© 2007 Schattauer GmbH, Stuttgart 156 Formation of mixed platelet-PMN leukocyte aggregates in the platelet function analyzer (PFA-100) device Norma Maugeri*, Maria Benedetta Donati, Giovanni de Gaetano, Chiara Cerletti Laboratory of Cell Biology and Pharmacology of Thrombosis, Research Laboratories, “John Paul II” Center for High Technology Research and Education in Biomedical Sciences, Catholic University, Campobasso, Italy Letters to the Editor Correspondence to: Dr. Chiara Cerletti Center for HighTechnology Research and Education in Biomedical Sciences Catholic University – Head Laboratory of Cell Biology and Pharmacology of Thrombosis Research Laboratories Largo Gemelli,1 Campobasso Italy 86100 Tel.: +39 0874 312277, Fax: +39 874 312710 E-mail: ccerletti@rm.unicatt.it *Present address: Clinical Cardiovascular Biology Research Centre, Cardio-Thoracic and Vascular Department, UniversityVita-Salute San Raffaele, Milan, Italy. E-mail: normamaugeri2003@yahoo.it. Financial support: This work was partially supported by MIUR (ProgrammaTriennale Ricerca, decreto 1588). Received October 11, 2006 Accepted after revision November 22, 2006 Prepublished online December 8, 2006 doi:10.1160/TH06–10–0581 Thromb Haemost 2007; 97: 156–157 Dear Sir, Platelet-polymorphonuclear leukocyte (PMN) mixed aggregates were described by Giulio Bizzozero in blood flowing from a bleeding injury, during the process of hemostasis (1). In the last two decades, interest for mixed platelet-PMN ag- gregates has been renewed by the possible relevance of this phe- nomenon to thrombosis and vascular ischaemic injury (2–10). We report here that mixed platelet-PMN aggregates are de- tectable in the blood emerging from the Platelet Function Ana- lyzer (PFA-100), a device currently used to evaluate platelet dis- orders and platelet function (11). The system mimics the high shear of small arterioles and some biochemical interactions oc- curring when damaged subendothelium is exposed. In the PFA-100, citrated whole blood is challenged by standardized shear forces (negative pressure through a capillary) and by a “biochemically” active membrane (cartridge). We present here the results obtained with collagen-ADP cartridges. Preliminary data obtained with collagen-epinephrine cartridges were too variable, possibly due to the high inter-individual variability of platelet response to epinephrine (12). Further studies with the latter cartridge are currently under way. Venous blood was collected on 3.8% trisodium citrate (9:1, v:v) from nine healthy volunteers (all blood donors from the Transfusion Centre of the “Cardarelli” Hospital, Campobasso) (5 males, 4 females, aged 44 ± 3 years). All subjects, previously informed that part of their blood would be collected for research purposes and that their privacy would be protected, signed a written consent. They were all non-smokers, denied having re- ceived any medication (including oral contraceptives) during the last two weeks and had hemato-chemical values within the nor- mal ranges. The blood group was 0 in five subjects (four Rh negative, one positive),A in two (one negative, one positive), and B in two (both positive). Aliquots of the blood emerging from the PFA-100 device, as- pirated through the capillary and the aperture of the PFA-100, were fixed and labelled with specific antibodies to determine cell aggregates and platelet P-selectin expression (7, 10, 13). Briefly, platelet-PMN aggregates were determined by three- color flow-cytometry as the percent of CD61-positive platelets in PMN cluster (CD45 positive and according to their forward and scatter characteristics). Platelet P-selectin expression was determined by double-color flow cytometry as the percent of P-selectin-positive platelet cluster (200,000 CD61-positive events). Blood samples were simultaneously labeled with spe- cific MoAb and fixed with Thrombofix ® (Instrumentation Lab- oratory, Milan, Italy), and analyses were performed four hours after sample preparation. We found that platelet-PMN aggregates increased from 10.3 ± 2.4% (in the residual blood that was not aspirated from the “reservoir” through the PFA-100 device) to 30.3 ± 6.8% (in the blood emerging from the device) (mean ± sem, n=9; p<0.05) (Fig. 1). The formation of mixed platelet-PMN aggregates might be even greater than suggested by these data. Indeed, the back- ground levels of platelet-PMN complexes in the samples exam- ined for this study were higher than expected (4.4 ± 1.1%, n=9) as in our previous experience (13). Moreover, these data do not take into account the possible capture of activated platelets and/ or PMN into forming a hemostatic plug within the aperture of the cartridge. Further studies will indicate whether a proportion of mixed (possibly larger) aggregates are formed and retained with- in the cartridge, thus escaping detection in the emerging blood. The percent of mixed aggregates apparently did not correlate with the PFA-100 closure time (91 ± 6 sec; mean ± sem, n=9), suggesting that in the device, platelet-PMN interaction does not substantially contribute to the closure time. This conclusion is, however, indirect and needs further investigation, including leu- kocyte-depleted or -enriched blood samples. This phenomenon is mainly, if not exclusively, mediated by platelet aggregation triggered by the ADP- and collagen-rich cartridge (11). Platelet P-selectin expression was indeed increased from 2.6 ± 0.5% (in the residual blood that was not aspirated through the PFA-100 device) to 43.1 ± 2.8% (in the blood emerging from the device) (mean ± sem, n=5; p<0.01) (Fig. 1). The platelet P-selectin valu- For personal or educational use only. 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