[Determination of the cytokine level during storage of partially leukocyte-depleted (<0.5 x 10(9)) or totally leukocyte-depleted (<0.5 x 10(6)) platelets].
OBJECTIVE
To evaluate the levels of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) during the storage of pools of PCs obtained after removing the buffy-coat, in comparison with the amount of leukocytes present in these components.


METHODS
Blood was collected in quadruple-bag-system containing 63 mL of CPD as anticoagulant and 100 mL of SAG-M solution as additive for the red cells. Approximately twelve hours after collection, blood separation was made automatically by Compomat (NPBI) and platelet concentrates were prepared from the buffy-coat fraction. Five or six PCs were mixed to obtain a pool (n = 28). Eight pools were WBC reduced by filtration (PXL-8, Pall España). Before storage a sample of each pool was obtained in order to count platelets and WBC as basal values. All kind of pools were stored at room temperature with continuous agitation during a period of 10 days. Volumes were measured by weight and specific gravity. Platelets and leukocytes were counted in the Coulter-counter (STKR Counter. Izasa) or in Nageotte chamber for the filtered products. On days 1, 4 and 7 interleukins were measured by ELISA (EASIA Kits, Medgenix Diagnostics, Brussels), Lecture was done at 450 nm using a spectophotometer (ANTHOS 2001). Lower limits of sensitivity were 2 pg/mL for IL-1 beta and 3 pg/mL for IL-6 and TNF-alpha according to the manufacturer. Wilcoxon test was used for statistical analysis.


RESULTS
The average volume of the pools was 460 mL and 374 for the filtered ones. The total platelet amount was 3.63 x 10(11) and 2.8 x 10(11) respectively, with a WBC contamination of 380 x 10(6) and 0.54 x 10(6) for the filtered. The yield of platelets after filtration was 84% with a loss of 99.90% of WBC (3-log). The measures of interleukins were not homogeneous, but a great variability was found shown among the different pools, not always in relationship with the amount of leukocytes. The levels of IL-1 beta were 3.58 pg/mL on day 1, 6.36 pg/mL on day 4 and 8.76 pg/mL on day 7 (p < 0.005). For the IL-6 we found 13.08 pg/mL on day 1, 15.43 pg/mL on day 4 and 19.77 on day 7 (p < 0.05). For the TNF-alpha, 13.65 pg/mL an day 1,24.33 pg/mL on day 4 and 30.10 pg/mL on day 7 (p < 0.05). In the filtered pools the detections of IL-1 beta and IL-6 were always under the sensibility threshold, but there was an increment of TNF-alpha on day 7 (16.91 pg/mL). Microbiologic cultures were always negative.


CONCLUSIONS
The accumulation of IL-1 beta, IL-6 and TNF-alpha is not prevented by the fact of removing the buffy-coat layer when preparing PCs, although these levels are considerably lower in comparison with those obtained by the PRP technic. Filtration of pooled PCs avoids the presence of these cytokines except TNF-alpha, where a low amount can be detected.
Algora
M
M
Centro de Transfusión Madrid, España.
Barbolla
L
L
Zamora
C
C
Moreno
C
C
Rodríguez
M A
MA
Merino
J L
JL
Torres
P
P
D001792:Blood Platelets; D001793:Blood Preservation; D002469:Cell Separation / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay; D005374:Filtration; D006801:Humans; D007375:Interleukin-1 / Q000097:blood*; D015850:Interleukin-6 / Q000097:blood*; D007958:Leukocyte Count; D013696:Temperature; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis*
D007375:Interleukin-1; D015850:Interleukin-6; D014409:Tumor Necrosis Factor-alpha