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Antigen rapid diagnostic tests (RDTs) for SARS coronavirus 2 (SARS-CoV-2) are quick, widely available, and inexpensive. Consequently, RDTs have been established as an alternative and additional diagnostic strategy to quantitative reverse transcription polymerase chain reaction (RT-qPCR). However, reliable clinical and large-scale performance data specific to a SARS-CoV-2 virus variant of concern (VOC) are limited, especially for the Omicron VOC. The aim of this study was to compare RDT performance among different VOCs.This single-centre prospective performance assessment compared RDTs from three manufacturers (NADAL, Panbio, MEDsan) with RT-qPCR including deduced standardized viral load from oropharyngeal swabs for detection of SARS-CoV-2 in a clinical point-of-care setting from November 2020 to January 2022.Among 35 479 RDT/RT-qPCR tandems taken from 26 940 individuals, 164 of the 426 SARS-CoV-2 positive samples tested true positive with an RDT corresponding to an RDT sensitivity of 38.50% (95% CI, 34.00-43.20%), with an overall specificity of 99.67% (95% CI, 99.60-99.72%). RDT sensitivity depended on viral load, with decreasing sensitivity accompanied by descending viral load. VOC-dependent sensitivity assessment showed a sensitivity of 42.86% (95% CI, 32.82-53.52%) for the wild-type SARS-CoV-2, 43.42% (95% CI, 32.86-54.61%) for the Alpha VOC, 37.67% (95% CI, 30.22-45.75%) for the Delta VOC, and 33.67% (95% CI, 25.09-43.49%) for the Omicron VOC. Sensitivity in samples with high viral loads of ≥10RDT sensitivity for detection of the Omicron VOC is reduced in individuals infected with a high viral load, which curtails the effectiveness of RDTs. This aspect furthert: limits the use of RDTs, although RDTs are still an irreplaceable diagnostic tool for rapid, economic point-of-care and extensive SARS-CoV-2 screening.

Erythrocytes undergo a well-defined switch from fetal to postnatal circulation, mainly reflected by stage-specific expression of hemoglobin chains. Perinatal alterations of thrombopoiesis remain poorly understood. We assessed the ontogenesis of platelet phenotype and function from early prematurity to adults. We recruited 64 subjects comprising 7 extremely preterm (27-31 weeks gestational age), 25 moderately preterm (32-36 weeks) and 10 term neonates, 8 infants (13a). Blood was withdrawn at up to 3 different time points in neonates (t1: 0-2, t2: 3-7, and t3: 8-14 days after birth). We found that expression levels of the major surface receptors for fibrinogen, collagen, vWF, fibronectin, and laminin were reduced, but correlated with decreased platelet size indicating a normal surface density. While CD62P and CD63 surface exposure upon stimulation with TRAP-6, ADP or U46619 was unaltered or just slightly reduced in neonates, GPIIb/IIIa inside-out and outside-in activation was blunted, but showed a continuous increase until adulthood, correlating with expression of the GPIIb/IIIa regulating tetraspanin CD151. Platelet subpopulation analysis by automated clustering revealed that neonates presented with a CD63+/PAC1- pattern, followed by a continuous increase of CD63+/PAC-1+ platelets until adulthood. Our findings reveal that the number of platelet-monocyte and -neutrophil aggregates, but not platelet-lymphocyte aggregates is increased in neonates and that neonatal aggregate formation depends in part on CD62P activation. Our PLatelets In Neonatal Infants Study (PLINIUS) provides several lines of evidence that platelet phenotype and function evolve continuously from neonates until adulthood.

BackgroundRapid antigen detection tests (RDT) are an easily accessible, feasible, inexpensive, and point-of-care method in SARS-CoV-2 diagnostics – established in adults as well as in children and adolescents. Despite this, large-scale data of clinical performance in the paediatric population especially regarding the influence of SARS-CoV-2 virus variants of concern (VOC) and COVID-19 vaccination on test accuracy is rare.MethodsThis single-centre prospective diagnostic study evaluates three RDT (NADAL®, Panbio™, MEDsan®) in comparison to quantitative reverse transcription polymerase chain reaction (RT-qPCR). 9,760 oropharyngeal screening samples regarding SARS-CoV-2 VOC and COVID-19 vaccination in paediatric hospitalised patients aged younger than 18 years were enrolled.FindingsRDT sensitivity was 44·7% (157/351, 95% CI 39·6%–50·0%) compared to the reference standard RT-qPCR, specificity 99·8% (9,392/9,409, 95% CI 99·7%–99·9%). Most SARS-CoV-2 infections considered were caused by Omicron VOC. Diagnostic accuracy of RDT depended on specimen containing viral load with a decreasing RDT sensitivity by descending viral load, corresponding with a significantly impaired sensitivity in asymptomatic children. A sensitivity of 71·0% was obtained for a viral load higher than 106SARS-CoV-2 RNA copies per ml suggested as infectivity threshold. No significant differences in RDT sensitivity could be observed regarding gender, symptoms, COVID-19 vaccination status, and VOC.InterpretationIn a paediatric population, RDT have proven to reliably detect potentially highly infectious patients with a viral load of at least 106SARS-CoV-2 RNA copies per ml. Due to the low sensitivity in asymptomatic individuals, the usefulness of RDT seems limited in large-scale SARS-CoV-2 screening programs.FundingFederal Ministry for Education and Science (BMBF), Free State of Bavaria

Background: Hereditary pyruvate kinase (PK) deficiency results in lifelong hemolytic anemia and several significant comorbidities, the epidemiology of which are not well characterized. Among these is reduced bone mineral density (BMD), which can result in premature osteopenia, osteoporosis, and fractures. To better characterize the bone density abnormalities in patients with PK deficiency, this study evaluated pooled pre-treatment baseline data from 3 clinical trials involving patients with PK deficiency investigating mitapivat, an allosteric activator of PK: DRIVE-PK (NCT02476916), ACTIVATE (NCT03548220), and ACTIVATE-T (NCT03559699). This is the first large PK deficiency cohort in which dual-energy x-ray absorptiometry (DXA) scores were systematically and consistently assessed. Methods: DRIVE-PK is a completed phase 2, global, randomized, open-label study. ACTIVATE is an ongoing phase 3, global, randomized, double-blind, placebo-controlled study. ACTIVATE-T is an ongoing phase 3, global, open-label, single-arm study. In all 3 studies, patients ≥ 18 years of age with a confirmed diagnosis of PK deficiency were eligible to participate. Patients were eligible to participate in DRIVE-PK and in ACTIVATE if they were not regularly transfused (DRIVE-PK: ≤ 3 units of red blood cells in the prior 12 months; no transfusions in the prior 4 months; ACTIVATE: ≤ 4 transfusion episodes in the previous year; no transfusions in the prior 3 months) and in ACTIVATE-T if they were regularly transfused (≥ 6 transfusion episodes in the previous year). BMD was measured using DXA scans at baseline; scans were obtained locally for all 3 studies. Scans were interpreted locally for DRIVE-PK and centrally for ACTIVATE and ACTIVATE-T. Osteopenia and osteoporosis were identified on DXA scanning according to standard definitions, and the prevalence of each was compared to the prevalence ascertained via medical history. Results: Full demographics and characteristics of patients at baseline are shown in the Table. Of 159 patients evaluated (DRIVE-PK, n = 52; ACTIVATE, n = 80; ACTIVATE-T, n = 27), the median age was 34 years (range, 18-78) and the majority were female (n = 88; 55.3%). Of 155 patients who had baseline T-scores for total femur, spine, and femoral neck, 38 (24.5%) had a T-score of ≥ -1.0 at all locations, indicating normal BMD; 91 (58.7%) had a T-score of < -1.0 to > -2.5 at 1 or more locations, indicating osteopenia; and 26 (16.8%) had a T-score of ≤ -2.5 at 1 or more locations, indicating osteoporosis. The proportion of patients in each T-score range for each of the 3 locations is shown in the Figure. In contrast to the DXA scan findings, only 28 (17.6%) patients had a known medical history of osteopenia and 23 (14.5%) had a known medical history of osteoporosis. Taking together DXA scan results and medical history for all 159 patients, 85 patients (53.5%) had osteopenia and 33 patients (20.8%) had osteoporosis. The median age for patients with either osteopenia or osteoporosis (n = 118) was 36 years (range, 18-78). Of these, 20 patients (16.9%) were regularly transfused and 98 patients (83.1%) were not regularly transfused. Conclusions: In this large cohort, universal DXA scanning revealed that over three-quarters of adults with PK deficiency had osteopenia or osteoporosis, irrespective of transfusion requirements. Given the young median age of the cohort (34 years), these findings have considerable significance and implications for the screening and care of patients with PK deficiency throughout their adult lives. Early monitoring of these patients with DXA scans in order to ensure a prompt diagnosis of bone density abnormalities and indicated treatment may be warranted. Disclosures Al-Samkari: Argenx: Consultancy; Agios: Consultancy, Research Funding; Dova: Consultancy, Research Funding; Rigel: Consultancy; Amgen: Research Funding. Grace:Novartis: Research Funding; Agios: Research Funding; Dova: Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding. Glenthoej:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bluebird: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexicon: Research Funding; Novo Nordisk: Honoraria. Barcellini:Agios: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: invited speaker , Research Funding; Novartis: Honoraria, Other: invited speaker , Research Funding; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Galactéros:Addmedica: Membership on an entity's Board of Directors or advisory committees. Kuo:Bluebird Bio: Consultancy; Novartis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Apellis: Consultancy; Celgene: Consultancy; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Pfizer: Consultancy, Research Funding. Layton:Cerus: Membership on an entity's Board of Directors or advisory committees; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Morado:Sanofi Genzyme: Honoraria, Other: Grants. Viprakasit:BMS, Novartis: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios Pharmaceuticals, Ionis Pharmaceuticals, La Jolla Pharmaceuticals, Protagonist Therapeutics, Vifor Pharma: Consultancy, Research Funding. Dong:Agios Pharmaceuticals: Current Employment, Current equity holder in private company. Tai:Agios Pharmaceuticals: Current Employment, Current equity holder in private company. Hawkins:Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company; Bristol-Myers Squibb: Current equity holder in publicly-traded company; Infinity Pharmaceuticals: Current equity holder in publicly-traded company; Jazz Pharmaceuticals: Current equity holder in publicly-traded company. Gheuens:Agios Pharmaceuticals: Current Employment, Current equity holder in private company. Bowden:Agios Pharmaceuticals: Current Employment, Current equity holder in private company. Porter:Silence Therapeutics: Honoraria; La Jolla Pharmaceuticals: Honoraria; Vifor Pharmaceuticals: Honoraria; Protagonist Therapeutics: Honoraria; Agios Pharmaceuticals: Consultancy, Honoraria; bluebird bio, Inc.: Consultancy, Honoraria; BMS: Consultancy, Honoraria. van Beers:Novartis: Research Funding; Pfizer: Research Funding; RR mechatronics: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees, Research Funding.

BackgroundAntigen rapid diagnostic tests (RDT) for SARS-CoV-2 are fast, broadly available, and inexpensive. Despite this, reliable clinical performance data is sparse.MethodsIn a prospective performance evaluation study, RDT from three manufacturers (NADAL®, Panbio™, MEDsan®) were compared to quantitative reverse transcription polymerase chain reaction (RT-qPCR) in 5 068 oropharyngeal swabs for detection of SARS-CoV-2 in a hospital setting. Viral load was derived from standardized RT-qPCR Cycle threshold (Ct) values. The data collection period ranged from November 12, 2020 to February 28, 2021.FindingsOverall, sensitivity of RDT compared to RT-qPCR was 42·57% (95% CI 33·38%–52·31%), and specificity 99·68% (95% CI 99·48%–99·80%). Sensitivity declined with decreasing viral load from 100% in samples with a deduced viral load of ≥108SARS-CoV-2 RNA copies per ml to 8·82% in samples with a viral load lower than 104SARS-CoV-2 RNA copies per ml. No significant differences in sensitivity or specificity could be observed between the three manufacturers, or between samples with and without spike protein variant B.1.1.7. The NPV in the study cohort was 98·84%; the PPV in persons with typical COVID-19 symptoms was 97·37%, and 28·57% in persons without or with atypical symptoms.InterpretationRDT are a reliable method to diagnose SARS-CoV-2 infection in persons with high viral load. RDT are a valuable addition to RT-qPCR testing, as they reliably detect infectious persons with high viral loads before RT-qPCR results are available.FundingGerman Federal Ministry for Education and Science (BMBF), Free State of BavariaResearch in contextEvidence before this studyWe searched PubMED an MedRxiv for articles including “COVID-19”, “COVID”, “SARS-CoV-2”, “coronavirus” as well as “antigen detection”, “rapid antigen test”, “Point-of-Care test” in title or abstract, published between January 1, 2020 and February 28, 2021. The more than 150 RDT on the market at the end of February 2021 represent a huge expansion of diagnostic possibilities.1Performance of currently available RDT is evaluated in several international studies, with heterogeneous results. Sensitivity values of RDT range from 0·0%2to 98·3%3, specificity from 19·4%4to 100·0%.2,5–14. Some of this data differs greatly from manufacturers’ data. However, these previously published performance evaluation studies were conducted under laboratory conditions using frozen swabs, or in small cohorts with middle-aged participants. Comparable RDT performance data from large-scale clinical usage is missing.5–19Added value of this studyBased on previous examinations the real life opportunities and limitations of SARS-CoV-2 RDT as an instrument of hospital infection detection and control are still unclear as well as further study results are limited in transferability to general public. Our findings show that RDT performance in daily clinical routine is reliable in persons with high viral for punctual detection and isolation of infectious persons before RT-qPCR become available. In persons with lower viral load, or in case of asymptomatic patients SARS-CoV2 detection by RDT was unsuccessful. The general sensitivity of 42·57% is too low to accept the RDT in clinical use as an alternative to RT-qPCR in diagnosis of COVID-19. Calculated specificity was 99.68%. The results are based on a huge study cohort with more than 5 000 participants including a representative ages structure with pediatric patients up to geriatric individuals, which portrays approximately the demographic structure of the local society.Implications of all the available evidenceDue to the low general sensitivity RDT in clinical use cannot be accepted as an alternative but as an addition to RT-qPCR in SARS-CoV-2 diagnosis. The benefit of early detection of highly infectious persons has to be seen in context of the effort of testing and isolation of false positive tested persons.

Der Pyruvatkinase(PK)-Mangel der Erythrozyten wird autosomal-rezessiv vererbt und durch Mutationen im PKLR-Gen verursacht. Das Gen kodiert fur das letzte Enzym der anaeroben Glykolyse der Erythrozyten, die Pyruvatkinase (PK-R [Pyruvatkinase-R-Isoform]). Die Folge ist eine nichtautoimmunvermittelte, nichtspharozytare hamolytische Anamie. Dieser Beitrag beleuchtet die klinischen, laborchemischen und molekulargenetischen Charakteristika von Patienten mit PK-Mangel in Deutschland. Mithilfe von standardisierten Fragebogen wurden von den 28 deutschen Patienten mit PK-Mangel, die alle an der PKD Natural History Study teilnahmen, retrospektiv Daten erhoben. Die Patienten wurden auserdem prospektiv uber einen Zeitraum von 2 Jahren beobachtet. Besondere Schwerpunkte wurden auf die Genotyp-Phanotyp-Korrelation sowie die Analyse der Lebensqualitat der Patienten mit PK-Mangel gelegt. Ziel dieses Beitrags ist es, die vielfaltigen klinischen und molekulargenetischen Eigenschaften dieser seltenen Erkrankung darzustellen, um durch eine erhohte Wachsamkeit die Erkrankung fruher zu diagnostizieren und adaquat zu behandeln.

Storage pool disease (SPD) covers a group of platelet defects in which α- and/or delta-granules are reduced or cannot be secreted adequately in response to agonists. The detection of delta-granule release defects is hampered by a lack of fast and feasible tests. We aimed to implement a flow cytometry–based kinetic mepacrine assay to better identify and subgroup childhood patients with a mild to moderate bleeding diathesis and compare our method to established laboratory tests. We analysed 50 children with suspected SPD whose initial parameters were re-assessed in a second site visit. Mepacrine uptake and release patterns were correlated with CD63 exposure, platelet ADP/ATP release and content, and the bleeding score ascertained by the ISTH-BAT. Mepacrine release was overall significantly reduced in investigated patients compared with controls. Summarizing, our time-resolved approach proved to be a quick and inexpensive tool that was additionally able to distinguish between mepacrine uptake, mepacrine release, and combined defects. Classification of patients using such a kinetic assay makes it feasible to sensitively detect frequently missed SPD and to group these patients for further analyses and clinical correlations.

Background: Hereditary pyruvate kinase (PK) deficiency results in chronic hemolysis that may lead to reduced bone mineral density (BMD). Mitapivat (AG-348) is an investigational, first-in-class, allosteric activator of PKR that has been shown to improve hemoglobin and other hemolysis markers in non-regularly transfused patients (pts) and reduce transfusion burden in regularly transfused pts. Mitapivat has mild aromatase inhibition effects; however, a recent study of 30 non-regularly transfused pts with PK deficiency showed that mitapivat did not appear to promote progression of BMD abnormalities for up to 56 months (mos). The large-pooled analysis presented here assessed BMD over time in a broader PK deficiency population including both non-regularly transfused and regularly transfused pts receiving long-term treatment with mitapivat. Methods: This study included pts enrolled in DRIVE-PK (NCT02476916), ACTIVATE (NCT03548220), ACTIVATE-T (NCT03559699), and the long-term extension (LTE) study (NCT03853798). All trials included pts ≥ 18 years (yrs) with a confirmed diagnosis of PK deficiency treated with mitapivat. DRIVE-PK (N = 52) was a Phase (Ph.) 2, global, randomized open-label study. ACTIVATE (N = 80) was a Ph. 3, randomized, double-blind, placebo-controlled study. ACTIVATE-T (N = 27) was a Ph. 3, open-label, single-arm study. Pts in DRIVE-PK and ACTIVATE were not regularly transfused, defined as ≤ 3 and ≤ 4 units of red blood cells in the prior 12 mos, and no transfusions in the prior 4 and 3 mos, respectively. Pts in ACTIVATE-T were regularly transfused, defined as ≥ 6 transfusion episodes in the prior 12 mos. Data from 90 pts who received mitapivat for > 12 mos were pooled from these 3 trials along with the LTE study of pts from ACTIVATE and ACTIVATE-T who elected to continue or crossover (if on placebo) to mitapivat. BMD changes over time were measured using dual-energy X-ray absorptiometry (DXA) scans at 3 locations (total femur [combined femoral neck and total hip], femoral neck, and spine). Based on DXA T-scores, pts were classified according to standard definitions as having normal BMD (≥ -1.0 at all locations), osteopenia (> -2.5 to < -1.0 at ≥ 1 locations), or osteoporosis (≤ -2.5 at ≥ 1 locations). DXA scans were obtained locally for all 3 studies and interpreted locally for DRIVE-PK and centrally for ACTIVATE, ACTIVATE-T, and LTE. Scans were collected at baseline (BL), between week (wk) 24 and 28, every 6 mos through mo 30, and then annually for DRIVE-PK; at BL and wk 24 for ACTIVATE; at BL, wk 16, and wk 40 for ACTIVATE-T; and at BL, every 24 wks until wk 96, and then every 48 wks for the LTE study. Results: Of 90 pts assessed (median age 34.5 yrs [range 18-71]; 50.0% female), median exposure to mitapivat was 20 mos (range 12.2-64.9). Eight pts (8.9%) had concomitant use of bisphosphonate medications, 8 pts (8.9%) had a prior medical history of fracture, and 6 pts (6.7%) reported fractures or joint injuries during the study period. BL BMD T-scores were collected for 88 pts. At BL, 24 pts (26.7%) had a T-score ≥ -1.0 (normal BMD), 48 pts (53.3%) had a worst T-score > -2.5 to < -1.0 (osteopenia), 16 pts (17.8%) had a worst T-score ≤ -2.5 (osteoporosis), and 2 pts (2.2%) were missing T-score data. At last BMD assessment (data cut-off 03Mar2021), 76 of 88 pts (86.4%) remained stable, 7 pts (7.5%) improved, and 5 pts (5.7%) worsened (Figure 1a). As of the last assessment, 27 (30.0%), 46 (51.1%), and 17 (18.9%) of 90 pts had a T-score indicating normal BMD, osteopenia, and osteoporosis, respectively (Table 1b). Among 24 pts with BL T-scores ≥ -1.0, 21 pts remained stable, and 3 pts showed worsening BMD. Of 48 pts with a BL T-score > -2.5 to < -1.0, 6 pts improved to a T-score indicating normal BMD, 40 pts remained stable, and 2 pts worsened. Of 16 pts with a BL T-score of ≤ -2.5, 1 pt improved to a T-score of > -2.5 to < -1.0. Conclusions: This large-pooled analysis showed that BMD remained largely stable over time in adult pts with PK deficiency receiving long-term treatment with mitapivat for up to 65 mos. As these pts had a substantial degree of reduced BMD at BL which did not progress, it is hypothesized that mitapivat may halt bone loss in PK deficiency patients via its mechanism of action by decreasing hemolysis and ineffective erythropoiesis and stabilizing iron homeostasis. BMD data will continue to be monitored as part of the ongoing LTE study to further understand this process. Figure 1 Figure 1. Disclosures Al-Samkari: Novartis: Consultancy; Dova/Sobi: Consultancy, Research Funding; Argenx: Consultancy; Rigel: Consultancy; Amgen: Research Funding; Agios: Consultancy, Research Funding; Moderna: Consultancy. Grace: Dova: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios: Research Funding; Principia: Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Glenthoej: Novo Nordisk: Honoraria; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Consultancy, Membership on an entity's Board of Directors or advisory committees; Calgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Alexion: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Barcellini: Alexion Pharmaceuticals: Honoraria; Novartis: Honoraria; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding. Galactéros: Addmedica: Membership on an entity's Board of Directors or advisory committees. Kuo: Bluebird Bio: Consultancy; Apellis: Consultancy; Novartis: Consultancy, Honoraria; Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Alexion: Consultancy, Honoraria; Pfizer: Consultancy, Research Funding. Layton: Cerus: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Agios Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees. Morado: Sanofi Genzyme: Honoraria. Viprakasit: La Jolla Pharmaceuticals: Consultancy, Research Funding; Ionis Pharmaceuticals,: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Agios: Consultancy, Research Funding; Protagonist Therapeutics: Consultancy, Research Funding; Vifor Pharma: Consultancy, Research Funding. Dong: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Bristol-Myers Squibb: Current equity holder in publicly-traded company; Infinity Pharmaceuticals: Current equity holder in publicly-traded company; Jazz Pharmaceuticals: Current equity holder in publicly-traded company. Tai: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Grekas: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Khoja: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Gheuens: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. McGee: Agios Pharmaceuticals: Current Employment, Current equity holder in publicly-traded company. Porter: Protagonism: Honoraria; Agios: Consultancy, Honoraria; La Jolla Pharmaceuticals: Honoraria; Celgene (BMS): Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Vifor: Honoraria, Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Van Beers: RR Mechatronics: Research Funding; Novartis: Research Funding; Pfizer: Research Funding; Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Defects of platelet intracellular signaling can result in severe platelet dysfunction. Several mutations in each of the linked genes FERMT3 and RASGRP2 on chromosome 11 causing a Glanzmann‐like bleeding phenotype have been identified so far. We report on novel variants in two unrelated pediatric patients with severe bleeding diathesis—one with leukocyte adhesion deficiency type III due to a homozygous frameshift in FERMT3 and the other with homozygous variants in both, FERMT3 and RASGRP2 . We focus on the challenging genetic and functional variant assessment and aim to accentuate the risk of obtaining misleading results due to the phenomenon of genetic linkage.

Patient: Male, 3-day-old Final Diagnosis: Neonatal leukemia Symptoms: Skin lesions Medication: — Clinical Procedure: Biopsy • stem cell transplant Specialty: Hematology • Pediatrics and Neonatology Objective: Rare disease Background: Neonatal acute leukemia is a rare condition. Little is known about its incidence and outcomes, and treatment options have not been standardized. Case Report: A 3-day old, apparently healthy male newborn was referred to the pediatric intensive care unit with multiple violaceous macules and a few papules on his face and upper trunk. After initial spontaneous regression, the lesions reappeared. Skin biopsy and bone marrow aspirate revealed a diagnosis of acute lymphoblastic leukemia (ALL). ALL induction therapy was initiated on day 24, resulting in morphological remission at the end of induction therapy. ALL chemotherapy was guided by sequential PCR-based monitoring of minimal residual disease (MRD). The patient received a transplant from an unrelated HLA high-resolution matched (10/10 loci) permissive donor. He was followed-up after transplant conducted by sequential PCR-based measurements of MRD in bone marrow. Conclusions: Neonatal leukemia often presents as congenital skin lesions known as blueberry muffin rash. ALL induction therapy was started at the end of the neonatal period. Treatment was well-tolerated and effective. Early donor search and PCR-MRD guided treatment surveillance can help to achieve and maintain molecular remission.

Hereditary spherocytosis (HS) is characterised by increased osmotic fragility and enhanced membrane loss of red blood cells (RBC) due to defective membrane protein complexes. In our diagnostic laboratory, we observed that pyruvate kinase (PK) activity in HS was merely slightly elevated with respect to the amount of reticulocytosis. In order to evaluate whether impaired PK activity is a feature of HS, we retrospectively analysed laboratory data sets from 172 unrelated patients with HS, hereditary elliptocytosis (HE), glucose-6-phosphate dehydrogenase (G6PD) or PK deficiency, sickle cell or haemoglobin C disease, or β-thalassaemia minor. Results from linear regression analysis provided proof that PK activity decreases with rising reticulocyte counts in HS (R2 = 0·15; slope = 9·09) and, less significantly, in HE (R2 = 0·021; slope = 8·92) when compared with other haemolytic disorders (R2 ≥ 0·65; slopes ≥ 78·6). Reticulocyte-adjusted erythrocyte PK activity levels were significantly lower in HS and even declined with increasing reticulocytes (R2 = 0·48; slope = -9·74). In this report, we describe a novel association between HS and decreased PK activity that is apparently caused by loss of membrane-bound PK due to impaired structural integrity of the RBC membrane and may aggravate severity of haemolysis in HS.

Background HD is a B-lineage lymphoma characterized, depending on subtype, by a prominent inflammatory infiltrate and fibrosis. Clinically, inflammatory symptoms like fever, weight loss and night sweats (B-symptoms) and increased blood biomarkers of inflammation, including ESR and CRP, are characteristic of more advanced disease. The clinical trial EURONET-PHL-C2 (Second International Inter-Group Study for Classical Hodgkin's Lymphoma in Children and Adolescents) is a randomized, prospective trial that compares chemo- and radiotherapy treatment concepts of different intensities in patients with intermediate and advanced HD. Patients are stratified by risk into 3 therapy groups (TL-1 to TL-3). An ESR> 30mm/h had been a risk factor for relapse in previous studies and leads to upstaging from the lowest (TL-1, Ann Arbor stage I and IIa without additional risk factors) to the intermediate risk group TL-2 in the current study. This addon pilot study tested urine proteomic patterns from pediatric patients with HD at diagnosis and compared them to the patterns of normal children. The questions were: Is there a HD-specific pattern, a pattern that identifies high risk or a pattern that correlates with inflammatory markers? Patients and methods Capillary electrophoresis coupled to mass spectrometry (CE-MS) was used to compare the peptide profiles in the mass range of 0.8 to 20 kDa of urine samples (N=34) from 16 children with pediatric Hodgkin lymphoma (PHL) as case and 32 age-matched children with no evidence of a disease (N=28) or with urinary tract infection (N=4) as control groups. Marker selection was based on a two-step strategy. First, a group-wise comparison of rank sum differences was performed on a set of 2418 annotated peptides with distribution frequencies above 30% in at least one of the groups with subsequent adjustment for multiple testing by the method of Bonferroni. In the second step marker candidates were further restricted to those demonstrating a significant positive or negative Spearman rho correlation coefficient (≥0.34 or ≤-0.34) to the Ann-Arbor classification criteria. From the resulting peptides a multivariate peptide marker classifier was established by support vector machine modeling and applied to an independent confirmation set of PHL (N=16, 31 urine samples) and control (N=18, 18 urine samples) patients to determine classification accuracy in receiver operating characteristics (ROC) analysis. Peptides included in the PHL classifier were resolved in their amino acid sequence by tandem mass spectrometry to identify the proteins from which the peptide markers are derived. Results The established multivariate peptide marker model consisting of 40 naturally occurring urinary peptides enabled absolute differentiation between PHL patients and children without signs of disease or urinary tract infection in independent validation as revealed by an area under the ROC curve value of 1.0 (95% confidence interval: 0.93 to 1.00, p Conclusions and perspective We were able to identify a proteomic pattern characteristic for HD, markers for relapse risk group or HD-associated inflammation were not yet identified. Disclosures Metzger: Mosaiques Diagnostics: Employment.

Exact diagnosis of hereditary spherocytosis (HS) is widely considered unreliable around birth. However, early postnatal diagnosis at the beginning of congenital hemolysis may be essential for managing neonatal anemia and hemolytic icterus, identifying those at high risk for severe hyperbilirubinemia, irreversible kernicterus, or sudden need for red cell transfusion. We analyzed 37 blood samples from neonates or infants up to six weeks of life that had been collected in-house or shipped to our laboratory due to suspected red cell membrane disorder. By combining assessment of red cell morphology, acidified glycerol lysis test (AGLT), and eosin-5'-maleimide (EMA) binding assay, we were able to clearly exclude HS in 22 and confirm HS in 10 patients, of which one had undergone red cell transfusion prior to blood sampling. Assessment of red cell morphology and normal test results allowed diagnosis of infantile pyknocytosis or Heinz body anemia in three neonates. Re-evaluation of five patients with inconsistent results of AGLT and EMA binding led to confirmation of HS in two cases. Automated analysis of hematologic parameters revealed elevated proportion of hyperdense cells to be a highly significant indicator for HS in neonatal infants. We showed that assessment of red cell morphology in combination with AGLT and EMA binding assay is a reliable basis for confirming or rejecting suspected diagnosis of HS even in neonates. Our data underline the necessity for blood sampling and laboratory exploration in suspected red cell membrane or enzyme defects at the earliest occasion.

Acute Graft-versus-Host-Disease (GvHD) is a potentially life-threatening complication after allogeneic stem cell transplantation. If not treated early and adequately, the complex immunological mechanisms may lead to a self-perpetuating cycle of alloreactivity, which is then associated with a high mortality. Here we assessed the cytokine profile on a daily basis in a patient with grade 4 skin GvHD, demonstrating a signature resembling cytokine-release-syndrome. After multimodal immunosuppressive intervention, including treatment with the IL6 receptor-blocking antibody tocilizumab, the severe clinical symptoms unexpectedly resolved within 48 hr.

ZusammenfassungAngeborene Thrombozytopathien sind komplexe Erkrankungen. Zur optimalen Blutungsprophylaxe und -behandlung empfehlen wir eine Abklärung und Diagnose der zugrunde liegenden Funktionsstörung. Dazu steht eine interdisziplinäre Leitlinie zur Verfügung (AWMF # 86–003 S2K; Hämostaseologie 2014; 34: 201–212).Die Behandlung ist auf den Defekt, die Thrombozytenzahl, das Lebensalter und die klinischen Umstände abzustimmen. Die Transfusion von Thrombozyten allein kann wegen der damit verbundenen Risiken unangemessen sein. Empfohlen wird ein stufenweiser und individualisierter Behandlungsplan. Antifibrinolytika sind generell anwendbar, besonders bei der Quebec-Erkrankung. Desmopressin ist älteren Kindern vor behalten und vor allem zur Behandlung der Storage- Pool-Erkrankungen und nicht klassifizierter Defekte geeignet. Rekombinanter FVIIa ist zwar nur für die Behandlung der Thrombasthenie Glanzmann mit Alloantikörpern zugelassen, wird jedoch in der klinischen Praxis auch bei Thrombozytendefekten mit schwerer Blutungsneigung eingesetzt. Das Ziel dieser Leitlinie ist die Behandlung von Patienten mit angeborenen Thrombozytenfunktionsstörungen bestmöglich zu unterstützen.

Haemophiliacs and extremely premature infants are both at an increased risk for intracranial haemorrhage; both conditions might be further elevating the risk. We report a case of a very immature preterm-infant of 26 gestational weeks (birth weight 635 g) with severe haemophilia A. Furthermore, we provide an overview of the published literature on this subject matter. Until now, deficiency of factor VIII or IX as a potential risk factor for ICH in preterm infants remains controversial. However, prophylactic substitution of factor VIII or IX in preterm infants with haemophilia may minimize the risk of bleeding complications and neurologic sequelae.

Acute Graft-versus-Host-Disease (GvHD) is a potentially life-threatening complication after allogeneic stem cell transplantation. If not treated early and adequately, the complex immunological mechanisms may lead to a self-perpetuating cycle of alloreactivity, which is then associated with a high mortality. Here we assessed the cytokine profile on a daily basis in a patient with grade 4 skin GvHD, demonstrating a signature resembling cytokine-release-syndrome. After multimodal immunosuppressive intervention, including treatment with the IL6 receptor-blocking antibody tocilizumab, the severe clinical symptoms unexpectedly resolved within 48 hr.

Gamma interferon (IFN-γ) has been shown to be increased in sera from patients with acute measles and after vaccination, to exhibit protective functions in brains of patients with subacute sclerosing panencephalitis, and to mediate a noncytolytic clearance of measles virus (MV) from rodent brains. In order to reveal a possible intracellular antiviral activity in the absence of antigen presentation and cytotoxic T cells, we investigated IFN-γ-induced effects on MV replication in various tissue culture cells. While attenuated MV strains are more sensitive to IFN-α/β than are wild-type strains, IFN-γ inhibits the replication of all MV strains in epithelial, endothelial, and astroglial cells, but not in lymphoid and neuronal cell lines. The antiviral activity induced by IFN-γ correlates with the induction of indoleamine 2,3-dioxygenase (IDO), an enzyme of the tryptophan degradation pathway known to mediate antiviral as well as antibacterial and antiparasitic effects. The IFN-γ-induced antiviral activity can be overcome by the addition of excess amounts of l -tryptophan, which indicates a specific role of IDO in the anti-MV activity. Our data suggest that the IFN-γ-induced enzyme IDO plays an important antiviral role in MV infections of epithelial, endothelial, and astroglial cells.

SummaryPlatelets are not only centrally involved in haemostasis, but also in antimicrobial defence and inflammation. Since evaluation of platelet physiology in the particular patient group of preterm and term neonatal infants is highly restricted for ethical reasons, there are hardly any data available in healthy and much less in extremely immature or ill neonates. By summarising current knowledge and addressing both platelet researchers and neonatologists, we describe neonatal platelet count and morphology, report on previous analyses of neonatal platelet function in primary haemostasis and provide insights into recent advances in platelet immunology that considerably impacts our clinical view on the critically ill neonatal infant. We conclude that neonatal platelets, originating from liver megakaryocytes, substantially differ from adult platelets and may play a pivotal role in the pathophysiology of neonatal sepsis or intraventricular haemorrhage, both complications which seriously augment perinatal morbidity and mortality.

Background Reliable central venous access (CVC) is essential for hematology–oncology patients since frequent puncture of peripheral veins—e.g., for chemotherapy, antibiotic administration, repeated blood sampling, and monitoring—can cause unacceptable pain and psychological trauma, as well as severe side effects in cases of extravasation of chemotherapy drugs. However, CVC lines still carry major risk factors, including thrombosis, infection (e.g., entry site, tunnel, and luminal infections), and catheter dislocation, leakage, or breakage. Methods Here we performed a retrospective database analysis to determine the incidence of CVC-associated thrombosis in a single-center cohort of 448 pediatric oncologic patients, and to analyze whether any subgroup of patients was at increased risk and thus might benefit from prophylactic anticoagulation. Results Of the 448 patients, 269 consecutive patients received a CVC, and 55 of these 269 patients (20%) also had a thrombosis. Of these 55 patients, 43 had at least one CVC-associated thrombosis (total number of CVC-associated thrombosis: n = 52). Among all patients, the median duration of CVC exposure was 464 days. Regarding exposure time, no significant difference was found between patients with and without CVC-associated thrombosis. Subclavia catheters and advanced tumor stages seem to be the main risk factors for the development of CVC-associated thrombosis, whereas pharmacologic prophylaxis did not seem to have a relevant impact on the rate of thrombosis. Conclusions We conclude that pediatric surgeons and oncologists should pay close attention to ensuring optimal and accurate CVC placement, as this appears the most effective tool to minimize CVC-associated complications.

A 9-month-old infant presented with fatal pneumococcal sepsis and attenuated inflammation indices. Even in septic conditions, flow cytometry-based CD62L shedding test on granulocytes proved to be a fast and reliable diagnostic tool for the detection of a defect in the innate immunity. Confirmatory immunologic and genetic assays identified an autosomal-recessive interleukin-1 receptor-associated kinase-4 deficiency due to compound heterozygous mutations.

SummaryBackground Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disease characterized by oculocutaneous albinism and platelet dysfunction. We report on a novel HPS6 homozygous frameshift variant (c.1919_1920delTC; p.Val640Glyfs*29) in a nonconsanguineous Caucasian family with two affected siblings (index patients) who presented with oculocutaneous albinism at birth and a mild bleeding phenotype during childhood and adolescence. Procedure Genetic analysis was conducted by panel-based next-generation sequencing (NGS) and Sanger sequencing. Platelets of the index patients, their parents, and the unaffected sister were then comprehensively evaluated by luminoaggregometry, whole blood flow cytometry, immunoblotting, immunofluorescence, and transmission electron microscopy. Results The homozygous frameshift variant in HPS6 gene detected by panel-based NGS and its segregation in the family was confirmed by Sanger sequencing. Flow cytometric analysis of the patients’ platelets revealed a substantially decreased mepacrine uptake and release upon activation with a thrombin receptor agonist. Electron microscopy of resting platelets confirmed diminished dense granule content and enhanced vacuolization. Reduced release of adenosine triphosphate and CD63 neoexposition upon activation indicated not only a lack of dense granule content, but even an impairment of dense granule release. Conclusions Our results demonstrate that the novel loss-of-function variant in the HPS6 subunit of biogenesis of lysosome-related organelles complex 2 is pathologic and leads to a reduced platelet dense granules and their release. The findings are compatible with an impaired platelet function and hence an enhanced bleeding risk. In future, a valid genotype–phenotype correlation may translate into best supportive care, especially regarding elective surgery or trauma management.

Growth factor independent 1 (GFI1) and Growth Factor Independent 1B (GFI1B) are zinc finger transcriptional repressors that share about 90% homology on amino acid sequence and are expressed during hematopoiesis. While GFI1 is most important for granulocyte-monocyte lineage commitment, GFI1B is an essential master regulator of erythroid and megakaryocytic lineages. Mice lacking Gfi1b are embryonic lethal due to anemia and thrombocytopenia. In humans, alternative splicing leads to a shorter p32 isoform that lacks the first 2 of 6 zinc fingers. GFI1B germline mutations have been reported to cause autosomal-dominant macrothrombocytopenia with a grey-platelet syndrome phenotype, implying that the mutant protein acts in a dominant-negative manner. We report on a Chechen family from eastern Georgia whose affected family members all present with severe, life-threatening bleeding diathesis. The female index patient had recurrent hematomata and multiple petechiae since childhood. Both of her children (age 9 and 7) present with very low platelet counts (below 45/nL) and a similar cutaneous bleeding pattern like her mother. The brother also had thrombocytopenia and died at age 33 in reponse to a spontaneous cerebral hemorrhage. In contrast, the index patient's husband, her parents and the children of the deceased brother were clinically unaffected. Blood smears of affected patients showed macrothrombocytopenia with reduced May-Grünwald-Giema staining and decreased staining for alpha-granule markers von Willebrand factor (vWF) and P-selectin (CD62P). Platelet function testing revealed reduced responses to ADP, collagen, TRAP-6 and arachidonic acid. White and red blood cell parameters were overall normal in the index patient and the two affected children. We analyzed DNA from the index patient by targeted next generation sequencing for 59 genes relevant for platelet formation or function. We found a novel homozygous single nucleotide insertion in GFI1B (NM_004188.5; c.551insG), which was confirmed by Sanger sequencing and is expected to cause a premature stop-codon. The homozygous mutation co-segregated with the phenotype. The unaffected mother, the husband and two unaffected nephews were heterozygous, suggesting a local founder variant and an unexpected autosomal-recessive trait. Bone marrow analysis showed unaffected myeloid and erythroid cells, but dysplastic micromegakaryocytes with increased CD34 staining. Peripheral blood platelets were also positive for CD34. We performed quantitative real-time PCR of platelet RNA and found residual homozygous c.551_G insertion in the p37 transcript and an unexpected expression of the p32 variant. The p37 transcript was markedly reduced in context with an increased p32/p37 ratio compared to controls. Our findings indicate that the mutated transcript was not completely degraded by nonsense-mediated decay, but mostly subjected to alternative splicing skipping the mutated exon 9. Our findings imply that the first two zinc fingers of GFI1B are dispensable for human erythropoiesis, but essential for normal megakaryopoiesis and the production of functional platelets. While previous mutations affect both isoforms, the insertion variant presented here, results in a premature stop-codon and affects only the p37 isoform due to alternative splicing. This splice variant defines an important node at the megakaryocytic-erythroid progenitor stage and we conclude that the transcriptional regulation of erythropoiesis is uncoupled from that of megakaryopoiesis through alternative splicing of GFI1B. Disclosures No relevant conflicts of interest to declare.

Measles virus (MV) infects endothelial cells of the skin, the brain and other organs during acute or persistent infections. Endothelial cells are supposed to play an important role in virus spread from the blood stream to surrounding tissues. CD46 and CD150 (signalling lymphocytic activation molecule, SLAM) have been described as cellular receptors for certain MV strains. We found that human umbilical vein and brain microvascular endothelial cells (HUVECs and HBMECs) were CD46-positive, but did not express SLAM. Wild-type MV strains, which do not use CD46 as a receptor at the surface of transfected Chinese hamster ovary cells, infected HUVECs and HBMECs to varying extents in a strain-dependent way. This infection was not inhibited by antibodies to CD46. These data suggest the presence of an additional unidentified receptor for MV uptake and spread in human endothelial cells.