Validation of a Parvovirus B19 NAT Assay for Screening of Umbilical Cord Blood for Allogenic Hematopoietic Stem Cell Donation.

Introduction: Parvovirus B19 transmitted by umbilical cord blood (UCB) products may cause severe disease in allogenic hematopoietic stem cell transplant recipients. Thus, commercially available nucleic acid test (NAT) assays for highly sensitive detection of parvovirus B19 DNA validated for the specimen cord blood plasma (CBP) are required to avoid parvovirus B19 transmission by umbilical hematopoietic stem cell preparations. Methods: The multiplex cobas DPX NAT assay was validated for detection of parvovirus B19 DNA in CBP derived from citrate anticoagulated UCB units which have been processed by the Rubinstein method. In total, 363 retained CBP samples pretested negative for parvovirus B19 DNA were prepared for analyzing sensitivity, specificity, and interference of that NAT assay. The 3rd WHO International Standard for parvovirus B19 DNA was used for determining the 95% limit of detection (LOD95) by probit analysis. Results: The validation of the parvovirus B19 NAT assay for CBP demonstrated high sensitivity, specificity, intra- and inter-assay precision. Dilution series and replicate analyses showed a high linearity of the assay with a coefficient of determination above 0.99 and revealed a LOD95 of 17 International Units (IU)/mL (95% confidence interval, 14-44 IU/mL) for parvovirus B19 DNA in CBP samples. Conclusion: The validation of a commercially available parvovirus B19 NAT assay for the specimen CBP demonstrated a high assay performance fulfilling German guidelines and international regulations.

Principles of Therapeutic Apheresis in Neurological Disease.

Background: Therapeutic plasma exchange (TPE) is a well-known apheresis technology since many years and is available worldwide. Myasthenia gravis is one of the first neurological diseases successfully treated with TPE. TPE is also frequently applied in acute inflammatory demyelinating polyradiculoneuropathy (Guillain-Barré syndrome). Both neurological disorders are immunologically mediated and might cause life-threatening symptoms in patients. Summary: There is a large body of evidence from many randomized controlled trials (RCTs) that the application of TPE in myasthenia gravis crisis or in acute Guillain-Barré syndrome is effective and safe. Thus, TPE is recommended as first-line therapy with a grade 1A recommendation during the critical course of these neurological diseases. Even chronic inflammatory demyelinating polyneuropathies characterized by complement-fixing autoantibodies to myelin are successfully treated with TPE. The plasma exchange reduces inflammatory cytokines, complements activating antibodies, and leads to an improvement of neurological symptoms. TPE is no standalone treatment but often combined with immunosuppressive therapy. Recent studies (clinical trials, retrospective analysis, meta-analysis, and systematic reviews) evaluate special apheresis technology (i.e., immunoadsorption [IA], small volume plasma exchange), compare different treatments of these neuropathies, or report on the therapy of rare immune-mediated neuropathies in case reports. Key Messages: TA is a well-established treatment and is safe in acute progressive neuropathies (myasthenia gravis, Guillain-Barré syndrome) with an immune etiology. TPE has been applied for decades and thus has the best evidence so far. The indication for IA depends on the availability of that technology and the evidence by RCTs in special neurological diseases. The treatment with TA should improve the clinical outcome of patients, reducing acute or chronic (chronic inflammatory demyelinating polyneuropathies) neurological symptoms. The informed consent of the patient should carefully weight risks and benefits of the apheresis treatment and consider alternative therapies.

Monitoring of direct oral anticoagulants plasma levels for secondary stroke prevention.

BACKGROUND: Patients with atrial fibrillation have a relevant risk for ischemic stroke despite the recommended use of direct oral anticoagulants (DOAC). The risk correlates with the functional DOAC plasma levels in clinical trials, but the value of their measurement in community use remains undetermined. OBJECTIVES: We aim to investigate the clinical implications and the prognostic value of DOAC plasma level measurement during steady state. METHODS: In this observational clinical cohort study among patients with ischemic stroke and atrial fibrillation, 397 individuals on oral anticoagulants for secondary stroke prevention were included between 2016 and 2020. The functional DOAC plasma levels were measured during steady state. Early stroke recurrence within 3 months was recorded as the main outcome parameter. RESULTS: Three hundred ninety-seven patients (201 female, mean age 78 [±9] years, median CHA2 DS2 VASc-Score 6 [interquartile range 5-7]) were included. Mean DOAC plasma trough level was 95.9 (±66.9) ng/ml. A high glomerular filtration rate (GFR) was an independent predictor of lower levels in a multivariate model (R coefficient: -0.174, P = .014). During follow-up, 10 patients (3%) suffered from early ischemic stroke recurrence despite the use of DOAC, while 10 clinically relevant bleeding complications occurred (3%). Ischemic stroke recurrence was associated with numerical lower plasma levels for patients on apixaban and dabigatran after propensity score matching. CONCLUSIONS: Monitoring of DOAC plasma levels could help to identify patients with increased risk for stroke recurrence and should be considered for certain subgroups, including patients with high GFR.

Head and neck tumor cells treated with hypofractionated irradiation die via apoptosis and are better taken up by M1-like macrophages.

PURPOSE: The incidence of head and neck squamous cell carcinomas (HNSCC) is increasing worldwide, especially when triggered by the human papilloma virus (HPV). Radiotherapy has immune-modulatory properties, but the role of macrophages present in HNSCC and having contact with irradiated tumor cells remains unclear. The influence of irradiated (2â€¯× 5Gy) HNSCC cells on the (re-)polarization and phagocytosis of human macrophages, either non-polarized or with a more M1 or M2 phenotype, was therefore investigated. METHODS: Human monocytes were differentiated with the hematopoietic growth factors M­CSF (m) or GM-CSF (g) and additionally pre-polarized with either interleukin (IL)-4 and IL-10 or interferon (IFN)-γ and lipopolysaccharides (LPS), respectively. Subsequently, they were added to previously irradiated (2â€¯× 5Gy) and mock-treated HPV-positive (UD-SCC-2) and HPV-negative (Cal33) HNSCC cells including their supernatants. RESULTS: The HNSCC cells treated with hypofractionated irradiation died via apoptosis and were strongly phagocytosed by M0m and M2 macrophages. M0g and M1 macrophages phagocytosed the tumor cells to a lesser extent. Irradiated HNSCC cells were better phagocytosed by M1 macrophages compared to mock-treated controls. The polarization status of the macrophages was not significantly changed, except for the expression of CD206 on M2 macrophages, which was reduced after phagocytosis of irradiated HPV-negative cells. Further, a significant increase in the uptake of irradiated HPV-positive cells by M0g macrophages when compared to HPV-negative cells was observed. CONCLUSION: HNSCC cells treated with hypofractionated irradiation foster phagocytosis by anti-tumorigenic M1 macrophages. The data provide the first evidence on the impact of the HPV status of HNSCC cells on the modulation of the macrophage response to irradiated tumor cells.

Validation of a SARS-CoV-2 RNA RT-PCR assay for high-throughput testing in blood of COVID-19 convalescent plasma donors and patients.

BACKGROUND: The frequency of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNAemia in blood donors is uncertain. Thus, assays for SARS-CoV-2 RNA detection in blood, validated on commercially available polymerase chain reaction (PCR) systems, are required to allow a good comparability of data. STUDY DESIGN AND METHODS: The cobas SARS-CoV-2 dual-target reverse transcriptase PCR (RT-PCR) assay, licensed for respiratory swab SARS-CoV-2 RNA testing, was validated for detection of viral RNA in blood. For the validation panel, SARS-CoV-2-positive plasma samples were prepared by spiking SARS-CoV-2-positive respiratory specimens in negative human plasma. The 95% limit of detection (LOD95) was determined by probit analysis. For clinical validation, coronavirus disease 2019 (COVID-19) convalescent plasma (CCP) donors and patients with COVID-19 with a severe disease course treated in an intensive care unit (ICU) were included. RESULTS: The validation of the SARS-CoV-2 RT-PCR assay for blood demonstrated high sensitivity and specificity and intra- and inter-assay precision and efficiency. The LOD95 for SARS-CoV-2 RNA was 5.0 genome copies/mL (95% confidence interval [CI], 3.3-12 copies/mL) for target 1 and 4.3 genome copies/mL (95% CI, 2.9-10 copies/mL) for target 2. In a cohort of 39 CCP donors with 66 CCP donations no SARS-CoV-2 RNA in plasma was detected. Screening of 25 blood samples of 19 ICU patients with COVID-19 showed six positive results for SARS-CoV-2 RNA in at least one target of the assay. CONCLUSION: The SARS-CoV-2 RNA assay, only licensed for respiratory swabs, performed on a PCR system for high-throughput testing, showed a good assay performance for blood testing.

Thrombin Generation in Fresh and Frozen-Thawed Platelet Poor Plasma - Is there a Difference?

BACKGROUND: Applicability of thrombin generation tests to clinical routine has been sought for many years. The aim of this study was to compare thrombin generation measured in fresh platelet poor plasma (f-PPP) and frozen-thawed platelet poor plasma (ft-PPP) to prove the consistency of results. METHODS: In this prospective study, thrombin generation was measured in twenty-fold repetitions in 3.2% citrate PPP obtained from male healthy blood donors aged 19 - 39 years (n = 54 donations). The tests were performed with fresh PPP and repeated after storing the PPP at -60°C. In two subgroup analyses, the effect of higher and lower normal baseline platelet counts on the Calibrated Automated Thrombogram (CAT) assay and the influence of ABO blood groups on thrombin generation were analyzed. RESULTS: Referring to the parameters of thrombin generation most frequently used in studies, peak thrombin of f-PPP and ft-PPP agreed in about 50% of the samples. Endogenous thrombin potential (ETP) of f-PPP and ft-PPP agreed in nearly two-thirds of the samples. A slightly but significantly slower kinetic was found in the thrombin generation of ft-PPP compared with f-PPP. At least in f-PPP, ETP correlates with baseline platelet counts of the whole blood sample. Peak thrombin was significantly higher in non-O blood groups compared to O blood group. CONCLUSIONS: A low level of agreement between the results of f-PPP and ft-PPP is shown. In terms of practicability of sample collection using semi-automated thrombin-generation assays ft-PPP should be preferred over f-PPP. We therefore recommend using ft-PPP in clinical studies.

ILC2 Lung-Homing in Cystic Fibrosis Patients: Functional Involvement of CCR6 and Impact on Respiratory Failure.

Cystic fibrosis patients suffer from a progressive, often fatal lung disease, which is based on a complex interplay between chronic infections, locally accumulating immune cells and pulmonary tissue remodeling. Although group-2 innate lymphoid cells (ILC2s) act as crucial initiators of lung inflammation, our understanding of their involvement in the pathogenesis of cystic fibrosis remains incomplete. Here we report a marked decrease of circulating CCR6+ ILC2s in the blood of cystic fibrosis patients, which significantly correlated with high disease severity and advanced pulmonary failure, strongly implicating increased ILC2 homing from the peripheral blood to the chronically inflamed lung tissue in cystic fibrosis patients. On a functional level, the CCR6 ligand CCL20 was identified as potent promoter of lung-directed ILC2 migration upon inflammatory conditions in vitro and in vivo using a new humanized mouse model with light-sheet fluorescence microscopic visualization of lung-accumulated human ILC2s. In the lung, blood-derived human ILC2s were able to augment local eosinophil and neutrophil accumulation and induced a marked upregulation of pulmonary type-VI collagen expression. Studies in primary human lung fibroblasts additionally revealed ILC2-derived IL-4 and IL-13 as important mediators of this type-VI collagen-inducing effect. Taken together, the here acquired results suggest that pathologically increased CCL20 levels in cystic fibrosis airways induce CCR6-mediated lung homing of circulating human ILC2s. Subsequent ILC2 activation then triggers local production of type-VI collagen and might thereby drive extracellular matrix remodeling potentially influencing pulmonary tissue destruction in cystic fibrosis patients. Thus, modulating the lung homing capacity of circulating ILC2s and their local effector functions opens new therapeutic avenues for cystic fibrosis treatment.

Ischemic stroke and dose adjustment of oral Factor Xa inhibitors in patients with atrial fibrillation.

BACKGROUND: Oral Factor Xa inhibitors for the prevention of stroke in atrial fibrillation require dose adjustment based on certain clinical criteria, but the off-label use of the reduced doses is common. METHODS: Data from an observational registry including patients admitted with acute cerebral ischemia while taking oral Factor Xa inhibitors for atrial fibrillation between April 2016 and December 2018 were investigated. The dose regimen of the Xa inhibitor was classified as "appropriate", "underdosed" and "overdosed" in conformity with the European Medicines Agency labelling. The effect of underdosing on the functional factor Xa plasma level on admission, the clinical stroke severity and the functional outcome after 3 months were investigated. RESULTS: 254 patients with cerebral ischemia while on Factor Xa inhibitors were included. The dose regimen of the Factor Xa inhibitor was appropriate in 166 patients (65%), underdosed in 67 patients (26%) and overdosed in 21 patients (8%). Underdosing was associated with female sex, diabetes mellitus and higher CHA2DS2-Vasc scores. Underdosing independently predicted lower anti-Xa plasma levels on admission [median 69.4 ng/ml (IQR 0.0-121.6) vs. 129.2 ng/ml (65.5-207.2); p < 0.001], was associated with higher NIHSS scores on admission [median 5 (IQR 1-10) vs. 3 (1-7); p = 0.041] and worse functional outcome after 3 months (favorable outcome 26.9% vs. 46.9%; p = 0.025). CONCLUSION: One in three patients with ischemic stroke during treatment with oral Xa inhibitors used inappropriate dose regimens. Underdosing was associated with lower functional plasma levels, higher clinical stroke severity and worse functional outcome.

Thrombocyte apheresis cassettes as a novel source of viable peripheral blood mononuclear cells.

BACKGROUND: Traditionally, white blood cells (WBCs) are collected from buffy coats or freshly drawn blood. However, the increasing demand for peripheral blood mononuclear cells (PBMCs) in the research phases of immunological therapy development makes it necessary to identify alternative sources of these cells. STUDY DESIGN AND METHODS: Leukapheresis products are cost intensive and not offered by all blood banks. Therefore, thrombocyte apheresis cassettes (TACs), plateletpheresis waste products, were investigated as a possible low-cost and easily accessible blood source for research laboratories. The recovery rate, phenotype, and functionality of WBC subsets from TAC are unknown and were investigated in comparison to frequently used blood resources via flow cytometry. RESULTS: On average, TACs provide 30.3 × 106 /mL PBMCs, situating themselves between peripheral whole blood (WB; 5.35 × 106 /mL) and leukoreduction system chamber (LRSC; 163.9 × 106 /mL) yields. Frequencies of CD14, CD3, CD4, CD8, CD56, CD19, and CD11c positive cells in TACs correlate with normal proportions of WBC populations. Stimulation of TAC-derived PBMCs by lipopolysaccharide (LPS) and resiquimod (R848) showed no significant differences in expression levels of human leukocyte antigen (HLA)-DR, DQ, DP, and CD86 or cytokine secretion compared to other blood source derived PBMC. Following stimulation with LPS or R848, comparable levels of tumor necrosis factor-α, interleukin-10, and interleukin-1ß could be measured between TAC, LRSC, and WB. Additionally, TAC-derived T cells retained their proliferation capability and were able to produce interferon-γ following T-cell receptor stimulation. CONCLUSION: TACs provide a cost-effective source of viable and functional human blood cells that can readily be used for clinical and laboratory investigations after plateletpheresis preparation.

Blood counts in adult and elderly individuals: defining the norms over eight decades of life.

The blood count is one of the most common tests used for health assessment. In elderly individuals, selection of a 'healthy' reference population for laboratory assessment is difficult due to the high prevalence of chronic morbidities, leading to uncertainty regarding appropriate reference intervals. In particular, age-specific lower haemoglobin reference limits to define anaemia are controversial. Here, we applied a data mining approach to a large dataset of 3 029 904 clinical routine samples to establish blood count reference intervals. We excluded samples from units/specialists with a high proportion of abnormal blood counts, samples from patients with an unknown or decreased estimated glomerular filtration rate, and samples with abnormal test results in selected other analytes. After sample exclusion, 566 775-572 060 samples from different individuals aged 20-100 years were available for analysis. We then used an established statistical algorithm to determine the distribution of physiological test results and calculated age- and sex-specific reference intervals. Our results show substantial trends with age in haematology analytes' reference intervals. Most notably, haemoglobin and red cell counts decline in men with advanced age, accompanied by increases in red cell volume in both sexes. These findings were confirmed in an independent dataset, and suggest an at least partly physiologic cause.

Direct Oral Anticoagulant Plasma Levels for the Management of Acute Ischemic Stroke.

INTRODUCTION: The management of acute ischemic stroke in patients on direct oral anticoagulants (DOACs) is challenging. However, the substance-specific plasma level could guide treatment decisions on recanalization therapies. We present a plasma-level-based protocol for emergency treatment of stroke patients on oral anticoagulants. Bleeding complications and clinical outcome for patients on DOACs are reported and compared to patients on vitamin K antagonists (VKAs). METHODS: In patients with acute ischemic stroke and suspected use of DOACs within 48 h prior to hospital admission, plasma levels were measured using the calibrated Xa-activity (apixaban, edoxaban, rivaroxaban) or the Hemoclot®-assay (dabigatran). Levels <50 ng/mL were supportive for thrombolysis, while high values >100 ng/mL excluded patients from recombinant tissue plasminogen activator use. For patients on VKAs, the cutoff was set at international normalized ratio of 1.7. Endovascular thrombectomy of a large vessel occlusion was performed independently from coagulation testing. Consecutive patients were included in an observational registry. RESULTS: Five hundred and twenty-two patients (261 on VKAs and 261 on DOACs) were included. Thirty patients (11.5%) on VKAs and 24 (9.2%) on DOACs received thrombolysis, followed by mechanical thrombectomy in 10 and 14 patients, respectively. Seventeen patients in each group received thrombectomy only. Symptomatic intracranial hemorrhage associated with thrombolysis occurred in 1 patient on VKA (3.3%) and 1 on DOAC (4.2%; p = 0.872). The turnaround time of specific assays did not show a significant delay in comparison to standard coagulation parameters. CONCLUSION: DOAC plasma levels could support decisions on emergency treatment of ischemic stroke. Systemic thrombolysis below suggested thresholds appears preliminary feasible and safe without an excess in bleeding complications.

Data mining of reference intervals for coagulation screening tests in adult patients.

BACKGROUND: Appropriate reference intervals are essential when evaluating laboratory test results. However, establishment of reference intervals is challenging, especially for coagulation screening tests, and uncertainty exists regarding age- and sex-dependency of test results. Data mining of laboratory information systems is an emerging approach to reference interval determination, and we evaluated its applicability to coagulation tests. METHODS: We analyzed measurements of activated partial thromboplastin time (aPTT), prothrombin time (PT), international normalized ratio (INR), thrombin time (TT), and fibrinogen performed during clinical care in the University Hospital Erlangen, Germany (1,778,738 samples from 116,754 adult patients, 45,577-509,859 samples per analyte). We identified the proportion of samples from healthy individuals using an established statistical approach (Reference Limit Estimator), in which the distribution of physiological test results is approximated using a parametrical function, and used for the calculation of reference intervals. RESULTS: We established age- and sex specific reference intervals for aPTT, PT, INR, TT, and fibrinogen, and created batch- and reagent-specific aPTT-reference intervals. Additionally, we evaluated the sensitivity of the established aPTT reference intervals for the detection of factor VIII, IX, XI, XII deficiencies. CONCLUSION: Data mining of laboratory test results allows the creation of age- and sex-reference intervals for coagulation tests that are specific to the examined population, analytical framework, and reagent. This approach can complement conventional methods when establishing reference intervals and improve clinical decision-making based on coagulation tests. The reference intervals established in this study show only minor variation with sex and age, supporting the practice of providing a common reference interval for adult women and men.

Multivariate Platelet Analysis Differentiates Between Patients with Alzheimer's Disease and Healthy Controls at First Clinical Diagnosis.

BACKGROUND: Early diagnosis of Alzheimer's disease (AD) is challenging, and easily accessible biomarkers are an unmet need. Blood platelets frequently serve as peripheral model for studying AD pathogenesis and might represent a reasonable biomarker source. OBJECTIVE: In the present study, we investigated the potential to differentiate AD patients from healthy controls (HC) based on blood count, platelet morphology, and function as well as molecular markers at the time of first clinical diagnosis. METHODS: Blood samples from 40 AD patients and 29 age-matched HC were included for determination of 78 parameter by blood counting, platelet morphometry, aggregometry, flow cytometry (CD62P, CD63, activated fibrinogen receptor), protein quantification of nicotinic acetylcholine receptor α7 (nAChRα7) and caveolin-1 (CAV-1), and miRNA quantification (miR-26b, miR-199a, miR-335). Group comparison between patients and controls was performed in univariate and multivariate statistical analyses. RESULTS: AD patients showed significantly lower aggregation response to ADP and arachidonic acid and significantly decreased CD62P and CD63 surface expression induced by ADP and U46619 compared to HC. Relative nAChRα7 and CAV-1 expression was significantly higher AD platelets than in HC. Multivariate analysis of 63 parameter revealed significant differences between AD patients and healthy controls. The best performing feature model revealed a sensitivity of 96.6%, a specificity of 80.0%, and a positive predictive value of 89.3%. No grouping could be achieved by using single parameter groups. CONCLUSION: Significant differences between platelet characteristics from AD patients and HC at the time of first clinical diagnosis were observed. The best performing parameter can be used as a blood-based biomarker for AD diagnosis in a multivariate model in addition to the standardized mental tests.

Cerebral Ischemia in Patients on Direct Oral Anticoagulants.

Background and Purpose- In patients with ischemic stroke on therapy with vitamin K antagonists, stroke severity and clinical course are affected by the quality of anticoagulation at the time of stroke onset, but clinical data for patients using direct oral anticoagulants (DOACs) are limited. Methods- Data from our registry including all patients admitted with acute cerebral ischemia while taking oral anticoagulants for atrial fibrillation between November 2014 and October 2017 were investigated. The activity of vitamin K antagonists was assessed using the international normalized ratio on admission and categorized according to a threshold of 1.7. DOAC plasma levels were measured using the calibrated Xa-activity (apixaban, rivaroxaban, and edoxaban) or the Hemoclot-assay (dabigatran) and categorized into low (<50 ng/mL), intermediate (50-100 ng/mL), or high (>100 ng/mL). Primary objective was the association between anticoagulant activity and clinical and imaging characteristics. Results- Four hundred sixty patients were included (49% on vitamin K antagonists and 51% on DOAC). Patients on vitamin K antagonists with low international normalized ratio values had higher scores on the National Institutes of Health Stroke Scale and a higher risk of large vessel occlusion on admission. For patients on DOAC, plasma levels were available in 75.6% and found to be low in 49 (27.7%), intermediate in 41 (23.2%), and high in 87 patients (49.2%). Low plasma levels were associated with higher National Institutes of Health Stroke Scale scores on admission (low: 8 [interquartile range, 3-15] versus intermediate: 4 [1-11] versus high: 3 [0-8]; P<0.001) and higher risk of persisting neurological deficits or cerebral infarction on imaging (85.7% versus 75.6% versus 54.0%; P<0.001). Low DOAC plasma levels were an independent predictor of large vessel occlusion (odds ratio, 3.84 [95% CI, 1.80-8.20]; P=0.001). Conclusions- The activity of anticoagulation measured by specific DOAC plasma levels on admission is associated with stroke severity and presence of large vessel occlusion.

Recommendations for Therapeutic Apheresis by the Section "Preparative and Therapeutic Hemapheresis" of the German Society for Transfusion Medicine and Immunohematology.

The section "Preparative and Therapeutic Hemapheresis" of the German Society for Transfusion Medicine and Immunohematology (DGTI) has reviewed the actual literature and updated techniques and indications for evidence-based use of therapeutic apheresis in human disease. The recommendations are mostly in line with the "Guidelines on the Use of Therapeutic Apheresis in Clinical Practice" published by the Writing Committee of the American Society for Apheresis (ASFA) and have been conducted by experts from the DACH (Germany, Austria, Switzerland) region.

Automated closed-system manufacturing of human monocyte-derived dendritic cells for cancer immunotherapy.

Dendritic cell (DC)-based vaccines have been successfully used for immunotherapy of cancer and infections. A major obstacle is the need for high-level class A cleanroom cGMP facilities for DC generation. The CliniMACS Prodigy® (Prodigy) represents a new platform integrating all GMP-compliant manufacturing steps in a closed system for automated production of various cellular products, notably T cells, NK cells and CD34+ cells. We now systematically tested its suitability for producing human mature monocyte-derived DCs (Mo-DCs), and optimized it by directly comparing the Prodigy approach to our established standard production of Mo-DCs from elutriated monocytes in dishes or bags. Upon step-by-step identification of an optimal cell concentration for the Prodigy's CentriCult culture chamber, the total yield (% of input CD14+ monocytes), phenotype, and functionality of mature Mo-DCs were equivalent to those generated by the standard protocol. Technician's labor time was comparable for both methods, but the Prodigy approach significantly reduced hands-on time and high-level clean room resources. In summary, using our optimized conditions for the CliniMACS Prodigy, human Mo-DCs for clinical application can be generated almost automatically in a fully closed system. A significant drawback of the Prodigy approach was, however, that due to the limited size of the CentriCult culture chamber, in contrast to our standard semi-closed elutriation approach, only one fourth of an apheresis could be processed at once.

Impact of collection programs for the generation of monocyte apheresis products on product quality and composition as starting material for the generation of cellular therapeutics.

BACKGROUND: With the discontinuation of the last generation of apheresis machines, new options for monocyte apheresis became available. As apheresis products play a crucial role in the generation of new cellular therapeutics (e.g., generation of dendritic cells [DCs] or precursor for T-cell experiments) we sought to compare two different collection programs for potential benefits or disadvantages due to different composition of the cellular products. STUDY DESIGN AND METHODS: Composition of discontinuously and continuously collected monocyte products from the same 13 donors was analyzed. For further evaluation as starting material for manufacturing of cellular therapeutics typically used steps such as Ficoll Isopaque, cryoconservation and monocyte isolation, with subsequent generation of mature DCs (mDCs) and assessment of T-cell function, were performed on seven of these apheresis pairs. RESULTS: Yield of total cells, monocytes, and mDCs was equal with both methods. T-cell composition did not differ significantly in content of CD3+, CD4+, and CD8+ cells. Differentiation status and cytokine production of CD8+ T cells upon stimulation with cytomegalovirus pp65 antigen was not significantly different. CONCLUSION: Both methods seem comparably suited for the generation of cellular products. If the intended use is "fresh" (without cryoconservation), continuously harvested cells show better cell numbers, while discontinuously harvested cells show better recovery after cryoconservation.

First comparative analysis concerning the plasma platelet contamination during MNC collection.

BACKGROUND AND OBJECTIVES: Monocytes can be cultured into dendritic cells with addition of autologous plasma, which is highly prone to platelet contamination due to the apheresis process. Since platelets affect the maturation process of monocytes into dendritic cells and might even lead to a diminished harvest of dendritic cells, it is very important to reduce the platelet contamination. A new collection device (Spectra Optia) was analyzed, compared to two established devices (COM.TEC, Cobe Spectra) and evaluated regarding the potential generation of source plasma. MATERIALS AND METHODS: Concurrent plasma collected during leukapheresis was analyzed for residual cell contamination in a prospective study with the new Spectra Optia apheresis device (n=24) and was compared with COM.TEC and Cobe Spectra data (retrospective analysis, n=72). Donor pre-donation counts of platelets were analyzed for their predictive value of contaminating PLTs in plasma harvests. RESULTS: The newest apheresis device showed the lowest residual platelet count of the collected concurrent plasma (median 3.50×109/l) independent of pre-donation counts. The other two devices and sets had a higher platelet contamination. The contamination of the plasma with leukocytes was very low (only 2.0% were higher than 0.5×109/l). CONCLUSIONS: This study showed a significant reduction of platelet contamination of the concurrent plasma collected with the new Spectra Optia device. This plasma product with low residual platelets and leukocytes might also be used as plasma for fractionation.

Twelve-year survival and immune correlates in dendritic cell-vaccinated melanoma patients.

BACKGROUND: Reports on long-term (≥10 years) effects of cancer vaccines are missing. Therefore, in 2002, we initiated a phase I/II trial in cutaneous melanoma patients to further explore the immunogenicity of our DC vaccine and to establish its long-term toxicity and clinical benefit after a planned 10-year followup. METHODS: Monocyte-derived DCs matured by TNFα, IL-1ß, IL-6, and PGE2 and then loaded with 4 HLA class I and 6 class II-restricted tumor peptides were injected intradermally in high doses over 2 years. We performed serial immunomonitoring in all 53 evaluable patients. RESULTS: Vaccine-specific immune responses including high-affinity, IFNγ-producing CD4+ and lytic polyfunctional CD8+ T cells were de novo induced or boosted in most patients. Exposure of mature DCs to trimeric soluble CD40 ligand, unexpectedly, did not further enhance such immune responses, while keyhole limpet hemocyanin (KLH) pulsing to provide unspecific CD4+ help promoted CD8+ T cell responses - notably, their longevity. An unexpected 19% of nonresectable metastatic melanoma patients are still alive after 11 years, a survival rate similar to that observed in ipilimumab-treated patients and achieved without any major (>grade 2) toxicity. Survival correlated significantly with the development of intense vaccine injection site reactions, and with blood eosinophilia after the first series of vaccinations, suggesting that prolonged survival was a consequence of DC vaccination. CONCLUSIONS: Long-term survival in advanced melanoma patients undergoing DC vaccination is similar to ipilimumab-treated patients and occurs upon induction of tumor-specific T cells, blood eosinophilia, and strong vaccine injection site reactions occurring after the initial vaccinations. TRIAL REGISTRATION: ClinicalTrials.gov NCT00053391. FUNDING: European Community, Sixth Framework Programme (Cancerimmunotherapy LSHC-CT-2006-518234; DC-THERA LSHB-CT-2004-512074), and German Research Foundation (CRC 643, C1, Z2).

Association of an In-House Blood Bank with Therapy and Outcome in Severely Injured Patients: An Analysis of 18,573 Patients from the TraumaRegister DGU®.

INTRODUCTION: Hemorrhagic shock remains one of the most common causes of death in severely injured patients. It is unknown to what extent the presence of a blood bank in a trauma center influences therapy and outcome in such patients. MATERIAL AND METHODS: We retrospectively analyzed prospectively recorded data from the TraumaRegister DGU® and the TraumaNetzwerk DGU®. Inclusion criteria were Injury Severity Score (ISS) ≥ 16, primarily treated patients, and hospital admission 2 years before or after the audit process. RESULTS: Complete data sets of 18,573 patients were analyzed. Of 457 hospitals included, 33.3% had an in-house blood bank. In trauma centers with a blood bank (HospBB), packed red blood cells (PRBCs) (21.0% vs. 17.4%, p < 0.001) and fresh frozen plasma (FFP) (13.9% vs. 10.2%, p <0.001) were transfused significantly more often than in hospitals without a blood bank (Hosp0). However, no significant difference was found for in-hospital mortality (standard mortality ratio [SMR, 0.907 vs. 0.945; p = 0.25). In patients with clinically apparent shock on admission, no difference of performed transfusions were present between HospBB and Hosp0 (PRBCs, 51.4% vs. 50.4%, p = 0.67; FFP, 32.7% vs. 32.7%, p = 0.99), and no difference in in-hospital mortality was observed (SMR, 0.907 vs. 1.004; p = 0.21). DISCUSSION: In HospBB transfusions were performed more frequently in severely injured patients without positively affecting the 24h mortality or in-house mortality. Easy access may explain a more liberal transfusion concept.