Human erythrocyte separation according to age on a discontinuous ‘Percoll’ density gradient

293

Clinica Chimica Acta, 122 (1982) 293-300 Elsevier Biomedical Press

CCA 2148

Brief technical

Human

note

erythrocyte separation according to age on a discontinuouS ‘Percoll’ density gradient Giuseppe Salvo *, Patrizia Caprari, Paola Samoggia, Gualtiero Mariani and Anna Maria Salvati

Reparto di Biochitiica

Ematologica,

(Received

Laboratorio di Patologra non Infettiva, Roma, Rome (Italy)

November

17th. 1981; revision

February

Istituto Superiore di Samtir

1 Ith, 1982)

Introduction It is well known that during ageing, erythrocyte density increases [l-3]. Density gradient centrifugation is the technique most generally used for fractionating erythrocytes of different mean age. Materials employed to construct density gradients, such as bovine serum albumin (BSA) [4], Stractan II [5], Ficoll [6] and Dextran [7], have many disadvantages. The development of Percoll (colloidal silica particles coated with polyvinylpyrrolidone) has overcome these problems. Owing to its low viscosity, low osmotic pressure and non-toxicity this material can be easily adjusted to physiological conditions. Percoll methods allow separation of erythrocytes on discontinuous gradients [&lo], linear gradients [ 111, or self-generated gradients [ 121. In this work, red cell separation in a discontinuous gradient similar to that reported by Alderman et al. [9] is described. To obtain Percoll with the closest approximation to physiological conditions, a suitable modification of Rennie’s buffer system [ 1 l] has been used. Separated red cell populations have been tested for cell recoveries, reticulocyte count and some age-dependent erythrocyte enzyme activities (pyruvate kinase, hexokinase, glucose-6-phosphate dehydrogenase, aldolase) and indices (mean cell volume and red cell volume distribution width index, mean cell haemoglobin concentration). Two non age-dependent measurements were also made (phosphoglycerate kinase and mean cell haemoglobin). The effectiveness and reliability of the method have been evaluated.

* Address correspondence to: Dr. Giuseppe Salvo, Laboratorio Superiore di SanitB Viale Regina Elena 299, 00161 Rome Italy.

di Patologia

non

Infettiva,

lstituto

294

Materials and methods Collection and storage of blood samples Blood samples were collected by healthy donors with informed consent.

venepuncture on K ,EDTA from Samples were used immediately.

20 male

Isolation of red cells from blood samples Blood samples were filtered through an cu-cellulose-microcrystalline cellulose (1 : 1. w/w) column in HEPES buffered isotonic saline [ 111. The haematocrit of filtered cells was adjusted to about 50% with the same buffer. Preparation

of discontinuous

gradients

2.66 mol/l HEPES buffered stock soiution (NBS stock) 0.2 mol/l HEPES (N-2-hydroxyethylpiperazine-N-2-ethane

NaCl, 0.09 mol/i KCl, sulphonic acid), pH 7.4.

Solutions BSA-HEPES buffered solution, pH 7.4, 7r 265 mOsm/kg H,O at 3.5% BSA final concentration. This solution is obtained by adding 19 ~01s. of BSA (Cohn fraction V, Sigma Chem. Co.) in water (pH 7.4) to 1 vol. of HBS stock. SolutionB BSA-Percoll-HEPES buffered solution, H,O at 3.5% BSA final concentration. This solution is of BSA in Percoll (Pharmacia Fine Chemicals) to 1 vol. Solutions A and B were mixed to form four solutions tions of 60% 63%, 66% and 69% (d 1.075-1.100, pH H,@ Discontinuous four-step gradients were prepared Percoll concentration using a peristaltic pump.

pH 7.2, rr 330 mOsm/kg obtained by adding 19 ~01s. of HBS stock. at final Percoll concentra7.3, r 310-320 mOsm/kg

by superimposing

6 ml of each

Erythrocyte separation Four ml of filtered red cell suspension containing about 20-24 X lo9 cells were layered on top of the gradient. Centrifugat~on was carried out in a Sorvall RC-2B in an angle rotor (SS-34 rotor) at 1000 X g for 10 min at 20°C. Cell fractions were collected by aspiration with a peristaltic pump and washed three times with HEPES buffered isotonic saline at 4°C to remove Percoll. Reticulocytes Reticulocyte counts were performed 1000 cells stained with new methylene

within 2 h of drawing the blood by counting blue (MediChem, reticulocyte stain set}.

Red cell parameters Mean cell volume (MCV), mean cell haemoglobin (MCH), mean cell haemoglobin con~ntration (MCHC) and red cell volume distribution width index (RDW) were determined using a Couiter Counter Model S Plus.

295

Erythrocyte enzymes Assays of pyruvate kinase (PK), hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PD), aldolase and phosphoglycerate kinase (PGK) activities were performed as described by Beutler et al [13]. Determinations were performed at 37’C using a Beckman Acta M VI spectrophotometer adapted for continuous measurements. Density and osmolality Relative density was measured at 20°C by weighing the solutions precalibrated with distilled water on an analytical balance. Osmolality was measured in a Roebling Micro-Osmometer.

in a 50 ml flask,

Results

The discontinuous Percoll gradient produces five red cell fractions; Fig. 1 shows an example of replicated separations on the same blood sample. Fig. 2 shows the enzyme activities and erythrocyte indices in the fractions. Mean and standard deviation values (n = 20) obtained for each fraction are reported. A marked decrease of PK and HX activities is observed between the top and bottom layers; G6PD and aldolase decrease less markedly and PGK is steady. MCHC tends to increase, MCV to decrease, except in the 5th fraction, while MCH remains virtually constant. RDW decreases in the first three fractions but increases in the others; moreover it shows greater variation in the lst, 2nd and 5th fractions. The enrichment in reticulocytes obtained in the fractions is illustrated in Fig. 3. The precision of the method, evaluated by duplicate measurements on 20 samples, and the mean pooled coefficients of variation (pooled CV) are given in Table I. The

Fig.

1. Red cell separation on four-step Percoll density gradient. Replicates of the same sample.

296

Fig. 2. Behaviour of red cell PK, HX, G6PD, aldolase activities (% of whole blood activity), mean cell volume (MCV), mean cell haemoglobin concentration (MCHC), mean cell haemoglobin (MCH) and red-cell volume distribution width index in five fractions (X 2 SD).

total variation, including methodological and individual variation was evaluated by the mean coefficients of variation (m) of the haematological measurements (Table II). The mean recovery of cells in these five bands (% of total cells applied to the gradient) was respectively: 2.9, 7.1, 32.6, 24.0, 33.4 and their mean pooled coefficient

% of total

RETICULOCYTES

1

7 i I

I

I

1

1

2

3

Fig. 3. The reticulocyte reticulocyte separation

i

*

4

5 Cell fractions

enrichment in five fractions of the gradient. obtained by one-step Percoll gradient.

The figure also shows an example

of

297

TABLE

I

MEAN TIONS

VALUES

OF

POOLED

Erythrocyte

COEFFICIENTS

OF VARIATION

IN THE

RED

CELL

FRAC-

Red cell parameters

enzymes

PK

HX

G6PD

Aldolase

PGK

MCV

MCH

MCHC

RDW

9.9

8.9

1.3

6.0

6.1

1.7

3.4

4.9

6.4

Pooled 3 (%)

of variation (pooled s) and mean coefficient of variation (CV) were 9.5% and 49.8%. To evaluate the differences between the fractions and the discriminating power of each measurement, analysis of variance and simple contrasts between subsequent fractions were performed (Table III). The analysis of variance shows significant differences for all measurements, except PGK and MCH; however the simple contrasts between subsequent bands underline the different behaviour of the indices. Discussion The method described employs a discontinuous gradient of buffered Percoll containing physiological levels of sodium and potassium. Divalent ions were avoided to prevent cell clumping and Hepes-BSA buffer has been used since it helps to maintain normal cell morphology [ 111. A similar method for red cell separation was described by Alderman [9], but Dulbecco’s buffer was employed. Further modifications in our procedure are: (a) maximum sample loading is less than 25 X lo9 cells per tube to avoid overlapping between fractions and mixing between steps; (b) red cells are obtained free of leucocytes and platelets by filtration on microcrystalline cellulose [ 11,131, since the removal of “buffy coat” should bring about a depletion of the least dense RBC subpopulation [9]. Our procedure does not damage cells, is simple and inexpensive. Tube size is not critical and a short centrifugation time is required.

TABLE

II

MEAN

VALUES

OF COEFFICIENTS

Erythrocyte

cv

(%)

OF VARIATION

enzymes

IN THE RED CELL

FRACTIONS

Red cell parameters

PK

HX

G6PD

Aldolase

PGK

MCV

MCH

MCHC

RDW

19.2

28.2

14.3

14.1

7.6

7.0

6.8

8.4

13.2

I.4 6.9 13.0

NS, not significant.

co.05

NS
3.0

<0.001 <0.001 10.001 CrO.001 NS

MCH MCHC RDW

61.9 18.2 15.3 12.8 1.2

P

I-2 l-2 1-2 I-2

1-2 l-2 l-2 1-Z l-2

p
NS NS NS p CO.01

p -==0.05 p
2-3 2-3 2-3 2-3 2-3 NS NS p
3-4 3-4 3-4 3-4

3-4 3-4 3-4 3-4 3-4

NS NS NS NS

p co.01 NS NS NS NS

AND SIMPLE CONTRASTS

p
IN THE FRACTIONS

Simple contrasts (Student’s t test)

OF RED CELL PARAMETERS

MCV

PK HX G6PD Aidolase PGK

F

Analysis of variance

ANALYSIS OF VARIANCE

TABLE III

4-5 4-S 4-5 4-5

4-5 4-5 4-5 4-5 4-5

NS NS NS p
p
BETWEEN SUBSEQUENT

BANDS

299

The characteristics of the method produce a virtually constant band position (Fig. 1). The effectiveness of the method is demonstrated by the most expressive age-related measurements [3,15,16]: reticulocyte count, HX and PK activities (Figs. 2 and 3). Furthermore, the analysis of variance and simple contrasts (Table III) performed on HX and PK values show significant differences between our five fractions. A satisfactory reproducibility is demonstrated by the pooled CV of recoveries and of red cell indices in the fractions (Table I). In all cases these values are less than 10%. These results cannot be compared with the data reported by other authors using Percoll in similar conditions [9,11], since they did not perform a statistical evaluation. However the reticulocyte enrichment and the behaviour of red cell determinations are in agreement with previous findings [3,5,11,17]. The analysis of total variation (Table II), the analysis of variance and simple contrasts (Table III) indicate the main features which must be taken into account when red cell age-separation is employed. The high CV value (49%) obtained for red cell recoveries in the fractions demonstrates marked differences between normal individuals. This finding is well known [4] and might be explained by even small differences in individual frequency distribution of red cell densities. Enzyme activities show CV values (Table II) which seem to increase with the rate of the decline with ageing. In fact HX and PK have the highest CV, whereas PGK has the lowest one. The analysis of variance and simple contrasts of these 5 fractions are in agreement with a different decline [2,3] with ageing of the enzyme activities studied (PK = HX > G6PD > aldolase). Our data on red cell measurements confirm previous observations [3,5,11,17], but some new features are pointed out. With regard to enzyme activities, PK and HX have a comparable age dependence, the former being more suitable as an index of actual age fractionation, and presenting less variation due to the better precision of the assay [ 181. MCV does not show such a marked age dependence and should be used with care as a marker of red cell age [ 111. Furthermore it decreases from the first to the fourth fraction, then increases in the last one. Opposite behaviour was observed for MCHC. This is probably due to the fact that normal older red cells tend to become spherical [19,20]. The MCV changes could also explain the discordant results obtained when ‘top’ and ‘bottom’ separations were employed [6]. RDW shows a similar trend (Fig. 2) to MCV, but greater variation in the first, second and fifth fractions. These fractions are rich in reticulocytes and the oldest cells. The procedure described here can be useful for haematological studies on normal and pathological blood samples. The number and density of steps can be suitably chosen in order to obtain either reticulocyte (Fig. 3) or oldest cell enrichment. The reliability of the method is strictly dependent on the standardization of working conditions (temperature, reagents, pH, osmolarity, density). PK seems to be the most useful marker for erythrocyte age fractionation.

300

Acknowledgements The authors

are indebted

to Ms. Caterina

Tripodi

for secretarial

assistance.

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