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.

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.

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.

Comparison of flow cytometry vs. a haematology cell analyser-based method to guide the optimal time-point for peripheral blood stem cell apheresis.

BACKGROUND AND OBJECTIVES: For timing the onset of apheresis, parameters obtained by flow cytometry and by a haematological cell analyser were compared. MATERIALS AND METHODS: Haematopoietic cell counts (n = 159) were performed by two different methods; CD34 analyses by flow cytometry, immature myeloid information (IMI) and human progenitor cell counts (HPC) by a haematological cell analyser. RESULTS: Comparing the IMI total results with CD34+ analyses (n = 159) revealed a correlation of r = 0.46 (P < 0.05). Similar results were obtained for HPC (r = 0.44; P < 0.05). CONCLUSION: The haematology analyser-based method does not allow the precise determination of absolute haematopoietic stem cell numbers and is thus not able to replace flow cytometry for the monitoring of peripheral blood stem cell counts.

Detection of disseminated epithelial cancer cells by liquid culture--factors interfering with the standardization of assays.

BACKGROUND: Immunocytochemistry is the standard method for detection of disseminated breast-cancer cells. Tumor-cell enrichment by cell culture has been used by several investigators, however assays published have not been well-standardized. METHODS: Breast-cancer cells from two lines were diluted in hemopoietic cells of varying origins and cultured in different media and different flasks. Factors influencing successful tumor-cell amplification by liquid culture were identified by investigation of 277 cultures. Parallel clinical samples, consisting of BM aspirations, leukapheresis samples and peripheral blood samples obtained from women with breast cancer, were investigated in 113 cultures. Cancer-cell detection by cell culture could be compared to immunocytochemistry in 101 cases. RESULTS: The frequency of tumor-cell detection was not improved by liquid culture, but a significant correlation between conventional tumor-cell detection and detection after liquid culture was found. Factors influencing tumor-cell amplification in the dilution assay could not be transferred to the investigation of clinical samples. It was concluded that culture-enrichment of disseminated cancer cells was very complex, and could be influenced by a variety of factors-even when a model system was used. DISCUSSION: It should be recognized that culture-enriched cancer cells probably represent a highly selected population of disseminated cancer cells, despite the significant correlation between tumor cells detected by conventional methods and following conventional methods after liquid culture. There is currently no evidence to suggest that cancer-cell amplification by cell culture could become a standardized technique for the detection of disseminated epithelial tumor cells.

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.

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.

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.

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.

Disseminated breast cancer cells prior to and after high-dose therapy.

Women with breast cancer in a distinct stage of disease can benefit from high-dose therapy (HDT) with autologous stem cell support; however, a significant number of these patients relapse despite this intensive treatment. This study investigates the persistence of malignancy on the single-cell level. A total of 194 data sets consisting of bone marrow and blood samples obtained prior to and after HDT and of aliquots of apheresis products were searched with immunocytochemistry and reverse transcriptase polymerase chain reaction (RT-PCR) for disseminated cancer cells. Presence of cancer cells in the marrow is frequent prior to and after HDT, but HDT reduces the amount of malignant cells in marrow significantly. In contrast, there was no effect on the number of circulating cancer cells. Reinfusion of contaminated apheresis products was surprisingly associated with a low number of malignant cells in bone marrow after HDT and vice versa. The impact of disseminated tumor cells in bone marrow, apheresis, and peripheral blood on disease-free survival after HDT could be investigated in a total of 165 samples. Surprisingly, neither the presence of tumor cells in marrow or blood nor in apheresis was associated with a bad prognosis in Kaplan-Meyer survival analysis. These results suggest that apheresis products and bone marrow should be regarded as different biological compartments for epithelial cancer cells. It can be concluded that complete elimination of disseminated cancer cells by HDT is not always possible. The theory of reinduction of metastatic breast cancer by accidentally reinfused contaminants is not supported by this study so far. However, further research is necessary to identify distinct cell populations with the potentially capacity to metastasize.

Xenon does not affect human platelet function in vitro.

We sought to determine whether xenon affects platelet glycoprotein expression and platelet-related hemostasis in vitro at a clinically relevant concentration. Human whole blood was stimulated with either adenosine diphosphate or the thrombin receptor agonist peptide (TRAP)-6 after incubation with 65% xenon. Halothane at 2 minimum alveolar anesthetic concentration was used as a positive control. Platelet function and activation were evaluated with two-color flow cytometry. The expression of the platelet glycoproteins GPIIb/IIIa, GPIb, and P selectin were detected with fluorochrome-conjugated monoclonal antibodies. In vitro measurement of platelet-related hemostasis under conditions of high shear stress was performed in citrated whole blood with a platelet function analyzer (PFA-100((R))) by using collagen/epinephrine and collagen/adenosine diphosphate cartridges. Xenon did not affect basal or agonist-induced expression of platelet membrane glycoproteins, activation-dependent conformational changes of the GPIIb/IIIa receptor, expression of P selectin, or PFA closure times. In contrast, halothane reduced TRAP-6-induced activation of the GPIIb/IIIa complex. Furthermore, collagen/epinephrine-induced PFA closure time was significantly prolonged. These results demonstrate that xenon does not affect the unstimulated or agonist-induced platelet glycoprotein expression, activation of GPIIb/IIIa, or platelet-related hemostasis.

Influence of preharvest tumor cell contamination in bone marrow or blood does not predict resultant tumor cell contamination of granulocyte colony-stimulating factor mobilized stem cells.

Tumor cell contamination of stem cell collections harvested from breast cancer patients is a common phenomenon described by several investigators but with findings that vary among reports. Although so-called co-mobilization of these cells has been hypothesized, the origin of tumor cell contamination in stem cells is still unknown. A total of 47 G-CSF mobilized stem cell grafts from patients with nodal-positive (n = 30), chemosensitive metastatic (n = 11), and 5 women with inflammatory breast cancer were evaluated for cancer cells by immunocytochemistry. Additionally, 40 bone marrow aspirations and 23 peripheral blood samples collected prior to apheresis and after one to two cycles of conventional chemotherapy were available for examination. Tumor cell contamination of leukapheresis correlated best with preharvest blood state. This was valid when the nominal (positive/negative) presence of tumor cells in blood was compared to the nominal presence of tumor cells in apheresis samples and when the it was correlated to the tumor cell load of apheresis samples (TCL = tumor cells per 10(6) nucleated cells investigated). The correlation between blood and stem cells was better (nominal and quantitative) than that between marrow and stem cells, despite the larger sample size of marrow aspirations. The presence or absence of cancer cells in apheresis samples could not be safely predicted by the presence or absence of tumor cells in marrow or blood alone. Diagnostic specificity seems to improve from a combination of results from marrow and blood analysis. No correlation was found in quantitative analysis of tumor cell contamination between marrow and blood. In conclusion, the results suggest that blood and bone marrow represent different compartments for epithelial cancer cells and that contaminating tumor cells in stem cell harvests may be derived from the blood and/or marrow compartment. The tumor cell contamination of a stem cell harvest cannot be safely predicted by a preceding blood or marrow analysis.

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 microg/kg G-CSF (n = 25) compared with twice injections daily of 5 microg/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 x 10(6)/kg donor weight (range 2.8-13.3) in the 2 x 5 microg/kg group compared with 4.0 x 10(6)/kg (range 0.4-8.8) in the 1 x 10 microg/kg group (P = 0.007). The target of collecting > 3.0 x 10(6) CD34(+) cells/kg donor weight with one apheresis procedure was achieved in 24/25 (96%) donors in the 2 x 5 microg/kg group and in 17/25 (68%) donors in the 1 x 10 microg/kg group. The target of collecting > 5.0 x 10(6) CD34(+) cells/kg in the first apheresis was achieved in 64% in the 2 x 5 microg/kg group, but in only 36% in the 1 x 10 microg/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 x 5 microg/kg group than in the 1 x 10 microg/kg group (7.0 vs. 3.5 x 10(5)/kg, P = 0.01; 6.6 vs. 5.0 x 10(5)/kg; P = 0.1). Administering G-CSF (filgrastim) at a dosage of 5 microg/kg twice daily rather than 10 microg/kg once daily is recommended; this leads to a higher CD34(+) cell yield and requires fewer apheresis procedures without increasing toxicity or cost.

False-positive results of plasma PCR for cytomegalovirus DNA due to delayed sample preparation.

Positive results by cytomegalovirus (CMV) PCR of plasma are considered predictive of active CMV infection in kidney allograft recipients. To assess whether contamination with leukocyte-derived CMV DNA can distort the results, aliquots of whole-blood samples from 60 CMV immunoglobulin G-positive patients with leukocyte CMV DNAemia were stored for up to 24 h at room temperature (RT) and at 4 degrees C before plasma preparation. Native and ultrafiltered plasma samples were tested by CMV and beta-globin PCRs. Among 30 latently infected patients (negative for CMV pp65 antigens), low baseline rates (10%) and levels (median number of copies, 10 [per 10 microl]) of CMV plasma DNAemia in native plasma samples increased significantly over time (after 4 h at RT, 37% [P < 0.001]; median number of copies, 45 [P < 0.001]). Similar effects were found during storage at 4 degrees C. Ultrafiltration reduced the levels of CMV plasma DNAemia, but by 6 h of storage the levels were significantly elevated as well. CMV and beta-globin DNA kinetics in plasma were parallel. In contrast, 30 actively infected patients (pp65 positive) had high baseline rates (87% in native samples) and levels (median number of copies, 75) of CMV plasma DNAemia. No significant effects of storage or ultrafiltration and no concordance with beta-globin DNA kinetics were seen. In conclusion, delayed preparation of plasma samples bears a significant risk of false-positive CMV PCR results, probably due to leukocyte lysis. This has important implications in the clinical setting and for PCR standardization.

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.

Flow cytometry: the standard for monitoring the onset of apheresis and for the evaluation of stem and progenitor cell graft quality.

Several high-quality protocols exist that describe the methodological and technical aspects of the flow cytometric analyses of stem and progenitor cells. In addition, recent advances in automation and quantitative analyses show promising results. For further improvement of the test quality and more objective control, internal as well as external quality control is mandatory. In addition, cost aspects move into the focus of interest and have to be considered. Alternative methods such as volumetric capillary cytometry or haematological analyzers are of limited use for stem and progenitor cell analyses. Currently, flow cytometry represents the gold standard for monitoring the onset of apheresis and to evaluate the quality of stem and progenitor cell grafts. In the near future, only in vitro diagnostics that are CE-labelled or FDA-approved should be used for these analyses.

Cytomegalovirus cultured from different major leukocyte subpopulations: association with clinical features in CMV immunoglobulin G-positive renal allograft recipients.

Cytomegalovirus (CMV) cultured from peripheral blood mononuclear cells (PBMCs) was shown to be associated more closely with clinical manifestations than infectious CMV in polymorphonuclear leukocytes (PMNLs) of renal allograft recipients with secondary CMV infection. Shell vial culture was carried out with ficoll-purified PBMCs and PMNLs of 71 CMV IgG-positive patients after kidney transplantation. Thirty-six patients experienced active CMV infections. Of these, 17 developed clinical symptoms. The diagnostic value of PMNLs and PBMCs viremia was determined in comparison to pp65 antigenemia, leukoDNAemia, plasma DNAemia, and detection of cytomegalic endothelial cells. In both PMNLs and PBMCs (with or without detectable endothelial cells), frequencies and levels of viremia were significantly higher among symptomatic patients. Regarding the occurrence of clinical CMV manifestations, the sensitivity of culture from PMNLs and from PBMCs fractions was 100%. Viremia in PBMCs, however, was far more specific (94%) than in PMNLs (74%). Cutoff values established previously for pp65 antigenemia and leukoDNAemia, standard markers in the laboratory, had similar specificity (96% each) to PBMCs viremia, but were less sensitive (88% each). Plasma DNA-emia was both less sensitive (82%) and less specific (69%) than PBMCs viremia. Detection of endothelemia showed maximal specificity (100%), but inferior sensitivity (47%). All patients had PBMCs viremia before the onset of symptoms. In conclusion, infectious CMV present in PBMCs may prove to be a determinant of clinical CMV manifestations in seropositive immunocompromised individuals. Factors involved in PBMCs tropism may help to understand the pathogenetic mechanisms of CMV dissemination in this group of patients.

Extracorporeal plateletpheresis induces the interaction of activated platelets with white blood cells.

BACKGROUND AND OBJECTIVES: In this study we investigated whether platelet activation during apheresis results in the binding of platelets to white blood cells. MATERIAL AND METHODS: Analysis of platelet-leukocyte interaction was performed using multiparameter, three-color flow cytometry. RESULTS: Over the duration of the procedure, there was an increase in the surface expression of CD62p (P-selectin) and CD63 (p<0.05), and also in the binding of platelets to monocytes (p<0.05), neutrophilic granulocytes (p<0.05) and to CD3+ cells (initially to a low degree; p<0.05). Platelet binding to CD19+ cells did not change significantly. CONCLUSION: This study demonstrates that platelets become activated during apheresis and that following this process, interaction with monocytes and neutrophilic granulocytes occurs.