Plasma macrophage colony-stimulating factor levels during cardiopulmonary bypass with extracorporeal circulation

Leukocytosis and thrombocytopenia occur during cardiopulmonary bypass (CPB) with extracorporeal circulation (ECC). Elevated circulating concentrations of macrophage colony-stimulating factor (M-CSF) are reported during thrombocytopenia and leukopenia of different origins. We have assessed M-CSF concentrations in 40 patients undergoing CPB with ECC. Plasma M-CSF concentrations were stable during ECC and increased at the 6th (7.3 ± 0.7 IU/μg protein) and 24th (8.6 ± 0.8 IU/μg protein) postoperative hour compared with pre-ECC values (4.9 ± 0.5 IU/μg protein). A deep thrombocytopenia was found during ECC and until the 24th postoperative hour. A drop of leukocyte counts was found during ECC followed by an increase after ECC weaning. While no correlation was found between M-CSF concentrations and the leukocyte counts, M-CSF values were positively correlated with platelet counts only before and during ECC. Thus, M-CSF is not implicated in the thrombocytopenia and the leukopenia generated during CPB with ECC. However the elevated levels of M-CSFa few hours after the end of ECC might play a role in the inflammatory process often observed after CPB.


Introduction
Cardiopulmonary bypass (CPB) with extracorporeal circulation (ECC) results in major haematological changes including leukocytosis and thrombocytopenia. The release of several inflammator compounds including cytokines, eicosanoids, platelet-activating factor (PAF), and activation of the coagulation and the complement cascade are suspected to play a role in these haematological disorders and in the pathogenesis of the inflammatory syndrome observed after CPB. [2][3][4][5][6] Monocte/macrophage activation accompanies CPB. Macrophage colony-stimulating factor (M-CSF), also known as CSF-1, is a major regulator of the production as well as functional state of cells of the mononuclear-phagocytic lineage. 8'9 M-CSF is produced by numerous cell types such as endothelial cells, monocytes/ macrophages, Tand B-cells spontaneously or after induction. 8-1 M-CSF acts on mature monocytes/macrophages, stimulating production of tumour necrosis factor (TNF), interferon, interleukin-6 (IL6), prostaglandins and CSFs11-13.
Some reports have highlighted the role of M-CSF in the pathogenesis of thrombocytopenia and leukopenia of various origins. [14][15][16] The purpose of this study was to assess plasma M-CSF concentrations during and after cardiac surgery with ECC. We also investigated the relationship between platelet and leukocyte counts and M-CSF levels during and after CPB.

Patients and Methods
After ethical committee approval, 40 patients scheduled to undergo coronary artery bypass graft were included in this prospective study. All the patients gave written informed consent.

Anaesthesia
All patients had a preoperative ejection fraction above 40%. Upon arrival on operating room, all patients received their usual oral cardiac medication, flunitrazepam (2mg) and morphine (lOmg) intramuscularly. Anaesthesia was induced and maintained with titrated doses of fentanyl and flunitrazepam. Muscular relaxation was achieved with pancuronium (0.1 mg/kg). Bubble or membrane oxygenators were used according to the planed number of graft and surgical difficulties. The blood was harvested from surgical field and from the cell saver at the microtitre plates. Standard dilutions of M-CSF end of ECC and reinfused to all patients. All (400 to 6 international units per ml (IU/ml) of patients (except two with uneventful surgery) human M-CSE international standard, NIBSC, received high doses aprotinin. All the patients Potters Bar, UK) and samples were transferred were managed by the same surgical anaesthetic into wells and incubated overnight at 4C. team.
Plates were washed and anti-M-CSF-biotin conjugate was added to all wells and incubated Blood sampling and haematologic overnight at 4C. Plates were washed and measurements avidine phosphatase (1 Ig/ml) was added to wells and incubated for 1 h at 37C. The Blood samples were collected from the radial revelation was performed with para-nitropheartery catheter at the following times: before nyl-phosphate disodium (4 g/ml) for 15 min at vascular cannulation and after opening the 37C. The reaction was blocked by addition of chest (To), during ECC just before (T1) and after 50 bl of NaOH l ON to the wells. Colour cross-clamp release (T2), after weaning from development was measured at OD 405 nm. M-ECC (T3), at the 6th (T4) and 24th post-CSF levels in samples were calculated with operative hour (T5) (Fig. 1). Two ml of blood respect to the calibration curve obtained with were placed in EDTA then centrifuged to obtain standard M-CSE The sensitivity of the assay plasma which was stored at -80C until assay,  were found at the 6th (7.3 +0.7IU/ml/btg protein) and 24th (8.6 -q-0.8 IU/ml/gg protein) postoperative hour compared with To (4.9-+-0.5 IU/ml/pg protein).
A positive correlation was observed before (To) and during ECC (T1) between platelet counts and M-CSF concentrations ( Table 2). After cross-clamp release no correlation was found between platelet counts and M-CSF concentrations. No correlation was found between M-CSF concentrations and leukocyte counts at any blood sampling time (Table 2).
No difference was documented between M-CSF concentrations according to the grade of haematological complications at T3, T4 and T5 (Fig. 3).

Discussion
This study shows that plasma M-CSF concentrations are stable during ECC and increase at the 6th and 24th postoperative hour compared with pre-ECC values. While lipidic mediators are produced chiefly during ECC, 7'8 elevated cytokine concentrations are often found after few hours. 5'6'1 The time-course of M-CSF concentrations during cardiac surgery fits well with those of the other cytokines so far assessed. We may speculate that the monocytes, lymphocytes, and polymorphonuclear neutrophils 'primed' by CPB might be implicated in these elevated M-CSF concentrations. However a lowering M-CSF degradation might also explain our results. M-CSF receptors is almost totally cleared from the M-CSF is suspected to play a role in the fall of platelet counts during pregnancy, 14  The concept that there is a marked synergy between the effects of the various mediators released during cardiac surgery is now widely accepted. 2 A combination of small amounts of them may be more active than a large release of one by itself. M-CSF might play a role in the inflammatory process observed after cardiac surgery since the key cells of the inflammatory response to CPB (i.e. monocytes, polymorphonuclear neutrophils and lymphocytes) produce M-CSF in response to cytokines and produce cytokines in response to M-CSE [8][9][10][11][12][13] In addition elevated circulating amounts of granulocyte-CSF (G-CSF) have been recently found after cardiac surgery, 2 strengthening that the involvement of growth factors after CPB deserves further evaluations.