Effect of the colloids gelatin and HES 130/0.4 on blood coagulation in cardiac surgery patients: a randomized controlled trial.

OBJECTIVE: The choice of the prime solution for cardiopulmonary bypass can play an important role in limiting the effect on blood coagulation, but it is still unclear what the effect of colloids on blood coagulation is. The aim of this study was to investigate the effect of synthetic colloids on blood loss and blood coagulation in patients after on-pump coronary artery bypass graft (CABG) procedures. METHODS: Sixty elective, on-pump CABG patients were randomly assigned to receive the prime solutions lactated Ringer's solution combined with hydroxyethyl starch 130/0.4 (HES, 6% Volulyte, Fresenius Kabi Nederland BV, Zeist, the Netherlands) (HES group) or gelatin (Gelofusin(®), B Braun Melsung AG, Melsungen, Germany) (Gelo group). Blood loss was assessed using post-operative chest tube output; secondary endpoints were number of blood component transfusions, routine coagulation test values and rotation thromboelastometry values (Rotem(®) delta, Pentapharm GmbH, Munich, Germany). RESULTS: Total post-operative chest tube output was 500 ± 420 ml in the HES group versus 465 ± 390 ml in the Gelo group (p = 0.48). No significant differences were observed in any of the routine coagulation tests values, thromboelastometry parameters or number of blood component transfusions between the groups. CONCLUSIONS: In this randomized, controlled trial of adults after on-pump CABG procedures, there was no significant difference in blood loss or blood coagulation between the HES group and the Gelo group.

Processing and transfusion of residual cardiopulmonary bypass volume: effects on haemostasis, complement activation, postoperative blood loss and transfusion volume.

The aim of this prospective randomized study was to compare the effects of the transfusion of unprocessed and cell saver-processed residual cardiopulmonary bypass (CPB) volume on haemostasis, complement activation, postoperative blood loss and transfusion requirements after elective cardiac surgery. Blood samples were taken at eight points in time, perioperatively. Haematological data, including haemoglobin, haematocrit and platelet counts as well as coagulation parameters, including activated partial thromboplastin time, prothrombin time, thrombin time, fibrinogen and the fibrinolytic parameter D-dimers, were measured from each blood sample. For the assessment of complement activation, the total complement CH50 was analysed. In addition, postoperative blood loss and transfusion requirements were measured during the first 24 hours, postoperatively. The results of the study showed impaired haemostasis after the transfusion of both unprocessed and processed CPB volume. No significant differences were found between the groups in the measured coagulation parameters. Nor was a significant difference found in the complement concentration. However, in patients transfused with unprocessed CPB volume, a significantly (p = 0.019) higher amount of blood loss was found, postoperatively. In the same group of patients, the number of units of allogeneic erythrocyte concentrate suspension transfused was also significantly (p = 0.023) higher during the first 24 hours, postoperatively, compared to the patients transfused with processed CPB blood. The number of units of fresh frozen plasma and platelet suspension transfused was not significantly different between the groups. In conclusion, processing CPB volume in combination with processing peroperative blood loss may result in reducing the volume of transfusion needed of allogeneic blood products.

T helper frequencies in peripheral blood reflect donor-directed reactivity in the graft after clinical heart transplantation.

We describe the usefulness of a fast (48-h) limiting dilution assay (LDA) for the enumeration of human alloreactive helper T lymphocytes (HTL) in the peripheral blood, in relation to histologically defined rejection grades after heart transplantation. HTL frequencies (HTLf) in pretransplant samples varied from patient to patient, ranging from 106 to 625 HTL/106 peripheral blood mononuclear cells (PBMC). In the first week after heart transplantation (HTx), when immunosuppression was instituted, HTLf were significant lower (range 30-190 HTL/106). The level of HTL in the first week after HTx when rejection grade was 0 or 1A (ISHLT score) was considered to be the baseline frequency. This frequency did not correlate with the number of subsequent rejection episodes. During rejection (grade 3), donor-specific HTLf were increased above their baseline frequencies (P = 0.01). Expressed as percentage of baseline frequencies, HTLf increased significantly during acute rejection (AR) compared with 1-2 weeks before rejection (P = 0.003). The increase was specific, since viral infections did not result in a rise of donor-specific HTL, while also HTLf specific for third party HLA antigens were not elevated during rejection. Monitoring HTLf in peripheral blood with a shortened (48-h) assay may serve as a non-invasive method for detecting intragraft immunological reactivity. Demonstrating absence of donor-specific reactivity may limit the number of invasive endomyocardial biopsy (EMB) procedures and allow tapering of immunosuppressive treatment.

Kinetics of circulating cytotoxic T lymphocyte precursors that have a high avidity for donor antigens: correlation with the rejection status of the human cardiac allograft.

Studies on graft infiltrating cells demonstrated that accumulation of cytotoxic T lymphocytes (CTL) with high avidity for donor antigens (Ag) coincided with acute cardiac rejection. In the present study, we analyse whether such high-avidity CTL are present within the peripheral blood of cardiac transplant recipients and whether their kinetics correspond with the rejection status of the allograft. Using limiting dilution analysis (LDA), donor-specific CTL were enumerated in serial blood samples of seven patients. From each patient, 7-11 samples were obtained during the first year after transplantation and up to three samples were obtained at a later date. Enumerated donor-specific CTL were divided into CTL with high or low avidity for donor Ag, depending on their sensitivity to CD8-blocking. In contrast to the situation in the graft, the donor-specific CTL present within the peripheral blood were CTL precursors (pCTL) and not fully mature CTL (cCTL). The number of donor-specific pCTL among peripheral blood cells fluctuated irrespective of the rejection grade of the allograft, indicating that the frequency of circulating donor-specific CTL does not reflect the immunological status of the allograft. During acute cardiac rejection, 66% (median) of the circulating donor-specific pCTL had a high avidity for donor Ag. This percentage significantly exceeded pre- and postrejection values obtained during the first year post-transplantation (median, 39% and 37%, respectively). The disparity in avidity increased even further more than 1 year after transplantation, when stable engraftment was achieved. Among donor-specific pCTL in peripheral blood, those with a high avidity were absent (median, 0%). Hence the avidity of circulating donor-specific CTL might inform us about the immune status of the cardiac allograft.

The avidity of allospecific cytotoxic T lymphocytes (CTL) determines their cytokine production profile.

Donor-specific CTL present within the cardiac allograft during a rejection episode are distinct from those that populate the cardiac allograft in the absence of rejection. Whereas the former generally have a high avidity for donor cells, the latter mainly have a low avidity for donor cells. This observation made us reason that high-avidity CTL are implicated in transplant rejection, whereas low-avidity CTL are not. In the present study, we analyse whether both CTL subsets were distinct with respect to their IL-2, IL-4, IL-6 and interferon-gamma (IFN-gamma) secretion pattern. CTL clones with either a high or a low avidity for donor antigens were stimulated with donor cells, third party cells, or immobilized anti-CD3 MoAb and the amount of cytokine released was measured. High- and low-avidity CTL clones were found to differ with respect to their IFN-gamma production profile. Stimulation with donor cells resulted in IFN-gamma secretion by high-avidity CTL clones, but not by low-avidity CTL clones. CD3 stimulation, in contrast, led to secretion of equivalent amounts of IFN-gamma by both CTL subsets. These observations indicate that low-avidity CTL are fully capable of producing IFN-gamma, but, in contrast to high avidity CTL, fail to do so when they encounter donor cells. As IFN-gamma favours the occurrence of transplant rejection, this observation emphasizes the relevance of high-avidity CTL in the rejection process. Additionally, the data show that the cytokine production profile of CTL depends on the nature of the stimulus.

Kinetics of IL-2 and IL-4 mRNA and protein production by graft-infiltrating lymphocytes responsible for rejection after clinical heart transplantation.

During cardiac rejection we studied the kinetics of IL-2 and IL-4 mRNA and subsequent protein production by in vivo primed graft-infiltrating lymphocytes (GIL), using semiquantitative RT-PCR and ELISA. Following in vitro stimulation with either donor or third-party antigens, mRNA expression of IL-2 and IL-4 were already detectable 1-2 h after stimulation, while their protein production could be measured from 4 h onwards at least until 48 h. At both the mRNA and protein level, we measured a donor-specific signal for IL-2 and for IL-4 production (p = 0.02), while the relative donor-specific IL-2 mRNA level was significantly higher than the relative IL-4 mRNA level (p = 0.002). These observations suggest that after in vitro challenge with donor antigens, GIL obtained from rejecting cardiac allografts predominantly produce IL-2 mRNA and protein.

Different patterns in donor-specific production of T-helper 1 and 2 cytokines by cells infiltrating the rejecting cardiac allograft.

BACKGROUND: Cytokines play an important role in allograft rejection. The local production of cytokines by T-helper 2 cells within an allograft could influence the induction of graft rejection. METHODS: Therefore we studied the in vitro production of cytokines by cells infiltrating the graft. graft-infiltrating cell cultures derived from human endomyocardial biopsy specimens more often produced interleukin-2 (p < 0.001), interferon-gamma (p < 0.001), interleukin-4 (p = 0.02), and interleukin-6 (p = 0.04) after stimulation with a B-cell line obtained from the heart donor than after stimulation with a third-party B-cell line. Furthermore, the levels of these cytokines were significantly higher after donor stimulation than after third-party stimulation (p < 0.001). RESULTS: Within the first 90 days after heart transplantation, significantly higher levels of interleukin-2 (p = 0.050 and interferon-gamma (p = 0.02) were produced by donor-stimulated lymphocyte cultures derived from biopsy specimens taken during a rejection episode compared with cultures from biopsy specimens taken during a period without rejection. After 90 days, the levels of T-helper 1 cytokine (interleukin-2 and interferon-gamma) production were, irrespective of the rejection grade, comparable with those found in the cultures from rejection biopsy specimens taken early after transplantation. With regard to T-helper 2 cytokines (interleukin-4 and interleukin-6), no relation was found with the presence of rejection at any time after transplantation. CONCLUSIONS: These data suggest that in the first 3 months after heart transplantation, acute rejection is associated with the production of increased levels of T-helper 1 cytokines but not of T-helper 1 cytokines but not of T-helper 2 cytokines by donor stimulated graft-infiltrating lymphocytes. Thereafter, the T-helper 1 cytokine production of graft-infiltrating lymphocytes remained high, suggesting a continuous state of immunologic activity even in the absence of rejection.

Prophylactic therapy with OKT3 does not affect donor specific reactivity of peripheral blood lymphocytes from heart transplant recipients.

To avoid the nephrotoxic effect of high-dose cyclosporin A (CsA) immediately posttransplant, heart transplant recipients received as prophylactic therapy intravenous OKT3 for seven days instead of intravenous CsA. Patients receiving OKT3 were compared with patients receiving CsA with respect to specific proliferation and cytotoxicity of their peripheral blood mononuclear cells (PMNC) against donor antigens, at different times within three months post-transplant. No effect of the initial immunosuppressive therapy was observed on these parameters. Acute rejection did not induce a consistent effect on the relative proliferation. PMNC from patients who experienced one or more rejection episodes showed a decrease in donor specific relative proliferative response in time after transplantation, while nonrejectors did not or only slightly, independent of the initial immunosuppressive protocol. Within the group of patients not receiving OKT3, this effect of rejection reached significance. In conclusion, no effect of prophylactic OKT3 therapy compared with prophylactic CsA therapy was observed on donor reactivity of PMNC in vitro during the subsequent three to four months post-transplant.

Discrepancy between mRNA expression and production of IL-2 and IL-4 by cultured graft infiltrating cells propagated from endomyocardial biopsies.

We studied whether acute rejection correlated with the cytokine production pattern and mRNA expression of interleukin-2 (IL-2) and interleukin-4 (IL-4) in lymphocyte cultures derived from endomyocardial biopsies (EMB) that were stimulated with B cell lines of donor origin. Unstimulated biopsy cultures neither expressed mRNA nor produced IL-2 or IL-4. All stimulated biopsy cultures contained mRNA transcripts for IL-2 and IL-4. In contrast, we found different IL-2 and IL-4 production patterns. Within the first 90 days after heart transplantation (HTx), higher levels of IL-4 were measured in cultures derived from EMB with myocytolysis than in cultures from EMB without signs of myocytolysis. More than 90 days after HTx, this phenomenon was reversed and more IL-4 was produced in cultures derived from EMB without myocytolysis. These differences were not detected for IL-2 production.

Monitoring of cardiac graft recipients: comparison of in vivo activated, committed T lymphocytes in peripheral blood and in the graft.

The proliferative and cytotoxic capacity of peripheral blood lymphocytes (PBL) and the cytotoxic activity of lymphocytes propagated from endomyocardial biopsies (EMB) towards donor cells was used to identify in vivo activated, committed T cells. A series of 39 PBL samples and 38 EMB simultaneously taken from 20 patients after heart transplantation was cultured in interleukin 2 (IL-2) conditioned medium. The cytotoxic capacity of these cultures against donor cells was tested in a 4-h chromium-51 release assay. From a comparable patient group, 224 samples were evaluated for donor reactivity by a primed lymphocyte test (PLT). Analysis showed that PBL cultures hardly ever contained committed cytotoxic T lymphocytes (cCTL, 2/39) or committed proliferative T lymphocytes (cPTL, 1/224). In contrast, significantly more EMB cultures (17/38, P < 0.001, chi2 test) demonstrated donor-directed cytotoxicity. This was especially found during rejection (11/17 vs 6/21 without rejection, P = 0.05). These results show that after heart transplantation, committed cells are mainly found in the graft.

Lysis of heart endothelial cells from donor origin by cardiac graft infiltrating cells.

Endothelial cells may be involved in the acute rejection of allografts. In the present study, graft infiltrating lymphoid cell lines were propagated from a heart graft at the time of histological diagnosis of rejection. The cell lines containing CD8+ cells lysed donor-derived BLCL and endothelial cells (EC) but not third party BLCL or random EC, suggesting that HLA antigens were recognized. The cell lines containing CD4+ cells only did not lyse any target cells. The lysis of EC without preincubation with gamma interferon (gIFN) indicated that the HLA antigens recognized were class I antigens. These results suggested that lysis of donor EC may be one of the mechanisms involved in rejection.

Donor directed cytotoxicity of cardiac graft infiltrating cells during cytomegalovirus infection.

We investigated whether cytomegalovirus (CMV) infection had an effect on donor directed cytotoxicity of cardiac graft infiltrating cells. The group we studied comprised 89 heart transplant recipients. Thirty eight showed signs of CMV infection, and in 27 of them cytolytic activity of biopsy-derived cultures could be tested during the infection. Fifty-one patients had never had CMV infection, and they were used as the control group. Eight patients had a primary, and 19 a secondary infection. We found that during CMV infection, both primary and secondary, a significantly higher proportion of the biopsy-derived cultures showed cytotoxicity against donor antigens (P < 0.01 when compared to the control group). In secondary infections, this was only due to an increase in donor class I directed cytotoxicity, while in primary infections a significant increase of class II directed cytotoxicity was also found (P < 0.005 when compared to secondary infection).