q 2001 Blackwell Science 389

Transfusion Medicine, 2001, 11, 389±390

CORRESPONDENCE

Interleukins 1b, 6, 8 and tumour necrosis factor a do not induce

platelet activation

Concentrations of cytokines, IL-1b, IL-6, IL-8 and TNF-a,

progressively increase during storage of nonfiltered platelet

concentrates (PCs) containing residual leucocytes, reaching

high levels on Day 5 of storage [median: 0´6±12 ng mL21;

maximum: 1´9±200 ng mL21 (Muylle et al., 1993; Stack &

Snyder, 1994)]. It was demonstrated that the plasma rather

than cellular components of leucocyte-replete PCs causes

febrile, nonhaemolytic transfusion reactions in thrombocy-

topenic patients; a strong positive correlation was observed

between the concentrations of IL-1b and IL-6 and the

incidence of the adverse reactions, and between the levels of

these cytokines and the leucocyte count in PCs (Heddle

et al., 1994). Some reports indicated that IL-6 and IL-8

induce activation of platelets in fresh platelet-rich plasma

(Oleksowicz et al., 1995; Lumadue et al., 1996), suggesting

that cytokines liberated from residual leucocytes in

nonleukoreduced PCs may potentially contribute to platelet

storage lesion by inducing platelet activation.

In this study, we assessed the effect of IL-1b, IL-6, IL-8

and TNF-a on platelet activation in prestorage-leukode-

pleted PCs by measuring P-selectin (CD62) expression, a

very sensitive marker of platelet activation in stored PCs

(Leytin et al., 2000a). Five PCs were prepared by the

Canadian Blood Services using the platelet-rich plasma

(PRP) technique and citrate phosphate double dextrose

(CP2D) anticoagulant, stored under standard blood

banking conditions up to 5 days, and diluted to a final

concentration of 2 � 106 platelets mL21 with the incuba-

tion buffer (phosphate-buffered saline, 10 mmol L21

HEPES, 0´1% bovine serum albumin, 1 mmol L21 MgCl2,

20 mmol L21 CaCl2, 2´5 mmol L21 tetrapeptide GPRP,

pH 7´4). A specific cytokine was added in final concentra-

tions of 1±270 ng mL21 to incubation mixtures containing

105 platelets and antibodies against P-selectin (anti-CD62-

PE) and glycoprotein Ib (anti-CD42b-FITC), and incubated

for 5 min at 37 8C; then buffer or human a-thrombin was

added to the final concentration of 0´025 U mL21 and

incubated for additional 10 min at 37 8C. Samples were

fixed with paraformaldehyde and analysed by flow

cytometry (Leytin et al., 2000b).

Our data show that none of four cytokines at very high

concentrations up to 270 ng mL21 affected CD62

expression in PCs stored up to 5 days, neither in the

absence nor in the presence of submaximal dose of

Fig. 1. Cytokines do not affect CD62 (P-selectin) expression

in prestorage-leukodepleted PCs in the absence and in the

presence of thrombin. Five PCs stored for 2±5 days were

studied, means ^ SD are presented; nucleic acid testing

(NAT) implemented in Canada prevent us from analysing

Day 1 PCs. (a) Data for IL-1b are shown; similar results

were obtained for IL-6, IL-8 and TNF-a. (b) Combined

results for all storage days are shown. Note that prestorage-

leukodepleted PCs maintain their functional response to

submaximal (0´025 U mL21) concentration of thrombin at

any day of storage (P , 0´01) but do not respond to very

high (100 ng mL21) concentration of cytokines (R & D

Systems, Minneapolis, MN), neither in the absence nor in

the presence of thrombin (P . 0´05). The other parameters

of CD62 expression [the mean fluorescence of CD62-

positive and total cells, and the index of platelet activation

of positive cells (Leytin et al., 2000b)] also do not reveal the

effect of cytokines.

thrombin (Fig. 1). We found that the cytokines at

concentrations of 1±100 ng mL21 do not affect

CD42b (glycoprotein Ib) expression in stored PCs, as

measured by flow cytometry. Furthermore, these

cytokines, in contrast to ADP (20 mmol L21), did not

induce platelet aggregation and shape change in PCs, as

measured by aggregometry.

We also studied the effects of IL-1b, IL-6, IL-8 and

TNF-a on fresh platelets in whole blood and in PRP;

these platelet preparations are characterized by signifi-

cantly lower basal level of CD62 expression than in

stored PCs. None of the cytokines at concentrations of

1±270 ng mL21 affected platelet activation, suggesting

that the cytokines do not induce activation of fresh

resting platelets either directly or indirectly through

substances that can be released from white and red blood

cells as the result of cytokine impact.

In summary, we demonstrated that IL-1b, IL-6, IL-8

and TNF-a released from residual leucocytes during

storage of nonleukodepleted PCs do not induce activa-

tion of resting platelets and do not potentiate storage-

induced and agonist-induced platelet activation.

V. Leytin, S. Shakoor, M. Mody, D. Allen, B.Hannach,* B. Garvey and J. Freedman

Department of Transfusion Medicine, St. Michael's

Hospital, Toronto, Ontario, Canada and

*The Toronto Centre of Canadian Blood Services

REFERENCES

Heddle, N.M., Klama, L., Singer, J., Richards, C., Fedak, P.,

Walker, I. & Kelton, J.G. (1994) The role of the plasma from

platelet concentrates in transfusion reactions. New England

Journal of Medicine, 331, 625±628.

Leytin, V., Allen, D., Mody, M., Garvey, B. & Freedman, J.

(2000a) Characterization of platelet activation in vitro

during storage of prestorage-leukodepleted platelet concen-

trates (Abstract). Blood, 96, 108b.

Leytin, V., Mody, M., Semple, J.W., Garvey, B. & Freedman,

J. (2000b) Quantitation of platelet activation status by

analyzing P-selectin expression. Biochemical and Biophysi-

cal Research Communications, 273, 565±570.

Lumadue, J.A., Lanzkron, S.M., Kennedy, S.D., Kuhl, D.T. &

Kickler, T.S. (1996) Cytokine induction of platelet activa-

tion. American Journal of Clinical Pathology, 106, 795±798.

Muylle, L., Joos, M., Wouters, E., De Bock, R. & Peetermans,

M.E. (1993) Increased tumor necrosis factor a (TNF-a),

interleukin 1, and interleukin 6 (IL-6) levels in the plasma

of stored platelet concentrates: relationship between TNF-

a and IL-6 levels and febrile transfusion reactions.

Transfusion, 33, 195±199.

Oleksowicz, L., Mrowiec, Z., Isaacs, R., Dutcher, J.P. &

Puszkin, E. (1995) Morphologic and ultrastructural evidence

for interleukin-6 induced platelet activation. American

Journal of Hematology, 48, 92±99.

Stack, G. & Snyder, E.L. (1994) Cytokine generation in stored

PCs. Transfusion, 34, 20±25.

Six hundred reasons to donate blood

I walked back from an economics lecture, passed an

advertisement for a weight-loss clinic, and arrived at my

appointment to donate blood. A thought came to mind:

why does a rational economist ever donate blood? The

activity takes time, requires planning, involves trans-

portation, causes pain, and indirectly leads to lost

personal productivity. Additionally, some risk of

financial cost is assumed if sloppy application of the

disinfectant causes a bad clothing stain.

I then had an idea. Perhaps blood donation could be

promoted as one way to lose a bit of unwanted weight.

Taking into account the composition and energy in each

component (Rechcigl, 1978; Kratz et al., 1998), I

estimated that one unit of blood reflects about 600 cal

of food intake. Hence, a single donation can off-set

either 2 hamburgers, 3 donuts, or 5 granola bars.

Awareness of these statistics might increase the appeal

of blood donation, particularly among healthy adults

who are concerned about obesity.

D. A. Redelmeier

de Souza Chair in Trauma, University of Toronto

REFERENCES

Rechcipl, M. [ed.] (1978) CRC Handbook Series in Nutrition

and Food Section E Nutritional Disorders, Vol II. CRC Press

Inc., p402.

Kratz, A., Kent, B. & Lewandrowski, K.B. (1998) Case

Records of the Massachusetts General Hospital (Normal

Reference Laboratory Values). New England Journal of

Medicine, 339, 1063±1072.

390 Correspondence

q 2001 Blackwell Science Ltd, Transfusion Medicine, 11, 389±390