Validation of cord blood split products prepared by an automated method.

OBJECTIVES: In this study, we studied whether the contents of the two compartments of automatically processed cord blood (CB) units are comparable with respect to cell counts and viability and therefore suitable for clinical therapy. BACKGROUND: CB-derived stem cells are increasingly used for allogeneic transplantation. Many centres prepare the transplants by automated methods allowing to split the product into two portions. METHODS: CB was collected at different sites in Germany and transported to a single centre for processing. Before and after cryopreservation laboratory analyses were performed to compare the quality of the two CB segments. RESULTS: In total, 33 products were processed [mean collection volume: 18·6 ± 1·2 mL (range 15·2-20·2 mL) segment A; mean: 4·7 ± 0·3 mL (range 4·2-5·2 mL) segment B]. CD34+ cell counts, viability of CD34+ cells and many other haematological parameters showed a good comparibility between the two segments. However, lymphocyte counts and results of clonogenic assays were significantly different between the two segments of the split product. CONCLUSION: We conclude that the preparation of the cord blood unit by the automated process results in a homogenous distribution of stem and progenitor cells. However, our findings show that the clonogenic capacity differs between the two segments.

Direct volumetric flow cytometric quantitation of CD34+ stem and progenitor cells.

OBJECTIVES: In this study, we compared a classic single-platform (SP) method applying beads for enumeration of CD45+ or CD34+ cells with a new device allowing direct volumetric measurements of stem and progenitor cells. BACKGROUND: Following apheresis and cyropreservation, the precise enumeration of CD34+ cells as key parameter of graft quality is mandatory for the clinical course after transplantation. Currently, flow cytometry with SP technique represents the 'gold standard' for such determinations. METHODS/MATERIALS: Fresh samples, 14 from mobilised peripheral blood (PB), 9 from apheresis products (AP) and 13 samples from frozen-thawed (FT) haematopoietic progenitor cell grafts, were analysed for CD34+ cells, CD45+ cells, and in frozen-thawed samples for viability by a bead-based flow cytometric method and in parallel by a direct, volumetric flow cytometric method. RESULTS: Comparison of CD34+ analyses revealed a significant correlation (P < 0·01) for each material between both techniques with r = 0·95 (PB), r = 0·933 (AP) and r = 0·929 (FT). Also, for analysis of CD45+ cells µL(-1) , the measured numbers evaluated with the different techniques did not significantly differ for all three materials analysed. In frozen-thawed samples, the analysis of viability was comparable for both techniques. CONCLUSIONS: The results of this study demonstrate that a direct volumetric analysis of CD34+ cells µL(-1) or CD45+ cells µL(-1) is feasible. This technique represents a simple and economical approach for standardisation of progenitor and stem cell analyses.

Diagnostic accuracy of noninvasive polymerase chain reaction testing for the determination of fetal rhesus C, c and E status in early pregnancy.

OBJECTIVE: The aim of the study was to determine the sensitivity, specificity and accuracy of noninvasive tests for the fetal rhesus CcEc (RHCE) alleles C, c and E in early pregnancy. DESIGN: A prospective clinical trial was carried out to evaluate diagnostic accuracy. SETTING: Women were recruited at four centres specialising in prenatal diagnosis. Peripheral blood and amniotic fluid samples were obtained and sent to a single laboratory for analysis. SAMPLE: A total of 233 tests (46 for C, 87 for c and 100 for E) were performed on 181 specimens obtained from pregnant women at weeks 12 to 28 (median week 16) of gestation. METHODS: Following automated extraction of fetal DNA from maternal plasma, two different real-time polymerase chain reaction (PCR) protocols were used for the detection of the C, c and E alleles of RHCE. The results of the PCR were compared with genotyping results for the amniotic fluid. MAIN OUTCOME MEASURES: Failure rate, sensitivity, specificity and accuracy were the main outcome measures. RESULTS: Unequivocal results were obtained for all specimens. With the first PCR protocol, the sensitivity was 100% for C, 38% for c and 59% for E. In contrast, with the second protocol, the sensitivity for C, c and E was 100%. The specificity for all assays was found to be between 99% and 100%. CONCLUSIONS: A highly accurate protocol has been identified for the detection of fetal RHCE alleles in maternal plasma in early pregnancy. This noninvasive approach can be considered as a useful test in the management of pregnancies with anti-c, anti-E or anti-C alloimmunisation.

Karyotype in multiple myeloma and plasma cell leukaemia.

Between October 1988 and October 1991, 104 patients with multiple myeloma and 6 with plasma cell leukaemia were studied cytogenetically. Abnormal karyotypes were found in bone marrow cells of 33 patients (30%). Most pathological karyotypes were complex with numerous modal and structural anomalies. Numerical anomalies most frequently involved chromosome 11 and structural aberrations occurred most often in chromosomes 1, 11 and 14. The most consistent structural aberration was a 14q+ chromosome (10 patients) resulting from a t(11;14)(q13;q32) in 4 patients and a t(8;14)(q24;q32) in 1 patient. Sequential cytogenetic studies were performed in 15 patients. In 5 of 8 cases with a normal karyotype at diagnosis, chromosomal anomalies were detected when disease progressed. In concomitant cytogenetic/cytological studies it was found that in the majority of patients with normal karyotype the mitoses originated from contaminating normal bone marrow cells. Pathological karyotypes were detected more frequently in pretreated than in untreated patients, in patients with plasma cell leukaemia than in patients with multiple myeloma, in patients with stage III and dense bone marrow infiltration than in patients with stage I. Patients with abnormal karyotype, irrespective if pretreated or not, had a significantly shorter median survival than those with normal karyotype. These findings suggest that karyotype is an independent prognostic factor in multiple myeloma.

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

We investigated the schedule dependency of G-CSF (10 microg/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, > or = 10+ Ln n = 36; locally advanced/inflammatory n = 8, stage IV (NED) n = 5) underwent PBPC collection after steady-state mobilization either with 1 x 10 microg/kg (n = 27) or with 2 x 5 microg/kg (n = 22) G-CSF daily for 4 consecutive days until completion of apheresis. Apheresis was started on day 5. Priming with 2 x 5 microg/kg resulted in a higher median number of CD34+ cells (5.8 vs 1.9 x 10(6)/kg, P = 0.003), MNC (6.6 vs 2.6 x 10(8)/kg, P < 0.001) and CFU-GM (6.5 vs 1.3 x 10(4)/kg, P = 0.001) in the first apheresis than with 1 x 10 microg/kg. Also the overall number of collected BFU-E was higher in the 2 x 5 microg group (9.2 vs 3.1 x 10(4)/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 x 10(6)/kg reinfused CD34+ cells ensured rapid platelet engraftment (12 vs 17 days; P = 0.12). Therefore, the target of collecting > 2.5 x 10(6) CD34+ cells was achieved in 21/27 (80%) patients of the 1 x 10 microg group and in 21/22 (95%) patients of the 2 x 5 microg/kg group with a median of two aphereses (range, 1-4). None in the 10 microg/kg group, but 6/22 (28%) patients in the 2 x 5 microg/kg group required only one apheresis procedure, resulting in fewer apheresis procedures in the 2 x 5 microg/kg group (mean, 1.8 vs 2.3, P = 0.01). These results demonstrate that priming with 10 microg/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 microg/kg twice daily (2 x 5 microg) leads to a higher harvest of CD34+ cells and required fewer apheresis procedures than when given 10 microg/kg once daily (1 x 10 microg).

Stem cell mobilisation with 16 microg/kg vs 10 microg/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 microg/kg G-CSF (filgrastim) given as 5 microg/kg twice daily, and group II (n = 44) received 16 microg/kg, given as 8 microg/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 x 8 microg/kg group only. One serious non-fatal event with non-traumatic spleen rupture occurred in the 2 x 5 microg/kg group. The CD34(+)cell count in the first apheresis of all donors was 5.1 x 10(6)/kg donor weight (range, 1.5-19.3). The CD34(+) cell harvest was higher in the 2 x 8 microg/kg group than in the 2 x 5 microg/kg group (7.1 x 10(6)/kg vs 4.9 x 10(6)/kg; P = 0.09). The target of collecting >5.0 x 10(6) 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 microg/kg twice daily leads to a higher CD34(+) cell yield than a dosage of 2 x 5 microg/kg, but is associated with increased toxicity and higher cost.

Purging and haemopoietic progenitor cell selection by CD34+ cell separation.

Tumour cell contamination of autologous peripheral blood stem cell samples (PBSC) and bone marrow (BM) is frequent. Enrichment of CD34+ stem cells is a promising approach to purging tumour cells from autografts without damaging progenitor cells. Breast cancer cells were seeded (10(-3)-10(-7)) into mononuclear cells from G-CSF-mobilised PBSC and BM harvests from patients without breast cancer. CD34+ cells were enriched from mixtures either by immunomagnetic separation (Isolex-50, and MiniMACS) or by biotin-streptavidin immunoaffinity columns (Ceprate-LC). CD34+ cell fractions were determined by FACS, cancer cells were detected immunocytochemically with an anti-pancytokeratin antibody. The CD34+ cells were enriched with a median purity of 92.2% (43.5-96.1) (n = 17) (Isolex-50), 96.5% (66.6-99.2) (n = 17) (MiniMACS) and 77.9% (31.4-93.6) (n = 15) (Ceprate-LC) from PBSC and BM harvests. The percentages of median recovery of CD34+ cells were 30.8% (18.6-71.8) (Isolex-50), 69.9% (39.1-100) (MiniMACS) and 42.9% (23.7-100) (Ceprate-LC). The median tumour cell reductions in log steps were 3.7 (2.9-4.3) (n = 13) (Isolex-50), 3.5 (2.6-4.3) (n = 13) (MiniMACS) and 1.5 (0.9-2.9) (n = 17) (Ceprate-LC). Results were compared statistically by univariate analysis. Purity was significantly (P < 0.05) better after MiniMACS selection. Recovery rates were significantly different between all devices tested. Tumour cell purging was superior after immunomagnetic separation (P < 0.001). Tumour cell purging is a main objective of CD34+ selection in the autologous setting. Our in vitro data clearly indicate that immunomagnetic separation is more efficient in the prevention of accidental reinfusion of contaminating tumour cells compared to immunoaffinity. However, it is not yet known if the same results can be obtained with fresh contaminating tumour cells.

Detection of cancer cells in peripheral blood stem cells of women with breast cancer by RT-PCR and cell culture.

The benefit of high-dose therapy and blood stem cell reinfusion for women with high-risk breast cancer is currently under investigation. Contaminations of autologous blood stem cells with cancer cells have been described. Cancer micrometastases may be detected by immunocytochemistry, culture techniques and cytokeratin-19 mRNA reverse transcriptase PCR. Women with breast cancer received adjuvant HD-CTM with peripheral blood stem cell (PBSC) support after surgical therapy and 4 cycles conventional chemotherapy. Peripheral blood stem cells were mobilised by G-CsF and harvested after the third or fourth cycle of standard therapy. Aliquots of PBSC-collections (10(7)-2*10(7) cells) were subjected to CK19-mRNA reverse transcriptase PCR. RNA was extracted by standard methods and reverse transcription was performed with MMV-RT. Integrity of RNA was checked by coamplification of housekeeping sequences. Aliquots of the RT-mix were subjected to PCR-amplification with outer and inner primer pairs, subsequently. A second aliquot of 2*10(7) cells was cultured over 42 days in liquid culture. Cytospins were prepared weekly from cultured cells and evaluated by light microscopy with or without prior immunocytochemistry. Ten leukaphereses from 6 women were available for PCR-analysis and cell culture. Six leukaphereses were negative for CK19-mRNA and for detection of cancer cells by culture technique, two samples were positive for CK19-mRNA and culturally enriched cells and two samples were positive for CK19-mRNA and negative for cultured cancer cells. No sample was positive for cultured cells and negative for CK19-mRNA. Overall, the results corresponded in 80%. Two sensitive techniques for the detection of cancer micrometastases were applied to aliquots from 10 leukaphereses of six breast cancer patients with corresponding results in 80%. PCR-mediated detection of cancer cells was confirmed by culture technique and light microscopy, however, further comparison of CK19-PCR with standard techniques like cell culture and immunocytochemistry is still necessary.

Increase of mobilized CD34-positive peripheral blood progenitor cells in patients with Hodgkin's disease, non-Hodgkin's lymphoma, and cancer of the testis.

G-CSF (filgrastim) can effectively mobilize peripheral blood progenitor cells (PBPC) when administered during steady-state hematopoiesis. In this single center study, we compared the effectiveness of two different doses of G-CSF on the mobilization of peripheral blood stem cells in patients with Hodgkin's disease, non-Hodgkin's lymphoma, and cancer of the testis. A first group including 33 patients received 10 micrograms G-CSF/kg BW per day (group A), whereas a second group comprising 34 patients was treated with 24 (2 x 12) micrograms G-CSF/kg body weight (BW) per day (group B) prior to the leukapheresis. A significant difference (P = 0.015) in the total number of CD34+ cells between group A: 11.32 x 10(7) (range 0.34-110.2) and group B: 48.25 x 10(7) (range 1.33-447.4) has been observed in the first leukapheresis product. Moreover, the total number of CFU-GM increased significantly from 34.79 x 10(4) (range 1.07-300.9) to 147.69 x 10(4) (range 1.03- 1204.0) (P < 0.005), and the number of MNC increased from 1.35 x 10(10) (range 0.41-3.09) group A) to 2.93 x 10(10) (range 0.66-9.7) (group B) (P < 0.001). Comparable results were obtained in the second leukapheresis. Our data indicate, that the application of higher doses of G-CSF can significantly improve the effectiveness of mobilizing PBPC during steady-state conditions, and thereby considerably contribute to a safe and fast engraftment as well as a reduced number of leukapheresis procedures to achieve sufficient number of PBPC.

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.

Enhanced platelet aggregation with TRAP-6 and collagen in platelet aggregometry in patients with venous thromboembolism.

The role of platelet hyperaggregability as a possible risk factor for venous thromboembolism is not well defined. Some authors described enhanced maximal platelet aggregation in platelet aggregometry as a contributing factor for arterial and venous thrombosis. This syndrome has been termed "sticky-platelet syndrome" (SPS). The diagnosis of SPS is based on the demonstration of platelet hyperaggregability in aggregometry after stimulation with epinephrine (EPI) and/or adenosine diphosphate (ADP). We investigated platelet hyperaggregability in platelet-rich plasma (PRP) of patients (n = 34) with unexplained venous thromboembolism in comparison to healthy individuals (n = 53). For analysis, platelet aggregometry was performed and the influence of epinephrine, adenosine diphosphate, collagen (Coll) and thrombin receptor-activated peptide (TRAP-6) as agonist were determined. Compared to the control group, patients with venous thromboembolism showed an enhanced maximal platelet aggregation with low concentrations of TRAP-6 (2 microM) and collagen (0.05 microM). In contrast, we could not detect an increased platelet aggregation with EPI or ADP. Our results indicate that platelet hyperaggregability may represent an independent risk factor in patients with otherwise unexplained venous thromboembolism. In our study, low concentrations of TRAP-6 and collagen are superior to EPI and ADP to define platelet hyperreactivity in platelet aggregometry.

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.

In vitro analyses of diamond-like carbon coated stents. Reduction of metal ion release, platelet activation, and thrombogenicity.

Coronary artery stents can induce platelet activation by shear forces, contact to the biomaterial, and release of metal ions. This activation is one important trigger for thrombosis. Coating of stents is a possible approach to prevent this side effect. The purpose of this study was to evaluate in vitro the biocompatibility of stents coated with diamond-like carbon (DLC). Semiquantitative energy-dispersive X-ray microanalyses showed a complete coverage of the DLC stents. Flow cytometric analyses revealed a significantly higher increase of mean channel fluorescence intensity for the activation-dependent antigens CD62p and CD63 in non-coated compared to DLC-coated stents (p<0.05). Atomic adsorption spectrophotometry analyses revealed a significant release of nickel and chromium metal ions by non-coated stents over a storage period of 96 hours in human plasma (p<0.05). In contrast, only minimal concentrations of released ions could be detected in the case of DLC-coated stents. Similar observations were made with inductively coupled plasma mass spectrometry analyses. Here, high concentrations of molybdenum and manganese ions were released from non-coated stents (p<0.05), while release of these ions from DLC-coated stents was virtually undetectable (p=0.1 for molybdenum and p=0.4 for manganese). Coating of intracoronary stents with diamond-like carbon significantly improves biocompatibility. This biocompatible coating may therefore contribute to a reduction in thrombogenicity.

Anticoagulant fucoidan fractions from Fucus vesiculosus induce platelet activation in vitro.

Anticoagulant fucoidan fractions of different molecular weight and sulfate content were prepared and investigated for their effects on platelet function in vitro. The fucoidan fractions were incubated with human platelet rich plasma (PRP) at concentrations of 5, 10 and 50 micrograms/ml. Platelet activation was subsequently studied by a standard aggregation assay and flow cytometric determination of the activation dependent platelet-surface markers CD62p (P-selectin, GMP-140) and CD63 (GP53). All fucoidan fractions induced irreversible platelet aggregation in a dose-dependent manner. Comparing fractions of identical molecular weight (100 kDa) the low sulfate content fucoidan FF5 (S = 7.6%) exerted a significantly greater effect than the highly sulfated fucoidan FF7 (S = 10.2%) over the whole concentration range (n = 5, P < 0.05). Among fractions of identical sulfate content fucoidan-induced platelet aggregation was also found to depend on the molecular weight of the fucoidan. At concentrations of 10 and 50 micrograms/ml the high molecular weight fraction FF7/1 (150 kDa) showed a significantly greater effect than the 50 kDa fraction FF7/3 (24.8 +/- 6.7 vs. 7.0 +/- 3.5 and 54.6 +/- 13.5 vs. 15.0 +/- 9.0%, respectively; mean +/- SD, n = 5, P < 0.05). The molecular weight dependence of the fucoidan effect was also reflected by the flow cytometric data. Coincubation of FF7/1 and FF7/3 (10 micrograms/ml) with PRP increased the number of CD62p and CD63 positive platelets by 9.0 +/- 3.3 vs. 2 +/- 1.9 and 7.1 +/- 2.4 vs. 3.2 +/- 2.6% over control values, respectively (n = 5, P < 0.05). In conclusion, our results show that the low molecular weight fucoidan FF7/3 combines potent anticoagulant and fibrinolytic properties with only minor platelet activating effects and is therefore a suitable substance for further pharmacological studies.

Influence of stent length and heparin coating on platelet activation: a flow cytometric analysis in a pulsed floating model.

Platelets are involved in acute and subacute thrombotic occlusions of coronary stents and also may play a role in the pathophysiology of in-stent restenosis. This study sought to investigate the expression of activation dependent glycoproteins on platelets by flow cytometry and time until stent thrombosis in an in vitro model of stent thrombosis. Coronary stents were placed in parallel silicon tubings with circulating citrated platelet rich plasma to measure 1) influence of stent length on platelet antigens; 2) influence of heparin coating on platelet antigens; and 3) time until stent thrombosis. After recalcification aliquots of platelet-rich plasma were taken over 10 minutes in 2-minute intervals and immediately fixed and stabilized. For flow cytometric analysis monoclonal antibodies to CD41a (glycoprotein IIb/ IIIa), CD42b (glycoprotein Ib-V-IX), CD62p (P-selectin), and CD63 (glycoprotein 53) were used. Within 2 minutes after start of circulation, the expression of CD62p and CD63 increased. Longer stents resulted in more platelet activation than shorter stents (25 mm vs. 15 mm; p<0.001. Time until stent thrombosis was reduced (25 mm vs. 15 mm; p<0.05). Heparin coating did not significantly influence flow cytometry detectable platelet activation but prolonged time until stent thrombosis (coated vs. uncoated; p<0.005). In control tubing systems without stents platelet activation was less pronounced (p<0.0001). Antibodies to CD41a and CD42b did not show significant changes. In this model platelet activation detected by flow cytometry and time until stent thrombosis were dependent on stent length and coating. In vitro testing could be useful to optimize stent design and material.

Expression of NK-associated surface-antigens cd-56 and cd-16 on aml-cells.

Monoclonal antibodies Leu 11a (CD16) and Leu 19 (CD56) were tested for reactivity with cells from 36 patients with acute myelogenous leukemia (AML) using two-colour flow cytometry. Blast cells were identified by a broad panel of monoclonal antibodies. In 33% (12/36) the monoclonal antibody Leu 19, which has been demonstrated to bind to the 140 kD isoform of the human neural cellular adhesion molecule N-CAM, found on peripheral natural killer (NK)-cells, neuroectodermal cells, activated T-cells, and myeloma cells, was shown to bind strongly to the leukemic cells. The monoclonal antibody Leu 11a, which recognizes a surface differentiation antigen associated with the low affinity FcRIII for IgG, expressed on NK-cells, granulocytes and macrophages were found to bind to leukemic cells of four of the 12 Leu-19 positive cases. 50% (6/12) of Leu-19 positive patients were classified as having M4 according to the French-American-British (FAB) morphology criteria. The potential diagnostic and clinical importance of CD 56 and/or CD 16 expression in acute myelogenous leukemia is presently under investigation.

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.

Tumour cell detection in G-CSF mobilised stem cell harvests of patients with breast cancer.

Peripheral blood stem cells were mobilised with G-CSF from steady-state haemopoiesis after previous anthracyclin-containing standard dose chemotherapy in patients with high-risk breast cancer. 48 samples were obtained from patients with stage II-III breast cancer and > or = 10 lymph nodes, 15 samples from patients with chemotherapy sensitive metastatic disease, and 13 samples from women with inflammatory breast cancer. 44 samples were first or single leukaphereses and 32 samples were second or third harvests. Aliquots were searched for contaminating tumour cells by immunocytochemistry (IC) and cytokeratin-19 reverse transcriptase polymerase chain reaction rtPCR). The median count of MNCs examined by IC was 2 x 10(6); cDNA prepared from 2 x 10(7) cells was subjected to PCR. Fifty-nine samples were examined by immunocytochemistry, 36 samples by rtPCR, and 19 samples by both techniques. Samples investigated by IC and rtPCR were judged as positive if there was at least one positive test. On the whole, 42/79 (55.3%) of the samples were positive with an insignificant trend to a higher positivity rate in second or subsequent leukaphereses (52.3% vs 59.3%). The median tumour cell load per 10(6) MNCs was low with 0.5 (0-7) cells in all, and a total of 2.2 (0.5-7) cells in positive specimen. Differences in the cancer cell load of first and subsequent leukaphereses and between subgroups of patients were not found. PCR and IC gave consistent results in 63.2%. This phenomenon can be explained by the greater sensitivity of the molecular method and by a Poisson distribution of coharvested tumour cells in samples. Tumour cell contamination in G-CSF mobilised stem cells from patients with breast cancer from steady state haemopoiesis after preceding anthacyclin-containing chemotherapy is frequent, but the tumour cell load is low. To allow a comparison of different studies dealing with cancer cell contamination in stem cells, standardisation of assays is necessary.

Anti-TNF antibody treatment has no positive effect on survival in a model of pneumococcal sepsis in pigs.

Gram-positive organisms causing sepsis have gained more significance in the past years. Especially patients with acquired immunodeficiency have been shown to be at risk for gram-positive infections. The mortality in Streptococcus pneumoniae bacteremia has been shown to be as high as 20%. Tumor necrosis factor-alpha (TNF-alpha) plays a crucial role in the "sepsis cascade." The previously described positive effect of monoclonal TNF antibody (anti-TNF-mAb) in gram-negative sepsis should be controlled in gram-positive pneumococcal sepsis. In a porcine model, pneumococcal sepsis was induced, and the course and outcome of a group treated with anti-TNF-mAb were compared to those of an untreated control. Streptococcus pneumoniae serotype 6 B was isolated from patients with systemic infection. The isolates were prepared, cryopreserved at -80 degrees C, and recultivated in a standardized fashion as needed. Then 10(9) bacteria were injected intravenously. Pigs of the German Landrace type with a weight of 20-30 kg were anesthetized using standardized midazolam and ketamine intravenous anesthesia. After introduction of central venous, arterial, and urinary catheters, bacteria were injected intravenously via the ear vein. In the therapy group, animals were treated with anti-TNF-mAb (5 mg/kg body weight) intravenously immediately prior to pneumococci injection. Survival and survival times were primary endpoints. Biochemical and vital parameters were also compared. In the anti-TNF-mAb group, 4/11 animals died (35%), compared to 6/11 (55%) in the control group. The mean survival times were 11 and 10 h, respectively (n.s.). TNF levels were significantly different. The TNF peak at 90-240 min was not present in the anti-TNF group (340 pg/ml vs. 19 pg/ml, p = .034). Leukocyte counts differed also significantly. After an initial drop in both groups, we observed a leukocytosis of up to 32.8 +/- 5.0 g/L in the anti-TNF-group, while in the control group leukocyte counts remained below 15.0 g/L (13.3 +/- 3.0 g/L, p = .007). All other parameters did not differ significantly. Thus, anti-TNF-mAb effectively suppresses the TNF peak following gram-positive septicemia. In the presented setting, these effects did not influence overall survival or survival times.

Automated, semi-automated, and manual analyses of anti-cardiolipin and anti-ß2-glycoprotein I antibodies in women with a history of miscarriage.

INTRODUCTION: Anti-cardiolipin and ß2-glycoprotein I antibodies represent important diagnostic parameters in routine hematology. In this study, five different automated, semi-automated, and manual immunoassays detecting IgG/IgM anti-cardiolipin and anti-ß2 -glycoprotein I antibodies were tested. METHODS: A total of 162 samples from women with a history of miscarriage were recruited from 110 different G&O outpatient centers in Germany. RESULTS: For both anti-cardiolipin and anti-ß2 -glycoprotein I antibodies, considerable differences in the percentage of positive results were seen between all five methods, and itemization of all positive test results revealed a poor accordance. These findings were confirmed by Cohen's kappa coefficients. CONCLUSION: Our study revealed a moderate to poor accordance between five different test systems for anti-cardiolipin and anti-ß2 -glycoprotein I antibodies. Such deviations may result in clinical misinterpretation of data and may lead to wrong therapeutic consequences. Therefore, further standardization of all tests for anti-phospholipid antibodies should be achieved.