Kai Gutensohn

A randomized comparison of once versus twice daily recombinant human granulocyte colony-stimulating factor (filgrastim) for stem cell mobilization in healthy donors for allogeneic transplantation.

To evaluate the schedule dependency of granulocyte colony‐stimulating factor (G‐CSF) (filgrastim) for stem cell mobilization, we conducted a randomized comparison in 50 healthy donors, with one subcutaneous daily injection of 10 µg/kg G‐CSF (n = 25) compared with twice injections daily of 5 µg/kg G‐CSF (n = 25). The two groups were well balanced for age, body weight and sex. G‐CSF application was performed on an out‐patient basis and leukapheresis was started in all donors on day 5. The most frequent side‐effects of G‐CSF were mild to moderate bone pain (88%), mild headache (72%), mild fatigue (48–60%) and nausea (8%) without differences between the two groups. The CD34+ cell count in the first apheresis was 5·4 × 106/kg donor weight (range 2·8–13·3) in the 2 × 5 µg/kg group compared with 4·0 × 106/kg (range 0·4–8·8) in the 1 × 10µg/kg group (P = 0·007). The target of collecting > 3·0 × 106 CD34+ cells/kg donor weight with one apheresis procedure was achieved in 24/25 (96%) donors in the 2 × 5 µg/kg group and in 17/25 (68%) donors in the 1 × 10 µg/kg group. The target of collecting > 5·0 × 106 CD34+ cells/kg in the first apheresis was achieved in 64% in the 2 × 5µg/kg group, but in only 36% in the 1 × 10 µg/kg group. The progenitor cell assay for granulocyte–macrophage colony‐forming units (CFU‐GM) and erythroid burst‐forming units (BFU‐E) was higher in the 2 × 5 µg/kg group than in the 1 × 10 µg/kg group (7·0 vs. 3·5 × 105/kg, P = 0·01; 6·6 vs. 5·0 × 105/kg; P = 0·1). Administering G‐CSF (filgrastim) at a dosage of 5 µg/kg twice daily rather than 10 µg/kg once daily is recommended; this leads to a higher CD34+ cell yield and requires fewer apheresis procedures without increasing toxicity or cost.

The determination of the fetal D status from maternal plasma for decision making on Rh prophylaxis is feasible

BACKGROUND: Noninvasive fetal RHD genotyping might become a valuable tool in decision making on antenatal Rh prophylaxis, which is currently in routine practice for all D− pregnancies in several countries. This study provides a large‐scale validation study of this technology to address questions concerning feasibility and applicability of its introduction into clinical routine.

Stem cell mobilisation with 16 μg/kg vs 10 μg/kg of G-CSF for allogeneic transplantation in healthy donors

We compared two doses of recombinant human granulocyte-stimulating factor (G-CSF) for stem cell mobilisation in 90 healthy donors for allogeneic stem cell transplantation in a retrospective analysis. Group I (n = 46) received 10 μg/kg G-CSF (filgrastim) given as 5 μg/kg twice daily, and group II (n = 44) received 16 μg/kg, given as 8 μg/kg twice daily with a 12-h interval. The groups were well-balanced for age and body-weight. G-CSF application was performed on an out-patient basis, and leukapheresis was started in all donors on day 5. The most frequent side-effects of G-CSF were grade I/II, bone pain, headache and fatigue in both groups, whereas grade III of bone pain, headache and fatigue occurred in the 2 × 8 μg/kg group only. One serious non-fatal event with non-traumatic spleen rupture occurred in the 2 × 5 μg/kg group. The CD34+cell count in the first apheresis of all donors was 5.1 × 106/kg donor weight (range, 1.5–19.3). The CD34+ cell harvest was higher in the 2 × 8 μg/kg group than in the 2 × 5 μg/kg group (7.1 × 106/kg vs 4.9 × 106/kg; P = 0.09). The target of collecting >5.0 × 106 CD34+ cells/kg donor weight with one apheresis procedure was achieved in 45% of group I and in 61% of group II, respectively. Administering G-CSF at a dosage of 8 μg/kg twice daily leads to a higher CD34+ cell yield than a dosage of 2 × 5 μg/kg, but is associated with increased toxicity and higher cost.

Sulfatides Activate Platelets Through P-Selectin and Enhance Platelet and Platelet–Leukocyte Aggregation

Objective— Sulfatides are sulfated glycosphingolipids present on the surface of a variety of cells; however, their exact physiological function is not known. Recently, we have shown that the inhibition of sulfatide–P-selectin interactions leads to disaggregation of platelet aggregates. Methods and Results— In this study, we show that sulfatides activated platelets as they increased activation of GPIIb/IIIa (PAC-1 epitope) and expression of P-selectin on the platelet surface. Furthermore, sulfatides aggregated washed platelets in a dose-dependent manner and enhanced platelet aggregation in platelet-rich plasma. Previous activation of platelets was necessary for this effect. Monoclonal anti–P-selectin antibodies inhibited not only sulfatide-induced PAC-1 binding to platelets but also sulfatide-induced platelet aggregation, suggesting that sulfatides activate platelet GPIIb/IIIa via signaling through P-selectin. The proaggegatory effect of sulfatides was also observed in an ex vivo thrombosis model using whole blood and pulsatile flow at 37°C. In this model, sulfatides significantly enhanced platelet aggregation and the formation of platelet–leukocyte aggregates. Conclusions— We show that sulfatide-P-selectin interactions lead to subsequent platelet activation and P-selectin expression, forming a positive feedback loop that can potentiate formation of stable platelet aggregates. In addition, sulfatides enhance the aggregation of platelet–leukocyte aggregates. These mechanisms may play a significant role in hemostasis and thrombosis.

Immunomagnetic tumor cell selection—implications for the detection of disseminated cancer cells

BACKGROUND: The optimal method for the detection of disseminated epithelial cancer cells has not yet been found. The standard method, using immunocytochemistry, offers a sensitivity of up to 10−6. Molecular methods such as cytokeratin‐19 RT‐PCR are about 10 times as sensitive, but they are hampered by interference such as illegitimate gene expression.

Stem cell mobilization with G-CSF alone in breast cancer patients: higher progenitor cell yield by delivering divided doses (2 × 5 μg/kg) compared to a single dose (1 × 10 μg/kg)

We investigated the schedule dependency of G-CSF (10 μg/kg) alone in mobilizing peripheral blood progenitor cells (PBPC) in breast cancer patients. After a median of three cycles (range, 2–6) of anthracycline-based chemotherapy, 49 patients with breast cancer (stage II/III, ⩾10+ Ln n = 36; locally advanced/ inflammatory n = 8, stage IV (NED) n = 5) underwent PBPC collection after steady-state mobilization either with 1 × 10 μg/kg (n = 27) or with 2 × 5 μg/kg (n = 22) G-CSF daily for 4 consecutive days until completion of apheresis. Apheresis was started on day 5. Priming with 2 × 5 μg/kg resulted in a higher median number of CD34+ cells (5.8 vs 1.9 × 106/kg, P = 0.003), MNC (6.6 vs 2.6 × 108/kg, P < 0.001) and cfu-gm (6.5 vs 1.3 × 104/kg, P = 0.001) in the first apheresis than with 1 × 10 μg/kg. Also the overall number of collected BFU-E was higher in the 2 × 5 μg group (9.2 vs 3.1 × 104/kg; P = 0.01). After high-dose chemotherapy with cyclophosphamide/thiotepa/mitoxantrone (n = 46) hematopoietic engraftment with leukocyte count >1.0/nl was reached in both groups after a median of 10 days (range, 8–15) and with platelets count >50/nl after 12 (range, 9–40) and 13 days (range, 12–41), respectively. A threshold of >2.5 × 106/kg reinfused CD34+ cells ensured rapid platelet engraftment (12 vs 17 days; P = 0.12). Therefore, the target of collecting >2.5 × 106 CD34+ cells was achieved in 21/27 (80%) patients of the 1 × 10 μg group and in 21/22 (95%) patients of the 2 × 5 μg/kg group with a median of two aphereses (range, 1–4). None in the 10 μg/kg group, but 6/22 (28%) patients in the 2 × 5 μg/kg group required only one apheresis procedure, resulting in fewer apheresis procedures in the 2 × 5 μg/kg group (mean, 1.8 vs 2.3, P = 0.01). These results demonstrate that priming with 10 μg/kg G-CSF alone is well tolerated and effective in mobilizing sufficient numbers of CD34+ cells in breast cancer patients and provide prompt engraftment after CTM high-dose chemotherapy. G-CSF given 5 μg/kg twice daily (2 × 5 μg) leads to a higher harvest of CD34+ cells and required fewer apheresis procedures than when given 10 μg/kg once daily (1 × 10 μg).

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.

Cell-free fetal DNA in specimen from pregnant women is stable up to 5 days.

Before noninvasive prenatal diagnosis on the fetal Rhesus D status (NIPD RhD) can be implemented on a mass‐scale, it is crucial to define requirements regarding sample transport. The aim of this study was to determine the relation between the transport time of samples for NIPD and the concentration of fetal DNA in maternal plasma.

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.