Paroxysmal Nocturnal Hemoglobinuria: Grasping the Flow of Diagnostic Dilemmas

2019 ◽  
Vol 152 (Supplement_1) ◽  
pp. S88-S88
Author(s):  
Phuong-Lan Nguyen
Keyword(s):  
Flow Cytometry ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Meta Analysis ◽  
Research Literature ◽  
Limit Of Quantification ◽  
University Of Kentucky ◽  
Hematopoietic Stem ◽  
Diagnostic Threshold ◽  
Interlaboratory Validation

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare life-threatening condition due to an acquired somatic mutation of the PIGA gene, leading to nonmalignant clonal expansion of hematopoietic stem cells, which are deficient in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). Fluorescein-labeled proaerolysin (FLAER) and flow cytometry are key tools in the diagnosis of PNH. While clonal detection of PNH in both tests has a sensitive diagnostic threshold of 0.01% in erythrocytes and 0.05% to 1% in leukocytes, one must be cautious in ruling out the possibilities of myelodysplastic syndrome (MDS) or aplastic anemia. We propose guidelines in the differential diagnosis and evaluation of PNH from these and other hematologic disorders that can arise from GPI-AP deficient cells. These guidelines are based on a meta-analysis of five research literature sources, including four case studies. We also compare and contrast our limits of quantification of the in-house PNH assay at University of Kentucky Healthcare with those of an interlaboratory validation of 11 institutions within the United Kingdom. Our report advocates for thorough evaluation of multiple laboratory and clinical variables affecting sensitivity and accuracy of flow cytometry and FLAER in PNH. Furthermore, we recommend lowering of the in-house limit of quantification from the current 1% to 0.01%. This allows for the critical consideration of conditions such as MDS and aplastic anemia and their disease courses, all of which can present with PNH clones as low as 0.01% on flow cytometry and FLAER.

scholarly journals Paroxysmal Nocturnal Hemoglobinuria Clones Are Infrequent in Hepatitis-Associated Aplastic Anemia at the Time of Diagnosis

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5016-5016
Author(s):  
Wenrui Yang ◽  
Xin Zhao ◽  
Guangxin Peng ◽  
Li Zhang ◽  
Liping Jing ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Clonal Evolution ◽  
Extrinsic Factors ◽  
Clone Size ◽  
Hematopoietic Stem ◽  
Over Time ◽  
Pnh Clone

Aplastic anemia (AA) is an immune-mediated bone marrow failure, resulting in reduced number of hematopoietic stem and progenitor cells and pancytopenia. The presence of paroxysmal nocturnal hemoglobinuria (PNH) clone in AA usually suggests an immunopathogenesis in patients. However, when and how PNH clone emerge in AA is still unclear. Hepatitis associated aplastic anemia (HAAA) is a special variant of AA with a clear disease course and relatively explicit immune pathogenesis, thus serves as a good model to explore the emergence and expansion of PNH clone. To evaluate the frequency and clonal evolution of PNH clones in AA, we retrospectively analyzed the clinical data of 90 HAAA patients that were consecutively diagnosed between August 2006 and March 2018 in Blood Diseases Hospital, and we included 403 idiopathic AA (IAA) patients as control. PNH clones were detected in 8 HAAA patients (8.9%,8/90) at the time of diagnosis, compared to 18.1% (73/403) in IAA. Eight HAAA patients had PNH clone in granulocytes with a median clone size of 3.90% (1.09-12.33%), and 3 patients had PNH clone in erythrocytes (median 4.29%, range 2.99-10.8%). Only one HAAA patients (1/8, 12.5%) had a PNH clone larger than 10%, while 24 out of 73 IAA patients (32.9%) had larger PNH clones. Taken together, we observed a less frequent PNH clone with smaller clone size in HAAA patients, compared to that in IAAs. We next attempted to find out factors that associated with PNH clones. We first split patients with HAAA into two groups based on the length of disease history (≥3 mo and < 3mo). There were more patients carried PNH clone in HAAA with longer history (21.4%, 3/14) than patients with shorter history (6.6%, 5/76), in line with higher incidence of PNH clone in IAA patients who had longer disease history. Then we compared the PNH clone incidence between HAAA patients with higher absolute neutrophil counts (ANC, ≥0.2*109/L) and lower ANC (< 0.2*109/L). Interestingly, very few VSAA patients developed PNH clone (5%, 3/60), while 16.7% (5/30) of non-VSAA patients had PNH clone at diagnosis. We monitored the evolution of PNH clones after immunosuppressive therapy, and found increased incidence of PNH clone over time. The overall frequency of PNH clone in HAAA was 20.8% (15/72), which was comparable to that in IAA (27.8%, 112/403). Two thirds of those new PNH clones occurred in non-responders in HAAA. In conclusion, PNH clones are infrequent in HAAA compared to IAA at the time of diagnosis, but the overall frequency over time are comparable between the two groups of patients. In SAA/VSAA patients who are under the activated abnormal immunity, longer clinical course and relatively adequate residual hematopoietic cells serve as two important extrinsic factors for HSCs with PIGA-mutation to escape from immune attack and to expand. Disclosures No relevant conflicts of interest to declare.

Frequency of Paroxysmal Nocturnal Hemoglobinuria Clones by Multiparametric Flow Cytometry in Pediatric Aplastic Anemia Patients of Indian Ethnic Origin

2015 ◽  
Vol 63 (1) ◽  
pp. 93-97 ◽  
Author(s):  
Sreejesh Sreedharanunni ◽  
Man Updesh Singh Sachdeva ◽  
Parveen Bose ◽  
Neelam Varma ◽  
Deepak Bansal ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Ethnic Origin ◽  
Multiparametric Flow Cytometry

Screening Patients with Myelodysplastic Syndrome and Aplastic Anemia for Paroxysmal Nocturnal Hemoglobinuria Clones: A Retrospective Study,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3426-3426 ◽  
Author(s):  
Andrew Shih ◽  
Ian H. Chin-Yee ◽  
Ben Hedley ◽  
Mike Keeney ◽  
Richard A. Wells ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Retrospective Study ◽  
Aplastic Anemia ◽  
Board Of Directors ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Cell Surface Expression ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Pnh Clone

Abstract Abstract 3426 Introduction: Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare disorder due to a somatic mutation in the hematopoietic stem cell. The introduction of highly sensitive flow cytometric and aerolysin testing have shown the presence of PNH clones in patients with a variety of other hematological disorders such as aplastic anemia (AA) and myelodysplasic syndrome (MDS). It is hypothesized that patients with these disorders and PNH clones may share an immunologic basis for marrow failure with relative protection of the PNH clone, due to their lack of cell surface expression of immune accessory proteins. This is supported by the literature showing responsiveness in AA and MDS to immunosuppressive treatments. Preliminary results from a recent multicenter trial, EXPLORE, notes that PNH clones can be seen in 70% of AA and 55% of MDS patients, and therefore there may be utility in the general screening of all patients with bone marrow failure (BMF) syndromes. Furthermore, it has been suggested that the presence of PNH cells in MDS is a predictive biomarker that is clinically important for response to immunosuppressive therapy. Methods: Our retrospective cohort study in a tertiary care center used a high sensitivity RBC and FLAER assay to detect PNH clones as small as 0.01%. Of all patients screened with this method, those with bone marrow biopsy and aspirate proven MDS, AA, or other BMF syndromes (defined as unexplained cytopenias) were analysed. Results from PNH assays were compared to other clinical and laboratory parameters such as LDH. Results: Overall, 102 patients were initially screened over a 12 month period at our center. 30 patients were excluded as they did not have biopsy or aspirate proven MDS, AA, or other BMF syndromes. Of the remaining 72 patients, four patients were found to have PNH clones, where 2/51 had MDS (both RCMD, IPSS 0) [3.92%] and 2/4 had AA [50%]. The PNH clone sizes of these four patients were 0.01%, 0.01%, 0.02%, and 1.7%. None of the MDS patients with known recurrent karyotypic abnormalities had PNH clones present. Only one of the four patients had a markedly increased serum LDH level. Conclusions: Our retrospective study indicates much lower incidence of PNH clones in MDS patients or any patients with BMF syndromes when compared to the preliminary data from the EXPLORE trial. There is also significant disagreement in other smaller cohorts in regards to the incidence of PNH in AA and MDS. Screening for PNH clones in patients with bone marrow failure needs further study before adoption of widespread use. Disclosures: Keeney: Alexion Pharmaceuticals Canada Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wells:Alexion Pharmaceuticals Canada Inc: Honoraria. Sutherland:Alexion Pharmaceuticals Canada Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Upfront Alternative Donor Transplant Versus Immunosuppressive Therapy in Patients with Severe Aplastic Anemia Who Lack Fully HLA Matched Related Donor: Systematic Review and Meta-Analysis of Retrospective Studies. on Behalf of the Severe Aplastic Anemia Working Party of European Group for Blood and Marrow Transplantation (SAAWP of EBMT)

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-7
Author(s):  
Hind Alotaibi ◽  
Mahmoud Aljurf ◽  
Regis Peffault De Latour ◽  
Shahid Iqbal ◽  
Andrea Bacigalupo ◽  
...  
Keyword(s):  
Aplastic Anemia ◽  
Board Of Directors ◽  
Older Patients ◽  
Research Funding ◽  
Retrospective Studies ◽  
Meta Analysis ◽  
Severe Aplastic Anemia ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Meta Analyses

Introduction: WIdiopathic aplastic anemia is a rare and life threatening disorder characterized by immune mediated hematopoietic stem cells dysfunction. The standard treatment strategy of severe aplastic anemia (SAA) has been hematopoietic stem cell transplant (HSCT) for children and adults younger than the age of 40 if an HLA matched sibling donor (MSD) is available. Immunosuppressive therapy (IST) is the mainstay of treatment for older patients or when MSD is not available. The response rate to IST with the use of horse anti-thymocyte globulin (ATG) is around 70%. Despite that, many patients suffer from relapse or clonal evolution. The use of alternative donor transplant (ADT) from matched unrelated donor (MUD) or HLA haploidentical donor (HID) is not commonly used in frontline setting. We herein, conducted a systematic review and meta-analysis of retrospective studies to compare the outcome of IST versus ADT as upfront therapy for SAA. Methods: WWe conducted a comprehensive search in PUBMED/MEDLINE and EMBASE (1998-2019) for retrospective studies that compared the outcome of ADT with IST as upfront therapy in patients with SAA. The study was conducting in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. We included the studies with 10 patients or more in each arm. Studies that included patients with inherited aplastic anemia or PNH are excluded. The primary outcome is the 5-year overall survival. Two authors independently screened the studies, extracted the data, and evaluated the quality of included studies and discrepancies were resolved by a third author. Study quality was evaluated by description of study characteristics, patients' characteristics, treatment details, and outcome. The odd ratio (OR) for 5-year survival was measured by Mantel-Haenszel test using random effect model. We also conducted another search and meta-analysis to compare upfront with salvage ADT. The meta-analyses were performed using Review Manager software version 5.3. Result: WWe screened a total of 697 articles (506 EMBASE, 191 PUBMED/MEDLINE). Five studies met our inclusion criteria included a total of 343 patients (176 in ADT group and 167 in IST group) for upfront ADT versus IST comparison and 6 studies with a total of 298 patients (198 in upfront ADT group and 100 in salvage ADT group) for upfront versus salvage ADT comparison. Included patients were of pediatric age group in 4 out of 5 studies. Xu ZL et al, included adult patients with median age 28 (18-49) years in upfront ADT arm and 32 (18-62) years in IST arm. Of those, only 10 patients in ADT group and 12 patients in IST group were above age of 40. In ADT versus IST comparison, the type of transplant was HID in three studies (total of 124 patients) and MUD/MMUD in two studies (total of 52 patients). The rabbit ATG was used in three studies, horse ATG in one study, and both types were used in one study (total of 68 patients received horse ATG and 99 patients received rabbit ATG). In term of disease severity, all included patients were SAA and very SAA (VSAA). Five studies were included in meta-analysis for 5-year overall survival. The pooled OR is statistically significant at 0.44 [95% CI 0.23-0.85] in favor of upfront ADT (Fig 1-A). The survival outcome was compared between upfront versus salvage ADT in 6 studies. The pooled OR is statistically significant at 0.31 [95% CI 0.15-0.64] in favor of upfront ADT (Fig 1-B). Conclusion: WThe pooled analysis of this study showed a potential survival advantage of upfront ADT over IST in patients with SAA who lack an HLA identical sibling donor. The use of ADT earlier in the disease course rather than as a salvage in young patients with severe disease, may improve survival. Data in older patients are limited, and at present, we cannot recommend ADT upfront in older patients. Given the limitations of our study including various types of IST, heterogeneity of patient population, health care systems, and retrospective nature of included studies; further studies are needed to confirm our findings. Disclosures Peffault De Latour: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Amgen: Research Funding; Apellis: Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Schrezenmeier:Alexion Pharmaceuticals Inc.: Honoraria, Research Funding. Hoechsmann:Apellis: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria, Research Funding; Roche: Consultancy, Honoraria. Risitano:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Alnylam: Research Funding; Alexion: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Jazz: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees; Samsung: Membership on an entity's Board of Directors or advisory committees; Amyndas: Consultancy; RA pharma: Research Funding; Biocryst: Membership on an entity's Board of Directors or advisory committees; Apellis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Achillion: Membership on an entity's Board of Directors or advisory committees; Pfizer: Speakers Bureau. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees.

High-Sensitivity Flow Cytometry Testing for Paroxysmal Nocturnal Hemoglobinuria in Children with Cytopenia: A Single Center Study

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2398-2398
Author(s):  
Choladda V. Curry ◽  
M. Tarek Elghetany ◽  
Andrea M. Sheehan ◽  
Alison A. Bertuch ◽  
Ghadir S. Sasa
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Aplastic Anemia ◽  
Immunosuppressive Therapy ◽  
Hemolytic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
High Sensitivity ◽  
Deficiency Anemia ◽  
Unknown Etiology ◽  
Flow Cytometry Assay

Abstract Abstract 2398 Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by expansion of cells with complete or partial loss of glycosyl phosphatidyl-inositol-anchored proteins. PNH usually presents with one or more of three clinical manifestations: intravascular hemolysis, thrombosis, or acquired bone marrow failure [aplastic anemia (AA) or myelodysplastic syndrome (MDS)]. Flow cytometry has become the gold standard for the diagnosis of PNH, particularly with the recent publication of guidelines for the diagnosis and monitoring of PNH and related disorders in 2010. PNH occurs rarely in children, and, consequently, the published literature regarding PNH in this pediatric population consists only of small case series, making it difficult to extrapolate the frequency of which PNH clones are identified. Moreover, no studies are available on the incidence of PNH clones in children with MDS and acquired aplastic anemia (AAA). We, therefore, sought to determine how frequently a high sensitivity FLAER-based assay, with a sensitivity of 0.01%, would detect PNH clones in children with cytopenias. Method and Results: The study period was from December 2010 to July 2011. PNH testing was performed using a high sensitivity FLAER based assay according to published guidelines using the combination of FLAER/CD64/CD15/CD33/CD24/CD14/CD45 for WBC testing and CD235a/CD59 for RBC testing. There were 31 peripheral blood samples from 29 patients (17 males/12 females) ranging in age from 4 months to 17 years (median, 10 years). All patients were tested for PNH because of cytopenia [pancytopenia (n = 14) and uni- or bicytopenia (n = 15)]. Patients had a mean Hgb of 10.7 gm/dL, mean ANC of 2.66 X103/uL and mean platelet of 115 X103/uL. Review of medical charts revealed the following clinical diagnoses: classic PNH - episodic hemolytic anemia with persistent thrombocytopenia (1), severe AA (SAA, 8), SAA with myelofibrosis (1), MDS (1), Fanconi anemia (1), chronic thrombocytopenia (2), refractory iron deficiency anemia (1), bone marrow suppression likely due to virus/medication (1), parvovirus infection (1), Copper deficiency (1), systemic lupus erythematosus (SLE, 1), and cytopenia of unknown etiology (10). Of note, all patients with AAA had SAA. PNH clones were identified in 6 out of 29 patients (20%): minor clones (<1% PNH population) in 3 patients: average clone sizes 0.12% [range 0.02–0.25] granulocytes (G), 0.51% [0.20–0.99] monocytes (M), and 0.08% [0.04–0.14] red blood cells (RBCs), and major clones (>1% PNH population) in 3 patients: average clone sizes 31.11% [3.98–67.58] G, 31.98% [6.15–71.1] M, and 14.76% [1.19–38.03] RBC, respectively, with ages ranging from 4 to 17 years. Patients who were identified to have minor PNH clones all presented with pancytopenia. Two were diagnosed with SAA; the cause of pancytopenia in the third patient is currently under investigation. None of patients with minor PNH clones had evidence of hemolysis or thrombosis. The three patients with major PNH clones had the following: Classic PNH with hemolytic anemia (1), SAA with PNH clones detected at the time of SAA diagnosis (1), and SAA with PNH clones detected 20 months after immunosuppressive therapy (1). The latter two patients did not have evidence of hemolysis or thrombosis. Of the 10 patients with a diagnosis of SAA or MDS, PNH clones were identified in 4 (40%) patients (2 with minor clones, 2 with major clones). Conclusions: This is the first study to describe the utility of using a standardized high-sensitivity FLAER-based flow cytometry assay to identify PNH clones in children. This is also the first study describing the prevalence of PNH clones in children with MDS and AAA. The identification of a PNH population in 40% of the MDS and AAA cases emphasizes the need for PNH testing in all children with these disorders using a high-sensitivity FLAER based flow cytometry assay. A low sensitivity assay would have missed 2 patients with minor PNH clones. This finding may be of significance considering SAA or MDS patients with PNH clones are more likely to respond to immunosuppressive therapy. Further studies are needed to investigate the prevalence of PNH clones in this setting and its impact on disease manifestations, course, and outcomes in children. Disclosures: No relevant conflicts of interest to declare.

Ham Test For Therapeutic Monitoring Of Eculizumab In Paroxysmal Nocturnal Hemoglobinuria

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4876-4876
Author(s):  
Miriam Arcavi ◽  
Fernanda Ceballo ◽  
Andres L. Brodsky ◽  
Nora Silvia Halperin ◽  
Norma Cantenys ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Stem Cell ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Alternative Pathway ◽  
Specific Treatment ◽  
Chimeric Antibody ◽  
Intravascular Hemolysis ◽  
Hematopoietic Stem ◽  
Gold Standard Method

Abstract Introduction Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal disease, caused by an inactivating mutation in the PIG-A gene in a hematopoietic stem cell. The PIG-A gene encodes an enzyme required for glycosylphosphatidylinositol (GPI) anchor synthesis. Its inactivation results in a deficiency of many plasma membrane GPI-anchored proteins -including CD55 and CD59, natural inhibitors of the complement cascade- in the involved stem cell and all its progeny (the PNH clone). Intravascular hemolysis, anemia, thrombosis, acute and chronic renal damage, pulmonary hypertension, abdominal pain, esophagic spasm, erectile dysfunction -among others manifestations- are consequences of complement mediated damage of the sensitive PNH blood cells. In 2007 both the FDA and the EMA approved eculizumab, a monoclonal chimeric antibody targeted against C5 fraction of complement, as the first specific treatment of complement mediated PNH manifestations. Flow cytometry (FC) is the gold standard method for diagnosis. The former diagnostic test -the Ham test- is based on the susceptibility of PHN red blood cells (RBC), when they are incubated with both normal and patient sera to lysis mediated by the alternative pathway of complement (APC). APC is activated, in the Ham test, through sera acidification. Despite its physiopathological value, Ham test has been replaced with flow cytometry to diagnose PNH due to a much higher sensitivity and reproducibility. Aims To evaluate the Ham test in PNH treated patients, to monitor the eculizumab-mediated blockade of APC. Patients and methods Ham test was used to monitor APC blockade in the patient serum, testing the ability of the acidified patient serum to lyse his or her own PNH-RBC. Eight patients were diagnosed as PNH by FC and were treated with eculizumab. Six had a good therapeutic response, with decreased levels of both, LDH and the serum total complement hemolytic capacity (CH50). Ham test, in these six patients, showed hemolysis when PNH-RBC were mixed with normal acidified serum but absence of hemolysis when the acidified serum of eculizumab treated patient was added to the PNH-RBC. This result was called “blockade profile” and shows the “ex vivo” APC blockade, confirming thus the eculizumab success. The remaining two patients showed a persistent positivity of the Ham test at day 14 of eculizumab administration (as PNH-RBC lysis continued taking place with both normal and patient acidified sera). One patient demonstrated break through hemolysis occurring near the end of eculizumab dosing period as indicated by increase in LDH. As LDH may increase due to other possible factors (ie hepatic lesions) the positive Ham test confirmed that intravascular hemolysis was taking place, possibly due to a shorter eculizumab half life. An increase of the eculizumab dose to 1,200 mg/14 days reinstated lower LDH levels and the blockade profile in the Ham test (Table). There has been a single patient treated with eculizumab where LDH did not reduce. There was a persistently positive Ham test, elevated LDH and free hemoglobin levels and normal CH50 values despite a dose of 1,200 mg of eculizumab every 14 days (Table). A genetic study found in this case a C5 mutation, which seems responsible of the lack of response to eculizumab. Conclusions In our experience, the Ham test has proved to be a useful and economic method to monitor the effectiveness of eculizumab treatment in cases with high LDH levels due to either a) other causes than intravascular hemolysis, or b) no responsive patients due to pharmacokinetic (inadequate eculizumab concentration) or pharmacodynamic causes. Disclosures: Brodsky: Alexion Pharmaceuticals: Consultancy, Speakers Bureau. Colin:Alexion Pharmaceuticals: Consultancy.

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