Bone Marrow Histology in Patients with Paroxysmal Nocturnal Hemoglobinuria.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4215-4215
Author(s):  
Sandra van Bijnen ◽  
Konnie Hebeda ◽  
Petra Muus
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Plasma Cells ◽  
Bone Marrow Failure ◽  
Clone Size ◽  
Immune Mediated ◽  
Lymphoid Nodules ◽  
Pnh Clone

Abstract Abstract 4215 Introduction Paroxysmal Nocturnal Hemoglobinuria (PNH) is a disease of the hematopoietic stem cell (HSC) resulting in a clone of hematopoietic cells deficient in glycosyl phosphatidyl inositol anchored proteins. The clinical spectrum of PNH is highly variable with classical hemolytic PNH at one end, and PNH in association with aplastic anemia (AA/PNH) or other bone marrow failure states at the other end. It is still largely unknown what is causing these highly variable clinical presentations. Immune-mediated marrow failure has been suggested to contribute to the development of a PNH clone by selective damage to normal HSC. However, in classic PNH patients with no or only mild cytopenias, a role for immune mediated marrow failure is less obvious. No series of trephine biopsies has been previously documented of patients with PNH and AA/PNH to investigate the similarities and differences in these patients. Methods We have reviewed a series of trephine biopsies of 41 PNH patients at the time the PNH clone was first detected. The histology was compared of 27 patients with aplastic anemia and a PNH clone was compared to that of 14 patients with classic PNH. Age related cellularity, the ratio between myeloid and erythroid cells (ME ratio), and the presence of inflammatory cells (mast cells, lymphoid nodules and plasma cells) were evaluated. The relation with clinical and other laboratory parameters of PNH was established. Results Classic PNH patients showed a normal or hypercellular marrow in 79% of patients, whereas all AA/PNH patients showed a hypocellular marrow. Interestingly, a decreased myelopoiesis was observed not only in AA/PNH patients but also in 93% of classic PNH patients, despite normal absolute neutrophil counts (ANC ≥ 1,5 × 109/l) in 79% of these patients. The number of megakaryocytes was decreased in 29% of classic PNH patients although thrombocytopenia (< 150 × 109/l) was only present in 14% of the patients. Median PNH granulocyte clone size was 70% (range 8-95%) in classic PNH patients, whereas in AA/PNH patients this was only 10% (range 0.5-90%). PNH clones below 5% were exclusively detected in the AA/PNH group. Clinical or laboratory evidence of hemolysis was present in all classical PNH patients and in 52% of AA/PNH patients and correlated with PNH granulocyte clone size. Bone marrow iron stores were decreased in 71% of classic PNH patients. In contrast, increased iron stores were present in 63% of AA/PNH patients, probably reflecting their transfusion history. AA/PNH patients showed increased plasma cells in 15% of patients and lymphoid nodules in 37%, versus 0% and 11% in classic PNH. Increased mast cells (>2/high power field) were three times more frequent in AA/PNH (67%) than in PNH (21%). Conclusion Classic PNH patients were characterized by a more cellular bone marrow, increased erythropoiesis, larger PNH clones and clinically by less pronounced or absent peripheral cytopenias and more overt hemolysis. Decreased myelopoiesis and/or megakaryopoiesis was observed in both AA/PNH and classic PNH patients, even in the presence of normal peripheral blood counts, suggesting a role for bone marrow failure in classic PNH as well. More prominent inflammatory infiltrates were observed in AA/PNH patients compared to classical PNH patients. Disclosures: No relevant conflicts of interest to declare.

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.

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.

A Spontaneous Reduction of Clone Size in Paroxysmal Nocturnal Hemoglobinuria Patients Treated with Eculizumab for Greater Than 12 Months.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1992-1992 ◽  
Author(s):  
Richard Kelly ◽  
Stephen Richards ◽  
Louise Arnold ◽  
Gemma Valters ◽  
Matthew Cullen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Maintenance Dose ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Hemopoietic Stem Cells ◽  
Intravascular Hemolysis ◽  
Complement Protein ◽  
Clone Size ◽  
Pnh Clone

Abstract Abstract 1992 Poster Board I-1014 Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal disorder of hemopoietic stem cells that is characterized by bone marrow failure, intravascular hemolysis and venous thrombosis. Eculizumab is a humanized monoclonal antibody that specifically binds to the complement protein C5 preventing its cleavage thereby inhibiting the formation of the terminal components of the complement cascade. Eculizumab was approved by the FDA in 2007 after clinical trials showed it was efficacious in treating patients with hemolytic PNH. Prior to eculizumab therapy treatment options were mainly supportive in nature. Historical data shows that a third of patients who survive greater than 10 years undergo spontaneous recovery. We present data on 38 patients with hemolytic PNH treated at a single centre with eculizumab for 12 months or longer. Thirty six of these patients were treated with a loading dose of 600mg every week for 4 doses followed by 900mg the following week and then a maintenance dose of 900mg dose every 14 day. The other 2 patients required a higher maintenance dose of eculizumab, 1200mg every 14 days, due to symptomatic intravascular hemolysis on the standard regime. All our patients had a high PNH granulocyte clone size at the initiation of eculizumab treatment from 52.90% to 99.95% with a median of 96.38%. The duration of eculizumab therapy varied from 12 to 84 months with a median treatment duration of 50 months. Granulocyte clone size was used as it is not subject to as much variation as the erythrocyte clone size which changes both due to blood transfusions and to the extent of intravascular hemolysis present. The proportion of PNH granulocytes probably most accurately reflects the true size of the PNH clone. Seven out of these 38 patients (18.4%) have had a 10% or greater reduction in their granulocyte clone size during the course of their eculizumab treatment. These patients have had a steady and continued decline in their granulocyte clone size throughout their treatment with eculizumab. This may actually be due to an increase in the residual normal cells in some patients (see Table). Two of these patients (U.P.N. 5 and 7) have had such a dramatic reduction in their clone size that they have been able to stop their eculizumab treatment without any observed detriment to their health.TableChange in PNH clones in patients on eculizumabU.P.N.Months on eculizumabNeutrophils PNH clone size (%)Normal neutrophils (%)Pre-treatmentMost recent on treatmentPre-treatmentMost recent on treatment15097.242.82.85724878.063.222.036.335596.484.13.615.941592.577.07.523.051261.732.438.367.664788.362.511.737.578552.98.547.191.5 Two of these 7 patients were treated with ciclosporin for underlying aplasia as compared to 3 of the 31 of those who haven't had a decrease in their clone size. There was no difference in the white cell or platelet count in these 7 patients from when they started eculizumab treatment to the present day indicating that the degree of bone marrow failure present has not changed dramatically during this time course. 5 of the 7 patients had neutrophil clone sizes of less than the median perhaps indicating that recovery requires a certain number of residual normal stem cells to be present. There were no other observed differences to distinguish between patients whose clone size fell and those that did not. It is unlikely that eculizumab has a direct effect on clone size in hemolytic PNH. A more probable hypothesis is that the immune selection in favour of the PNH clone expires over time allowing normal hemopoietic stem cells to repopulate the bone marrow. Whether eculizumab has any influence on this rather than just allowing patients to survive and remain well until recovery occurs is not clear. Our data suggests that there needs to be some normal hematopoietic activity in order for the normal marrow cells to expand and clone size under 95% predicts for recovery. In conclusion, a significant minority of patients with PNH on eculizumab have a progressive reduction in the size of their PNH clone during therapy and in some of these patients the clone falls to a level at which eculizumab can safely be stopped. Disclosures: Kelly: Alexion Pharmaceuticals: Honoraria. Richards:Alexion Pharmaceuticals: Honoraria. Hill:Alexion: Honoraria. Hillmen:Alexion Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals When does a PNH clone have clinical significance?

Hematology ◽  
2021 ◽  
Vol 2021 (1) ◽  
pp. 143-152
Author(s):  
Daria V. Babushok
Keyword(s):  
Bone Marrow ◽  
Clinical Significance ◽  
Bone Marrow Failure ◽  
Clone Size ◽  
Complement Inhibitors ◽  
Immune Mediated ◽  
Clinical Presentations ◽  
Practical Guidelines ◽  
The Common ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired blood disease caused by somatic mutations in the phosphatidylinositol glycan class A (PIGA) gene required to produce glycophosphatidyl inositol (GPI) anchors. Although PNH cells are readily identified by flow cytometry due to their deficiency of GPI-anchored proteins, the assessment of the clinical significance of a PNH clone is more nuanced. The interpretation of results requires an understanding of PNH pathogenesis and its relationship to immune-mediated bone marrow failure. Only about one-third of patients with PNH clones have classical PNH disease with overt hemolysis, its associated symptoms, and the highly prothrombotic state characteristic of PNH. Patients with classical PNH benefit the most from complement inhibitors. In contrast, two-thirds of PNH clones occur in patients whose clinical presentation is that of bone marrow failure with few, if any, PNH-related symptoms. The clinical presentations are closely associated with PNH clone size. Although exceptions occur, bone marrow failure patients usually have smaller, subclinical PNH clones. This review addresses the common scenarios that arise in evaluating the clinical significance of PNH clones and provides practical guidelines for approaching a patient with a positive PNH result.

Increased frequency of HLA-DR2 in patients with paroxysmal nocturnal hemoglobinuria and the PNH/aplastic anemia syndrome

Blood ◽  
2001 ◽  
Vol 98 (13) ◽  
pp. 3513-3519 ◽  
Author(s):  
Jaroslaw P. Maciejewski ◽  
Dean Follmann ◽  
Ryotaro Nakamura ◽  
Yogen Saunthararajah ◽  
Candido E. Rivera ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Clinical Manifestations ◽  
Normal Incidence ◽  
Primary Diagnosis ◽  
Hla Alleles ◽  
Predictors Of Response ◽  
Pnh Clone

Abstract Many autoimmune diseases are associated with HLA alleles, and such a relationship also has been reported for aplastic anemia (AA). AA and paroxysmal nocturnal hemoglobinuria (PNH) are related clinically, and glycophosphoinositol (GPI)–anchored protein (AP)–deficient cells can be found in many patients with AA. The hypothesis was considered that expansion of a PNH clone may be a marker of immune-mediated disease and its association with HLA alleles was examined. The study involved patients with a primary diagnosis of AA, patients with myelodysplastic syndrome (MDS), and patients with primary PNH. Tests of proportions were used to compare allelic frequencies. For patients with a PNH clone (defined by the presence of GPI-AP–deficient granulocytes), regardless of clinical manifestations, there was a higher than normal incidence of HLA-DR2 (58% versus 28%; z = 4.05). The increased presence of HLA-DR2 was found in all frankly hemolytic PNH and in PNH associated with bone marrow failure (AA/PNH and MDS/PNH). HLA-DR2 was more frequent in AA/PNH (56%) than in AA without a PNH clone (37%;z = 3.36). Analysis of a second cohort of patients with bone marrow failure treated with immunosuppression showed that HLA-DR2 was associated with a hematologic response (50% of responders versus 34% of nonresponders; z = 2.69). Both the presence of HLA-DR2 and the PNH clone were independent predictors of response but the size of PNH clone did not correlate with improvement in blood count. The results suggest that clonal expansion of GPI-AP–deficient cells is linked to HLA and likely related to an immune mechanism.

Natural History of Paroxysmal Nocturnal Hemoglobinuria Clones In Patients Presenting as Aplastic Anemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4428-4428
Author(s):  
Jeffrey J Pu ◽  
Galina Mukhina ◽  
Hao Wang ◽  
William Savage ◽  
Robert A Brodsky
Keyword(s):  
Bone Marrow ◽  
Natural History ◽  
Aplastic Anemia ◽  
Median Time ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Clone Size ◽  
Hematopoietic Stem ◽  
History Of ◽  
Pnh Clone ◽  
Ldh Value

Abstract Abstract 4428 Introduction: Acquired aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) are closely related bone marrow failure disorders. Most AA results from an autoimmune attack directed against hematopoietic stem/progenitor cells. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a PIG-A mutation. The PIG-A gene mutation leads to glycosylphosphatidylinositol-anchor protein (GPI-AP) biosynthesis deficiency and subsequent hemolysis secondary to the absence of complement regulatory proteins (CD55 and CD59). Both PNH and AA can be cured by allogeneic bone marrow transplantation (alloBMT), but only a minority of patients is offered this approach due to the potential morbidity and mortality. AA can be treated with immunosuppressive therapy (IST) and PNH can be controlled by eculizumab. It has been estimated that more than 50% of AA patients harbor small, but expandable PNH populations at diagnosis. The natural history of PNH clones in AA patients following non-transplant therapy is not well studied. The purpose of this study is to determine the fate and clinical relevance of these PNH clones in patients with AA who did not receive an alloBMT. Patients and Method: Twenty-seven patients with AA and a detectable PNH clone were monitored for a median of 5.3 years (range,1.5 to 11.5 years). The PNH granulocyte clone sizes were measured using flow cytometry and analyzed via CellQuest software. PE-conjugated anti-CD15 and fluoresceinated aerolysin variant (FLAER) staining were used to define granulocytes and GPI-AP deficient cells respectively. Serum lactate dehydrogenase (LDH) value was used as a surrogate for monitoring hemolysis and 1.5× the upper limit of normal LDH value (330mg/dL) as a cut-off point to define clinically apparent hemolysis. A PNH size change <2.5% was considered as stable. Patients were treated with IST, HiCy, or both. Result: We found a linear relationship between PNH granulocyte clone size and LDH values (Pearson correlation coefficient=0.73; P<0.0001). A PNH clone size above 23% was the threshold to identify hemolysis as measured by LDH (ROC analysis with AUC=0.88). Higher LDH values over the period of follow-up were associated with larger PNH granulocyte clone size at diagnosis (P=0.03). Patients with small (≤15%) initial PNH granulocytes had lower LDH levels at 5 years after diagnosis (mean±SD: 236.9±109.9 vs 423.1±248.8; P=0.02), and were less likely to develop hemolysis (13.3% vs 55.6%, P=0.06) comparing to those with larger (>15%) initial PNH granulocytes. Of 9 patients who initially were treated with traditional IST (ATG, CsA, and prednisone), 7 did not respond to treatment and eventually received high-dose cyclophosphamide (HiCy) salvage therapy, 2 achieved a remission and did not require further treatment though one demonstrated PNH clone size expansion to 50% after 37 months. After HiCy salvage, all 7 patients became transfusion independent and 4 of them had no further PNH clone expansion. PNH clone expansion was observed in 7 of 9 patients at a median time of 3 (range: 2 to 87) months after treatment. Of 15 patients who received HiCy as initial therapy, 14 achieved remissions. Later expansion of PNH size was observed in 7 patients, of which 5 eventually required intermittent blood transfusion but only 1 developed symptomatic hemolysis necessitating eculizumab therapy. The median time to PNH granulocyte clone expansion after HiCy was 52 (range: 18 to 106) months. In 5 patients who received HiCy and then relapsed, their PNH clone size only increased (1±0.7)% in (71±31) months observation during post treatment remission; however, their PNH clone size increase accelerated to (38±14)% in (34±21) months after AA relapse (P=0.04). Two nSAA patients with an initial PNH clone size ≤15% spontaneously recovered hematopoiesis at 84 and 56 months respectively, neither had PNH clone size expansion. In this study, 25.9% patients kept a stable PNH size, 48.1% patients increased the size, and 26% patients decreased the size. The group with small initial PNH clone sizes (≤15%) was the most stable over time. Conclusion: The risk of developing clinically significant PNH over 10 years appears to be low in AA patients with PNH clones, especially for those with small initial PNH granulocyte clones (≤15%) and for those who maintain remission following therapy. Disclosures: No relevant conflicts of interest to declare.

Paroxysmal Nocturnal Hemoglobinuria Clones in Children with Acquired Aplastic Anemia: A Multicentric Study

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1269-1269
Author(s):  
Fabio Stefano Timeus ◽  
Nicoletta Crescenzio ◽  
Alessandra Doria ◽  
Luiselda Foglia ◽  
Sara Pagliano ◽  
...  
Keyword(s):  
Stem Cell ◽  
Aplastic Anemia ◽  
Immunosuppressive Therapy ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Clonal Expansion ◽  
Becton Dickinson ◽  
Clone Size ◽  
Favourable Response ◽  
Immune Mediated ◽  
Pnh Clone

Abstract Abstract 1269 Introduction. Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic disorder characterized by the clonal expansion of a PIG-A mutated stem cell and consequent defective synthesis of glycosil phosphatidyl-inositol-anchored proteins, complement-mediated hemolysis, increased incidence of thrombosis, bone marrow failure. PNH and acquired aplastic anemia (AA) are closely related and a reciprocal progression is possible. A relative resistance of the PNH stem cell to the immune-mediated damage can explain the PNH clonal expansion in AA. High resolution flow cytometry analysis (FCA) has revealed a high incidence of minor PNH clones in adult AA patients at diagnosis, predictive for some Authors of a favourable response to the immunosuppressive therapy (IST) (Maciejevki et al, 2001; Ishiyama et al, 2003; Sugimori et al, 2006). “Pure” PNH is a very rare disease in children. Only a few studies have so far evaluated longitudinally PNH clones in pediatric AA patients. Materials and Methods. Ninety AA patients diagnosed in 8 AIEOP (Italian Association of Pediatric Hematology-Oncology) Centers (age at diagnosis 1–20 years, median =10.8, 51 severe AA, 30 very severe AA, 9 non severe AA) were studied: forty-one since diagnosis, 25 during IST, 20 off therapy and 4 selected cases after hematopoietic stem cell transplantation (HSCT). Among the patients followed since diagnosis, 8 received an HLA matched sibling donor HSCT as first line therapy, whereas the other 33 patients were treated with IST according to EBMT protocols (anti-lymphocyte globulin/anti-thymocyte, ciclosporin ± granulocyte colony stimulating factor). The study started in 1998. Peripheral blood PNH cells were detected by lack of CD59 expression on granulocytes by a two-color FCA for CD59 (clone p282-FITC Becton-Dickinson) and CD11b (clone D12-PE Becton-Dickinson); at least 105 cells were analyzed, for a total of 1104 tests. The presence of a population CD11b+/CD59- > 0.15% was defined as abnormal; the cut off value was established in 1998 by evaluating 87 normal controls (PNH clones: median = 0.001%, mean+2SD=0.10%). Since 2009 FCA results were confirmed by more sensitive techniques with three or six-color sequential gating analysis for CD45/33/66b or CD45/33/15/24/14/FLAER. Results. A PNH+ clone was observed in 15 patients (36.6%) at diagnosis (clone size 0.17–10.4%), in 10 patients (40%) during IST (clone size 0.16–12.6%) and in 8 patients (40%) off-therapy (clone size 0.16–4.0%). The presence of a PNH+ clone at diagnosis did not predict a favourable response to IST, both in ALG and ATG-treated patients. In 33 patients (16 at diagnosis, 9 in IST, 8 off therapy), the presence of the PNH clone was sporadic or intermittent, whereas in 13 patients (9 at diagnosis, 3 in IST, 1 off therapy) the clone persisted for more than 3 following controls (follow up 6–60 months). Among the 26 PNH- patients at diagnosis, in 10 a PNH clone (clone size 0.16–1.7%) appeared later during IST. Among the 25 patients studied during IST, in one patient PNH clone appearance was associated with the tapering of cyclosporine (figure 1), in two with the relapse when off therapy. In one out of 4 patients treated with HSCT, a PNH clone appeared at time of relapse and disappeared after starting IST with cyclosporine (figure 2). A mild hemolysis was observed in the only 2 patients with a major PNH clone (clone size 12.6 and 10.4% respectively). No thrombotic events were reported. Conclusions. We have observed a significant incidence of minor PNH clones in pediatric AA at diagnosis, as reported in adults. Whereas previous studies in adults correlated the presence of pre-treatment minor PNH clones with a favourable response to IST, we do not confirm those observations both in the present multi-centre as in our previous single-centre study (Timeus et al, 2010), in agreement with Yoshida et al (2008) and Scheinberg et al (2010). The appearance of a PNH clone in a PNH- patient at diagnosis is described as uncommon (Sugimori et al, 2009), however in our series this was observed in 38% of previously PNH- patients. In AA the presence of PNH clones seems related to complex interactions between stem cells, immune-mediated damage and immunosuppressive therapy. A periodic screening for PNH clones in patients with AA is recommended, permitting modulation in IST, early identification of major PNH clones and prompt diagnosis of a frank PNH. Disclosures: Timeus: Alexion Pharma Italy s.r.l.: Research Funding. Dufour:Pfizer: Consultancy.

Clinical Reflection of Immunophenotyping in Paroxysmal Nocturnal Hemoglobinuria: Experience of a Single Center

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5150-5150
Author(s):  
Ugur Sahin ◽  
Mustafa Merter ◽  
Pinar Ataca ◽  
Erden Atilla ◽  
Berna Atesagaoglu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myelodysplastic Syndrome ◽  
Aplastic Anemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Treatment Modalities ◽  
Serum Lactate Dehydrogenase ◽  
Clone Size ◽  
Bone Marrow Disorders ◽  
Pnh Clone

Abstract Introduction: Flow cytometric assays (FCA), especially FLAER based methods, have become important recently in means of diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). In this retrospective study, we aimed to describe diagnostic features of PNH patients along with disease characteristics. Methods: FCA results of patients ordered for PNH diagnosis between November 2007 and July 2014 were retrospectively evaluated. FCA results were expressed as percentage of granulocytes and monocytes negative for CD55/59. The FLAER/CD24-CD14 assay has been available in our center since December 2011 and used for confirmation of FCA results since then. The distribution of erythrocyte types according to CD59 expression (type 1, 2 and 3) were also given in percentages. Results: FCA for PNH diagnosis was performed for a total of 175 patients. FLAER method was available in 136 of these assays (77.7%). A PNH clone was not detected in 136 (77.7%) of the FCAs. PNH clone free patients were diagnosed with unclassified anemia (n=34, 19.4%), other unclassified cytopenias (n=30, 17.2%), non-PNH hemolytic anemias (n=7, 4.0%), myelodysplastic syndrome (n=23, 13.1%), aplastic anemia (n=9, 5.1%), primary myelofibrosis (n=1, 0.6%), leukemia (n=7, 4.0%), lymphoma (n=3, 1.7%), thrombotic incidents (n=14, 8.0%), rheumatological disorders (n=4, 2.3%) and unknown diagnosis (n=4, 2.3%). A PNH clone was detected in 39 patients (22.3%). Of these, 27 (69.2%) constituted classical PNH and 12 (30.8%) PNH accompanying other bone marrow disorders. In the PNH clone positive group, the accompanying bone marrow disorders were aplastic anemia (n=9) and myelodysplastic syndrome (n=3). Median age at diagnosis was 31 (10-82) for classical PNH and 35 (18-77) for PNH accompanying other bone marrow disorders. Erythrocyte transfusion requirement was prominent in 48.7% (n=19) of the patients and thrombotic incidents occured in 20.5% (n=8). Leukemic transformation did not take place in any of the patients. In one patient with aplastic anemia, the FCA became positive for PNH clone within one year of follow-up. Among classical PNH patients, thrombosis was observed only among patients with a clone size of > 50%. Clone size (both for granulocyte and monocyte clones) correlated with corrected reticulocyte and serum lactate dehydrogenase levels (for granulocytes p=0.001 and p<0.001, for monocytes p=0.001 and p<0.001, respectively). Treatment modalities of the PNH clone positive patients included eculizumab (n=12, 30.8%), allogeneic stem cell transplantation (n=13, 33.3%) and none/other (CsA, steroids, ATG, danazol) (n=16, 41.0%). Conclusion: FCA is an important modality for the diagnosis and follow-up of PNH patients. Clone size might be used to assess the severity of the disease and the risk of thrombosis in classical PNH. In patients with PNH accompanying other bone marrow disorders clone sizes are generally small and complications seen in classical PNH are rare. However, aplastic anemia and myelodysplastic syndrome might evolve into PNH during the disease course, which is reflected as an increase in clone size. Thus, FCA might be used in selected patients with aplastic anemia and myelodysplastic syndrome, especially when treatment failure or unexpected complications are suspected. Disclosures No relevant conflicts of interest to declare.

scholarly journals Aplastic anemia: immunosuppressive therapy in 2010

2011 ◽  
Vol 3 (2s) ◽  
pp. 7 ◽  
Author(s):  
Antonio M. Risitano ◽  
Fabiana Perna
Keyword(s):  
Bone Marrow ◽  
Aplastic Anemia ◽  
State Of The Art ◽  
Bone Marrow Failure ◽  
Standards Of Care ◽  
Bone Marrow Failure Syndrome ◽  
Clinical Observations ◽  
Immune Mediated ◽  
Experimental Works ◽  
Current Standards

Acquired aplastic anemia (AA) is the typical bone marrow failure syndrome characterized by an empty bone marrow; an immune-mediated pathophysiology has been demonstrated by experimental works as well as by clinical observations. Immunusuppressive therapy (IST) is a key treatment strategy for aplastic anemia; since 20 years the standard IST for AA patients has been anti-thymocyte globuline (ATG) plus cyclosporine A (CyA), which results in response rates ranging between 50% and 70%, and even higher overall survival. However, primary and secondary failures after IST remain frequent, and to date all attempts aiming to overcome this problem have been unfruitful. Here we review the state of the art of IST for AA in 2010, focusing on possible strategies to improve current treatments. We also discuss very recent data which question the equality of different ATG preparations, leading to a possible reconsideration of the current standards of care for AA patients.

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