Digital polymerase chain reaction to monitor platelet transfusions in cardiac surgery patients.

BACKGROUND AND OBJECTIVES: Corrected count increment (CCI) measurements monitor the effectiveness of platelet transfusions in haemato-oncology, but they usually fail in patients undergoing cardiac surgery. We investigated whether polymerase chain reaction (PCR) of mitochondrial single-nucleotide polymorphisms (SNPs) is able to monitor the survival of transfused platelets in these patients. MATERIALS AND METHODS: Leukocyte-free, platelet-rich plasma was prepared from patients' blood to measure platelet counts based on patient-/donor-specific SNPs by digital PCR after DNA extraction. Platelet counts in samples from patients with severe thrombocytopenia were analysed by both PCR and flow cytometry. Ten patients undergoing cardiac surgery with the use of heart lung machine and without overt bleeding received a single apheresis platelet concentrate because of either dual platelet inhibition during a non-elective intervention or a complex procedure. Blood samples were collected at nine defined intervals (0-120 h) post transfusion. RESULTS: The digital PCR of the seven SNPs reliably quantified levels ≥0.6 G/L platelets, in good agreement with flow cytometry and without interference by other SNPs or by platelet activation. A mean 24-h CCI of 11.8 (range: 5.6-19.8) and a mean 120-h area under the curve (AUC) of 1386 (915-1821) hxG/L were observed for the transfused platelets. The mean AUC of 14,103 (3415-27,305) hxG/L for the patients' endogenous platelets indicates that transfused platelets represented only 11% (5-25) of the total platelet counts during 120 h post transfusion. CONCLUSION: PCR of mitochondrial SNPs offers a tool to assess the survival of platelets from apheresis concentrates in cardiac surgery patients to facilitate the implementation of improved transfusion strategies.

Intensity of endogenous thrombocytopenia after autologous stem cell transplantation in patients prophylactically transfused with platelets.

BACKGROUND AND OBJECTIVES: Large clinical trials have demonstrated that some patient groups with hypoproliferative thrombocytopenia benefit from prophylactic platelet transfusions, while in others, a therapeutic transfusion regimen might be sufficient. The remaining capacity to generate endogenous platelets might be helpful to select the platelet transfusion regimen. We assessed whether the recently described method of digital droplet polymerase chain reaction (PCR) can be used to assess the endogenous platelet levels in two groups of patients undergoing high-dose chemotherapy with autologous stem cell transplantation (ASCT). MATERIALS AND METHODS: Multiple myeloma (n = 22) patients received high-dose melphalan alone (HDMA); lymphoma patients (n = 15) received BEAM or TEAM (B/TEAM) conditioning. Patients with a total platelet count <10 G/L received prophylactic apheresis platelet concentrates. Daily endogenous platelet counts were measured by digital droplet PCR for at least 10 days post-ASCT. RESULTS: Post-transplantation B/TEAM patients received their first platelet transfusion on average 3 days earlier than HDMA patients (p < 0.001) and required about twofold more platelet concentrates (p < 0.001). The endogenous platelet count fell ≤5 G/L for a median of 115 h (91-159; 95% confidence interval) in B/TEAM-treated patients compared to 12.6 h (0-24) (p < 0.0001) in HDMA-treated patients. Multivariate analysis confirmed this profound effect of the high-dose regimen (p < 0.001). The CD-34+ -cell dose in the graft was inversely correlated with the intensity of endogenous thrombocytopenia in B/TEAM-treated patients. CONCLUSION: Monitoring endogenous platelet counts detects the direct effects of myelosuppressive chemotherapies on platelet regeneration. This approach may help to develop a platelet transfusion regimen tailored to specific patient groups.

Long-term outcome in patients with mantle cell lymphoma following high-dose therapy and autologous stem cell transplantation.

BACKGROUND: Long-term clinical and molecular remissions in patients with mantle cell lymphoma (MCL) after autologous stem cell transplantation (ASCT) have been evaluated in only a few studies. DESIGN AND METHODS: Sixty-five patients with MCL received ASCT (54 first-line ASCT, 10 second-line ASCT, and 1 third-line ASCT). In the case of long-term remission (≥5 years; n = 27), peripheral blood was tested for minimal residual disease (MRD) by t(11;14)- and IGH-PCR at the last follow-up. RESULTS: Ten-year overall survival (OS), progression-free survival (PFS), and freedom from progression (FFP) after first-line ASCT were 64%, 52%, and 59% versus after second-line ASCT 50%, 20%, and 20%, respectively. Five-year OS, PFS, and FFP for the first-line cohort were 79%, 63%, and 69%, respectively. Five-year OS, PFS, and FFP after second-line ASCT were 60%, 30%, and 30%, respectively. Treatment-related mortality (3 months after ASCT) was 1.5%. So far 26 patients developed sustained long-term clinical and molecular complete remissions of up to 19 years following ASCT in first treatment line. CONCLUSION: Sustained long-term clinical and molecular remissions are achievable following ASCT.

Long-term remissions in patients with early relapse of diffuse large B-cell lymphoma following high-dose chemotherapy, autologous stem cell transplantation, and radiotherapy of residual disease.

PURPOSE: The prognosis of an early relapse of diffuse large B-cell lymphoma (DLBCL) appears to be poor following autologous stem cell transplantation (ASCT). The aim of this study is to contribute data to the open question on whether additional radiotherapy can improve the outcome. PATIENTS AND METHODS: Forty-eight patients with an early relapse (median 4 months after the end of initial immunochemotherapy, range 1-11) of DLBCL have been treated in our institution with high-dose therapy (usually the BEAM protocol) and ASCT since 2008 (median age 61 years, range 28-73). Twenty-three patients received ASCT in a second treatment line, 25 in a third line (19 refractory to second-line salvage therapy, 5 after second relapse). Fifteen of these 48 patients received radiotherapy (36-50 Gy, median 40) of residual masses after ASCT. RESULTS: Three-year overall survival (OS) and progression-free survival (PFS) after second-line ASCT were 61 and 57%, after third-line ASCT 47 and 44%, respectively, without significant differences. A prognostic factor was the International Prognostic Index (IPI) at the start of salvage therapy. Three-year OS and PFS in low-risk patients were 69 and 69%, in low-intermediate-risk 63 and 53%, and in high-intermediate-risk 23 and 23%, respectively (p = 0.033). Twenty-three patients achieved a sustained complete remission (13-146 months, median 62). CONCLUSION: Sustained long-term remissions can be achieved in patients with early relapse of DLBCL following ASCT in a second or third treatment line, particularly in patients with low- and low-intermediate-risk IPI, following radiotherapy of residual disease after ASCT. Further investigations are required to clarify which patients need an alternative therapy (potentially CAR T­cells or allogeneic transplantation).

New ultraviolet C light-based method for pathogen inactivation of red blood cell units.

BACKGROUND: Pathogen inactivation (PI) technologies for platelet concentrates and plasma are steadily becoming more established, but new PI treatment options for red blood cells (RBCs), the most commonly used blood component, still need to be developed. We present a novel approach to inactivating pathogens in RBC units employing ultraviolet C (UVC) light. METHODS: Whole blood-derived leukoreduced RBCs suspended in PAGGS-C, a third generation additive solution, served as test samples, and RBCs in PAGGS-C or SAG-M as controls. Vigorous agitation and hematocrit reduction by diluting the RBCs with additional additive solution during illumination ensured that UVC light penetrated and inactivated the nine bacteria and eight virus species tested. Bacterial and viral infectivity assays and in vitro analyses were performed to evaluate the system's PI capacity and to measure the RBC quality, metabolic, functional, and blood group serological parameters of UVC-treated versus untreated RBCs during 36-day storage. RESULTS: UVC treatment of RBCs in the PAGGS-C additive solution did not alter RBC antigen expression, but significantly influenced some in vitro parameters. Compared to controls, hemolysis was higher in UVC-treated RBC units, but was still below 0.8% at 36 days of storage. Extracellular potassium increased early after PI treatment and reached ≤70 mmol/L by the end of storage. UVC-treated RBC units had higher glucose and 2,3-diphosphoglycerate levels than controls. CONCLUSION: As UVC irradiation efficiently reduces the infectivity of relevant bacteria and viruses while maintaining the quality of RBCs, the proposed method offers a new approach for PI of RBC concentrates.

Herpes zoster prophylaxis with low-dose acyclovir in patients with malignant lymphoma and multiple myeloma treated with autologous stem cell transplantation.

BACKGROUND: Herpes zoster (HZ) is a frequent complication after autologous stem cell transplantation (ASCT). The option of zoster prophylaxis with an antiviral drug is described in the literature, but there is no consensus on the drug and the dosage. PATIENTS AND METHODS: We analyzed the records of 310 patients treated with ASCT who were controlled regularly regarding HZ inter alia for at least 24 months following ASCT. Since 01/2015 patients received prophylactic low-dose acyclovir (400 mg per day) during the first 12 months following discharge after ASCT (n = 107). RESULTS: Twenty percent of patients without this kind of prophylaxis and 2.8% of patients with prophylaxis developed HZ (p < .001). No patient with this prophylaxis developed HZ in the first year after ASCT, 2.8% of patients in the second year after ASCT. A prognostic factor was the kind of diagnosis: 30% of lymphoma patients and 14% of myeloma patients developed HZ in the first 24 months after ASCT without prophylaxis, but only 6.3% and 0% of patients with prophylaxis, respectively. Neither an increase of HZ cases following prophylaxis nor acyclovir refractory HZ cases were observed. CONCLUSIONS: Zoster prophylaxis with low-dose acyclovir over 12 months after ASCT is effective and well tolerated.

Efficacy of UVC-treated, pathogen-reduced platelets versus untreated platelets: a randomized controlled non-inferiority trial.

Pathogen reduction (PR) technologies for blood components have been established to reduce the residual risk of known and emerging infectious agents. THERAFLEX UVPlatelets, a novel UVC light-based PR technology for platelet concentrates, works without photoactive substances. This randomized, controlled, double-blind, multicenter, noninferiority trial was designed to compare the efficacy and safety of UVC-treated platelets to that of untreated platelets in thrombocytopenic patients with hematologic-oncologic diseases. Primary objective was to determine non-inferiority of UVC-treated platelets, assessed by the 1-hour corrected count increment (CCI) in up to eight per-protocol platelet transfusion episodes. Analysis of the 171 eligible patients showed that the defined non-inferiority margin of 30% of UVC-treated platelets was narrowly missed as the mean differences in 1-hour CCI between standard platelets versus UVC-treated platelets for intention-to-treat and perprotocol analyses were 18.2% (95% confidence interval [CI]: 6.4%; 30.1) and 18.7% (95% CI: 6.3%; 31.1%), respectively. In comparison to the control, the UVC group had a 19.2% lower mean 24-hour CCI and was treated with an about 25% higher number of platelet units, but the average number of days to next platelet transfusion did not differ significantly between both treatment groups. The frequency of low-grade adverse events was slightly higher in the UVC group and the frequencies of refractoriness to platelet transfusion, platelet alloimmunization, severe bleeding events, and red blood cell transfusions were comparable between groups. Our study suggests that transfusion of pathogen-reduced platelets produced with the UVC technology is safe but non-inferiority was not demonstrated. (The German Clinical Trials Register number: DRKS00011156).

Long-term outcome in patients with follicular lymphoma following high-dose therapy and autologous stem cell transplantation.

OBJECTIVE: To contribute data on long-term outcome and potential curative impact of ASCT in FL, especially following HDT with the BEAM protocol (BCNU, etoposide, cytarabine and melphalan), given very limited data on this topic in the literature. PATIENTS AND METHODS: Patients with FL (n = 76) were treated in our institution with HDT and ASCT. In the case of long-term remission (≥8 years), peripheral blood was tested for minimal residual disease by t(14;18)- and IGH-PCR, including the last follow-up. RESULTS: 10-year overall survival, progression-free survival, and freedom from progression (FFP) after first-line ASCT (n = 20) were 80%, 60%, and 69%, after second-line ASCT (n = 48, following BEAM) 66%, 38%, and 41%, after third/fourth-line ASCT (n = 8) 33%, 25%, and 25%, respectively. Prognostic factors for FFP were treatment line and FLIPI (Follicular Lymphoma International Prognostic Index). 10-year FFP for second-line ASCT and low-risk FLIPI at relapse was 69%, intermediate-risk 28%, and high-risk 25% (P < .05). 26 patients developed sustained long-term clinical and molecular remissions of up to 27 years. CONCLUSIONS: Sustained long-term clinical and molecular complete remissions up to 27 years can be achieved following ASCT (including HDT with BEAM in second treatment line), indicating a potential curative impact of ASCT in FL.

A nait-associated and previously unreported mutation in the ITGB3 gene with a low frequency in the local population.

BACKGROUND: Neonatal alloimmune thrombocytopenia is a rare but potentially severe postnatal complication caused by maternal allo-antibodies against platelet antigens of the newborn. In relatively few cases, immunisation against low-frequency antigens has been reported. METHODS: Platelet antigens of a newborn with severe thrombocytopenia and his family members were investigated by serological and molecular biological methods. A real-time PCR assay was developed to reliably detect this mutation in pools of DNA from up to seven individuals. RESULTS: Serological testing showed positive reactions of maternal plasma with paternal platelets but not with conventional platelet donor panels. Sequencing of the ITGB3 gene revealed a G > A polymorphism in position c.1915 of exon 12 for the father, the newborn and three of four paternal relatives. Screening of samples from a local population of 1575 Caucasian blood donors identified only a single individual with this mutation. CONCLUSION: This finding of a previously unreported private platelet antigen demonstrates that the identification of the target glycoprotein by MAIPA assay followed by sequencing of the affected gene can be combined with an efficient population screening by real-time PCR with pooling of DNA samples.

Digital droplet polymerase chain reaction to monitor ultraviolet C treatment of single-donor and buffy coat platelet units.

BACKGROUND: UVC illumination of agitated platelet concentrates (PCs) inactivates pathogens and white blood cells by modifications of their nucleic acids. Related effects on mitochondrial DNA (mtDNA) in platelets serve as a basis for an efficient monitoring suited for routine quality control (QC) of this purely physical pathogen reduction technology. STUDY DESIGN AND METHODS: Samples from PCs (n = 530) were tested with an established LightCycler PCR (LC PCR) for QC of the UVC procedure. RNR2 and TRNK/ATP8 genes were sequenced in the PCs (n = 21) with out-of-specification results in the LC PCR. A digital droplet PCR (ddPCR) was developed to minimize the outliers and cross-validated by testing the 530 PCs. The ddPCR was further evaluated in a subgroup of 300 PCs without mtDNA extraction and in samples from systematic variations of UVC dose and agitation speed. RESULTS: Apheresis PCs (n = 380) resulted in 5.3% outliers in LC PCR versus only 0.7% in buffy coat pool PCs (n = 150). Sequencing of these outliers revealed single-nucleotide polymorphisms in the primer- and probe-binding sites of LC PCR. The development of a ddPCR assay with modified probe sequences reduced the outliers to 0.4%. The ddPCR analysis of PCs both with and without mtDNA extraction demonstrated low intra- and interassay variabilities and congruent results also compared to LC PCR. Experiments varying the UVC dose and the agitation speed demonstrated that the ddPCR results closely reflect functional effects of the UVC treatment. CONCLUSION: The ddPCR assay offers a valid and reliable tool for QC of routine production of the UVC-treated PCs as well as for monitoring treatment conditions during optimization of the UVC procedure.

Inactivation of three emerging viruses - severe acute respiratory syndrome coronavirus, Crimean-Congo haemorrhagic fever virus and Nipah virus - in platelet concentrates by ultraviolet C light and in plasma by methylene blue plus visible light.

BACKGROUND: Emerging viruses like severe acute respiratory syndrome coronavirus (SARS-CoV), Crimean-Congo haemorrhagic fever virus (CCHFV) and Nipah virus (NiV) have been identified to pose a potential threat to transfusion safety. In this study, the ability of the THERAFLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation systems to inactivate these viruses in platelet concentrates and plasma, respectively, was investigated. MATERIALS AND METHODS: Blood products were spiked with SARS-CoV, CCHFV or NiV, and then treated with increasing doses of UVC light (THERAFLEX UV-Platelets) or with methylene blue (MB) plus increasing doses of visible light (MB/light; THERAFLEX MB-Plasma). Samples were taken before and after treatment with each illumination dose and tested for residual infectivity. RESULTS: Treatment with half to three-fourths of the full UVC dose (0·2 J/cm2 ) reduced the infectivity of SARS-CoV (≥3·4 log), CCHFV (≥2·2 log) and NiV (≥4·3 log) to the limit of detection (LOD) in platelet concentrates, and treatment with MB and a fourth of the full light dose (120 J/cm2 ) decreased that of SARS-CoV (≥3·1 log), CCHFV (≥3·2 log) and NiV (≥2·7 log) to the LOD in plasma. CONCLUSION: Our study demonstrates that both THERAFLEX UV-Platelets (UVC) and THERAFLEX MB-Plasma (MB/light) effectively reduce the infectivity of SARS-CoV, CCHFV and NiV in platelet concentrates and plasma, respectively.

Hepatitis E virus is effectively inactivated in platelet concentrates by ultraviolet C light.

BACKGROUND AND OBJECTIVES: As previous investigations have shown, THERAFLEX UV-Platelets, a UVC-based pathogen inactivation (PI) system, is effective against non-enveloped transfusion-relevant viruses such as hepatitis A virus (HAV), which are insensitive to most PI treatments for blood products. This study investigated the PI efficacy of THERAFLEX UV-Platelets against HEV in platelet concentrates (PCs). MATERIALS AND METHODS: Buffy coat-derived PCs in additive solution were spiked with cell culture-derived HEV and treated with the THERAFLEX UV-Platelets system using various doses of UVC (0·05, 0·10, 0·15 and 0·20 (standard) J/cm2 ). Titres of infectious virus in pre- and post-treatment samples were determined using a large-volume plating assay to improve the detection limit of the virus assay. RESULTS: THERAFLEX UV-Platelets dose-dependently inactivated HEV in PCs. The standard UVC dose inactivated the virus to below the limit of detection, corresponding to a mean log reduction of greater than 3·5. CONCLUSION: Our study demonstrates that the THERAFLEX UV-Platelets system effectively inactivates HEV in PCs.

Bacterial inactivation of platelet concentrates with the THERAFLEX UV-Platelets pathogen inactivation system.

BACKGROUND: The THERAFLEX UV-Platelets system (Maco Pharma) uses ultraviolet C (UVC) light for pathogen inactivation (PI) of platelet concentrates (PCs) without any additional photoactive compound. The aim of the study was to systematically investigate bacterial inactivation with this system under conditions of intended use. STUDY DESIGN AND METHODS: The robustness of the system was evaluated by assessing its capacity to inactivate high concentrations of different bacterial species in accordance with World Health Organization guidelines. The optimal use of the PI system was explored in time-to-treatment experiments by testing its ability to sterilize PCs contaminated with low levels of bacteria on the day of manufacture (target concentration, 100 colony-forming units/unit). The bacteria panel used for spiking experiments in this study included the World Health Organization International Repository Platelet Transfusion Relevant Reference Strains (n = 14), commercially available strains (n = 13), and in-house clinical isolates (n = 2). RESULTS: Mean log reduction factors after UVC treatment ranged from 3.1 to 7.5 and varied between different strains of the same species. All PCs (n = 12/species) spiked with up to 200 colony-forming units/bag remained sterile until the end of storage when UVC treated 6 hours after spiking. UVC treatment 8 hours after spiking resulted in single breakthrough contaminations with the fast-growing species Escherichia coli and Streptococcus pyogenes. CONCLUSION: The UVC-based THERAFLEX UV-Platelets system efficiently inactivates transfusion-relevant bacterial species in PCs. The comprehensive data from this study may provide a valuable basis for the optimal use of this UVC-based PI system.

Ultraviolet C light efficiently inactivates nonenveloped hepatitis A virus and feline calicivirus in platelet concentrates.

BACKGROUND: Nonenveloped transfusion-transmissible viruses such as hepatitis A virus (HAV) and hepatitis E virus (HEV) are resistant to many of the common virus inactivation procedures for blood products. This study investigated the pathogen inactivation (PI) efficacy of the THERAFLEX UV-Platelets system against two nonenveloped viruses: HAV and feline calicivirus (FCV), in platelet concentrates (PCs). STUDY DESIGN AND METHODS: PCs in additive solution were spiked with high titers of cell culture-derived HAV and FCV, and treated with ultraviolet C at various doses. Pre- and posttreatment samples were taken and the level of viral infectivity determined at each dose. For some samples, large-volume plating was performed to improve the detection limit of the virus assay. RESULTS: THERAFLEX UV-Platelets reduced HAV titers in PCs to the limit of detection, resulting in a virus reduction factor of greater than 4.2 log steps, and reduced FCV infectivity in PCs by 3.0 ± 0.2 log steps. CONCLUSIONS: THERAFLEX UV-Platelets effectively inactivates HAV and FCV in platelet units.

Mitochondrial DNA multiplex real-time polymerase chain reaction inhibition assay for quality control of pathogen inactivation by ultraviolet C light in platelet concentrates.

BACKGROUND: Several ultraviolet (UV) light-based pathogen inactivation (PI) technologies for platelet (PLT) products have been developed or are under development. Upon implementation of PI technologies, quality control measures are required to ensure consistent efficiency of the treatment process. Previous reports showed that amotosalen/UVA and riboflavin/UV-based PI technologies induce modifications of the PLT-derived mitochondrial DNA (mtDNA) that can be detected by polymerase chain reaction (PCR) inhibition assays. In this study, we sought to establish a PCR inhibition assay to document the impact of ultraviolet C (UVC) treatment with the THERAFLEX UV-Platelets system on the mitochondrial genome in PLT concentrates (PCs). STUDY DESIGN AND METHODS: A multiplex real-time PCR inhibition assay with simultaneous short-amplicon (143 bp) and long-amplicon (794 bp) amplification was developed to detect mtDNA modifications in PLTs after UVC treatment. Assay performance was tested in UVC-treated and untreated, plasma-reduced pooled PCs, and apheresis PCs and challenged using PCs manufactured for a clinical trial under routine-like conditions. RESULTS: UVC illumination of PLTs resulted in dose-dependent inhibition of mtDNA amplification for the larger amplicon. Amplification of the shorter amplicon was not affected by UVC treatment. Evaluation of 283 blinded apheresis and pooled PLT samples from routine-like PC production resulted in prediction of UVC treatment status with 100% accuracy. CONCLUSION: The proposed dual-amplicon size real-time mtDNA PCR assay effectively detects nucleic acid damage induced by UVC illumination of PLTs and could be useful as an informative indicator of PI quality of the THERAFLEX UV-Platelets system.

Inactivation of Ebola virus and Middle East respiratory syndrome coronavirus in platelet concentrates and plasma by ultraviolet C light and methylene blue plus visible light, respectively.

BACKGROUND: Ebola virus (EBOV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have been identified as potential threats to blood safety. This study investigated the efficacy of the THERAFLEX UV-Platelets and THERAFLEX MB-Plasma pathogen inactivation systems to inactivate EBOV and MERS-CoV in platelet concentrates (PCs) and plasma, respectively. STUDY DESIGN AND METHODS: PCs and plasma were spiked with high titers of cell culture-derived EBOV and MERS-CoV, treated with various light doses of ultraviolet C (UVC; THERAFLEX UV-Platelets) or methylene blue (MB) plus visible light (MB/light; THERAFLEX MB-Plasma), and assessed for residual viral infectivity. RESULTS: UVC reduced EBOV (≥4.5 log) and MERS-CoV (≥3.7 log) infectivity in PCs to the limit of detection, and MB/light decreased EBOV (≥4.6 log) and MERS-CoV (≥3.3 log) titers in plasma to nondetectable levels. CONCLUSIONS: Both THERAFLEX UV-Platelets (UVC) and THERAFLEX MB-Plasma (MB/light) effectively reduce EBOV and MERS-CoV infectivity in platelets and plasma, respectively.

Extended Donor Typing by Pooled Capillary Electrophoresis: Impact in a Routine Setting.

BACKGROUND: PCR with sequence-specific priming using allele-specific fluorescent primers and analysis on a capillary sequencer is a standard technique for DNA typing. We aimed to adapt this method for donor typing in a medium-throughput setting. METHODS: Using the Extract-N-Amp PCR system, we devised a set of eight multiplex allele-specific PCR with fluorescent primers for Fya/Fyb, Jka/Jkb, M/N, and S/s. The alleles of a gene were discriminated by the fluorescent color; donor and polymorphism investigated were encoded by product length. Time, cost, and routine performance were collated. Discrepant samples were investigated by sequencing. The association of new alleles with the phenotype was evaluated by a step-wise statistical analysis. RESULTS: On validation using 312 samples, for 1.1% of antigens (in 5.4% of samples) no prediction was obtained. 99.96% of predictions were correct. Consumable cost per donor were below EUR 2.00. In routine use, 92.2% of samples were successfully predicted. Discrepancies were most frequently due to technical reasons. A total of 11 previously unknown alleles were detected in the Kell, Lutheran, and Colton blood group systems. In 2017, more than 20% of the RBC units prepared by our institution were from donors with predicted antigen status. A steady supply of Yt(a-), Co(a-) and Lu(b-) RBC units was ensured. CONCLUSIONS: Pooled capillary electrophoresis offers a suitable alternative to other methods for extended donor DNA typing. Establishing a large cohort of typed donors improved transfusion support for patients.

Platelet recovery and survival measured in patients by quantitative polymerase chain reaction of mitochondrial DNA.

BACKGROUND: Mitochondrial (mt) DNA markers have been identified as potential targets for the quantification of endogenous and allogeneic platelets (PLTs) in the blood of individuals who received transfusions. Our goal was to develop a routine polymerase chain reaction (PCR) assay for ex vivo monitoring of PLT survival in patients after transfusion. STUDY DESIGN AND METHODS: Targets were selected for real-time (RT)-PCR of mt DNA based on the frequency distribution of nucleotide polymorphisms and assay sensitivity in vitro. The assays were then evaluated with ex vivo samples to measure PLT survival and recovery of therapeutic doses of apheresis PLTs in hematooncologic patients with thrombocytopenia. RESULTS: Nucleotides in two positions (73/310 hypervariable region [HVR] 2) and three positions (295 HVR 2, 16069/16311 HVR 1) had allele frequencies of approximately 0.5 and 0.85, respectively, in a population of 960 Caucasian PLT donors. They provided targets for sensitive assays detecting at least 1 × 10(3) PLTs per whole blood sample with adequate reproducibility (interassay coefficient of variation <4.0%). Transfusions of single-donor PLT concentrates in patients with thrombocytopenia (n = 30) were monitored with these markers. The mean 24-hour corrected count increment was 8.3 and the mean calculated survival time was 3.3 days. Results for a second marker were available for 13 transfusions. The survival time values derived from both markers for the same transfusion were almost identical (linear regression: r(2) = 0.957, slope = 0.87). CONCLUSION: This RT-PCR method detects mt DNA polymorphisms in Caucasians for a highly sensitive and reproducible quantification of endogenous and allogeneic PLT numbers in blood samples from transfused patients with thrombocytopenia.

Pathogen reduction by ultraviolet C light effectively inactivates human white blood cells in platelet products.

BACKGROUND: Residual white blood cells (WBCs) in cellular blood components induce a variety of adverse immune events, including nonhemolytic febrile transfusion reactions, alloimmunization to HLA antigens, and transfusion-associated graft-versus-host disease (TA-GVHD). Pathogen reduction (PR) methods such as the ultraviolet C (UVC) light-based THERAFLEX UV-Platelets system were developed to reduce the risk of transfusion-transmitted infection. As UVC light targets nucleic acids, it interferes with the replication of both pathogens and WBCs. This preclinical study aimed to evaluate the ability of UVC light to inactivate contaminating WBCs in platelet concentrates (PCs). STUDY DESIGN AND METHODS: The in vitro and in vivo function of WBCs from UVC-treated PCs was compared to that of WBCs from gamma-irradiated and untreated PCs by measuring cell viability, proliferation, cytokine secretion, antigen presentation in vitro, and xenogeneic GVHD responses in a humanized mouse model. RESULTS: UVC light was at least as effective as gamma irradiation in preventing GVHD in the mouse model. It was more effective in suppressing T-cell proliferation (>5-log reduction in the limiting dilution assay), cytokine secretion, and antigen presentation than gamma irradiation. CONCLUSIONS: The THERAFLEX UV-Platelets (MacoPharma) PR system can substitute gamma irradiation for TA-GVHD prophylaxis in platelet (PLT) transfusion. Moreover, UVC treatment achieves suppression of antigen presentation and inhibition of cytokine accumulation during storage of PCs, which has potential benefits for transfusion recipients.

In vitro variables of buffy coat-derived platelet concentrates with residual plasma of down to 10% are stably maintained in new-generation platelet additive solutions.

BACKGROUND: Platelet additive solutions (PASs) facilitate plasma recovery and may reduce the risk of plasma-associated adverse transfusion reactions. Whereas current apheresis techniques are able to produce platelet concentrates (PCs) with reduced residual plasma volumes, it is still technically challenging to prepare PCs with plasma levels less than 20% from whole blood. This study aimed to evaluate the feasibility of producing buffy coat (BC)-derived platelets (PLTs) with as little as 10% residual plasma and to test the ability of PASs to preserve PLT quality under these conditions. STUDY DESIGN AND METHODS: A pool-and-split design was used to evaluate the in vitro quality of semiautomatically prepared BC-derived PCs stored for 7 days in either SSP+ (PAS-IIIM) or Intersol-G (glucose-containing PAS) with 10% to 30% residual plasma in three phases: 1) 30% plasma (SSP+, Intersol-G), 2) 20 and 15% plasma (SSP+, Intersol-G), and 3) 10% plasma (Intersol-G). RESULTS: The different plasma-reduced PAS-PC types were comparable in volume, PLT concentration, and PLT yield and met all regulatory requirements. The in vitro quality variables of PLTs suspended in 20% or less residual plasma were comparable to those of PLTs stored in 30% plasma for both PAS types. PLT activation was slightly higher with SSP+ than with Intersol-G. The quality of PCs suspended in Intersol-G with 10% plasma was well maintained during storage. CONCLUSIONS: Preparation of BC-derived PCs with minimal plasma concentrations as low as 10% is feasible. Late-generation PASs satisfactorily preserve the in vitro quality of such PCs during storage.