ; the infectivity and respiratory activity oftrypanosomes (Trypanosotna rhodesiense) separated from rat blood by the sucrose gradient method, are essentially the same as after separation by normal centrifugation. A useful property of the sucrose gradient separation is its ability to concentrate gametocytes (up to 22-fold); this property is not shown by the dextran sedimentation or cellulose filtration methods, but similar concentration, with up to 50% yield, can be obtained by agarose filtration (KAss et al., 1971) Some concentration for the purposes of microscopic examination, can be achieved in the usual thick film preparation, but is much less than that obtained by gradient centrifugation. Our preliminary experiments

86

ISOLATION OF CONCENTRATED,WHITE-CELL-FREE PR3PARATIONS

suggest that the latter method could form the basis of a simple and useful means of obtaining enough viable gametocytes for metabolic and immunochemical analysis; little is known of these aspects of this important form of the parasite. Attempts to produce erythrocyte-free P. falciparum parasites by nitrogen cavitation showed primarily that the pressures required to burst red cells with the later and more mature stages of the infection were very much less than those needed to release equivalent stages of P. knowlesi from monkey cells; peripheral human blood cells carrying ring forms of P. falciparum, where weakening of the host red cell would not be extensive, were more resistant. As some enlargement of the host red cell occurs during maturation of P. falciparum (Fig. 2), rupture by cavitation is likely to be facilitated. Our experiments were not extensive enough to determine optimal pressures and exposure times for each of the 3 main parasite stages, but enough information was gained to show that nitrogen cavitation followed by gradient centrifugation could be used to prepare’erythrocytefree P. falciparum. Whether the red cell breakage is caused by a pure cavitation gas-release process, by shear at the outlet port, or by both, is not clear. In any event, pressure homogenization has much to commend it (AVIS, 1969), not least the adiabatic cooling which accompanies pressure release, and more detailed study of its potential for free parasite preparation seems eminently justified. ImmunodifIusion experiments conducted by Dr. R. J. M. Wilson with immune Gambian sera, showed that (a) considerable contamination with leucocytes (as in an IRBC sample derived from the bottom granulocyte “button” of a gradient separation) did not affect the precipitin pattern (WILSON, 1974), and (b) the most highly purified leucocyte-free preparation we obtained (P2A21; cf Table I) retained the complete spectrum of precipitinogenic activity given by the crude parent placental (P2) antigen as normally prepared (MCGREGORet al., 1966) and reconstituted (by Hughes press disintegration of lyophilized crude antigen). On the basis of the results presented here, further work on the bulk separation from infected human blood of P. falciparum material suitable for experiments in vaccine preparation, will require (a) trial of zonal centrifugation in a sucrose gradient (GUTTERIDGE et al., 1971), using placental blood free of sodium azide, (b) assessment of cellulose filtration for white cell removal, and (c) a more detailed study of methods of disrupting schizont-infected red cells and recovery of freed parasites. Summary

The sucrose gradient centrifugation method has been applied to representative samples of human (Gambian) blood infected with ring, trophozoite, schizont and gametocyte stages of P. falciparum in order to assessquantitatively the efficiency of recovery, white cell removal and the degree of enrichment of the infected cell fraction. Maximal white cell removal was 90%. (a.v.) Average infected cell recoveries varied with the level of white cell contamination, namely 47% (2-5% WBC), 34% (l-2% WBC) and 24% (< 1Y0 WBC); infected cells were enriched 2*9-fold on average, and up to 22-fold in the case of gametocytes. Preliminary attempts to prepare free parasites by nitrogen cavitation of infected cells showed that disruption took place at much lower pressures than those required to break normal red cells. Gel diffusion analyses showed that the most highly purified infected cell preparations retained the full precipitinogenic spectrum of the original crude preparation. REFERENCES ALI, S. N. & FLETCHER, K. A. (1971). Trans. R. Sot. trop. Med. Hyg., 65, 4. AVIS, P. J. G. (1969). Subcellular Components (Ed. Birnie, G. D. & Fox, S. M.), p. 1, London: Butterworths. AYAD, S. R., BONSALL, R. W. & HUNT, S. (1967). Science Tools, 14, 40. BAGGALEY, V. C. & ATKINSON, E. M. (1972). Trans. R. Sot. trop. Med. Hyg., 66, 4. BOZARTH, R. F., BROWNING, R. E. & GROSS, S. (1965). Contrib. Boyce Thompson Inst., 23, 141. BRITTEN, R. J. & ROBERTS,R. B. (1960). Science, 131, 32. CLARK, H. C. (1915). J. exp, Med., 22, 427. COOK, R. T., AIKAWA, M., ROCK, R. C., LITTLE, W. & SPRINZ, H. (1969). Milit. Med., 134, 866. CUTTS, J. H. (1970). CeZZSeparation. London: Academic Press. FIELD, J. W. (1940). Trans. R. Sot. trop. Med. Hyg., 34, 195. FITCH, C. D. (1969). Proc. nat. Acad. Sci., Wash., 64, 1181. FRIMMER, M. & LUTZ, F. (1969). Arch. Pharmakol., 263, 297. FULTON, J. D. & GRANT, P. T. (1956). Biochem. J., 63, 274. GUTTERIDGE,~. E.,TRIGG, I?. 1. 81 Co~~~,B.(1971). Trans. R. Sot. trop.Med. Hyg.,65, 418. HEIDELBERGER,M.,MAYER, M. M. & DEMAREST, C. R. (1946a).J. Zmmunol., 52, 325.

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COATES, W. A. & MAYER, M. (1946b). Ibid., 53, 101. PROUT, C., HINDLE, J. A. & ROSE, A. S. (1946~). Ibid., 53, 109. MAYER, M., ALVING, A. S., CRAIGE, Jr. B., JONES, Jr. R., PULLMAN, T. & WHARTON, C. M. (1946d). Ibid., 53, 113. HUNTER, M. J. & COMMERPORD, S. L. (1961). B&him. biophys. Actu, 47, 580. Us, L., WILLERSON, Jr., D., RIECKMANN, K. H., CARSON, P. E. & BECKER, R. P. (1971). Amer. J. trop. Med. Hyg., 20, 187. KONSTANSOPF, S. W. (1930). Zbl. B&t., 116, 241. MATHIAS, A. P., RIDGE, D. & TREZONA, N. St. G. (1969). Biochem. J., 111, 583. MCGREGOR, I. A., HALL, P. J., WILLIAMS, K., HARDY, C. L. S. & TURNER, M. W. (1966). Nature, 210, 1384. PHILLIPS, R. S., TRIGG, P. I., SCOTT-FINNIGAN, T. J. & BARTHOLOMEW, R. K. (1972). Parasitology, 65, 525. RICHARDS, W. H. G. & WILLIAMS, S. G. (1973). Ann. trop. Med. Par&t., 67, 179. ROCK, R. C., STANDEPER, J. C., COOK, R. T., LITTLE, W. & SPRINZ, H. (1971). Comp. Biochem. Physiol., 38b, 425. SAMIS, Jr., H. V. (1966). Anal. Biochem., 15, 355. TRAGER, W., RUDZINSKA, M. A. & BRADBURY, P. C. (1966). Bull. Wld Hlth Org., 35, 883. WHITBY, L. E. H. & BRITTON, C. J. C. (1947). Disorders of the Blood. London: J. & A. Churchill. WILLIAIMS, S. G. 81 RICHARDS, W. H. G. (1973). Ann. trop. Med. Purasit., 67, 169. WILLIAMS, T. W. & GRANGER, G. A. (1968). Nature, 219, 1076. 2, 85. WILLIAMSON, J. (1967). Protozoology (Suppl. J. Helminthology), & COVER, B. (1966). Trans. R. Sot. trop. Med. Hyg., 66, 425. -& (1971). Ibid., 65, 416. WILSON, R. J. M., MCGREGOR, I. A. & WILSON, M. E. (1973). Znt. 3. Purasitol., 3, 511. (1974) Ibid., 4, 537. ZUCICERMAN, A., SPIRA, D. & HAMBURGER, J. (1967). Bull. Wld Hlth Org., 37, 431.

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