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Eur J Cardiothorac Surg 2006;29:312-318
© 2006 Elsevier Science NL
a Department of Cardiac Surgery, Imperial College School of Medicine, University of London, Hammersmith Hospital, Du Cane Road, London W12 0NN, UK
b Department of Cardiac Surgery, Imperial College School of Medicine, University of London, St. Mary's Hospital, London, UK
Received 25 May 2005; received in revised form 25 October 2005; accepted 14 November 2005.
* Corresponding author: Tel.: +44 776 611 5590; fax: +44 208 740 7019. (Email: a.weerasinghe{at}ic.ac.uk).
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Key Words: Platelets • Monocyte • Cardiopulmonary bypass • Coronary artery bypass surgery
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Interestingly, only 10–20% of the total extractable tissue factor activity is expressed on the surface of intact monocytes. Thus, most TF is latent or encrypted in the cell membrane. Platelets, through platelet-derived microparticles are a source of tissue factor to circulating monocytes, resulting in rapid pro-coagulant responses. These involve the formation of thrombin via a TF/factor VII-dependent and factor XII-independent pathway [3].
In contrast, a delayed, transcriptionally mediated monocyte TF expression is thought to involve not only platelet interaction but also stimulation by circulating cytokines generated perioperatively. When co-incubated, leucocytes and platelets generate more TF activity than either cell type alone. Platelets play a pivotal role in decrypting TF activity of monocytes, generating a hybrid TF terrain, which both triggers and favours thrombogenesis [4].
Despite the awareness of these platelet–monocyte coagulant interactions, it is not known whether these monocytic and platelet count changes are related to the complexity of procedure performed and the use of cardiopulmonary bypass, and also if they have a clinically significant impact on haemostasis following conventional on-pump coronary surgery.
The specific objectives of this study were to
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Preoperative and postoperative white blood cell counts, monocyte counts and platelet counts were measured on all patients as part of their automated full blood count. Monocyte counts were expressed as a percentage of the total white cell count to give the monocyte percentage, thus aiming to detect if a relative monocytosis occurred following surgery and to differentiate this from a rise reflecting an overall leucocytosis. The preoperative and intraoperative variables used for the multivariate models in the study are shown in Table 1 along with the distribution of patients within each variable.
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2.2.2 On-pump technique, St. Mary's Hospital
CPB was instituted with aorto-caval cannulation. Standard bypass management included membrane oxygenators, arterial line filters and, non-pulsatile flow of 2.4 l/min/m2 with a mean arterial blood pressure 50–60 mmHg. Myocardial protection was achieved mainly by intermittent antegrade cold blood cardioplegia (4:1 blood to crystalloid ratio). Retrograde blood cardioplegia was used occasionally in addition, particularly if there was left main stem disease with tight right coronary artery stenosis, which might cause inadequate delivery of the cardioplegia to the targeted myocardium and consequently incomplete myocardial protection. Temperature management was again with moderate hypothermia.
2.2.3 Off-pump technique, St. Mary's Hospital
Off pump surgery was performed with proximal occlusion of the target coronary vessel with a silastic sling and the use of epicardial stabilising devices and apical suction devices [Octopus® 3 or 4 (Medronic Inc., Minneapolis, USA), StarfishTM (Medronic Inc.) or the Guidant Vortex Vacuum Assist (Cupertino, USA)]. Intracoronary shunts were not used routinely during the distal anastomoses. Systolic arterial pressures were maintained at a minimum of 70 mmHg during distal anastomoses utilising venous volume regulation, rate control, inotropes or vasoconstrictors. Proximal anastomoses were performed with a side-biting aortic clamp, with systemic pressures that were dictated by individual surgeon preference. The target ACT during surgery was 300. Normothermia was maintained by using warm intravenous fluids, heating mattress and a humidified airway, in addition to maintaining a warm operating theatre. A standby perfusionist with primed bypass circuit was available for all OPCAB cases.
2.3 Flow cytometry
2.3.1 Platelet–monocyte conjugate analysis
Blood was drawn preoperatively and at 2 h post-bypass and anti-coagulated with sodium citrate. Platelet–monocyte conjugate formation was analysed by tricolour flow cytometry using whole blood as described by us previously [5]. Briefly, whole blood was diluted (3:7) in a modified Tyrode's buffer and 100 µl aliquots were fixed with 1% paraformaldehyde and stained with the following monoclonal antibodies: pan-leucocyte marker CD45-FITC, the monocyte marker CD14-ECD and the platelet marker CD42b-PE (Beckman Coulter). The monocyte population was identified using CD45-FITC and CD14-ECD fluorescence, and then CD42b-PE was used to calculate the percentage of platelets adherent to the monocyte sub-population.
TF expression was determined using whole blood prepared as described above and stained with the pan-leucocyte marker CD45-PE, the monocyte marker CD14-ECD and the anti-human TF-FITC (America Diagnostica, Stamford, CT, USA). The monocyte population was identified using CD45-FITC and CD14-ECD fluorescence, and then mean TF fluorescence on the monocyte population was used to calculate the mean relative fluorescent intensity (RFI) of staining, calculated by dividing the mean fluorescent staining intensity of the anti-TF antibody at any given time point by the staining intensity of a class-matched and fluorochrome matched control antibody.
2.4 Statistical analysis
Continuous variables were expressed as mean ± SD. p-values 
0.05 were considered statistically significant. We evaluated the effect of several factors on blood loss by using stepwise multivariable regression analysis with both forward and backward variable selection. Changes in the monocyte and platelet counts over time were considered as repeated measures data and analysed by using Generalised Estimating Equations (GEE) [6,7]. Marginal models (unstructured correlation matrix) based on generalised estimation equations were used to perform regression analysis. Variables significant at the 5% level were retained in the final multivariable models. Analysis was conducted by using the statistical software SPSS version 12.0 for Windows (SPSS Inc., Chicago, IL, USA), and Intercooled Stata version 8.0 for Windows (Stata Corporation, USA).
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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3.1.2 In off-pump coronary surgery
We next ascertained how the above findings compared to the response of these cells in off-pump coronary surgery. It was apparent that at every postoperative time point, monocyte counts were significantly higher in the off-pump group when compared with the on-pump group having coronary surgery (beta coefficient = –0.07, p
= 0.03 and 95% CI –0.14 to –0.01) and significantly, higher platelet counts directly predicted a greater monocytic response (beta coefficient = 0.0004, p
= 0.003, 95% CI 0.0004 to 0.0007).
3.1.3 In first time on-pump coronary surgery
Changes in total white blood cell (WBC), monocyte and platelet counts are shown in Fig. 1
. The WBC significantly increased following surgery (p
< 0.001). A rapid peak by the second postoperative day was followed by a moderate drop on the third, fourth and fifth day. This was followed by a second rise by the sixth and seventh postoperative days. The monocyte count was seen to similarly increase on the first two postoperative days, but then in contrast to the WBC continued to rise steadily until the fifth postoperative day. Using the monocyte%, it was apparent that the initial rise in the monocyte count was a reflection of the rise in the total WBC. In contrast, over subsequent days an absolute monocytosis was seen, independent of total WBC. In comparison, the platelet count mirrored the WBC initially and showed a trough by the second postoperative day. This was followed by a subsequent rise that was significantly above the preoperative levels after the sixth postoperative day.
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Mo: the change in monocyte fraction, A: age in years, BMI: body mass index, DM: diabetic status (non-diabetic = 0, diabetic = 1), S: smoking status (non-smoker = 0, smoker = 1) and MP: the type of myocardial protection used (cross-clamp fibrillation = 0, cardioplegia = 1).
3.2 Platelet–monocyte conjugate formation and role of TF
Since monocytes exert some of their potential haemostatic effects in association with the formation of PMCs, we characterised actual PMC formation in association with on-pump coronary surgery. PMC formation (Fig. 2
) did not initially demonstrate a significant change although a slight downward trend following administration of heparin, followed by a subsequent peak at 5 min following the onset of CPB was seen. In contrast, 2 h after the onset of CPB, a significant decline in circulating PMCs was observed (p
= 0.04). A rise and return towards preoperative levels was then found between 24 h and 5 days postoperatively.
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Blood loss (ml) in 12 h = 1391.69 – 132.50(gender) – 8.26(core body temperature in °C) – 12.74(BMI) – platelet count(0.933) – 7.9(monocyte%) – 117.49(aprotinin) [gender = 1 for male or gender = 2 for female; platelet count as x109 l–1 if platelet count >160 x 109 l–1; aprotinin = 0 if not used and aprotinin = 1 if used].
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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A further observation was that patients with higher core body temperatures during coronary artery bypass grafting (relatively normothermic patients) were less likely to bleed in the postoperative period. Lower body temperatures may impair enzyme activity involved in the coagulation cascade. This is supported by in vitro studies, which have demonstrated that the clotting time of normal human plasma as measured by the activated partial thromboplastin times (APTT), pro-thrombin times (PT) and thrombin times (TT) is significantly prolonged by lower temperatures in an exponential manner [13]. Furthermore, it is also known that a lower body temperature during cardiopulmonary bypass causes increased platelet activation [14] and these preactivated platelets are dysfunctional in the postoperative period. However, hypothermia may also affect monocyte function. This possibility is supported by in-vitro human monocyte studies in which the temperature dependence of their active uptake of colloidal gold particles has been demonstrated by electron microscopy [15].
The late increase in PMCs detected in peripheral blood may be related to the observed increase in absolute numbers of available monocytes and platelets or due to their increased affinity as a result of cellular activation. In keeping with this, work by Wahba and Videm [16] has shown that platelets increase both in number and in state of activation towards the end of the first week after cardiac surgery. Although monocyte up-regulation of surface TF would theoretically further contribute to PMC formation through platelet–P-selectin interaction, we did not, in this study, detect any significant change in expression perioperatively. Therefore, the late rise in circulating PMCs following cardiac surgery is likely to be related to the observed monocytosis and thrombocytosis that occurs at the end of the first postoperative week and/or maybe mediated through TF-independent interactions. This increased number of circulating PMCs if reflective of an increased adhesive state, may potentially mediate thrombo-embolic phenomena that contribute to early graft occlusion as well as to neurological sequelae such as amaurosis fugax and other forms of transient ischaemic attack (TIAs) towards the end of the first postoperative week. The presence of significantly higher numbers of pro-thrombotic platelet–monocyte aggregates in patients with TIAs or stroke has previously been demonstrated in a non-surgical setting [17]. Interestingly, previous studies have demonstrated that the presence of circulating PMCs immediately postoperatively can act as a predictor of lower limb graft occlusion within the first six months following peripheral vascular surgery [18].
In agreement with earlier work, our study confirms that aprotinin decreases postoperative blood loss. This is thought to be in-part mediated by its anti-fibrinolytic affect. However, our group recently demonstrated that aprotinin also decreases thrombin-induced activation of platelets, whilst not inhibiting platelet aggregation induced by collagen and ADP [19]. Peak monocyte–platelet conjugate formation has been shown to be significantly reduced by aprotinin [20] and may protect both platelets and monocytes from activation by thrombin generated during cardiopulmonary bypass, allowing more effective haemostasis at the sites of surgical trauma postoperatively.
4.2 Potential impact of results on current practice
Leucocyte-depleting filters used in cardiac surgery have been shown to bind monocytes and platelets [21]. We observe platelet–monocyte conjugate formation to be at its lowest at 2 h after discontinuation of bypass, coinciding with the immediate postoperative period during which both platelet counts and function are depleted. The use of leucocyte depleting filters is likely to activate and entrap, and further decrease platelets and monocytes and PMCs, thereby reducing their availability for postoperative haemostasis [22]. Thus, it may be that they are best avoided in patients with preoperative risk factors likely to contribute to a higher risk of postoperative bleeding.
4.3 Potential future clinical use of results
In patients having postoperative bleeding diatheses despite transfusion of fresh frozen plasma and platelets and exclusion of a surgical cause, it is potentially conceivable that the contribution of monocytes to postoperative bleeding either directly or in conjunction with platelet interactions may be quantitatively more significant. The role of monocyte transfusions in these patients has not been explored despite leukapheresis having been recently shown to be a safe and efficient procedure for collecting large numbers of peripheral blood monocytes from donors [23]. Awareness of the complimentary effect between platelets and monocytes in reducing postoperative blood loss may thus be of use in developing new strategies for the management of patients having postoperative bleeding diatheses not controlled by standard measures.
4.4 Weaknesses of the study
Despite the monocyte count influencing postoperative blood loss, the absolute volume decrease appears small in relation to the contribution made by the platelet count. Nonetheless this interaction has not been studied previously. The monocytic mechanisms underlying these observations remain unclear, but do not appear to relate to monocyte TF up-regulation. Further mechanisms that may play a role will need to be addressed by a study entirely dedicated towards unravelling these processes. A further weakness is that the influence on postoperative bleeding seen in the study relates only to on-pump coronary surgery. We have mapped the response in off-pump surgery, but do not have the data to extrapolate the findings to postoperative blood loss in off-pump surgery.
The results of this study are clearly of a preliminary nature into platelet–monocyte behaviour after on-pump coronary surgery and we hope will stimulate further investigation.
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Acknowledgments |
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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This article has been cited by other articles:
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  P. Farneti, S Sbrana, D Spiller, A. Cerillo, F Santarelli, D Di Dario, P. Del Sarto, and M Glauber Reduction of blood coagulation and monocyte-platelet interaction following the use of a minimal extracorporeal circulation system (Synergy(R)) in coronary artery bypass grafting (CABG) Perfusion, January 1, 2008; 23(1): 49 - 56. [Abstract] [PDF]
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