Peter Kuehnl

Living donor for liver transplantation

Since living related liver transplantation was first performed in 1989, more than 150 cases have been performed worldwide, mostly in the United States and Japan. This paper reports the first series of living related liver transplantation in Europe. Twenty living related liver transplantation surgeries were performed over a 13‐mo period, with an overall patient survival of 85%. For patients who underwent elective transplantation (n=13), the survival rate was 100%. Technical complications included one arterial thrombosis necessitating retransplantation and five bile leaks requiring surgical revision. The technical improvements that permit avoidance of these complications are discussed. A detailed description of the living related liver procurement is given. All procurements yielded grafts of excellent quality. No intraoperative complications occurred, and no reoperations were necessary. No heterologous blood transfusion was needed. In two patients, incisional hernias developed after wound infection. Living related liver transplantation does not absolve the transplant community of efforts to promote cadaveric organ procurement. Nevertheless, living related liver transplantation does have the advantage of a readily available graft of excellent quality, permitting transplantation with optimal timing under elective conditions. Several centers are now preparing living related segmental liver transplants, following the model of our protocol, for three reasons: (a) to obtain superior results compared with cadaveric liver transplantation; (b) to overcome cadaveric organ shortage and further reduce pretransplantation mortality and (c) to provide viable organs in countries where cadaveric organ procurement is not established. When performed by a team experienced in pediatric liver transplantation and in adult liver resection, living related liver transplantation is an excellent modality for the treatment of end‐stage liver disease in children. (Hepatology 1994;20:49S‐55S.)

Flow cytometric analysis of coronary stent-induced alterations of platelet antigens in an in vitro model

One of the limitations of coronary stenting is the subacute thrombotic occlusion. In an in vitro model, we examined the effects of tantalum wire stents (n = 12) on platelet antigens. Platelet-rich plasma (PRP) was circulated in PVC tubing systems. At fixed intervals over a 10-min time course, aliquots of PRP were drawn, stained with monoclonal antibodies (CD41a, CD42b, CD62p, and CD63), and analyzed by flow cytometry. Within 2 minutes of the onset of circulation, expression of the activation-dependent antigens CD62p and CD63 increased in all tubing systems with stents. This early increase was followed by a progressive rise in fluorescence intensity of these neoantigens over the course of 10 minutes (p < 0.05 vs.. control system without stent). Antigens CD41a and CD42b did not show significant changes in either system. The artificial surfaces and shear forces of stent meshes induce alterations in platelet antigens. Flow cytometry provides a sensitive technique for in vitro testing of the thrombogenicity of coronary stents, and may be useful in further improving stent biocompatibility.

Flow cytometric analysis of platelet membrane antigens during and after continuous-flow plateletpheresis.

BACKGROUND: The influence, extent, and duration of changes in platelet antigen expression caused by blood‐biomaterial interaction in plateletpheresis were assessed. STUDY DESIGN AND METHODS: Twenty‐two apheresis donors were studied by using two automated continuous‐flow apheresis devices. Blood samples were taken before, during, and for 4 days after extracorporeal circulation. The platelet surface expression of glycoproteins CD41a, CD42b, CD62p, and CD63 was analyzed by flow cytometry. RESULTS: Over the course of plateletpheresis, there was a significant increase in mean channel fluorescence intensity (MCFI) of CD62p, from 25.1 +/− 7.9 (mean +/− SD) to 50.4 +/− 28.9, and of CD63, from 22.3 +/− 6.5 to 33.3 +/− 13.2. There was a significant decrease in CD41a expression as measured by the MCFI, from 1129.8 +/− 125.0 to 1066.6 +/− 102.2, and in CD42b MCFI, from 329.6 +/− 49.4 to 321.4 +/− 52.0. The two apheresis devices showed different platelet activation kinetics, but the overall MCFI of CD62p and CD63 did not significantly diverge after 60 minutes of apheresis. CD62p and CD63 expression as measured by the MCFI returned to preapheresis levels during the follow‐ up period in 25 and 25 of 44 procedures, respectively, within 24 hours; in 10 and 13 of 44 procedures after 48 hours; in 7 and 3 of 44 procedures after 72 hours; and in 2 and 3 of 44 procedures on Day 5. CONCLUSION: The varying kinetics of expression, as measured by the MCFI, of platelet antigens CD62p, CD63, CD41a, and CD42b during extracorporeal circulation may be useful for biocompatibility testing. Activated platelets continue to circulate in donors for several days after cytapheresis, which suggests that a sufficient interval between apheresis procedures is necessary to avoid the collection of activated platelets.

Binding of activated platelets to WBCs in vivo after transfusion

BACKGROUND : During preparation and storage of apheresis concentrates, platelets are being activated. One of the alterations that occur during this process is an increased expression of P‐selectin (CD62p) on the cytoplasmic surface of platelets. This neoepitope represents a ligand for the binding of platelets to WBCs. It has been suggested that the activation of platelets is associated with the sequestration of platelets after transfusion. In this in vivo study, the binding of platelets to WBCs was analyzed following transfusion of platelet concentrates (PCs).

Platelet function testing in apheresis products: flow cytometric, resonance thrombographic (RTG) and rotational thrombelastographic (roTEG) analyses.

During storage of platelet concentrates, quality control of the units is mandatory. This includes the important testing of the hemostatic function of platelets. So far, mostly platelet aggregation analyses have been performed. In this study, new approaches were tested to evaluate the applicability of modern techniques for quality monitoring. Plateletpheresis was performed with two different cell separators (AMICUS cell separator, Fenwal, Baxter Healthcare, Deerfield, USA; COBE Spectra, COBE BCT, Lakewood, USA). In each procedure split products (n = 22) were prepared and stored for 1-2 days (n = 22) or 3 5 days (n = 22). Platelet hemostatic capacity was tested by applying flow cytometry. platelet aggregation (platelet-rich-plasma [PRP]+agonist), resonance thrombography (RTG; PRP, no agonist) and rotational thrombelastography (roTEG; PRP+agonist). Flow cytometric analyses did not reveal significant changes in structural (CD41a. CD42b) or activation-dependent antigens (CD62p, CD63, LIBS, RIBS). Also, differences in the data from the flow cytometric reactivity tests were not significant between the two groups. In platelet aggregation assays, shape change (p = 0.8), maximum aggregation (p = 0.4), and maximum gradient (p = 0.8) did not show significant differences between the two groups. In the RTG test, differences between r-time (reaction time; p = 0.4), and f-time (clot formation time [fibrin influence]; p = 0.3), and in roTEG r-time (coagulation time; p = 0.1) and k-time (clot formation time; p = 1.0) were not significant. P-time (clot formation time [platelet influence]) and M (maximum amplitude) in RTG, and k-time and MA (maximum amplitude) in roTEG showed a slight decrease in platelet function (p < or = 0.05). We conclude that platelet function is well maintained during storage. This is reflected by the results of immunological and platelet function assays. Rotational thrombelastography (in the case of PRP) and especially resonance thrombography represent promising methods for quality control of platelet concentrates and rapidly provide information about the status of platelet function and the whole clotting process.

Platelet Hemostasis Capacity in Smokers: In Vitro Function Analyses with 3.2% Citrated Whole Blood

INTRODUCTION Platelet function may be influenced by cigarette smoking. We therefore examined the effect of smoking on platelet hemostasis capacity (PHC) with an in vitro analyzer (PFA-100). METHODS AND RESULTS Healthy blood donors (n=54) were included in the study and divided into four groups: nonsmoking males (n=14), nonsmoking females (n=14), smoking males (n=12) and smoking females (n=14). For in vitro analyses, in each participant citrated blood (3.2% buffered) was tested for PHC by two cartridges coated with collagen, and additionally with epinephrine (Col/Epi) or ADP (Col/ADP). Analyses were performed within 4 h after sample taking. PHC was expressed as the time in seconds to occlude the cartridge (closure time, CT). The average CT was significantly prolonged in female smokers compared to the female nonsmoking group for both types of cartridges (Col/Epi: P=.02; Col/ADP: P=.03). No significant differences were detected comparing the CT of smoking and nonsmoking males. After pooling male and female smokers and nonsmokers, no significant differences could be found, neither for the Col/Epi cartridges nor the Col/ADP cartridges. Plaletet aggregation assays performed in parallel showed no significant differences, except a reduced aggregability in male smokers compared to male nonsmokers using epinephrine 8.0 microM/ml as activating agent (P=.01). Furthermore, smoking volunteers presented with a significantly increased fibrinogen level compared to nonsmoking volunteers (P<.01). CONCLUSIONS The results of our study show that in habitual smokers PHC (PFA-100) and the capability of platelets to react upon agonist stimulation in aggregation assays is not significantly influenced or increased compared to healthy nonsmokers. However, an immediate effect of cigarette smoking cannot be excluded.

Semi-automated flow cytometric analysis of CD34-expressing hematopoietic cells in peripheral blood progenitor cell apheresis products

BACKGROUND: The measurement of CD34+ cells is the most important step in the quality control of peripheral blood progenitor cell apheresis products. For this purpose, flow cytometry is applied. Recently, a new test kit has been introduced for the enumeration of CD34‐expressing cells, in combination with software support for semi‐automation of data acquisition and analysis.

The complement factor properdin induces formation of platelet-leukocyte aggregates via leukocyte activation

Both the complement system and platelet-leukocyte aggregates are involved in chronic and acute stages of atherosclerosis. Properdin, a positive regulator of the complement system, is secreted by leukocytes and endothelial cells. In the present study, the role of properdin in the formation of platelet-leukocyte aggregates was investigated. Incubation of human whole blood with properdin (25–200 µg/ml) resulted in a dose-dependent formation of platelet-leukocyte aggregates, with an increase of up to 2.2-fold compared to controls (p < 0.05), as analysed by flow cytometry. In addition, properdin significantly amplified ADP-induced aggregation of platelets with leukocytes by 53% (p < 0.05), while it had no effect on ADP-induced aggregation of platelets alone. Consistent with these results, properdin did not activate platelets as shown by the expression of activated GPIIb/IIIa (PAC-1 epitope) and P-selectin (CD62P) on the platelet surface. However, properdin significantly induced expression of CD11b (MAC-1) on leukocytes by 12-fold (p < 0.05) as a measure of leukocyte activation. In conclusion, the complement system component properdin induces the formation of platelet-leukocyte aggregates via leukocyte activation. The data establish a link between the complement system and platelet-leukocyte aggregates with potential significance in atherosclerotic vascular disease.

Increasing the economic efficacy of peripheral blood progenitor cell collections by monitoring peripheral blood CD34+ concentrations

BACKGROUND: After mobilization, the collection of peripheral blood progenitor cells (PBPCs) can either be started a fixed number of days after having passed the white blood cell nadir (fixed‐day scheme) or be based on monitoring of CD34+ cells. This study was conducted to compare both approaches and to assess possible financial consequences.

The role of flow cytometry in improving biocompatibility in transfusion medicine.

In transfusion medicine, blood and blood components, donors and patients are increasingly confronted with biomaterials. The need to understand the response of human blood to contact with these artificial surfaces has led to multiple studies on the biocompatibility of biomaterials. Up to this time, these investigations have predominantly been performed using physical, immunological and biochemical methods. Many of these approaches are useful in investigating the multiple factors involved in blood-biomaterial interactions. However, they always reflect the overall behaviour of whole cellular populations in local or systemic reactions. The application of multiparameter flow cytometry, on the other hand, provides insight into antigenic expression and changes at the single-cell level. Therefore, the technique of flow cytometry represents a new and powerful way of analysing and improving the biocompatibility of these materials in blood-contacting applications in this field.