Vox Sang. 42: 145-150 (1982)

Leucocyte-Depleted Blood : A Comparison of Cell-Washing Techniques

Andrew Hughes, Valerie Mijovid, B. BrozoviC, T. D. Davies North London Blood Transfusion Centre, Edgware, UK

Abstract. Non-haemolytic febrile transfusion reactions are common in multi-transfused patients. It is generally accepted that the majority of these reactions occur when more than 0 . 5 ~ 1 0 ~ leucocytes are transfused. Values equal to or lower than this threshold, which we have called for convenience the critical antigenic load for leucocytes (CALL), can be achieved by decreasing the leucocyte content in one unit of whole stored blood by about 80%. We have compared the efficiency of leucocyte depletion of whole stored blood, using fixed-speed centrifugation (Haemonetics Model 15 and Model 115 cell washer), variable- speed centrifugation (Dideco Progress 90 Cell Separator), and serial centrifugation (IBM 2991 Blood Cell Processor). Fixed-speed centrifugation was least effective in depleting leu- cocytes; a reduction of 25 and 60% was achieved using the Haemonetics Model 15 and Model 115, respectively. Variable speed and serial centrifugation produced more satisfactory results with leucocyte depletions of82 and 89% using the Dideco Progress 90 Cell Separator and the IBM 2991 Blood Cell Processor, respectively. Platelet depletion of over 90% was achieved with all the cell washers, except the Haemonetics Model 15. Red cell losses varied from 3 to 30%. It seems unlikely that consistently high (over90%) leucocyte depletion can be obtained using cell-washing techniques without associated high red cell losses.

Introduction

Non-haemolytic febrile transfusion reac- tions are common in multi-transfused pa- tients. These reactions are usually caused by antibodies directed against leucocytes, often with HLA specificity. They can be pre- vented or modified by the transfusion of leu- cocyte-poor red cells. A variety of tech-

niques exist for depleting red cells of leuco- cytes, such as centrifugation, sedimentation, filtration, saline washing and freezing of red cells. Several cell-washing systems are now commercially available involving a number of different protocols, and these have been evaluated previously [Bryant et al., 1978 ; Buchholz et al., 1978 ; Meryman et al., 1980 ; Polesky et al., 1973 ; Reverberi and Fabbri,

146 Hughes/MijoviC/Brozovii./Davies

1979; Wenz et al., 19801. We have compared three red-cell-washing systems, each em- ploying different principles, with respect to their effectiveness in removing leucocytes.

Materials and Methods

Units of whole blood collected into Fenwal CPD Single Packs (FKR 0994), which were found unsuitable for hospital use, were stored at 4 ‘C for 4-13 days (mean 8 days) prior to washing.

The efftciency of the washing techniques was deter- mined by measuring the absolute numbers of leuco- cytes, platelets and red cells present in the unit ofblood before and after washing. The packs were weighed, thoroughly mixed, and sampled before and after wash- ing. Erythrocyte, total leucocyte and platelet counts were determined using an electronic cell counter (Coulter Model S Plus). Percent depletion was then calculated from the absolute cell numbers. Total pro- tein determinations were carried out before and after washing by the standard biuret method using a Vickers D.300 analyser.

Haemonetics Models 15 and 115 Cell Washers (Fixed-Speed Centrgugation) The Haemonetics cell-washing systems employ a

rigid disposable polycarbonate bowl of 375 ml capacity which is tilled with blood and saline under gravity through a central inlet port. The geometry of the bowl ensures that, when centrifuged at a constant speed of 4,800 rpm, the red cells and the buffy coat are sus- pended round the perimeter of the bowl by centrifugal force and continually perfused by incoming saline. The saline solution and accompanying plasma, some leuco- cytes and platelets, return to the waste bag through an outlet port located near the axis of rotation. The essen- tial difference between the model 15 and the model 115 is the shape of the washing bowl, the capacity being the same in both models. The manufacturer’s directions were followed and 1,500ml of saline was used to wash each unit of blood.

Dideco Progress 90 Cell Separator (Variable- Speed Centrifugation) This system also employs a rigid, disposable poly-

carbonate bowl and the principle of continuous cen-

trifugation. However, the facility to change the centri- fugation speed during the washing procedure allows the washing of the red cells in cycles [Reverberi and Fabbri, 19791. Whole stored blood was run into the bowl of 225 ml capacity (bowls of 125 and 325 ml capacity are also available) under gravity with the centrifugation speed set at 4,800 rpm. When all the blood had been run in, saline was added at approximately 20-30 ml/min under gravity until a stable buffy coat had been estab- lished. The saline flow was then rapidly increased to maximum gravity flow (approximately 200 ml/min) and the centrifugation speed s1owl.v (over 30-60 s) de- creased until the buffy coat became unstable and indis- tinct. This renders the interface between the leucocyte layer and the erythrocyte layer unstable and permits more effective depletion of white cells. The centrifuga- tion speed was then rapidly decreased and increased by about 200 rpm, below and above the instability speed, thereby allowing the buffy coat and some red cells to be washed over into the waste bag. This rapid oscillation above and below the instability speed was repeated 5 times, after which the centrifugation speed was rapidly returned to 4,800 rpm, the saline flow was simultane- ously decreased to 20-30ml/min. The buffy coat was then allowed to reform and stabilize. This whole pro- cedure comprises one washing cycle.

Three variations on this standard protocol were used: (A) three wash cycles with 5 min between each cycle for buffy coat stabilization; (B) six wash cycles with 5min between each cycle, and (C) three wash cycles with 1 min between each cycle.

IBM 2991 Blood Cell Processor (Serial Centrifugation) The IBM 2991 Blood Cell Processor utilises the

principle of serial centrifugation, with intermittent pe- nods of agitation during which saline is added. The supernatant is expressed from a flexible bag by hydrau- lically raising a rubber diaphragm in the centrifuge chamber. A photocell, sensitive to the passage of red cells, monitors the effluent washing solution and pre- vents the red cells from leaving the bag. However, a red cell override is incorporated which permits the empty- ing phase to be extended by up to 7 s, thus increasing the removal of leucocytes entrained in the top ofthe red cell layer. The procedure used has been previously de- scribed [Bryant et al., 1978; Eucholz et al., 1978; O’Connor Wooten, 19761. The IBM 2991 Blood Cell Processor was used in automatic mode, with the follow- ing dial settings: centrifugation speed: 3,000 rpm, dis-

Leucocyte Depletion of Blood by Washing Techniques 147

Table I. Leucocyte depletion ofblood

Cell-washing Number of Absolute number of leucocytes Depletion procedure blood units (Mean k SD) (Mean f SD)

processed % before after processing processing x 10’ x 109

Haemonetics Model 15 10 Model 115 10

Progress 90 Cell Separator

Protocol A Protocol B Protocol C

10 5

10

IBM 299 1 Blood Cell Processor

Protocol A 4 Protocol B 4 Protocol C 5 Protocol D 5

2.8 f 0.7 2.1 k 0.5 26 f 1 2 2.2 f 0.5 0.9 k 0.6 62 f 23

2.2 f 0.7 0.5 f 0.3 75 f I 1 2.6 f 0.6 0.4 f 0.2 82 f 14 2.3 f 0.6 0.6 f 0.4 75 f 19

3.0 f 0.5 0.5 f 0.2 8 5 f 7 3.1 f 0.6 0.3 f 0.05 8 9 + 4 3.2 f 0.7 0.6 k 0.3 8 2 + 7 2.9 f 0.7 0.4 f 0.2 8 7 + 7

charge rate of supernatant: 450 ml/min, minimum agitation time: 60 s, maximum supernatant volume:

protocols were used, each comprising three washing

RCO set at 3 s for each of the first two cycles only, i.e. expresion of the plasma and the supernatant from the first but not the second wash cycle (designated 3,3,0); (B) RCO set at 3 s for each of the last two cycles (desig- nated0,3,3);(C) RCOsetat 3sforall threecycles(desig- nated 3,3,3), and (D) RCO set at 4s for all 3 cycles (designated 4,4,4).

100

600m1, first spin 2.5 min and second spin 2.0min. Four 80

cycles with varying red cell override (RCO) times: (A) 60

40

20 c z

2 Y k

LEUCOCYTES RED CELLS PLATELETS REMOVED LOST REMOVED

Fig. 1. Leucocyte depletion of blood. Mean results Results obtained using Haemonetics Model 15 and Model 115

cell washers are represented by columns A and B. The

The results of erythrocyte, leucocyte and standard deviations are represented by vertical bars.

platelet depletion using the three cell-wash- ing techniques are presented in table I and in erate. All the protocols on the Progress 90 figures 1-3. The white cell depletion Cell Separator and the IBM 2991 Blood Cell achieved with the Haemonetics Model 15 Processor produced good leucocyte deple- and Model 115 cell washers was only mod- tion, bringing down the absolute number of

148 HugheslMijoviC/BrozoviC/Davies

100 r

I T ' ' T

.EUCOCYTES REMOVED

RED CELLS LOST

PLATELETS REMOVED

1

Fig. 2. Leucocyte depletion of blood using Dideco Progress 90 Cell Separator. Mean results obtained with protocols A, B and C are represented by columns, A, B and C . The standard deviations are represented by ver- tical bars.

loo I

20 I- z Y " Y

I

LEUCOCYTES REMOVED

I

I ! RED CELLS

LOST PLATELETS

REMOVED

D -

Fig. 3. Leucocyte depletion ofblood using IBM 2991 Blood Cell Processor. Mean results obtained using pro- tocols A, B, C and Dare represented by columns A, B, C and D. The standard deviations are represented by ver- tical bars.

leucocytes per unit of blood to about 0.5 x lo9.

Plasma protein removal which occurs during washing was also determined on a small number of units of blood. Using the Progress 90 Cell Separator and protocols A

and C, the mean protein loss was 90.1% (n=3) and 65.0% (n=3), respectively; using the IBM 2991 Blood Cell Processor and pro- tocol A, it was 85.3% (n=6).

Discussion

A major difficulty in the clinical evalua- tion of the in vitro techniques used for leu- cocyte depletion of blood is that there is a different absolute number of leucocytes re- quired to cause a non-haemolytic febrile transfusion reaction in each sensitized pa- tient [Perkins et al., 19661. However, Perkins et al. [1966] suggested that if less than 0 . 5 ~ 1 0 ~ leucocytes are transfused to a pre- viously sensitized patient, the majority of non-haemolytic febrile reactions, defined as a rise in temperature of at least 1 "C, can be prevented. We have arbitrarily called this threshold (0.5~10~) the critical antigenic load for leucocytes (CALL) in order to pro- vide a convenient reference point for relating in vitro leucocyte depletion to an in vivo end-point. In our study, the mean value for the absolute leucocyte count in stored whole CPD blood was 2.4 k 0.8 x lo9. Thus a leuco- cyte depletion of approximately 80% is re- quired to decrease the number of leucocytes per unit to below the CALL.

Semi-automated cell-washing techniques were originally introduced to remove the glycerol used as cryopreservative from red cells which had been preserved by freezing. These techniques were assumed to be capa- ble of depleting fresh blood of leucocytes. Manual washing techniques could remove up to 80% of leucocytes with a 15 -20% red cell loss [Polesky et al., 1973; Crowley and Valeri, 1974; Langfelder et al., 19701, but

Leucocyte Depletion of Blood by Washing Techniques I49

were tedious and time consuming. However, with semi-automated systems such as the Haemonetics Model 15 and the Elutramatic, leucocyte depletion was found to be less effective, removing 50 and 70% of leuco- cytes, respectively [Polesky et al., 1973; Re- verberi and Fabbri, 1979; Crowley and Va- leri, 19741. A modification of the Haemone- tics system, the Haemonetics Model 102, has produced variable results ranging from 68 to 85% leucocyte depletion [Meryrnan et al., 1980; Wenz et al., 19801. The introduction of serial centrifugation with the IBM 2991 Blood Cell Processor has increased leuco- cyte depletion to about 90% but with red cell losses ranging from 10 to 25% [Bryant et al., 1978; Bucholz et al., 19781. Recently a vari- able speed centrifugation system, as opposed to the previous fixed speed techniques, has been introduced (Dideco Progress 90 Cell Separator) with leucocyte depletion ap- proaching 90% [Reverberi and Fabbri, 19791.

The time taken to wash 1 unit of blood was similar for all protocols that were used (20-30 min per unit), except for protocol B with the Progress 90 Cell Separator, which comprises six wash cycles, where the time taken per unit was twice as long. The cost of disposable software for all techniques is comparable if 3-4 units are to be washed, but is less with the IBM 2991 Blood Cell Processor if only 1 unit is processed. The results we obtained are in good agreement with those previously reported by others. On the basis of our results, it appears unlikely that a further improvement in the efficiency of leucocyte depletion can be achieved with cell washers available at present. The differ- ence between specific densities of leucocytes and erythrocytes is too small to allow their complete separation, therefore protocols de-

signed to provide a high degree of leucocyte depletion would invariably incur high red cell losses. Although additional units of blood can be easily processed to make up for the loss of red cells, the practice may be pro- hibitively wasteful to use routinely. The ex- isting protocols, however, may be further improved by using buffy-coat-free packed cells, but this would increase the time and effort involved and would lead to further red cell losses.

Centrifugation of blood and manual re- moval of the buffy coat can produce leuco- cyte depletion of up to 80% [Meryrnan et al., 1980; Polesky et al., 1973; Tenczar, 19731, but although it is technically simpler and less expensive than cell-washing procedures, it produces less consistent results. In addi- tion, red cell losses are usually greater. The recently described filtration techniques [Ki- kugawa and Minoshima, 1978; Sirchia et al., 19801 can produce consistently greater than 95% leucocyte depletion with relatively small red cell losses, but cell washing is as- sociated with more efficient platelet and plasma removal.

It would appear from our results that fixed-speed continuous-centrifugation cell- washing techniques are the least effective as regards leucocyte depletion. Both variable- speed and serial centrifugation produce ac- ceptable leucocyte depletion with regard to CALL. However, the efficiency of the Prog- ress 90 Cell Separator depends to an extent on the skill of the operator, while the IBM 2991 Blood Cell Processor is fully auto- mated. This may result in a more consistent leucocyte loss. It seems unlikely that the cur- rently available cell-washing techniques can produce leucocyte depletion which is consis- tently greater than 90% without associated high red cell losses.

150 Hughes/MijoviClBrozoviC/Davies

Acknowledgements

We would like to thank Mr. F. Fellingham in the Department of Haematology, University College Hos- pital for the automated cell counts, and Mrs. R. E. Wen- zerul for typing the manuscript. We would also like to acknowledge the support provided by Haemonetics (UK) Ltd. Definox Ltd. and IBM (UK) Ltd.

References

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Received: July 14, 1981 Accepted: October 26, 1981

Dr. Andrew Hughes, Haemophilia and Haemostasis Unit, Royal Free Hospital, Pond Street, London NW3 (UK)