For the reconstitution of hematopoiesis after Myeloablative chemo-therapy the transplantation of peripheral blood progenitor cells (PBPC) is increasingly performed. The engraftment success is correlated with the number of infused CD34+ cells [2]. For the flow cytometric determination of these cells there are two Main clinical applications: The timing of the stem cell harvest and the determination of the harvest yield.

Storage of regular platelet concentrates (PCs) is limited to 5 days, predominantly due to the risk of possible bacterial contamination. Alternative approaches for platelet storage May result in a prolonged storage period. In this study PCs were cryopreserved with a new storage solution (ThromboSol; LifeCell Corp., Branchburg, USA) [1,2]. CD62p expression is a useful Measure for quality control during platelet storage [3]. To verify platelet activation flow cytometric analyses was performed in the cryopreserved PCs [4]. For this purpose the expression of an activationdependent neoepitope (CD62p) was analyzed on platelet surface in PCs before and after platelet cryopreservation.

In the past years prophylactic or therapeutic platelet transfusions have become a routine treatment. Therefore platelet storage prior to transfusion became necessary. One of the problems occurring during storage of platelet concentrates (PCs) is a decrease in platelet function, also known as platelet storage lesion [4]. It appears to be pronounced More with some forms of agitation of PCs during storage [1]. The platelet storage lesion is associated with an in vitro activation of platelets. The purpose of this study was to compare a new rotational storage device with the storage of PCs under routine conditions on a flat bed agitator. Quality control of PCs was performed Measuring the P-selectin expression (CD62p), that is used as a predictor of platelet activation in vitro, on the platelet surface by flow cytometry [2,3].

Acute occlusion of stented coronary vessels still occurs in up to 3%. Acitvated platelets have been found to play a major role in the pathogenesis of these complications. We therefore analyzed the efficacy of a heparin coating of coronary stents and investigated the ex vivo efficacy of different antiplatelet drugs. Each of seven healthy volunteers was treated with each of the following medications for 7 days: ASA 100 mg/day, ASA 300 mg/day, ticlopidine 250 mg/day, and ticlopidine 500 mg/day. Three standardized in vitro silicon tubing models, one of them containing an uncoated stent, one a heparin-coated stent, and one without a stent ( control) were filled with PRP and circulation was started. TOS in systems with heparin-coated stents was 2.4-times longer compared to systems with uncoated stents (P<0.001), and 1.5-times longer compared to the control (P<0.01). The increase of CD62p expression within the first 5 min was 2.5-times higher in systems with uncoated stents and 1.7-times higher in the control than in systems with heparin-coated stents (P<0.05). Aggregometry revealed significant medication- and dose-dependent inhibition of platelet aggregability for all medications. Heparin-coating of coronary stents reduces their thrombogenicity significantly. ASA and ticlopidine effectively reduce platelet activation ex vivo. The used in vitro system facilitates a reproducible method to estimate the thrombogenicity of coronary stents prior to in vivo trials.

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). STUDY DESIGN AND METHODS:Double apheresis concentrates were prepared with two different cell separators. One of the split products was stored for 1 to 2 days and the other one for 3 to 5 days. Flow cytometry was applied to analyze the degree of platelet activation in vitro, and also to measure the extent of platelet binding to WBC subclasses in vivo after transfusion into patients. RESULTS:The results of this study show that platelet activation occurs during apheresis and storage of PCs. After transfusion of the PCs, no significant binding of platelets to T or B-cells could be detected. However, a significant binding of platelets to monocytes and neutrophil granulocytes occurs. While in Baxter PCs stored for 1-2 days the amount of platelet-leukocyte aggregates in vivo was higher compared to COBE PCs, no such difference could be detected anymore for the PCs stored for 3-5 days. CONCLUSION:This study demonstrates that binding of activated platelets occurs to monocytes and neutrophil granulocytes but not to T- and B-cells in the circulation after transfusion. In addition, the interaction of platelets and WBCs is dependent on the degree of P-selectin expression. Platelets showing a higher degree of activation adhere to WBCs to a higher degree than nonactivated platelets.

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⩽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.

In human type 1 diabetes (T1D) autoantibodies to insulin precede clinical disease, while little is known about the contribution of insulin-specific T lymphocytes-in particular, T helper (Th) subsets. Here we have studied the in vivo primed cytokine response to preproinsulin in peripheral blood mononuclear cells (PBMCs) and two major Th cell subsets-CD45RO+ memory cells and CD45RA+ naive/resting cells-in 35 individuals with HLA-DRB1*04, DQB1*0302 diabetes risk marker: 12 patients with T1D, 12 autoantibody-positive (Ab+) individuals, and 11 healthy controls. Cytokine secretion (TNF-alpha, IFN-gamma, IL-2, IL-4, IL-5, and IL-10) was measured in the supernatants of the cultures stimulated with 21 overlapping preproinsulin peptides as well as proinsulin and insulin. In Ab+ individuals our results reveal higher IL-4 levels in CD45RO+ memory cells and higher IL-5 levels in CD45RA+ naive/resting cells, while higher IL-2 production was found in PBMCs. In contrast, in PBMCs of T1D patients higher IFN-gamma and IL-10 secretion was found. Our data delineate characteristic cytokine patterns in peripheral T lymphocytes from patients at different stages of the T1D development.

Today, transfusions of platelet products are important blood components for the management of patients suffering from congenital or acquired platelet or hemostatic disorders. Platelet products may be prepared by apheresis. Production of platelet concentrates by this method is usually performed in an extracorporal circuit. Due to the influence of the extracorporal system, different interactions may occur between the blood and artificial components of the apheresis device [1, 2].These effects are provoked mainly by the contact between the donor’s blood and biomaterials as well as shear stress.

To reconstitute hematopoiesis after myeloablative chemotherapy the transplantation of autologous or allogeneic peripheral blood progenitor cells (PBPC) is increasingly performed [6, 10]. Following G-CSF mobilization PBPC can be collected from peripheral blood by leukapheresis [3].

Background. Determining the onset of peripheral blood stem cell apheresis is known to be associated with several advantages. The method applied most commonly so far is the immunological analysis of cells expressing the CD34 antigen by flow cytometry. In this study a new parameter for monitoring was tested: the measurement of the so-called human progenitor cell (HPC) parameter by an automated hematology analyzer. Materials and Methods: Eleven multiple myeloma patients were included in this study. Following the white blood cell nadir monitoring of CD34+ cells by flow cytometry and of IMI-total (immature myeloid information) and HPC counts by a hematology analyzer (Sysmex SE-9000(TM)) were performed daily. The quality of the harvest product was determined by flow-cytometric analysis. Results: Monitoring was performed on 46 days (total). Comparing the IMI-total results with the immunological CD34+ cell analyses in peripheral blood, a correlation of r=0.609 (p<0.05) was obtained. When comparing the CD34+ cell analysis with the HPC count, a correlation of r=0.477 (p=0.05) resulted. A total of 16 PBSC aphereses was performed. When comparing the preapheresis peripheral blood measurements to the CD34+ cell content of the harvest product, a correlation of r=0.383 (p=0.14) and of r=0.254 (p=0.34) was calculated for the IMI-total counts, and the HPC counts, respectively. The CD34+ pre-apheresis results showed an excellent correlation with the quality of the peripheral blood stem cell apheresis product (r=0.937; p<0.05). Conclusion: The gold standard for determining the timing of PBSC apheresis remains the analysis of CD34+ stem and progenitor cells by flow cytometry. The determination of the timing of an apheresis is not possible with the IMI-total or HPC results. In addition, a predictive impression of the graft quality to be expected is only possible with flow cytometry. However, the HPC counts may be applicable for the determination of the onset of CD34+ cell flow-cytometric analyses. Further tests will have to be performed to prove this finding.

The transfusion of peripheral blood progenitor cells (PBPC), autologous or allogeneic, is increasingly being performed to provide rapid hematopoietic recovery after myeloablative high-dose chemotherapy [4, 6]. The application of G-CSF stimulates the proliferation of bone marrow stem cells so that they can be collected from peripheral blood by leukapheresis [2].