Validation of an apoptosis assay for extracorporeal photopheresis

Objectives This validation study investigated a flow cytometric apoptosis assay according to good manufacturing practice (GMP). Background Extracorporeal photopheresis (ECP) is a treatment for various immunological diseases and cutaneous T‐cell lymphomas. It is based on the induction of apoptosis by 8‐methoxypsoralene and ultraviolet A light. The quantification of apoptosis is therefore essential for ECP improvements. However, despite numerous publications on apoptosis, validated technical details are lacking. Methods and materials Mononuclear cells were collected by apheresis and treated by ECP or camptothecin. Samples taken before and after ECP were cultured for 24, 48 and 72 h and analysed for apoptosis and viability of T cells and monocytes by flow cytometry with Annexin V and 7‐AAD staining. Accuracy of the assay, intra‐ and inter‐assay precision and the pre‐analytical and analytical stability of the analytes were the investigated parameters. Results Our data indicate that the median intra‐ and inter‐assay precision coefficient of variation for T cells was 3.86% and 4.80%, respectively. Pre‐analytical stability of T cells and monocytes was ensured during short‐term storage for up to 2 h on ice. After staining, analytical stability was limited to 30 min, likely because of ongoing apoptosis and loss of monocytes due to plastic adhesion. Conclusion The results of this validation study show that the assay is GMP‐compliant and that its reliability, accuracy and precision are acceptable. While pre‐analytical stability of the cells was compatible with on‐site procedures, our analytical stability data indicate that this assay is not suited for batch mode analysis of ECP products

Total platelet donation count and donation frequency are determinants of plateletpheresis‐associated lymphopenia

Background Plateletpheresis using a leukocyte reduction system (LRS) traps donor WBCs in the LRS chamber, which may lead to lymphopenia, especially in frequent plateletpheresis donors. It seems plausible that this might cause adverse effects. However, current knowledge about potential confounders and donor health impacts is incomplete. Donors and methods Recent platelet donors and donations collected at University Hospital Regensburg from 2016 to 2019 using the Terumo BCT Trima Accel LRS system were retrospectively analyzed and compared with historical platelet donors and donations collected mainly with Fresenius Kabi Amicus non-LRS system from 2010 to 2013. Additionally, recent donors were prospectively surveyed using a health-related topics questionnaire. Results Analysis of 819 recent donors with 11,254 blood counts and 1464 questionnaires and 1011 historical donors with 12,848 blood counts revealed that increased annual platelet donation frequencies were associated with decreased lymphocyte counts in both groups. Median lymphocyte counts in recent donors with no versus ≥24 previous annual donations declined from 2.0 to 1.2 × 103/μL (p < 2.2 × 10−16), and those in historical donors with no versus ≥24 previous annual donations decreased from 2.0 to 1.5 × 103/μL (p = 6 × 10−4), respectively. The questionnaire results showed that donation frequency and lymphopenia were not associated with upper respiratory tract infection (URTI) incidence or duration, but platelet donors who concomitantly donated granulocytes had significantly shorter URTI durations than those who did not (p = .008). Conclusion This study confirmed that plateletpheresis-associated lymphopenia occurs in LRS and to a lesser degree in non-LRS platelet donors, but revealed no evidence of a negative impact on donor health

Bacterial contamination rates in extracorporeal photopheresis

BACKGROUND Extracorporeal photopheresis (ECP) is an immunosuppressive treatment that involves leukocyte apheresis, psoralen and UV light treatment, and subsequent reinfusion. Patients treated with ECP are usually immunosuppressed. Bacterial contamination therefore poses a much unwanted risk, but incidence data are lacking. PATIENTS AND METHODS We screened all 1922 consecutive ECP procedures scheduled within a roughly 3-year period for eligibility. Those with missing data on ECP method (inline or offline) or type of venous access (peripheral or central) were excluded. ECPs with complete aerobic and anaerobic microbial testing of baseline patient blood samples (n = 1637) and of ECP cell concentrates (n = 1814) were included in the analysis. RESULTS A test for microbial contamination was positive for 1.82% of the cell concentrates, with central venous access was the most significant risk factor for the contamination (odds ratio = 19). Patient blood samples were positive in 3.85% of cases, but no patients became septic. Staphylococcus spp. were most abundant, and products with bacterial contamination did not cause side effects after reinfusion. There were no significant differences in contamination rates between inline and offline ECP. CONCLUSION These findings stress the importance of sterile procedures and the benefits of using peripheral over central venous access for reducing the risk of bacterial contamination in ECP

A highly specific and sensitive serological assay detects SARS‑CoV‑2 antibody levels in COVID‑19 patients that correlate with neutralization

Objective The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic challenges national health systems and the global economy. Monitoring of infection rates and seroprevalence can guide public health measures to combat the pandemic. This depends on reliable tests on active and former infections. Here, we set out to develop and validate a specific and sensitive enzyme linked immunosorbent assay (ELISA) for detection of anti SARS-CoV-2 antibody levels. Methods In our ELISA, we used SARS-CoV-2 receptor-binding domain (RBD) and a stabilized version of the spike (S) ectodomain as antigens. We assessed sera from patients infected with seasonal coronaviruses, SARS-CoV-2 and controls. We determined and monitored IgM-, IgA- and IgG-antibody responses towards these antigens. In addition, for a panel of 22 sera, virus neutralization and ELISA parameters were measured and correlated. Results The RBD-based ELISA detected SARS-CoV-2-directed antibodies, did not cross-react with seasonal coronavirus antibodies and correlated with virus neutralization (R2 = 0.89). Seroconversion started at 5 days after symptom onset and led to robust antibody levels at 10 days after symptom onset. We demonstrate high specificity (99.3%; N = 1000) and sensitivity (92% for IgA, 96% for IgG and 98% for IgM; > 10 days after PCR-proven infection; N = 53) in serum. Conclusions With the described RBD-based ELISA protocol, we provide a reliable test for seroepidemiological surveys. Due to high specificity and strong correlation with virus neutralization, the RBD ELISA holds great potential to become a preferred tool to assess thresholds of protective immunity after infection and vaccination

Die transkriptionelle Regulation der Toll-like Rezeptoren (TLR2, TLR3 und TLR4) in mononukleären Phagozyten

Toll-like Rezeptoren (TLR) sind Transmembranproteine, die sowohl für die angeborene als auch für die erworbene Immunantwort eine wichtige Rolle spielen. Diese Dissertation beschäftigt sich mit der Regulation von drei TLR-Genen in mononukleären Phagozyten des Menschen und der Maus. Es wurden hauptsächlich die genomischen Strukturen und regulatorischen Bereiche, die sogenannten proximalen Promotoren definiert und regulierende Faktoren identifiziert, die sowohl bei der Regulation der basalen als auch der induzierten Expression des TLR2, TLR3 und TLR4 eine wichtige Rolle spielen. Zur Charakterisierung der regulatorischen Sequenz des humanen TLR2-Gens wurden Transkriptionsstartpunkte definiert und mittels Datenbankanalysen die Genstruktur ermittelt. Anhand von Monozyten mRNA wurden alternative Spleißvorgänge in Exon II detektiert, welche zu unterschiedlichen Isoformen der TLR2-mRNA führen. Der 5`-regulatorische Bereich des humanen TLR2-Gens wurde festgelegt und in Reporteranalysen weiter charakterisiert. Der proximale Promotor umfasst einen Bereich von 220 bp vor dem Transkriptionsstart. Da der humane TLR2-Promotor in einer CpG-Insel liegt, wurden Methylierungsuntersuchungen durchgeführt. Eine gewebespezifische Methylierung des Promotors war jedoch nicht festzustellen. Anhand von Mutationsanalysen, Gelshiftassays und Kotransfektionen konnte eine wichtige Rolle der Transkriptionsfaktoren Sp1 und PU.1 für die Expression von TLR2 in mononukleären Phagozyten nachgewiesen werden. Eine LPS-Aktivierung von humanen Monozyten führte, anders als in Makrophagen der Maus, zu keiner Induktion von TLR2-mRNA. Eine Hochregulation der humanen TLR2-mRNA nach 3 h war auf die Adhärenz der Zellen an die Plastikschale zurückzuführen und nicht auf eine Stimulation mit LPS. Die Regulation von TLR3 wurde in mononukleären Phagozyten aus Mensch und Maus untersucht. Anhand semiquantitativer PCR wurde in einem direkten Vergleich der TLR3-Expression beider Spezies eine zelltypspezifische Expression festgestellt. TLR3-mRNA des Menschen kommt spezifisch auf dendritischen Zellen vor, TLR3-mRNA der Maus wird hauptsächlich auf Makrophagen exprimiert. Induktionsanalysen ergaben, dass TLR3-mRNA in beiden Spezies durch IFN-b induziert wird. Die Genstrukturen von humanen und murinen TLR3 wurden ermittelt und anhand von Transfektionsanalysen wurden die regulatorischen Bereiche des TLR3-Gens beider Spezies definiert und charakterisiert. Die Struktur der TLR3-Promotoren hinsichtlich Architektur und möglicher Bindungsstellen von Transkriptionsfaktoren ist in beiden Spezies unterschiedlich, es befinden sich jedoch sowohl in humanen als auch in murinen TLR3-Promotor IFN-regulierte Elemente. Mutationsstudien, Gelshifts und Kotransfektionen ergaben, dass IRF1 und IRF2 die Expression des TLR3-Gens in Mensch und Maus regulieren. Anhand von Knockout-Mäusen konnte geklärt werden, dass die Induktion von TLR3-mRNA in der Maus über den Jak-STAT-IRF-Signalweg verläuft. Die Hochregulation von TLR3-mRNA der Maus durch LPS wird über autokrines IFN-b vermittelt. Im Menschen wird TLR3-mRNA durch LPS nicht induziert und in mit LPS vorstimulierten DC blieb eine durch IFN-b induzierte Hochregulation von TLR3-mRNA aus. Es bleibt noch zu klären, welche Faktoren durch LPS induziert werden, die eine negative Regulation von humanen TLR3-mRNA erklären. Die Genstrukturen und 5`-Bereiche des TLR4-Gens in Mensch und Maus waren von unserer Arbeitsgruppe bereits identifiziert und mittels Reporteranalysen wurden der humane und murine proximale TLR4-Promotor weiter charakterisiert und miteinander verglichen. Den Transkriptionsfaktoren PU.1 und ICSBP konnte anhand von Mutationsassays und Gelshifts eine wichtige Rolle bei der Regulation des TLR4-Gens sowohl in Mensch als auch in Maus nachgewiesen werden. Der humane TLR4-Promotor war in Transfektionen trotz eines hohen Konservierungsgrades wesentlich aktiver ist als der murine TLR4-Promotor. Um die regulatorischen Bereiche zu charakterisieren, die für die unterschiedliche Aktivität verantwortlich sind, wurden Reporteranalysen mit chimären Konstrukten aus humanen und murinen TLR4-Promotor durchgeführt. Die stärkere Aktivität des humanen TLR4-Promotors wurde vor allem durch Bereiche unterhalb der konservierten PU.1/ICSBP-Site vermittelt, die den humanen Transkriptionsstart enthalten. Das humane und murine TLR4-Gen besitzen durch die unterschiedliche Lage der Transkriptionsstartpunkte eine unterschiedliche Promotorarchitektur. In nachfolgenden Untersuchungen sollen die regulatorischen Sequenzen des TLR4-Gens in Mensch und Maus weiter charakterisiert werden

A method for the quantification of 8-methoxypsoralen by mass spectrometry for offline extracorporeal photopheresis

Background: Extracorporeal photopheresis (ECP) is an efficient method to treat various autoimmune diseases, cutaneous T-cell lymphoma, and graft-versus-host disease. It is based on the ex vivo inactivation of lymphocytes by 8-methoxypsoralen (8-MOP)/UV light treatment. Despite the adhesive, lipophilic nature of 8-MOP, no quality control is established for the ECP procedure. Methods: We developed a sensitive high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS) assay to monitor residual 8-MOP concentration after UVA irradiation in the whole blood supernatant after acetonitrile precipitation. Results: The preanalytical stability of 8-MOP exceeded 7 days, allowing batch mode analysis. Linearity was determined with R-2 above 0.99. The 8-MOP concentrations decreased exponentially after UV exposure, with decay constants of 0.0259 in plasma and 0.0528 in saline. The recovery of 8-MOP in photopheresates was about 68%, indicating binding to DNA as well as to plastic structures. UVA induced no 8-MOP fragmentation, but caused self-adducts under extreme conditions (10-fold UV dosage). Conclusions: Detection of 8-MOP proved to be feasible and demonstrated that the doses were in the pharmaceutically active range

Triple apheresis platelet concentrate quality after pneumatic tube system, conveyor box, and courier transport: An observational study

Background and Aims Platelets are prone to activation from handling; they are therefore transported as gently as possible, most commonly by courier. Speedier methods like pneumatic tube system (PTS) transport could improve patient care but may subject platelets to mechanical stress. To test the impact of mechanical stress caused by transport, we compared a PTS with a conveyor box and courier transport on apheresis platelet function. Methods Fourteen apheresis platelet concentrate triple donations were analyzed by light transmission aggregometry (LTA), rotational thrombelastometry (ROTEM), and flow cytometry before and after indoor transport over 800 m by PTS, conveyor, and courier, respectively, while recording shocks and vibrations with a high-frequency acceleration data logger. Shock index scores were calculated as shock intensity (g-force) times frequency. Results The shock index was 81 for courier, 6279 for conveyor, and 9075 for PTS. Flow cytometry revealed no significant difference in platelet surface expression of CD62p before (16%) and after transport via courier (15%), conveyor (14%), or PTS (16%). LTA with adenosine phosphate and thrombin receptor-activating peptide-6 resulted in comparable platelet aggregation for courier, conveyor, and PTS. ROTEM assays showed no relevant differences in coagulation time, clot formation time, and maximum clot firmness between transport modes. Conclusion Though the mechanical challenge was smallest with courier transport, there were no significant differences in platelet activation or aggregation between the three transport modes. These data contradict restrictions on the use of PTSs for platelet concentrate transport

Apoptosis induction by extracorporeal photopheresis is enhanced by increasing the 8‐methoxypsoralen concentration and by replacing plasma with saline

Background Extracorporeal photopheresis (ECP), an apheresis-based therapy for various immunological diseases, works mainly by inducing apoptosis in lymphocytes. Several factors influence the efficacy of ECP with the photosensitizer 8-methoxypsoralen (8-MOP) and ultraviolet light A (UVA). This study aimed to optimize treatment by varying the 8-MOP starting concentration and the cell suspension medium. Materials and Methods All patients (n = 13) included in this study received photopheresis as medically indicated. Cells collected with a Spectra Optia apheresis system were suspended in plasma or physiological saline (NaCl) and incubated with 200 ng/ml versus 340 ng/ml photosensitizer before UVA irradiation (Macogenic G2 or UVA PIT system). Lymphocyte apoptosis and caspase activity were analyzed by flow cytometry and fluorimetry, and residual 8-methoxypsoralen concentrations by liquid chromatography–mass spectrometry. Results Raising the 8-MOP starting concentration significantly increased lymphocyte apoptosis, with values of 22% versus 35% (plasma) and 28%–46% (NaCl) at 24 h post-ECP and 37% versus 86% (plasma) and 74% versus 97% (NaCl) at 48 h for 200 ng/ml versus 340 ng/ml. Pre-transfusion residual 8-MOP levels were 168 ng/ml (plasma) and 162 ng/ml (NaCl) versus 290 ng/ml (plasma) and 266 ng/ml (NaCl) for the lower versus higher dose, respectively. Discussion Hence, 8-MOP concentration influences the efficacy of photopheresis as lymphocyte apoptosis rates were significantly higher with the higher starting concentration and with NaCl versus plasma. This indicates that increased 8-MOP starting doses and saline as additional suspension medium could help in improving ECP's efficacy

Comparison of two column agglutination tests for red blood cell antibody testing.

BackgroundSeveral sensitive methods are available for red blood cell (RBC) antibody screening. Among these, gel and glass card systems have demonstrated comparably good performance in retrospective studies and are widely used in routine patient diagnostics, but their performance in prospective studies has not been sufficiently characterised.Patients and methodsGel card (Bio-Rad DiaMed) and glass bead-based (Ortho Clinical Diagnostics) column agglutination technologies were used to screen for antibodies prospectively (group A) and for antibody identification in stored and fresh samples known to contain RBC antibodies retrospectively (group B). Untreated reagent RBCs and either papain-treated (Bio-Rad) or ficin-treated panel C cells (Ortho) were used for antibody identification.ResultsRBC-reactive antibodies were detected in 22 of 1000 group A samples, three of which tested positive only by gel card agglutination, and four only by glass bead agglutination (including one false positive each). Group B comprised 202 sera with known antibodies: 33 of these samples contained 36 antibodies detected only by gel card agglutination, whereas 9 samples contained antibodies detectable only by glass bead-based agglutination. Discrepancies mostly involved weak antibodies reactive by enzyme only. Two sera contained antibody mixtures that neither system detected completely. Of note, in antibody differentiation batches one and two, anti-Lua was reactive in 7 of 7 and 1 of 8 samples, respectively.ConclusionBoth column agglutination tests for red cell antibodies had equal sensitivity and specificity with unstored samples. In stored samples, weak and enzyme-only antibodies were more frequently detected with the gel card system

Impact of hydroxyethyl starch and modified fluid gelatin on granulocyte phenotype and function

BACKGROUND Patients with neutropenia or granulocyte dysfunction may require granulocyte transfusions for adequate immune restoration. High-molecular-weight hydroxyethyl starch (HES) is the most commonly used sedimentation agent to enhance granulocyte collection efficiency. However, authorities recently restricted the use of HES due to its unfavorable risk-benefit profile. As modified fluid gelatin (MFG) is already used as an alternative sedimentation agent, we tested the hypothesis that MFG is not inferior to HES in terms of the functionality and viability of granulocytes. STUDY DESIGN AND METHODS Granulocytes from ten healthy donors were isolated, aliquoted and incubated in parallel for 2 hours with either 0% (control), 7.5%, 15%, or 30% MFG (Gelafundin) or HES (Hespan), respectively, and granulocyte migration, chemotaxis, reactive oxygen species (ROS) production, neutrophil extracellular trap formation (NETosis), antigen expression, and viability were subsequently investigated in vitro. RESULTS Relative to the controls, all three concentrations of HES compared to only 15% and 30% MFG lowered migration distances, and the 15% and 30% concentrations of both sedimentation agents reduced track straightness. HES resulted in lower CD11b expression and higher CD62L expression compared to MFG and the controls, whereas the differences for CD66b did not reach statistical significance. No significant differences in the timing of ROS production or NETosis, or in neutrophil viability or respiratory burst were observed. CONCLUSION These results indicate that MFG is not inferior to HES in terms of granulocyte function in vitro when used at equal concentrations, and that potential impairment of granulocyte function can occur with HES