119

Clinica

Chimica

Acta,

98 (1979)

119-125 Biomedical Press

@ Elsevier/North-Holland

CCA 1142

HUMAN ERYTHROCYTE GRADIENTS

CAROLYN M. RENNIE and ALUN MADDY a

FRACTIONATION

a, STEPHEN

THOMPSON

Departments of Zoology a and Haematology EH9 3JT, Scotland, U.K.

(Received

IN “PERCOLL”

b, ALISTAIR

b, University

DENSITY

C. PARKER

of Edinburgh,

b

Edinburgh

May Bth, 1979)

Summary A new rapid method for the age fractionation of human erythrocytes by centrifugation through a “Percoll” density gradient is described. The fractionation is demonstrated by density-related changes in the volume, haemoglobin concentration, pyruvate kinase and acetylcholine esterase activities and potassium contents of the erythrocytes, and the distribution of reticulocytes on the gradients.

Introduction The fractionation of a population of erythrocytes according to age has been achieved with varying degrees of success by exploiting age related alterations in the cell’s surface [ 1,2], fragility [ 31 and density [4-81. A universally acceptable, simple, rapid, method has, however, yet to be found. Bovine serum albumin [4], Stractan II [6] and phthalate esters [7,8] are perhaps the most suitable materials that have been employed to construct density gradients, while Murphy [ 91 has fractionated erythrocytes by centrifugation in their own plasma. Each of these methods has disadvantages: serum albumin is expensive, commercially available Stractan is unacceptably crude, phthalate esters damage the cells, and the Murphy method, although it is the gentlest of treatments, cannot be directly applied to pathological samples where differences in density are accompanied by differences in shape and flexibility. All methods, apart from that employing phthalate esters, require prolonged centrifugation. We here report the density fractionation of normal healthy human erythrocytes by isopycnic centrifugation through gradients of “Percoll”, a commercially available polyvinyl-pyrrolidonecoated colloidal silica. Correspondence Edinburgh

EH9

to: 3JT.

Dr.

A.H.

Scotland.

Maddy, U.K.

Department

of Zoology,

University

of Edinburgh.

West

Mains

Road.

1 20

It, is our intention in this report only to establish the effectiveness of thtJ method for the age fractionation of erythrocytes, and not to give a comprehensive analysis of the many aspects of c>ryt.hrocyte physiology related to ageing. WC have t,hcrefore studied a selectioll of parameters which have been assocniated with ageing. Earlier workers wit11 other tc>chniques of age fractionation havtl shown that mean cell volume (MC’V), mean cell haemoglobin c~onc~entration (MCHC), pyn1vat.e kinasp and a;,ct~7l(~holine est.erase activities and rrythrocytt3 l)otnssium content all change with age. We have found these differences bc+vt>en t/he fractions prepared h?; centrifugation through “Pr>rcoll” gradient.s. We thereforcl c~onclude that thr “Prrcoll” gradient is an clffecative niet,hod for age fractionation and it will b(l argurtl that it ~~ossesses c,ertain advant,agcs. Methods Rlood

collection

Blood was obtained by venepuncture tleparin was tlsed as an anticoagulant.

from

healthy

male

and

female

aduits.

Stock solutions of (a) 5.263% (w/v) bovine serum albumin (BSA) (Cohn fraction V, Sigma Chem. Co.) in water, pH 7.4 and (h) 5.263% BSA in “Percoll” (Pharmacia, Fine Chemicals) were made and stored at 4°C for no longer than 3 days. On the day of use, these solutions were equilibrated to room temperature (lS”C), and both solutions were made isotonic, and BSA brought to 5% by the addition to (a) of 1 ~012.66 mol/l NaCl, 0.09 mol/l KC], 200 mmol/l HEPES (N-2-hydroxyethylpiperazine-N’-2-ethane sulphonic acid) buffer pH 7.4, to 19 ~01s of the .5.263% BSA in water and to (b) of 1 vol OC 2.66 mol/l NaCl, 0.09 tnol/l KCI. 200 mmol/l HEPES. pH 8.5, (the mixture has a final pH of 7.4) to 19 vols of the 5.2634 RS:I in “Percoll”. 9.5 ml continuous density gradirnt.s were formed as required from equal volumes of appropriate mixtures of these two solutions using a gradient, former attached to a peristaltic pump. As cell density varied between individuals exploratory gradients were run to establish the optimum for each person. Suitable gradients were in the region of 60-70% “Percoll”. Males tend to have denser cells than females [ 81.

Preparation

of erythrocyte

suspensions

for fractionation

White cells were removed by what is essentially Beutler’s method [lo] by passing the blood through an a-cellulose: microcrystalline cellulose (2 : 1 w/w) column in HEPES buffered isotonic saline (133 mmol/l NaCl, 4.5 mmol/l KC], 10 mmol/l HEPES, pH 7.4). The haematocrit of the filtered blood was adjusted to approximately 30% and the suspension stored at 4°C until used. Blood was filtered within 2 h of collection and fractionated within 4 h of filtration.

Age fractionation 0.5 ml of red cell suspension layered

on

top

of a 9.5-m]

was brought to room temperature and carefully density gradient. Separation was accomplished hy

121

centrifugation at 1100 X g in a swing-out rotor for 5 min at room temperature. The centrifuge was slowly decelerated over 5 min to prevent disturbance of thr~ gradient. Cells do not change their position in the gradient if the centrifuge time is extended, but fractionation deteriorates as there is a tendency for c-ells to clump after longer time intervals. Plasma, which remains at the top of the gradient, was removed and the cell fract,ions then sequentially aspirated from the top of the gradient using a Pasteur pipette attached to a peristaltic pump and manoeuvred manually over the liquid surface. The cells were finally washed free of “Percoll” by 3 washes in HEPES-buffered saline. Tests of age fractionation Mean cell volume (MCV), mean cell haemoglobin (MCH) and mean cell haemoglobin concentration (MCHC) were determined by standard haematologlcal procedures using a Coulter-model S. Reticulocyte counts were perfcrtned by counting at least 1000 cells on films of cells stained supravitally with new methylene blue. A whole slide was scanned before a zero count was registered. Enzyme assays. Washed cells were lysed with sufficient ice cold 10 mmol/l phosphate buffer, pH 7.2, to give a haemolysate of approximately 1.5 g/d1 of until required for assay. haemoglobin. Haemolysates were stored at -20°C Haemoglobin was assayed by its Soret band absorption at 412 nm. Pyruvate kinase was determined as described by Beutler [ 111. Acetylcholine esterase was determined by the Ellman method [ 121 with dithiohisdinitrobenzoic acid and acetylthiocholine at pH 7.4 in a 0.1 mol/l phosphate buffer. Cell potassium was measured by flame photometry of lysates made by the addition of glass-distilled water to cells which had been rapidly washed free of buffered saline with isotonic sucrose. Results

and discussion

As “Percoll” vinylpyrrolidone,

is an alien we have

substance, albeit striven to remain

67.5%

allied to the blood expander polyas near as possible to physiological

Per

Fig. 1. The distribution of erythrocytes after centrifugation on a “Percoll” density gradient. F‘or distribution studies the gradients are separated into 16 fractions of equal volume. In this particular blood sample the mode has a density of 1.096. As there are so few cells in the fractions from the two tails these fractions are pooled to provide sufficient cell numbers for other investigations. With experience it IS passible to collect fractions from a gradient so that, if six fractions are desired, they contain approximately 5%. 10%. 25%. 25’S, 10% and 5% of the total number of cells on the gradient.

122

TOP Fractions

of

bncreosing

dmsity

-----

The distribution of reticulocytes in “Percoll” gradients. After separation and washing as described in the text. the cells were resuspended in autologous plasma prior to staining and preparing the slides for

Fig.

2.

counting.

conditions. Consequently we have adjusted tonicity with physiological levels of sodium and potassium (but have been obliged to omit divalent ions as they cause cell clumping) and added near physiological levels of BSA which assists in the maintenance of normal cell morphology. Young cells remain as discs and the older cells have a reduced central pallor. distribution of In common with other workers [4,5] we observe a normal cells along the gradient (Fig. 1). If single fractions are re-centrifuged on fresh gradients each shows a narrow distribution centred around the predicted density. Reticulocytes are concentrated towards the top of gradients [4,5,13] (Fig. 2), and only very rarely is an occasional reticulocyte seen in the bottom half of a gradient.

01

1 TOP

Total Fractions

Fig. 3. The gradient.

0’

,ncreos,ng

density

decrease in mean cell volume

-

(MCV)

of erythrocytes

fractionated

on a “PercoIl”

density

7 23

: .

5&_LLeLL, Fractions

of

Fig. 4. The increase in mean “Percoll” density gradient.

increos~ng

density

cell haemoglobin

Total

-c

concentration

(MCHC)

of erythrocytes

fractionated

on a

Mean cell volume, mean cell haemoglobin concentration, pyruvate kinase and acetylcholine e&erase activities and potassium contents all changed with age in the same ways and to the same extents as reported by other workers [6,14,15], using different methods (Figs. 3-7). Mean cell haemoglobin content remains constant. It may be concluded from these results that density fractionations by “Percoll” gradients are as good as those achieved by other methods. The major advantages of “Percoll” are: (1) the speed of attaining equilibrium (5 min as compared with 1-2 h); (2) it allows the density distribution of an erythrocyte population to be determined, in particular in pathological cells which may not only be of abnormal shape and flexibility, but may also be denser than normal

. .

.

TOP

Totai

Fractions

St incr-easing

Fig. 5. The correlation between cytes fractionated on a “Percoll”

density

-

increased cell density density gradient.

and decreased

pyruvate

kinase activity

of erythro-

and too dtlnse to be separated on other gradient materials without problems of high viscosity; (3) gradient density may be easily varied at will; and (~1) it is relatively inexpensive and available commercially in a pure. sterile state. The simplicity of the method suggests that it might be adapted for clinical diagnostic purposes, and the quantities of cells separated are sufficient for studying erythrocyte ageing both in intact cells and in cell membranes. Acknowledgements We are grat,eful to many members of the Department of Haematology, ticularly Dr. M.L. Stirling and Mr. Ian Abbott for many helpful discussions

parand

125

technical assistance, and to the Wellcome holds an S.R.C. Research Studentship.

Trust for financial

support.

C.M.R.

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