Assessing glucose and oxygen diffusion in hydrogels for the rational design of 3D stem cell scaffolds in regenerative medicine

Hydrogels are attractive biomaterials for replicating cellular microenvironments, but attention needs to be given to hydrogels diffusion properties. A large body of literature shows the promise of hydrogels as 3D culture models, cell expansion systems, cell delivery vehicles, and tissue constructs. Surprisingly, literature seems to have overlooked the important effects of nutrient diffusion on the viability of hydrogel‐encapsulated cells. In this paper, we present the methods and results of an investigation into glucose and oxygen diffusion into a silated‐hydroxypropylmethylcellulose (Si‐HPMC) hydrogel. Using both an implantable glucose sensor and implantable oxygen sensor, we continuously monitored core glucose concentration and oxygen concentration at the centre of hydrogels. We demonstrated that we could tune molecular transport in Si‐HPMC hydrogel by changing the polymer concentration. Specifically, the oxygen diffusion coefficient was found to significantly decrease from 3.4 × 10^?10 to 2.4 × 10^?10 m² s^?1 as the polymer concentration increased from 1% to 4% (w/v). Moreover, it was revealed during in vitro culture of cellularized hydrogels that oxygen depletion occurred before glucose depletion, suggesting oxygen diffusion is the major limiting factor for cell survival. Insight was also gained into the mechanism of action by which oxygen and glucose diffuse. Indeed, a direct correlation was found between the average polymer crosslinking node size and glucose parameters, and this correlation was not observed for oxygen. Overall, these experiments provide useful insights for the analysis of nutrient transport and gas exchange in hydrogels and for the development of future cellular microenvironments based on Si‐HPMC or similar polysaccharide hydrogels.     

Bayesian evaluation of the human immunodeficiency virus antibody screening strategy of duplicate enzyme-linked immunosorbent assay in Xuzhou Blood Center, China

BACKGROUND: Accurate estimation of the risk of human immunodeficiency virus (HIV) infection through transfusion is essential for monitoring blood safety. The risk, however, is so low that it can only be estimated by mathematical modeling. With the Bayesian dependence model, this study evaluates the HIV antibody screening strategy of duplicate enzyme‐linked immunosorbent assay (ELISA) in Xuzhou Blood Center and therefore estimates part of the total risks of transfusion‐transmitted HIV infection. STUDY DESIGN AND METHODS: Data from Xuzhou Blood Center between 2004 and 2008 were used. Information was obtained on donor profiles and screening and confirmatory test results. The portion of the risks of HIV infection through transfusion concerned was estimated by evaluating the screening algorithm in terms of its accuracy and predictive power with the Bayesian dependence model. RESULTS: A total of 234,602 donations from voluntary blood donors in Xuzhou Blood Center were screened for HIV antibody. For the study screening algorithm, its sensitivity, specificity, false‐positive predictive value (FPPV), and false‐negative predictive value (FNPV) were 0.9951 (95% Bayesian credible interval [BCI], 0.9763‐0.9997), 0.9991 (95% BCI, 0.9990‐0.9992), 0.9647 (95% BCI, 0.9018‐0.9923), and 1.52 × 10^?7 (95% BCI, 7.31 × 10^?9‐1.15 × 10^?6), respectively. For the positive detection rate (9.60 × 10^?4) and FPPV (0.9647), the differences between their own Bayesian median estimates and real values were 2.70 × 10^?5 and ?0.0033, respectively. CONCLUSIONS: The HIV antibody screening algorithm of duplicate ELISA is well evaluated in its accuracy and predictive power with the Bayesian dependence model. The FNPV measures the part of the risks of transfusion‐associated HIV transmission concerned.     

Inhibition of plasminogen activator inhibitor-1: antibody fragments and their unique sequences as a tool for the development of profibrinolytic drugs

Summary. Physiological inhibition of plasminogen activator inhibitor‐1 (PAI‐1) might improve the prevention and treatment of various cardiovascular diseases. To date, a variety of monoclonal antibodies that neutralize PAI‐1 have been generated. The current study presents the cloning, expression and characterization of four single‐chain variable fragments (i.e. scFv‐33B8, scFv‐33H1F7, scFv‐35A5 and scFv‐55F4C12) from the corresponding PAI‐1 neutralizing monoclonal antibodies. Surprisingly, affinity constants of scFv‐33B8, scFv‐33H1F7 and scFv‐55F4C12 for PAI‐1 (K_A = 1.4 ± 0.2 × 10¹⁰ m ^?1, 3.7 ± 0.1 × 10⁹ m ^?1, 1.0 ± 0.2 × 10⁹ m ^?1, respectively) were only 2‐ to 4‐fold lower compared to those of the respective monoclonal antibodies (MAs). In contrast, scFv‐35A5 exhibited a 6250‐fold decrease in affinity (K_A = 3.2 ± 0.8 × 10⁶ m ^?1 vs. 2.0 ± 0.8 × 10¹⁰ m ^?1 observed for MA‐35A5) with a concomitant absence of functional effects on PAI‐1 activity. Evaluation of the dose–response curves of the PAI‐1 neutralizing effect of the other scFvs revealed a shift towards slightly higher concentrations (in line with the small decrease in affinity) eventually resulting in a similar maximum effect as the corresponding MAs (i.e. 92 ± 2%, 34 ± 3% and 66 ± 5% PAI‐1 inhibition for scFv‐33B8, scFv‐33H1F7 and scFv‐55F4C12, respectively). In conclusion, the sequence information of the scFvs allows to humanize MAs with PAI‐1 inhibiting properties whereas the scFv constructs serve as an excellent starting point for structure based drug design, both aiming at the reduction of cardiovascular diseases.     

Risk factors for bleeding,including platelet count threshold,in newly diagnosed immune thrombocytopenia adults

Essentials

• Risk factors of bleeding in adult immune thrombocytopenia are not known.
• This multicenter study assessed risk factors of bleeding at immune thrombocytopenia onset.
• Platelet count thresholds associated with bleeding were < 20 × 10⁹ L^?1 and < 10 × 10⁹ L^?1.
• Exposure to anticoagulants was a major risk factor of severe bleeding.

Summary

Background

The aim of this cross‐sectional study was to assess risk factors for bleeding in immune thrombocytopenia (ITP) adults, including the determination of platelet count thresholds.

Methods

We selected all newly diagnosed ITP adults included in the Cytopénies Auto‐immunes Registre Midi‐PyrénéEN (CARMEN) register and at the French referral center for autoimmune cytopenias. The frequencies of any bleeding, mucosal bleeding and severe bleeding (gastrointestinal, intracranial, or macroscopic hematuria) at ITP onset were assessed. Platelet count thresholds were assessed by the use of receiver operating characteristic curves. All potential risk factors were included in logistic regression models.

Results

Among the 302 patients, the frequencies of any, mucosal and severe bleeding were 57.9%, 30.1%, and 6.6%, respectively. The best discriminant threshold of platelet count for any bleeding was 20 × 10⁹ L^?1. In multivariate analysis, factors associated with any bleeding were platelet count (< 10 × 10⁹ L^?1 versus ≥ 20 × 10⁹ L^?1, odds ratio [OR] 48.2, 95% confidence interval [CI] 20.0–116.3; between 10 × 10⁹ L^?1 and 19 × 10⁹ L^?1 versus ≥ 20 × 10⁹ L^?1, OR 5.2, 95% CI 2.3–11.6), female sex (OR 2.6, 95% CI 1.3–5.0), and exposure to non‐steroidal anti‐inflammatory drugs (NSAIDs) (OR 4.8, 95% CI 1.1–20.7). A low platelet count was also the main risk factor for mucosal bleeding. Exposure to anticoagulant drugs was associated with severe bleeding (OR 4.3, 95% CI 1.3–14.1).

Conclusions

Platelet counts of < 20 × 10⁹ L^?1 and < 10 × 10⁹ L^?1 were thresholds for major increased risks of any and mucosal bleeding. Platelet count, female sex and exposure to NSAIDs should be considered for assessment of the risk of any bleeding. Exposure to anticoagulant drugs was a major risk factor for severe bleeding.

     

Clinical efficacy and safety of Flebogammadif®, a new high-purity human intravenous immunoglobulin, in adult patients with chronic idiopathic thrombocytopenic purpura

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder characterized by a low platelet count and bleeding, whose incidence is approximately 6·2 for each 100 000 adults per year. Intravenous immunoglobulins (IVIG) can be useful in patients with ITP to prevent bleeding or prior to surgery. In this study, the efficacy and safety of Flebogammadif^®, a new high‐purity IVIG, were assessed by an open, multicentre, non‐controlled, prospective study in adult patients with chronic ITP. A total of 20 patients (enrolled if experiencing chronic ITP since at least 6 months before recruitment and if platelet count <20 ×10⁹L^?1 before treatment) received 0·4 g kg^?1‐bw of Flebogammadif^® for 5 consecutive days and were followed‐up for 3 months. Efficacy endpoints were three: proportion of patients who reached a platelet count ≥50 ×10⁹L^?1, time for the platelet count to reach that level and duration of response. Safety parameters [adverse events (AE), laboratory determinations and vital signs] and viral markers were regularly monitored. A total of 14 patients achieved a platelet count of ≥50 ×10⁹L^?1. The median time to platelet response was ≤2·5 days, and the median number of days in which the platelet count remained ≥50 ×10⁹L^?1 was ≥7 days. A regression of haemorrhages was reported for 17 patients on day 14. Eight patients presented 21 AEs (mostly mild) potentially related to the study drug. Neither abnormalities in laboratory values nor in viral markers were registered during the follow‐up period. Flebogammadif^® was well tolerated and succeeded in providing a haemostatic platelet count in patients with ITP.     

The kinetics of G-CSF mobilization of CD34+ cells in healthy people

When healthy people are given granulocyte colony stimulating factor (G-CSF) for 10 days the number of CD34+ cells in the peripheral blood begins to increase on the fourth day, reaches a maximum on the sixth day and then decreases. In this study, we further define the time and variability of peak mobilization of CD34+ cells. Twenty-two healthy people were given G-CSF (7.5 or 10 μg kg^?1 day^?1) subcutaneously each morning for 5 days and peripheral blood CD34+ cell counts were analysed immediately prior to the fourth (day 4) and fifth (day 5) G-CSF injection and 24 h after the fifth injection (Day 6). White blood cell (WBC) and neutrophil counts were greatest on day 6 [WBC = 43.8 ± 13.9 × 10⁹ L^?1 (mean ± 1 SD) and neutrophils = 36.6 ± 12.8 × 10⁹ L^?1]. In contrast the CD34+ cell counts on day 6 (107 ± 104 × 10⁶ L^?1) were less than on day 5 (128 ± 136 × 10⁶ L^?1) (P= 0.048) but still greater than on day 4 (60.7 ± 40.2 × 10⁶ L^?1) (P < 0.0001). The CD34+ cell counts of 10 donors were measured 2, 4 and 6 h after the fifth injection to determine if the counts increased further between days 5 and 6. The number of CD34+ cells in the blood on day 5 2 h after the fifth injection (193 ± 277 × 10⁶ L^?1) was greater than the number prior to the injection (158 ± 190 × 10⁶ L^?1), 4 h post-injection (139 ± 158 × 10⁶ L^?1) and 6 h post-injection (170 ± 236 × 10⁶ L^?1), but the differences were not significant (P= 0.29, 0.25 and 0.45). The number of CD34+ cells in the blood of 12 people were measured before and after the fourth G-CSF dose. Prior to the day 4 injection the CD34+ count was 61 ± 40 × 10⁶ L^?1. At 2, 4 and 6 h the counts were 60 ± 40, 61 ± 29 and 64 ± 30 × 10⁶ L^?1, respectively, and the differences were not significant (P= 0.99, P= 0.98, and P= 0.73). In conclusion, when healthy volunteers are given daily G-CSF injections, the number of mobilized CD34+ cells was the greatest on day 5, slightly less on day 6 and the least on day 4. If only one PBSC component is needed, PBSCs can be collected on day 5 after only 4 days of G-CSF. If PBSC components are collected on both days 5 and 6, the fifth dose can be given either before or after the collection of the first PBSC component.     

Cloning and paratope analysis of an antibody fragment, a rational approach for the design of a PAI-1 inhibitor

Summary. This study reports the cloning, characterization and paratope analysis of the plasminogen activator inhibitor‐1 (PAI‐1) neutralizing single‐chain variable fragment 56A7C10 (scFv‐56A7C10). ScFv‐56A7C10‐wt exhibits a similar affinity (K_A = 1.01 ± 0.3 × 10⁹ m ^?1) and PAI‐1 inhibitory capacity (90 ± 6% PAI‐1 inhibition at a 16‐fold molar excess and IC₅₀ = 44 ± 14 ng mL^?1) as MA‐56A7C10 (K_A = 1.43 ± 0.4 × 10⁹ m ^?1, 90 ± 2% PAI‐1 inhibition at a 16‐fold molar excess and IC₅₀ = 122 ± 26 ng mL^?1). Subsequently, alanine scanning of the six complementarity determining regions (CDRs) was performed and the scFv‐56A7C10‐mutants (n = 26) were analyzed for their PAI‐1 binding and PAI‐1 inhibitory properties. Mutation of the residues Y32 and V33 in the CDR1 of the heavy chain (HCDR1) and the residues R98, H99, W100 or F100a (HCDR3) resulted in reduced PAI‐1 inhibitory capacities (IC₅₀ ≥ 418 ng mL^?1), confirmed by reduced affinities (14‐, 17‐, 7‐, 9‐ and 16‐fold reduced, respectively, vs. scFv‐56A7C10‐wt). In the light chain, mutation of the residues W50 (LCDR2), H91, Y92, D93, or W96 (LCDR3) resulted in reduced PAI‐1 inhibitory properties (IC₅₀ ≥ 160 ng mL^?1) and decreased affinities (i.e. 4‐, 9‐, 3‐, 3‐ and 2‐fold reduced affinity, respectively, vs. scFv‐56A7C10‐wt). Furthermore, an overlapping peptide scan confirmed the importance of the HCDR3 region. These data, combined with a three‐dimensional model of scFv‐56A7C10, reveal the molecular and structural properties of the paratope and contribute to the rational design of PAI‐1 neutralizing compounds.     

Consanguineous hemolytic uremic syndrome secondary to Escherichia coli O157:H7 infection treated with aggressive therapeutic plasma exchange

We describe the successful management of an elderly husband and wife with Escherichia coli O157:H7 associated hemolytic uremic syndrome (HUS) treated with aggressive therapeutic plasma exchange (TPE) with replacement with fresh frozen plasma. Following twelve TPEs (three 1.5 volume; nine 1 volume), the husband's platelet count increased from 45 × 10⁹/L to 183 × 10⁹/L. Following ten 1.5 volume TPEs, the wife's platelet count increased from 30 × 10⁹/L to 193 × 10⁹/L. This is the first known occurrence of E. coli O157:H7 associated HUS in an elderly husband and wife successfully treated with aggressive TPE. We conclude that early, aggressive TPE should be considered and may be life‐saving for E coli O157:H7 associated HUS in the elderly. J. Clin. Aperesis 16:155–156, 2001 © 2001 Wiley‐Liss, Inc.     

β‐Cell subcellular localization of glucose‐stimulated Mn uptake by X‐ray fluorescence microscopy: implications for pancreatic MRI

Manganese (Mn) is a calcium (Ca) analog that has long been used as a magnetic resonance imaging (MRI) contrast agent for investigating cardiac tissue functionality, for brain mapping and for neuronal tract tracing studies. Recently, we have extended its use to investigate pancreatic β‐cells and showed that, in the presence of MnCl₂, glucose‐activated pancreatic islets yield significant signal enhancement in T₁‐weigheted MR images. In this study, we exploited for the first time the unique capabilities of X‐ray fluorescence microscopy (XFM) to both visualize and quantify the metal in pancreatic β‐cells at cellular and subcellular levels. MIN‐6 insulinoma cells grown in standard tissue culture conditions had only a trace amount of Mn, 1.14 ± 0.03 × 10^?11 µg/µm², homogenously distributed across the cell. Exposure to 2 m m glucose and 50 µ m MnCl₂ for 20 min resulted in nonglucose‐dependent Mn uptake and the overall cell concentration increased to 8.99 ± 2.69 × 10^?11 µg/µm². When cells were activated by incubation in 16 m m glucose in the presence of 50 µ m MnCl₂, a significant increase in cytoplasmic Mn was measured, reaching 2.57 ± 1.34 × 10^?10 µg/µm². A further rise in intracellular concentration was measured following KCl‐induced depolarization, with concentrations totaling 1.25 ± 0.33 × 10^?9 and 4.02 ± 0.71 × 10^?10 µg/µm² in the cytoplasm and nuclei, respectively. In both activated conditions Mn was prevalent in the cytoplasm and localized primarily in a perinuclear region, possibly corresponding to the Golgi apparatus and involving the secretory pathway. These data are consistent with our previous MRI findings, confirming that Mn can be used as a functional imaging reporter of pancreatic β‐cell activation and also provide a basis for understanding how subcellular localization of Mn will impact MRI contrast. Copyright © 2011 John Wiley & Sons, Ltd.     

Aortic Elastic Properties of Patients with Neurocardiogenic Syncope

Objective: Neurocardiogenic syncope (NCS) is a common clinical problem; however, hemodynamic mechanism is not clearly understood. Aim of the present study was to investigate aortic elastic parameters of patients with NCS provoked by head‐up tilt test. Material and Method: We conducted a prospective study of 40 cases referred to our institution for head‐up tilt testing. Group I constituted as 22 patients who developed mixed response and were enrolled for analysis. Hemodynamic data were compared with subjects of negative head‐up tilt test (Group II). Aorta‐diastolic and aorta‐systolic diameters, aortic strain, aortic distensibility, aortic elastic modulus, and aortic stiffness index were calculated from transthoracic echocardiographically derived diameters of thoracic aorta. Results: Aortic distensibility (mean ± SD; 2.7 ± 1.2 cm²× dyn^?1× 10^?6 vs 4.0 ± 1.2 cm²× dyn^?1× 10^?6, P = 0,003) and aortic strain index (mean ± SD; 7.0 ± 1.8% vs 8.7 ± 2.9%, P = 0.042) were lower, and aortic stiffness index (mean ± SD; 27.6 ± 10.9 vs 20.9 ± 6.18, P = 0.035) and aortic elastic modulus (mean ± SD; 0.94 ± 0.7 cm²× dyn^?1× 10^?6 vs 0.49 ± 0.1 cm²× dyn^?1× 10^?6, P = 0.009) were higher in patients in Group I compared with those in Group II. There was no difference between two groups for following clinical variables: aorta‐diastolic and aorta‐systolic diameters, systolic and diastolic blood pressure, pulse pressure, E/A, weight, height, and body mass index. Conclusions: Findings of this study have shown that elastic properties of aorta are impaired in patients with NCS. The data suggest that increase in aortic stiffness might be one of the determinants responsible for NCS. This proposal of novel link should be confirmed in further studies.     

Absolute immature platelet counts in the setting of suspected heparin-induced thrombocytopenia may predict anti-PF4-heparin immunoassay testing results

Background

Heparin-induced-thrombocytopenia (HIT) is a disease mediated by antibodies to platelet factor 4 (PF4)-heparin complexes. Immature platelet fraction (%-IPF) and absolute immature platelet count (A-IPC) measure newly-released platelets into circulation and can prove useful in differentiating patients with thrombocytopenic presentations due to consumptive or hypoproduction processes. Therefore, we evaluated utility of A-IPC in a cohort of thrombocytopenic patients suspected of HIT.

Development of large-scale manufacturing of adipose-derived stromal cells for clinical applications using bioreactors and human platelet lysate

In vitro expanded adipose-derived stromal cells (ASCs) are a useful resource for tissue regeneration. Translation of small-scale autologous cell production into a large-scale, allogeneic production process for clinical applications necessitates well-chosen raw materials and cell culture platform. We compare the use of clinical-grade human platelet lysate (hPL) and fetal bovine serum (FBS) as growth supplements for ASC expansion in the automated, closed hollow fibre quantum cell expansion system (bioreactor). Stromal vascular fractions were isolated from human subcutaneous abdominal fat. In average, 95?×?10⁶ cells were suspended in 10% FBS or 5% hPL medium, and loaded into a bioreactor coated with cryoprecipitate. ASCs (P0) were harvested, and 30?×?10⁶ ASCs were reloaded for continued expansion (P1). Feeding rate and time of harvest was guided by metabolic monitoring. Viability, sterility, purity, differentiation capacity, and genomic stability of ASCs P1 were determined. Cultivation of SVF in hPL medium for in average nine days, yielded 546?×?10⁶ ASCs compared to 111?×?10⁶ ASCs, after 17?days in FBS medium. ASCs P1 yields were in average 605?×?10⁶ ASCs (PD [population doublings]: 4.65) after six days in hPL medium, compared to 119?×?10⁶ ASCs (PD: 2.45) in FBS medium, after 21?days. ASCs fulfilled ISCT criteria and demonstrated genomic stability and sterility. The use of hPL as a growth supplement for ASCs expansion in the quantum cell expansion system provides an efficient expansion process compared to the use of FBS, while maintaining cell quality appropriate for clinical use. The described process is an obvious choice for manufacturing of large-scale allogeneic ASC products.     

H2-receptor antagonists are scavengers of oxygen radicals

Abstract. Potential oxygen radical scavenging properties of the H₂-receptor antagonists cimetidine, ranitidine and famotidine were investigated. These drugs, although ineffective against superoxide anion and hydrogen peroxide, can scavenge hydroxyl radical (OH·) with a very high rate constant, which is about tenfold higher than that of the specific scavenger mannitol for famotidine (1·7 × 10¹⁰ mol^?1 s^?1) and cimetidine (1·6 × 10¹⁰ mol^?1 s^?1), ranitidine displaying a rate constant of 7·5 × 10⁹ mol^?1 s^?1. These OH· scavenging effects are significant beginning from 10, 28 and 100 μmol 1^?1 concentration for famotidine, cimetidine and ranitidine, respectively, thus suggesting that the drugs may effectively act as OH· scavengers in vivo especially in the gastric lumen. Only cimetidine can apparently bind and inactivate iron, which further emphasizes its antioxidant capacity. Moreover, all drugs, even at 10 μmol 1^?1 concentration, show powerful scavenging effects on hypochlorous acid and monochloramine, which are cytotoxic oxidants arising from inflammatory cells, such as neutrophils. These data suggest that some therapeutical effects of H₂-receptor antagonists in peptic ulcer may also be related to their antiradical-antioxidant capacity, and that these drugs could potentially be used in other disease entities characterized by free radical-mediated oxidative stress in vivo.     

Comparison of hematopoietic progenitor cell collections using the COBE Spectra version 7 and Amicus version 3.1 for patients with AL amyloidosis

To address concerns about infused fluid volume during HPC collections in patients with AL amyloidosis, our institution has used a 26:1 anticoagulant (AC) ratio on the COBE Spectra and on the Fenwal Amicus. In this study, in a cohort of AL amyloid patients, we compared the Amicus version 3.1 to the Spectra version 7 MNC collections with regard to infused fluid volume, CD34+ cell yield, lymphocyte yield, cross‐cellular content, and adverse reactions. Both instruments used a 26:1 AC ratio but the Amicus delivered significantly less AC per procedure (Amicus 678 mL vs. Spectra 753 mL). With comparable baseline CD34+ cell counts (Amicus 33 cells/μL vs. Spectra 27 cells/μL); Amicus collected significantly more CD34+ cells (3.1 vs. 1.5 × 10⁶/kg) and equivalent lymphocytes (18.7 vs. 14.5 × 10⁹). Amicus collected significantly fewer WBC (51.8 vs. 72.7 × 10⁹), granulocytes (15.1 vs. 27.5 × 10⁹), and PLT (2.3 vs. 3.9 × 10¹¹) per procedure with equivalent RBC content (26 vs. 30 mL). CD34+ cell (5.0 vs. 4.4 × 10⁶/kg) and lymphocyte doses (32.7 vs. 33.9 × 10⁹) were equivalent in infused products collected on the Amicus and Spectra but the frequency of high volume products was lower for Amicus. Frequency and severity of adverse reactions during collection and infusion were similar for both. In this group of AL amyloid patients, Amicus was superior to Spectra with regard to fluid infused, CD34+ cell yield, and cross‐cellular contamination with equivalent lymphocyte yield and reaction incidence. © 2011 Wiley‐Liss, Inc.     

Analytical performance of automated platelet counts and impact on platelet transfusion guidance in patients with acute leukemia

The aim of this study was to evaluate the performance of automated impedance platelet counts by Beckman Coulter LH780 (PLT-LH), Sysmex XN-3000 (PLT-XNi) and fluorescence method by Sysmex XN-3000 (PLT-F) in patients with acute leukemia. Blood specimens were subjected to platelet measurements by evaluated methods and then compared against the international reference method (IRM). Eighty-two blood specimens were included. Bland–Altman plots of the differences between the evaluated methods and IRM demonstrated mean biases of PLT-LH, PLT-XNi and PLT-F of 9?×?10⁹/L, 11?×?10⁹/L and 2?×?10⁹/L, respectively. For platelet transfusion guidance, all evaluated methods had acceptable accuracy. For platelet transfusion guidance, the sensitivities of PLT-LH, PLT-XNi and PLT-F were 33.3, 25.0 and 83.3%, respectively, at a transfusion threshold of 10?×?10⁹/L, and 73.1, 61.5 and 84.6%, respectively, at transfusion threshold of 20?×?10⁹/L. High blast count was associated with inaccurate PLT-LH and PLT-XNi. In conclusion, the PLT-F demonstrated excellent performance for diagnosis of thrombocytopenia and for platelet transfusion guidance in the evaluated specimens from acute leukemia patients. With respect to clinical relevance, careful blood smear review is necessary in case of high blast counts.     

SI23Assessment of Bleeding in Patients with Haematological Malignancies and the Association between Platelet Count and Bleeding

Aim To evaluate methods to collect bleeding outcomes in thrombocytopenic patients, including by patient self‐assessment, and to investigate the association between platelet count and bleeding. Methods Consecutive patients with haematological malignancies were enrolled in a study of bleeding data collection, platelet counts and transfusions during the period of thrombocytopenia. An educational session and information sheet was designed for patient self‐assessment. Results A total of 19 patients were included in the study. 410 days of thrombocytopenia were eligible for assessment of bleeds. Self‐assessment was deemed feasible, as self‐assessment was successfully completed on 288/410 (70%) of thrombocytopenic days. There was 86% agreement between bleeding data collected by both self‐assessment and medical examination, using a structured assessment form. Examples of discrepancies included the duration of petechiae/bruises and the reporting of minor bleeding. There was no evidence for an association between patients’ morning platelet count and daily WHO bleeding grade. Bleeding events were compared for platelet counts of ≤10 × 10⁹ L^?1, 11–20 × 10⁹ L^?1, and >20 × 10⁹ L^?1. The incidence of WHO Grade one and two bleeds were similar and did not reveal higher rates of bleeding at lower counts¹. Conclusion Patient self‐assessment can help support comprehensive daily prospective monitoring of bleeding, specifically facilitating data collection following hospital discharge. Exact definitions for bleeding events remains problematic. The association between platelet count, risk of bleeding and role of prophylactic platelet transfusions needs further evaluating in larger prospective trials. Reference ¹Stanworth S.J., Dyer C, Casbard A & Murphy M. (2006) Vox Sang. 91 63–9     

Radiolabelled GLP‐1 analogues for in vivo targeting of insulinomas

Internalizing agonists are usually selected for peptide receptor targeting. There is increasing evidence that non‐internalizing receptor antagonists can be used for this purpose. We investigated whether the glucagon‐like peptide‐1 receptor (GLP‐1R) antagonist exendin(9–39) can be used for in vivo targeting of GLP‐1R expressing tumours and compared the in vitro and in vivo characteristics with the GLP‐1R agonists exendin‐3 and exendin‐4. The binding and internalization kinetics of labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) were determined in vitro using INS‐1 cells. The in vivo targeting properties of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) were examined in BALB/c nude mice with subcutaneous INS‐1 tumours. ^natIn‐labelled [Lys⁴⁰(DTPA)]exendin‐3, [Lys⁴⁰(DTPA)]exendin‐4 and [Lys⁴⁰(DTPA)]exendin(9–39) exhibited similar IC₅₀ values (13.5, 14.4 and 13.4 n m , respectively) and bound to 26 × 10³, 41 × 10³ and 37 × 10³ receptors per cell, respectively. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 and [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed rapid in vitro binding and internalization kinetics, whereas [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed lower binding and minimal internalization in vitro. In mice, high specific uptake of [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐3 [25.0 ± 6.0% injected dose (ID) g^?1] in the tumour was observed at 0.5 h post‐injection (p.i.) with similar uptake up to 4 h p.i. [Lys⁴⁰(¹¹¹In‐DTPA)]exendin‐4 showed higher tumour uptake at 1 and 4 h p.i. (40.8 ± 7.0 and 41.9 ± 7.2% ID g^?1, respectively). Remarkably, [Lys⁴⁰(¹¹¹In‐DTPA)]exendin(9–39) showed only low specific uptake in the tumour at 0.5 h p.i. (3.2 ± 0.7% ID g^?1), rapidly decreasing over time. In conclusion, the GLP‐1R agonists [Lys⁴⁰(DTPA)]exendin‐3 and [Lys⁴⁰(DTPA)]exendin‐4 labelled with ¹¹¹In could be useful for in vivo GLP‐1R targeting, whereas [Lys⁴⁰(DTPA)]exendin(9–39) is not suited for in vivo targeting of the GLP‐1R. Copyright © 2012 John Wiley & Sons, Ltd.     

Lenograstim with or without dexamethasone for neutrophil mobilization in healthy donors: short-term kinetics of white blood cells and effects of granulocyte apheresis

Objectives : To determine the optimal time schedule for neutrophil collection after single mobilization with glycosylated recombinant granulocyte colony‐stimulating factor (G‐CSF, lenograstim) with or without dexamethasone (DXM). Donors and Methods : In this prospective randomized trial, 26 healthy volunteers were randomly assigned to a single subcutaneous dose of lenograstim 6 μg/kg plus 8‐mg DXM (G‐CSF/DXM, n = 13) or placebo (G‐CSF/placebo, n = 13). Hematological and biochemical parameters were analyzed before and 12, 15, 18, 21, 24, 27, 29, 36, 48, 60, 72, and 84 h and 7 and 30 days after mobilization. Six G‐CSF/DXM subjects underwent standard neutrophil apheresis (NA) 12 and 36 h after mobilization. Results : Polymorphonuclear neutrophil (PMN) counts 12 and 21 h after mobilization were 22.7 (16.6?32.8) × 10⁹/L and 22.4 (18.6?30.6) × 10⁹/L for G‐CSF/placebo versus 33.1 (24.2–44.9) × 10⁹/L and 32.5 (17.4–39.6) × 10⁹/L for G‐CSF/DXM. This mobilization plateau was followed by slow normalization at 72–84 h. The six NA subjects had median PMN yields of 62 (47–101) × 10⁹ and 39 (23–42) × 10⁹ per therapeutic unit. After the first apheresis, PMN counts sharply decreased to 21.1 (14.8–26.3) × 10⁹/L and then temporarily recovered to 25.9 (18.9–36.5) × 10⁹/L (P ≤ 0.001) over the next 8 h. Conclusions : Single doses of lenograstim with or without DXM induced a PMN plateau that lasted 9 h (12–21 h after mobilization), with PMN counts suitable for neutrophil collection. Lenograstim plus DXM made it possible to perform NA twice, 12 and 36 h after mobilization. © 2011 Wiley Periodicals, Inc.     

Splenectomy and thrombosis: the case of thalassemia intermedia

See also Mannucci PM. Red cells playing as activated platelets in thalassemia intermedia. This issue, pp 2149–51. Summary. Background: Hypercoagulability in splenectomized patients with thalassemia intermedia (TI) has been extensively evaluated. However, clinical and laboratory characteristics of patients who eventually develop overt thromboembolic events (TEE) are poorly studied. Patients/Methods: Three Groups of TI patients (n = 73 each) were retrospectively identified from a registry involving six centers across the Middle East and Italy: Group I, all splenectomized patients with a documented TEE; Group II, age‐ and sex‐matched splenectomized patients without TEE; and Group III, age‐ and sex‐matched non‐splenectomized patients without TEE. Retrieved data included demographics, laboratory parameters, clinical complications, and received treatments that may influence TEE development, and reflected the period prior to TEE occurrence in Group I. Results: The mean age of Group I patients at development of TEE was 33.1 ± 11.7 years, with a male to female ratio of 33:40. TEE were predominantly venous (95%) while four patients (5%) had documented stroke. Among studied parameters, Group I patients were more likely to have a nucleated red blood cell (NRBC) count ≥ 300 × 10⁶ L^?1, a platelet count ≥ 500 × 10⁹ L^?1 and evidence of pulmonary hypertension (PHT), or be transfusion naïve. The median time to thrombosis following splenectomy was 8 years. Patients with an NRBC count ≥ 300 × 10⁶ L^?1, a platelet count ≥ 500 × 10⁹ L^?1, or who were transfusion naive also had a shorter time to thrombosis following splenectomy. Conclusion: Splenectomized TI patients who will develop TEE may be identified early on by high NRBC and platelet counts, evidence of PHT, and transfusion naivety.     

Specificity and binding capacity of human blood serum for tetrahydropterins

Binding behaviour of labelled biopterin, 6,7-dimethylpterin, 2,4-diamino-6,7-dimethylpteridin, and of their 5,6,7,8-tetrahydro derivatives was studied with human serum proteins. Binding capacity of serum proteins is in the range of 5 × 10^?10?10^?3 M without saturation for all these pteridines. Preincubation or competition with tetrahydrobiopterin does not influence binding of tetrahydro-6,7-dimethylpterin in these concentrations. The bulk of pteridines is bound through unspecific adsorption. Only at concentrations of less than 0.8 × 10^?8 M does high affinity binding seem to be possible, corresponding to about 300 pmol/g protein, whereas physiological biopterin concentration is near 2 × 10^?8 M. The binding proteins are very sensitive to ageing and lose their capacity during purification, whereas unspecific binding to serum proteins is only weakly influenced by alteration of salt concentration, pH, or temperature. Attempts to partially purify the binding proteins by ion exchange, dextran gel (Sephadex G-200), or affinity chromatography, demonstrate a specificity of tetrahydropterins for the α₂-macroglobulin fraction. Due to the high lability of this protein fraction and of pteridine binding, purification of a protein which specifically binds tetrahydrobiopterin was not achieved.