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Original Papers

Buffy Coat and Cell Separator Granulocyte Concentrates Comparison of Cell Content and in vitro Granulocyte Function

Andrew Hughesa, I . E. Addison", H . Al- Hadithya, B. BrozoviCh a Department of Haematology, University College Hospital. London. UK, and bNorth London Blood Transfusion Centre, Edgware, Middlesex, UK

Abstract. Buffy coat granulocyte concentrates were prepared manually from single fresh whole blood donations after centrifugation. The erythrocyte, granulocyte and platelet num- bers in these concentrates were compared to cell numbers in concentrates obtained by mechanical Ieukapheresis using intermittent flow centrifugation. Buffy coats contained approximately half the number of granulocytes and three times the number of erythrocytes per unit volume than cell separator concentrates. There was no difference in platelet num- bers. Thus to give a therapeutic dose of granulocytes equivalent to that obtained by cell separation, 2-3 times greater volume of buffy coat concentrates would be required.

In vitro granulocyte function, as measured by migration, phagocytosis, and Candida killing was also compared in buffy coat and cell separator concentrates. The only difference found was reduced Candida killing in the buffy coat granulocytes.

The possible clinical use of buffy coat concentrates, as a source of granulocytes, is discussed.

Introduction

The last decade has seen an increase in the median survival of many patients with malignant disease as a result of the more widespread use of treatment regimes involv- ing combination chemotherapy. Many of these regimes are myelosuppressive, and morbidity and mortality used to be asso- ciated with drug-induced thrombocytopenia and leukopenia. Since the advent and ready availability of prophylactic platelet concen-

trate transfusions, they are largely due to infection, mostly but not always, associated with leukopenia [l, 21. Several studies have demonstrated the beneficial effects of gran- ulocyte transfusions in granulocytopenic patients which proved bacterial infection and persistent bone marrow failure [3-51. This has led to their increasing use, therapeutically, and more recently, prophy- lactically [6, 71.

Initially, leucocytes obtained from pa- tients with chronic myelogenous leukaemia

2 Hughes/Addison/ Al-Hadi t h ylBrozoviC

were used [8] but now a variety oftechniques have been developed that allow the procure- ment of adequate numbers of granulocytes from normal donors. They include contin- uous [9] and intermittent flow centrifugation [lo], continuous-flow filtration [ll], and gravity sedimentation [12]. Recently, buffy coat concentrates, prepared from single whole fresh blood donations have been used as an alternative source of granulocytes for transfusion to febrile, granulocytopenic pa- tients. They are obtained during the course of manual platelet concentrate preparation, and contain large numbers of platelets and erythrocytes as well as leucocytes. Very few data are available regarding the cell content and granulocyte hnctions of these concen- trates, Therefore, we have investigated the numbers of erythrocytes, granulocytes and platelets present in these buffy coat prepara- tions and in granulocyte concentrates ob- tained by mechanical leukapheresis. We have also investigated in vitro granulocyte functions in cells obtained from both sources.

Materials and Methods

Buffy coat concentrates were prepared at the North London Blood Transfusion Centre. Fresh whole blood, collected into triple bags (Fenwal R1701) was centri- fuged in a Beckman 56 centrifuge at 4,200 rpm for 12 min at 20'C. Cell-free plasma was expressed manually into one of the satellite bags. The platelet-rich plasma and bufi coat were expressed into a second satellite bag, which was clipped off and issued as a single buffy coat unit.

Granulocyte concentrates were prepared at Univer- sity College Hospital, by mechanical leukapheresis us- ing intermittent-flow centrifugation (Haemonetics Model 30), and a method previously described [lo]. All donors were volunteers, either relatives of patients or non-related. They received 12 mg of dexamethasone

orally 6 h prior to leukapheresis. Hydroxyethyl starch was used as a red cell sedimenting agent during the procedure.

Red cell, total white cell and platelet counts were performed on samples from both buffy coat and cell separator concentrates using a Coulter Model S plus. White cell differential counts were performed manually on all samples following standard May-Griinwald- Giemsa staining.

In vitro granulocyte functions were tested using an adaptation of whole blood techniques [13, 141. Granu- locytes were added without prior treatment to recipient blood (essentially granulocyte free, as all patients were severely granulocytopenic with less than 0.1 x 109/litre granulocytes) to give white blood cell concentrations of 5 x 109/litre. Leucocyte containing blood was then mixed with Hanks's balanced salt solution in 1 : 1 ratio. Locomotion was measured by placing the diluted blood in plastic caps holding about 0.25 ml which were then inverted onto 3 y m pore size membranes resting on filter paper soaked in medium. After 24 h of incubation at .37 'C in a moisture chamber, the membranes were rinsed, fixed, stained and cleared so that migration could be estimated by the leading front method [IS]. Phagocytosis and killing were measured using Candida guilliermondiue [16]. 0.25 ml of diluted blood was mixed with 5011 of yeast to give a constant yeast: gran- ulocyte ratio. The mixture was incubated in plastic tubes with end-over-end rotation for 60 min at 37'C. Cytocentrifuge preparations were stained with May- Griinwald-Giemsa, and the number of yeast phagocy- tosed per 100 granulocytes and the percent of the latter showing colour changes indicating cell death esti- mated.

Results

Erythrocyte, granulocyte and platelet numbers in the buffy coat and cell separator concentrates are shown in table 1. The cell separator concentrates (average volume 500ml) contained more than double the number of granulocytes per unit volume compared to the buffy coats (mean volyme 61 ml) and one third the number of erythro- cytes. There was no difference in platelet numbers. In vitro granulocyte hnction is

Buffy Coat and Cell Separator Granulocytes 3

Table I. Erythrocyte, granulocyte and platelets numbers in buffy coat and cell separator concentrates (means f SD)

Erythrocytes x Io’*/litre

Granulocytes x 10’~itre

Platelets x 1o9/1itre

Buffy coat concentrates Cell separator concentrates

3.02 i 0.95 (n= 17) 1.2f 0.55* (n=16)

8.5 f 3.9 (n= 17) 19.4 f 1 1 * (n=18)

891 f 409 (n=16) 990 f 415 (n=13)

p < 0.001 (Student’s f test).

Table 11. In vitro granulocyte functions in buffy coat and cell separator concentrates (means f SD)

Migration Phagocytosis Killing distance through Candidall00 96 of ingested membrane, pm granulocytes Candida dead

Buffy coat concentrate 9 0 f 19 158 f 83 41 f 11 (n=5) Cell separator concentrate 89 f 19 160 f 76 64 f 10* (n=5)

~~

* p <0.001 (paired Student’s t test).

shown in table 11. The only difference was reduced Candida killing in the buffy coat granulocytes .

Discussion

It has been suggested that an adequate therapeutic dose ofgranulocytes should con- tain at least loio cells [17]. Our study indi- cates that this can be achieved by the ad- ministration of one donation obtained by mechanical leukapheresis (volume 500 ml), or by 20 single buffy coat units (volume

1,100-1,200 ml). Thus more than twice the volume of buffy coats would need to be administered, which could lead to problems with, first, fluid overload, and second, unde- sirable polycythaemia, especially in non- anaemic patients, as the buffy coats contain a considerable number of erythrocytes per unit volume.

Poon and Wilson [18] recently reported a method for preparing granulocytes man- ually from fresh, concentrated red cells using hydroxyethyl starch to enhance red cell sedi- mentation. Using this technique a donation of 1010 granulocytes could be harvested from 4-6 single donor units of red cell concen-

4 Hughes/Addison/Al-Hadit hy/Brozovii

trate. While it would appear to produce an acceptable granulocyte containing product, no information is given as to the fate of the red cells which are presumably discarded. Buffy coat preparation on the other hand allows the full utilisation ofboth plasma and red cells, and represents a more efficient use of whole blood.

Granulocytes collected by centrifugation techniques have been shown to have normal in vitro function [19]. Poon and Wilson in their study reported only that granulocytes appeared morphologically normal, and were NBT-positive at 24 h [18]. In our study we found no differences between buffy coat and cell separator granulocytes as regards migra- tion and phagocytosis, but there was reduced Candida killing in the buffy coat cells (ta- ble 11).

The clinical significance of this decrease in killing is not clear. However, in vitro func- tions in both preparations were of the same order as found in granulocytes prepared in the laboratory from normal subjects, and the whole blood technique employed in our study demonstrated the ability of the cells from both sources to function adequately in the recipients' plasma.

There is an increasing demand for the therapeutic and prophylactic use ofgranulo- cyte concentrates for patients with bone marrow failure. Currently, granulocytes are largely obtained using mechanical leuka- pheresis techniques. They involve high cap- ital expenditure and operating costs, and require highly trained personnel. The tech- niques are all time consuming and can be limited by the lack of availability of suitable donors. Donors are also exposed to the po- tential hazards of high molecular weight sedimenting agents and steroid premedica- tion. The use ofbuffy coat concentrates pre-

pared from single donor packs as an addi- tional source of granulocytes may help to meet this increasing demand. There is a potentially unlimited supply of donors who are not exposed to sedimenting agents or ste- roids; capital expenditure and running costs are low; the time involved is usually less; and personnel with no special training can do the work of buffy coat preparation.

At present there are two main disadvan- tages in using buffy coats. Firstly, their use would require the administration of large volumes of cell concentrates over a period of several days. However, buffy coats on aver- age contain 0 . 5 ~ 10" platelets per unit (equivalent to one single donor platelet unit), and thus their administration obviates the necessity to transfuse platelet concen- trates to the same patient. In addition the erythrocyte content could benefit patients where anaemia was present as well as throm- bocytopenia and granulocytopenia. Sec- ondly, the use of buffy coats prepared from multi-donor sources can be expected to stimulate the production ofHLA antibodies, even in immunosuppressed patients. This could result in transfusion reactions, and shortened survival and thus decreased effec- tiveness of the infused leucocytes. However, in our experience the use of buffy coats was not associated with an increase in post-trans- fusion reactions, and appeared as effective as single-donor granulocyte concentrates in lysing fever, whether the latter were HLA- matched or not. Prospective clinical studies remain to be camed out.

We have shown that buffy coats are an additional source of active granulocytes, provided that a sufficient number of units are administered, for despite their reduced killing ability buffy coat granulocytes retain a fair measure of functional integrity.

Buffy Coat and Cell Separator Granulocytes 5

Acknowledgement

We would like to thank Mrs. R.E. Wenzerul for typing the manuscript.

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Received: September 9, 1980 Accepted: February 2, 1981

Dr. Andrew Hughes, Haemophilia Unit, Royal Free Hospital, Pond Street, London, NW 3 (UK)