Paroxysmal Nocturnal Hemoglobinuria Type Research Articles

Ahemolytic PNH: Clinical Features of a Distinct Phenotype of Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem/progenitor cell disorder caused by PIGA mutations. All hematopoietic lineages derived from the affected clone exhibit the PNH phenotype [deficiency of glycosyl phosphatidylinositol linked membrane proteins (GPI-APs)]. Diagnosis is made by flow cytometric analysis of red blood cells (RBC), granulocytes, and monocytes. The percentage of granulocytes and monocytes with absent expression of GPI-APs is a measure of the size of the PNH clone. Typically, the percentage of GPI-AP deficient RBCs is somewhat less than that of granulocytes and monocytes due to selective destruction by complement-mediated intravascular hemolysis, manifested by high LDH, bilirubin, reticulocyte count and low haptoglobin. Herein, we report five patients with large PNH clones based on percentage of GPI-AP deficient neutrophils and monocytes, but with absent or near absent PNH RBCs and no biochemical evidence of hemolysis. We call this unique disease subtype, ahemolytic PNH. Methods: The medical records of PNH patients (174 patients with PNH granulocytes >20%) at the University of Texas Southwestern, Cleveland Clinic Foundation, and National Institutes of Health from 2000 to 2022 were examined. Information pertaining to their clinical, laboratory, and molecular attributes (NGS sequencing) was collected. Five patients with granulocyte clones >50% and RBC clones <5% were identified (Table). Results: Patient 1 is a 63-year-old male with a remote history of non-severe aplastic anemia (AA) diagnosed at 11 years old. The first PNH flow cytometry, obtained at age 61 years, showed 0.02% type II PNH RBC, 0.5% type III PNH RBC, 96% PNH monocyte and 97% PNH granulocytes (Table). There was no biochemical evidence of hemolysis at the time of diagnosis or in the preceding 5 years. Patient 2 is a 50-year-old male who presented with pancytopenia and was diagnosed with acute myeloid leukemia with myelodysplastic-related mutations. Flow cytometry showed 85% PNH monocytes and 59% PNH with no detectable PNH RBCs and no biochemical evidence of hemolysis at diagnosis or in 3 years of follow-up (Table). Patient 3 is a 73-year-old female who presented with pancytopenia and marrow aplasia with unremarkable morphology, and a PNH granulocyte clone of 93.6% with 1.3% type III PNH RBCs and 1.3% and 0.5% type II PNH RBCs, with no evidence of hemolysis (Table). Patient 4 is a 21-year-old female who presented with Budd-Chiari syndrome. Flow cytometry showed 73% PNH granulocytes with 3.6% type III PNH RBCs and 1.8% type II PNH RBCs. There was no biochemical evidence of hemolysis, but because of thrombosis, she was treated complement inhibitor therapy (Table). Patient 5 is a 61-year-old female with history of severe aplastic anemia treated with immunosuppression (IST) and eltrombopag. PNH flow cytometry showed PNH granulocytes of 92.4% and 2.1% PNH type II RBCs 2.1% post IST treatment with no biochemical evidence of hemolysis (Table). Conclusion: Herein, we present five patients with a distinct PNH phenotype that we call ahemolytic PNH. Except for the absence or near absence of PNH erythrocytes, this entity shares clinical and pathophysiological features with classic PNH, including thrombophilia (Budd-Chiari Syndrome, Table). The basis of this phenotype is speculative but may be determined by the genotype of the affected HSPC in which the somatic mutation of PIGA first occurred. According to this hypothesis, somatic mutations other than PIGA skew differentiation toward granulocyte/monocyte lineages. The International PNH Interest Group recognizes the following three categories of PNH: classic PNH, PNH in the setting of another define bone marrow abnormality, and subclinical PNH. Like ahemolytic PNH, subclinical PNH has no biochemical evidence of hemolysis. In this case, however, the absence of hemolysis is due to the small size of the PNH clone (median clone size <1%) as determined by flow cytometry analysis of peripheral blood granulocytes and monocytes. We propose that ahemolytic PNH, defined as patients having less than <5% PNH red cells and >50% PNH granulocytes/monocytes, be recognized as a distinct category of PNH.

A Nationwide Survey of Lower Risk Myelodysplastic Syndromes in Japan

A Nationwide Survey of Lower Risk Myelodysplastic Syndromes in Japan

Complement Blockade With a C1 Esterase Inhibitor In Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, clonal, hematopoietic stem cell disorder that manifests with a complement-mediated hemolytic anemia, bone marrow failure and a propensity for thrombosis. PNH confers deficiency of GPI anchored proteins that protect erythrocytes at different stages of the complement cascade. CD55 regulates the formation and stability of the C3 and C5 convertases, whereas CD59 blocks the formation of the membrane attack complex (MAC). These patients experience both intra- and extravascular hemolysis due to complement activation. Chronic treatment with eculizumab results in sustained control of intravascular hemolysis leading to transfusion independence for many. Eculizumab compensates for the CD59 deficiency on PNH erythrocytes; however, it does not compensate for the CD55 deficiency. PNH patients receiving eculizumab accumulate C3 on their erythrocytes; this leads to opsonization and extravascular hemolysis. There remains an unmet clinical need for a complement inhibitor with activity early in the complement cascade, which would not only reduce C3 accumulation on PNH erythrocytes, but also inhibit C3 activation by the alternative pathway (APC).C1 esterase inhibitor (C1 INH) is an endogenous human plasma protein that has broad inhibitory activity in the complement pathway. C1 INH inhibits the classical pathway of the complement system by binding C1r and C1s and inhibits the mannose-binding lectin-associated serine proteases in the lectin pathway. Thus, C1 INH is an ideal therapeutic for diseases of complement pathways. Nanofiltered plasma derived C1 INH (Cinryze®; ViroPharma) is FDA approved for routine prophylaxis against angioedema attacks in patients with hereditary angioedema (HAE), a disease characterized by constitutional deficiency or dysfunction of endogenous C1 INH. We hypothesized that this plasma-derived C1 INH would inhibit C3 deposition and the APC on PNH erythrocytes in patients treated with eculizumab.Peripheral blood of 3 healthy donors and 5 PNH patients (all with PNH type III erythrocyte proportion >5%) treated with eculizumab were obtained with written informed consent through a protocol approved by the local institutional review board. Clinical parameters for persistent extravascular hemolysis were noted at the time of patient sampling. Commercial vials of C1 INH were used for C1 inhibition assays ex vivo. Hemolysis experiments with acidified serum were performed in erythrocytes from PNH patients and healthy donors. We were able to show in the PNH patients that C1 inhibition is concentration dependent with increasing inhibition by serial dilutions of C1 INH up to 12 U/mL. The amount of hemolysis correlates with the percentage of CD55 deficient erythrocytes, but is attenuated by C1 INH in all patients. Flow cytometry was used to analyze deposition of C3 activation fragments on intact and lysed PNH erythrocytes. At time 0, small amounts of C3 deposition were observed on the PNH erythrocytes, but not the normal erythrocytes. The amount of C3 deposition was increased after incubation in acidified serum; however, co-incubation of C1 INH in the acidified serum markedly attenuated the amount of C3 deposition on the CD55 deficient erythrocytes. This demonstrated that C1 INH blocks APC-medicated deposition of C3 on the PNH erythrocytes in PNH patients.In this study, we show that the commercially available plasma derived C1-INH (Cinryze®) prevents lysis induced by the APC in PNH erythrocytes. The results presented here show that plasma-derived C1 INH can inhibit both APC- mediated hemolysis and C3 deposition. Importantly, C1-INH was able to block the accumulation of C3 degradation products on CD55 deficient erythrocytes from PNH patients who were being treated with eculizumab. This suggests a role for inhibition of earlier phases of the complement cascade than currently inhibited by eculizumab. A clinical trial to explore this hypothesis in vivo with patients who are suboptimal responders to eculizumab could be considered. Disclosures:Off Label Use: A nanofiltered plasma derived C1 INH (Cinryze®; ViroPharma) is FDA approved for routine prophylaxis against angioedema attacks in adolescent and adult patients with hereditary angioedema. Uknis:ViroPharma, Incorporated: Employment. Saye:ViroPharma, Incorporated: Employment. Brodsky:Alnylam Pharmaceuticals : Consultancy; Prolong Pharmaceuticals : Consultancy; ViroPharma, Incorporated: Research Funding; Alexion Pharmaceuticals : Membership on an entity's Board of Directors or advisory committees.

BCX9930, a Potent, Selective, Oral Factor D Inhibitor, Demonstrates Proof-of-Concept As Monotherapy in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

BCX9930, a Potent, Selective, Oral Factor D Inhibitor, Demonstrates Proof-of-Concept As Monotherapy in Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH)

Properdin Is a Key Player in Lysis of Red Blood Cells and Complement Activation on Endothelial Cells in Hemolytic Anemias Caused by Complement Dysregulation.

The complement system alternative pathway (AP) can be activated excessively in inflammatory diseases, particularly when there is defective complement regulation. For instance, deficiency in complement regulators CD55 and CD59, leads to paroxysmal nocturnal hemoglobinuria (PNH), whereas Factor H mutations predispose to atypical hemolytic uremic syndrome (aHUS), both causing severe thrombohemolysis. Despite eculizumab being the treatment for these diseases, benefits vary considerably among patients. Understanding the molecular mechanisms involved in complement regulation is essential for developing new treatments. Properdin, the positive AP regulator, is essential for complement amplification by stabilizing enzymatic convertases. In this study, the role of properdin in red blood cell (RBC) lysis and endothelial cell opsonization in these AP-mediated diseases was addressed by developing in vitro assays using PNH patient RBCs and human primary endothelial cells, where the effects of inhibiting properdin, using novel monoclonal antibodies (MoAbs) that we generated and characterized, were compared to other complement inhibitors. In in vitro models of PNH, properdin inhibition prevented hemolysis of patient PNH type II and III RBCs more than inhibition of Factor B, C3, and C5 (>17-fold, or >81-fold, or >12-fold lower molar IC90 values, respectively). When tested in an in vitro aHUS hemolysis model, the anti-properdin MoAbs had 11-fold, and 86-fold lower molar IC90 values than inhibition of Factor B, or C3, respectively (P < 0.0001). When comparing target/inhibitor ratios in all hemolysis assays, inhibiting properdin was at least as efficient as the other complement inhibitors in most cases. In addition, using in vitro endothelial cell assays, the data indicate a critical novel role for properdin in promoting complement activation on human endothelial cells exposed to heme (a hemolysis by-product) and rH19-20 (to inhibit Factor H cell-surface protection), as occurs in aHUS. Inhibition of properdin or C3 in this system significantly reduced C3 fragment deposition by 75%. Altogether, the data indicate properdin is key in promoting RBC lysis and complement activation on human endothelial cells, contributing to the understanding of PNH and aHUS pathogenesis. Further studies to determine therapeutic values of inhibiting properdin in complement-mediated diseases, in particular those that are characterized by AP dysregulation, are warranted.

CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria.

The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

Case Report: Paroxysmal nocturnal hemoglobinuria in a woman heterozygous for G6PD A-

We describe a case of paroxysmal nocturnal hemoglobinuria (PNH) in a woman who is heterozygous for the glucose-6-phosphate dehydrogenase A- ( G6PDA-) allele. PNH is associated with one or more clones of cells that lack complement inhibition due to loss of function somatic mutations in the PIGA gene. PIGA encodes the enzyme phosphatidylinositol glycan anchor biosynthesis, class A, which catalyses the first step of glycosylphosphatidylinisotol ( GPI) anchor synthesis. Two GPI anchored red cell surface antigens regulate complement lysis. G6PD catalyses the first step of the pentose phosphate pathway and enzyme variants, frequent in some populations have been because they confer resistance to malaria, are associated with hemolysis in the presence of oxidizing agents including several drugs. The patient had suffered a hemolytic attack after taking Bactrim, a drug that precipitates hemolysis in G6PD deficient individuals. Since both G6PD and PIGA are X-linked we hypothesized that the PIGA mutation was on the X-chromosome carrying the G6PDA- allele. Investigations showed that in fact the PIGA mutation was on the X-chromosome carrying the normal G6PD B allele. We speculate that complement activation on G6PD A- red cells exposed to Bactrim might have triggered complement activation inducing the lysis of G6PD B PNH Type II red blood cells or that the patient may have had a PNH clone expressing G6PDA- at the time of the hemolytic episode.

A Novel Evidence That PNH Affected Cells Residing in Bone Marrow (BM) Due to Impaired Incorporation of CXCR4 and VLA-4 Into Membrane Lipid Rafts Show Defective SDF-1- and VCAM-1-Mediated Retention in BM What Leads to Their Increased Motility and Impaired Interaction with the BM Stem Cell Niches

AbstractAbstract 1256 Background.Hematopoietic stem progenitor cells (HSPCs) are retained in bone marrow (BM) niches due to the stromal-derived growth factor-1 (SDF-1)–CXCR4 receptor axis and interactions between Very Late Antigen-4 (VLA-4 or a4b1integrin) and its ligand, Vascular Adhesion Molecule-1 (VCAM-1 or CD106). While HSPCs express CXCR4 and VLA-4, their corresponding ligands, SDF-1 and VCAM-1, are expressed by cells in the BM microenvironment (e.g., osteoblasts and fibroblasts). Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired hemolytic anemia that results from the expansion of hematopoietic stem cells with a mutation in one of the enzymes (PIG-A) responsible for glycosylphosphatidylinositol (GPI anchor) biosynthesis, which is a post-translation modification of proteins associated with lipid rafts on the cell membrane surface. Some of these proteins are involved in the resistance of erythrocytes to lysis by the final product of complement cascade (CC) activation, C5b-C9, also known as the membrane attack complex (MAC). Furthermore, as we reported, the CXCR4 receptor, which binds a-chemokine stromal derived factor-1 (SDF-1) is also associated with lipid rafts (Blood 2005;105:40–8) similarly as VLA-4 (a4b1integrin). Recently we demonstrated that the bioactive lipid sphingosine-1-phosphate (S1P), which is a major chemoattractant directing egress of HSPCs from bone marrow (BM) into peripheral blood (PB) during mobilization, is released from lyzed erythrocytes in C5b-C9/MAC-dependent manner (Leukemia 2010;24:976–85). We also demonstrated that PNH affected HSPCs are preferentially mobilized into PB which suggests their defective retention in BM microenvironment (Leukemia 2012;26:1722). Hypothesis.To explain preferential mobilization of PNH patients HSPCs into PB we hypothesized that BM residing HSPCs may have a defective adhesion in BM niches due to defect in lipid raft formation that are required for a proper function of CXCR4-SDF-1 and VLA-4-VCAM-1 retention axes. Experimental strategies.To address this issue we employed for adhesion, chemotaxis and lipid raft formation assays PIG-A mutated human K-562 and TF-1 cells and more importantly BM MNC derived from PNH patients (n=3). PNH affected HSPCs were sorted by FACS as CD34+FLAER− and normal unaffected HSPCs as CD34+FLAER+cell population. Using these cells we studied their i) adhesiveness to immobilized SDF-1 (CXCR4 ligand), VCAM-1 (VLA-4 ligand), fibronectin and BM stroma cells, ii) chemotactic responsiveness in Transwell assays to SDF-1 gradient, iii) ability to incorporate CXCR4 and VLA-4 receptors into membrane lipid rafts and iv) activation of signaling pathways relevant to cell adhesion/migration (MAPKp42/44 and AKT) in response to SDF-1 stimulation. Results.We found that both PIG-A mutated human K-562 and TF-1 cells and more important CD34+FLAER− HSPCs derived from BM of PNH patients in contrast to normal CD34+FLAER+HSPCs show defective adhesion to immobilized SDF-1, VCAM-I, fibronectin and BM stroma cells. This corresponded with weak responsiveness of these cells in chemotactic assays to SDF-1 gradient and weaker phosphorylation of MAPK042/44 and AKT after SDF-1 stimulation. Finally, direct confocal microscopy studies demonstrated defect in incorporation of CXCR4 and VLA-4 into membrane lipid rafts in CD34+FLAER− cells isolated from BM of PNH patients. Conclusions.Based on these observations, we propose a novel view of the pathogenesis of PNH and the expansion of PNH-affected cells in BM microenvironment. Accordingly, the lack of PIG-A protein, which plays an important role in lipid raft formation a proper function of CXCR4 and VLA-4, confers an adhesion defect and an advantage to PNH-affected HSPCs, which become more mobile. These PNH-mutated HSPCs over time may outcompete normal HSPCs for their niches in BM, due to their increased motility, and contribute to the PNH type of hematopoiesis. Defective retention of PNH affected HSPCs and they proper interaction with stem cell niches in BM may lead with time in some of the cases to their malignant transformation. We propose also that drugs that increase BM level of S1P and thus prevent egress of PNH HSPCs from their niches could be employed in PNH treatment along with CC inhibitors (Eculizumab). Disclosures:Maciejewski:NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding.

A Novel View of Paroxysmal Nocturnal Hemoglobinuria (PNH) Pathogenesis: Do Pathologic PNH Hematopoietic Stem/Progenitor Cells (HSPCs) Displace Normal HSPCs From Their Niches in Bone Marrow Because They Are More Motile Due to Defective Adhesion and Enhanced Migratory Properties?

Abstract 732 Background. Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon acquired hemolytic anemia that results from the expansion of hematopoietic stem cells with a mutation in one of the enzymes (PIG-A) responsible for glycosylphosphatidylinositol (GPI anchor) biosynthesis, which is a post-translation modification of proteins associated with lipid rafts on the cell membrane surface. Some of these proteins are involved in the resistance of erythrocytes to lysis by the final product of complement cascade (CC) activation, C5b-C9, also known as the membrane attack complex (MAC). As we reported, the CXCR4 receptor, which binds a-chemokine stromal derived factor-1 (SDF-1) in regulating the trafficking of hematopoietic stem/progenitor cells (HSPCs), is also associated with lipid rafts (Blood 2005;105:40-8). In addition, we recently demonstrated that the bioactive lipid sphingosine-1-phosphate (S1P), which is a major chemoattractant directing egress of HSPCs from bone marrow (BM) into peripheral blood (PB) during mobilization, is released from erythrocytes by C5b-C9/MAC (Leukemia 2010;24:976-85). Hypothesis. Based on this finding, we hypothesized that HSPCs are continuously mobilized from the BM of PNH patients due to the susceptibility of PIG-A-deficient erythrocytes to CC activation, which elevates the free S1P level in plasma, as well as to their defective adhesion in the BM microenvironment due to impaired lipid raft formation. Experimental strategies. To address this hypothesis, peripheral blood mononuclear cells (PBMNC) were isolated from 6 PNH patients and stained with the fluorescent variant of aerolysin (FLAER), which binds GPI anchor and thus identifies normal, but not PNH, cells in FACS analysis. PNH patient-derived cells were tested for i) the level of CD34 antigen expression, ii) chemotaxis in response to SDF-1 and S1P, and iii) adhesion to fibronectin and bone marrow stromal cells. Results. We observed in PNH patients ∼3-fold higher expression of CD34 antigen on FLAER– cells circulating in PB than FLAER+ cells, which suggests that PNH-mutated HSPCs are preferentially released/mobilized into PB. Next, in Transwell chemotaxis assays followed by in vitro clonogenic assays with cells collected from the lower Transwell chambers, we observed that FLAER– cells responding to SDF-1 are ∼20 times more enriched in migrating clonogenic BFU-E and CFU-GM progenitors than their normal FLAER+ counterparts. Moreover, in parallel experiments, FLAER– CFU-GM that were plated over BM-derived fibroblasts or fibronectin in the presence of SDF-1 and S1P (known activators of VLA-4–VCAM-1-mediated cell adhesion) exhibited impaired adhesion in comparison to normal FLAER+ CFU-GM cells. Conclusions. Based on these observations, we propose a novel view of the pathogenesis of PNH and the expansion of PNH-affected cells in BM. Accordingly, the lack of PIG-A protein, which plays an important role in lipid raft formation, confers an advantage to PNH-affected HSPCs, which become more mobile. These cells are preferentially mobilized into PB in response to S1P released from C5b-C9/MAC-lysed erythrocytes. Thus, PNH-mutated HSPCs over time may outcompete normal HSPCs for their niches in BM, due to their increased motility, and contribute to the PNH type of hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Identification and Clinical Significance of Type II Granulocytes Among Patients with Paroxysmal Nocturnal Hemoglobinuria (PNH) Identified Using Multiparameter High-Sensitivity Flow Cytometry.

Abstract 3015Poster Board II-991PNH is a hematopoietic stem cell disorder in which unregulated activation of terminal complement leads to impaired quality of life and significant ischemic morbidities with shortened lifespan. Life-threatening thromboembolism (TE) is the most feared complication of PNH, accounting for 45% of patient deaths. Thrombosis has been observed in PNH patients regardless of the level of hemolysis. Additionally, platelet activation with subsequent consumption and thrombocytopenia are observed more often in PNH patients at risk for thrombosis.Current laboratory PNH diagnostic methods rely on flow cytometry to characterize PNH clones. PNH granulocytes (Gran) are typically detected using antibodies to a variety of GPI-linked markers including CD55, CD59, CD16, CD24, and CD66b. Recently, FLAER, a fluorescent proaerolysin variant that binds directly to the GPI anchor, has been used to identify and quantify GPI-deficient WBCs at a very high level of sensitivity. Although these markers are well established to detect granulocytes with normal expression of GPI proteins (Type I cells) and complete loss of GPI proteins (Type III cells), less is known about their ability to detect granulocytes with partial loss of GPI proteins (Type II cells). The ability to detect both PNH Type II RBCs and WBCs would provide clinically important information since quantitation of only PNH RBC clones can be confounded by transfusion or hemolysis.We evaluated 2,921 consecutive patient peripheral blood samples submitted for PNH diagnostic testing with a high-sensitivity flow cytometry assay for granulocytes that includes the fluorescent proaerolysin variant (FLAER) with confirmatory lineage-specific antibodies to GPI-linked antigens to distinguish Type I, II and III Gran clones. In addition, standard CD235/CD59 analysis was performed on the RBCs and evaluation with FLAER, CD14 and lineage-specific antibodies was performed on the monocytes. 216 patient samples (7.4%) had a detectable PNH gran clone (≥ 0.01% PNH Type III granulocytes and an absolute count of at least 50 cells). Clinical information was available for 162 of these patients (Table I). Of these samples, nineteen (8.8 %) patients demonstrated a distinct Type II Gran population, ranging in size from 1.2 – 65% (median clone size = 7%). In 4/19 patients, this Type II Gran population represented >50% of the total (Type II + Type III) PNH cells. In 10/19 patients (53%), a type II monocyte population was identified. Evaluation of the granulocyte markers (Table II) showed that the type II gran population was detectable in all cases by FLAER and in decreasing percentage by CD66b (88%), CD55 (50%), CD24 (47%) and CD16 (0%). Patients with Type II Gran clones had a significantly larger median total Gran PNH clone size than those without Type II Gran clones (87% vs. 11%; p= 0.0003), as well as larger median Type II and Type III RBC clones, likely a reflection of the ability to detect type II gran PNH clones with overall larger PNH clone sizes. Patients with Type II Gran clones showed significantly lower median platelet (plt) counts (54 ×109/L) than patients without Type II Gran clones (116×109/L; p< 0.01). Patients with Type II Gran clones had similar peripheral WBC, peripheral RBC, absolute neutrophil count, and hemoglobin (Hgb) compared to patients without Type II Gran clones, suggesting that differences in platelet counts are likely not due to differences in underlying marrow blood cell production. [Display omitted] Type II PNH cells are an important component of the PNH diagnostic evaluation and both RBC and Gran Type II clones should be enumerated. In a large population of patients tested for the presence of PNH clones using a high sensitivity flow cytometry assay, a significant proportion of patients were identified with Type II PNH Gran clones. This study identified FLAER as the best reagent to identify type II Gran PNH clones and showed CD16 was least useful. This study also identified a clinical association between the presence of significant Type II clones and thrombocytopenia, potentially indicative of terminal complement-mediated platelet consumption. These findings are consistent with an increased risk of thrombosis in patients with significant Type II PNH clones. Disclosures:No relevant conflicts of interest to declare.

Autoantibodies Specific to hnRNP K: A New Diagnostic Marker for Immune Pathophysiology in Bone Marrow Failure Syndromes.

The pathophysiology of bone marrow failure syndromes (BMS) such as aplastic anemia (AA) and refractory anemia (RA) of myelodysplastic syndromes defined by the FAB classification involves immune mediated bone marrow injury and intrinsic defects of hematopoietic stem cells. The diagnosis of the immune pathophysiology is essential in providing the optimal treatment to patients with BMS. In order to identify a new diagnostic marker for the immune pathophysiology of BMS, the sera of AA patients characterized by the presence of small populations of paroxysmal nocturnal hemoglobinuria-type (PNH) cells were screened for the presence of antibodies (Abs) recognizing proteins derived from a megakaryocytic leukemia cell line, UT-7. Two-dimensional electrophoresis of UT-7 lysates followed by immunoblotting revealed a distinct spot of 65 kDa. Peptide mass fingerprinting identified this protein as a heterogeneous nuclear ribonucleoprotein (hnRNP) K. Although hnRNP K is known to be ubiquitously expressed, the expression levels based on a Western blotting analysis were greater in several leukemia cell lines such as UT-7, K562 and OUN-1 in comparison to peripheral blood mononuclear cells. When the sera of newly diagnosed 87 BMS patients (62 with AA and 25 with RA) were examined for the presence of anti-hnRNP K Abs using an enzyme-linked immunosorbent assay (ELISA) with recombinant hnRNP K protein, significantly higher titers of anti-hnRNP K Abs were detected in 13 (34%, 10 with AA and 3 with RA) of 38 patients with BMS displaying increased PNH-type cells (PNH+) and in 15 (31%, 8 with AA and 7 with RA) of 49 patients without increased PNH-type cells. The presence of hnRNP K Abs was significantly correlated with the presence of anti-diazepam-binding inhibitor-related protein-1 (DRS-1) Abs (r=0.7206) and anti-moesin Abs (r=0.8318). Of the 87 patients, 49 patients (39 with AA and 10 with RA) received antithymocyte globulin plus cyclosporine therapy after the Ab screening. All of the 22 patients with high anti-hnRNP K Ab titers responded to therapy while 10 (37%) of 27 patients without the high titer Abs responded (P < 0.005). When the three Ab titers were assessed, the response rate to immunosuppressive therapy in patients showing high titers in at least one Ab was 96%, while the response rate in patients not showing high titers in any of the auto-Abs was 39% (P < 0.005). These findings suggest that in patients with immune mediated BMS, a breakdown of immune tolerance to multiple self antigens including hnRNP K may take place and that the detection of these auto-Abs may help diagnose the immune pathophysiology of BMS.

Sensitive Detection of Survival of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Red Blood Cells, Granulocytes and Monocytes by High Resolution Multiparameter Flow Cytometry.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is an acquired clonal stem cell disorder characterized by intravascular hemolysis due to GPI-deficient red blood cells sensitive to complement-mediated lysis. Accurate and sensitive detection of PNH-type cells has become important not only to diagnose PNH but also because studies have shown PNH-type cells may indicate favorable response to therapy and favorable prognosis in patients with aplastic anemia and myelodysplastic syndrome. Previous studies have suggested optimal testing for PNH-type cells by flow cytometry should be limited to within 48 hours after collection of whole blood. Our laboratory has developed a very sensitive and specific high resolution flow cytometric method for detecting PNH-type cells based on testing over 3,000 patients with known PNH, aplastic anemia, myelodysplastic syndromes and other bone marrow failure syndromes. The aim for this study was to determine the longevity of PNH clones in whole blood samples, the day-to-day variability of these clones and the rate of deterioration of the PNH clones compared to normal blood cells. We analyzed 10 whole blood samples from patients known to have PNH-type cells on seven consecutive days utilizing a two-color assay with GPA-CD59 for the red blood cells, a 5-color assay with FLAER-CD24-CD14-CD15-CD45 for the granulocytes and a 5 color assay with FLAER-CD33-CD14-CD64-CD45 for the monocytes. The results are summarized in the table below. The initial PNH clone sizes ranged from 0.02% to 90.8%. The PNH cells showed an overall similar level of deterioration to the normal blood cells with even minor PNH clones of 0.02% able to be detected at day 7. The day-to-day variability of PNH clone sizes was generally less than 10%, with smaller clone sizes showing a higher degree of variation, up to 20%, due to their smaller absolute numbers. Interestingly, Type III PNH red blood cells showed slightly better overall survival than normal red blood cells and were detected in modestly increasing percentages throughout the study. Based on this data, we propose that accurate detection of PNH type cells can be achieved up to seven days after collection of whole blood when utilizing high resolution flow cytometry. PNH Clone Size on Sequential Days as Percentage of Original PNH Clone SizeOriginal PNH Clone SizesPNH Clone Sizes as Percentage of Original PNH Clone SizeCell TypeDay 1Day 2Day 3Day 4Day 5Day 6Day 7Type III RBCs.02%–58.6%102%106%107%104%108%103%Granulocytes.29%–90.8%100%100%93%89%79%86%Monocytes.52%–89.9%96%96%92%94%97%85%

Paroxysmale nächtliche Hämoglobinurie – Folgen eines fehlenden Ankers

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematological disorder characterized by the clonal expansion and differentiation of a multi-potent hematopoietic stem cell carrying a somatic mutation in the X-linked PIG-A gene. As a consequence of this mutation, glycosylphosphatidylinositol (GPI)-anchored proteins are lacking on the surface of blood cells derived from the mutated stem cell. This may result clinically in hemolytic anemia and a tendency for venous thrombosis and serious infection. Bone marrow failure is a frequently observed phenomenon associated with PNH. Reliable diagnosis of PNH is currently best achieved by flow cytometric analysis of GPI-anchored proteins on peripheral blood cells. Both the clinically relevant size of the PNH clone and type of GPI deficiency (complete or partial) can be reproducibly determined by this method. Most patients will benefit from supportive measures, albeit that allogeneic hematopoietic stem cell transplantation is currently considered the only potentially curative therapy. The development of a new therapeutic monoclonal antibody that reduces intravascular hemolysis and progress in diagnostic flow cytometry using a new GPI-specific marker may provide further benefit for PNH patients in the future.

Diazepam-binding inhibitor-related protein 1: a candidate autoantigen in acquired aplastic anemia patients harboring a minor population of paroxysmal nocturnal hemoglobinuria–type cells

AbstractTo identify candidate antigens in aplastic anemia (AA), we screened proteins derived from a leukemia cell line with serum of an AA patient and identified diazepam-binding inhibitor-related protein 1 (DRS-1). Enzyme-linked immunosorbent assay (ELISA) revealed high titers of anti–DRS-1 antibodies (DRS-1 Abs) in 27 (38.0%) of 71 AA patients displaying increased paroxysmal nocturnal hemoglobinuria (PNH)–type cells (PNH+), 2 (6.3%) of 32 PNH– AA patients, 5 (38.5%) of 13 PNH+ myelodysplastic syndrome (MDS) patients, and none of 42 PNH– MDS patients. DRS-1 gene was abundantly expressed in myeloid leukemia cell lines and in CD34+ cells derived from healthy individuals. Stimulation of T cells from an AA patient displaying high DRS-1 Abs with a putative CD4+ T-cell epitope (amino acid residues [aa's] 191-204) presented by HLA-DR15, which overlapped with a hot spot (aa's 173-198) of DRS-1 Ab epitopes, gave rise to T cells cytotoxic for L cells (murine fibroblasts) that were transfected with DRB1*1501 and DRS-1. Enzyme-linked immunospot assay demonstrated increased frequency of T-cell precursors specific to the DRS-1 peptide in other HLA-DR15+ AA patients displaying high DRS-1 Ab titers. These findings indicate that DRS-1 may serve as an autoantigen eliciting immune attack against hematopoietic stem cells in a subset of AA patients characterized by increased PNH-type cells.

Polyclonal hematopoiesis maintained in patients with bone marrow failure harboring a minor population of paroxysmal nocturnal hemoglobinuria–type cells

AbstractAlthough a minor population of paroxysmal nocturnal hemoglobinuria (PNH)–type blood cells is often detected in patients with aplastic anemia (AA) and refractory anemia (RA), the significance of such cells in the pathophysiology of bone marrow (BM) failure remains obscure. We therefore examined clonality in peripheral blood granulocytes from 118 female patients with AA or myelodysplastic syndrome using the X chromosome inactivation pattern. Clonality, defined as a clonal population accounting for 35% or more of total granulocytes, was confirmed in 22 of 68 (32.4%) AA patients, in 13 of 44 (29.5%) RA patients, in all 4 RA with excess blasts (RAEB) patients, and in 4 patients with PNH. When the frequency of patients with granulocyte clonality was compared with respect to the presence of increased PNH-type cells, the frequency was significantly lower in AA patients with (PNH+; 21.2%) than without (PNH–; 42.9%) increased numbers of PNH-type cells (P= .049). Clonality was absent in granulocytes from the 15 PNH+RA patients but present in 13 of 29 (44.8%) PNH–RA patients (P= .0013). The absence of clonality in AA and RA patients before treatment was strongly associated with positive response to immunosuppressive therapy (without clonality, 74.4%; with clonality, 33.3%;P= .0031) in all patients as well as in PNH+patients (without clonality, 96.2%; with clonality, 66.6%,P= .026). These results suggest that AA and RA with a minor population of PNH-type cells are benign types of BM failure with immune pathophysiology that have little relationship to clonal disorders such as RAEB or acute myeloid leukemia.

Clinical significance of a minor population of paroxysmal nocturnal hemoglobinuria–type cells in bone marrow failure syndrome

A minor population of blood cells deficient of glycosylphosphatidylinositol (GPI)-anchored membrane proteins is often detected in patients with aplastic anemia (AA), though the clinical significance of such paroxysmal nocturnal hemoglobinuria (PNH)-type cells remains unclear. To clarify this issue, we studied 164 patients with myelodysplastic syndrome (MDS) for the presence of CD55(-)CD59(-) granulocytes and red blood cells using sensitive flow cytometry. Among the different subgroups of MDS, a significant increase (ie, at least 0.003%) of PNH-type cells was detected in 21 of 119 patients with refractory anemia (RA); this frequency (17.6%) of RA patients with increased PNH-type cells (PNH(+) patients) was much lower than what we previously reported (52.0%) for AA patients. PNH(+) RA patients had distinct clinical features compared with RA patients without increased PNH-type cells (PNH(-) patients), such as less pronounced morphologic abnormality of blood cells, more severe thrombocytopenia, lower rates of karyotypic abnormality (4.8% vs 32.8%) and of progression to acute leukemia (0% vs 6.2%), higher probability of response to cyclosporine therapy (77.8% vs 0%), and higher incidence of HLA-DR15 (90.5% vs 18.5%). These data indicate that the presence of a minor population of PNH-type cells suggests a benign type of bone marrow failure, probably caused by an immunologic mechanism. To choose an appropriate therapy, peripheral blood should be tested using sensitive flow cytometry for the presence of PNH-type cells in all patients with bone marrow failure before treatment.

GATA1-Cre mediates Piga gene inactivation in the erythroid/megakaryocytic lineage and leads to circulating red cells with a partial deficiency in glycosyl phosphatidylinositol–linked proteins (paroxysmal nocturnal hemoglobinuria type II cells)

Patients with paroxysmal nocturnal hemoglobinuria (PNH) have blood cells deficient in glycosyl phosphatidylinositol (GPI)-linked proteins owing to a somatic mutation in the X-linked PIGA gene. To target Piga recombination to the erythroid/megakaryocytic lineage in mice, the Cre/loxP system was used, and Cre was expressed under the transcriptional regulatory sequences of GATA-1. Breeding of GATA1-cre (G) transgenic mice with mice carrying a floxed Piga (L) allele was associated with high embryonic lethality. However, double-transgenic (GL) mice that escaped early recombination looked healthy and were observed for 16 months. Flow cytometric analysis of peripheral blood cells showed that GL mice had up to 100% of red cells deficient in GPI-linked proteins. The loss of GPI-linked proteins on the cell surface occurred late in erythroid differentiation, causing a proportion of red cells to express low residual levels of GPI-linked proteins. Red cells with residual expression of GPI-linked proteins showed an intermediate sensitivity toward complement and thus resemble PNH type II cells in patients with PNH. Recombination of the floxed Piga allele was also detected in cultured megakaryocytes, mast cells, and eosinophils, but not in neutrophils, lymphocytes, or nonhematopoietic tissues. In summary, GATA1-Cre causes high-efficiency Piga gene inactivation in a GATA-1-specific pattern. For the first time, mice were generated that have almost 100% of red cells deficient in GPI-linked proteins. These animals will be valuable to further investigate the consequences of GPI-anchor deficiency on erythroid/megakaryocytic cells.

Resistance of Paroxysmal Nocturnal Hemoglobinuria Cells to the Glycosylphosphatidylinositol-Binding Toxin Aerolysin

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal stem cell disorder caused by a somatic mutation of the PIGA gene. The product of this gene is required for the biosynthesis of glycosylphosphatidylinositol (GPI) anchors; therefore, the phenotypic hallmark of PNH cells is an absence or marked deficiency of all GPI-anchored proteins. Aerolysin is a toxin secreted by the bacterial pathogen Aeromonas hydrophila and is capable of killing target cells by forming channels in their membranes after binding to GPI-anchored receptors. We found that PNH blood cells (erythrocytes, lymphocytes, and granulocytes), but not blood cells from normals or other hematologic disorders, are resistant to the cytotoxic effects of aerolysin. The percentage of lysis of PNH cells after aerolysin exposure paralleled the percentage of CD59+ cells in the samples measured by flow cytometry. The kinetics of red blood cell lysis correlated with the type of PNH erythrocytes. PNH type III cells were completely resistant to aerolysin, whereas PNH type II cells displayed intermediate sensitivity. Importantly, the use of aerolysin allowed us to detect PNH populations that could not be detected by standard flow cytometry. Resistance of PNH cells to aerolysin allows for a simple, inexpensive assay for PNH that is sensitive and specific. Aerolysin should also be useful in studying PNH biology.

Both Paroxysmal Nocturnal Hemoglobinuria (PNH) Type II Cells and PNH Type III Cells Can Arise From Different Point Mutations Involving the Same Codon of the PIG-A Gene

To the Editor: Paroxysmal nocturnal hemoglobinuria (PNH) is due to a somatic mutation of the PIG-A gene occurring in a hematopoietic stem cell. This results in a deficiency of all proteins that are normally attached to the cell membrane via a glycosyl phosphatidylinositol (GPI) anchor.[1][1] [2][2

Specific defect in N-acetylglucosamine incorporation in the biosynthesis of the glycosylphosphatidylinositol anchor in cloned cell lines from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal disorder arising in a multipotent hemopoietic stem cell. PNH manifests clinically with intravascular hemolysis resulting from an increased sensitivity of the red cells belonging to the PNH clone to complement-mediated lysis. Numerous studies have shown that surface proteins anchored to the membrane via a glycosylphosphatidylinositol (GPI) anchor (including proteins protecting the cell from complement) are deficient on the cells of the PNH clone, leading to the notion that GPI-anchor biosynthesis may be abnormal in these cells. To investigate the biochemical defect underlying PNH we have used lymphoblastoid cell lines (LCLs) with the PNH phenotype obtained by Epstein-Barr virus immortalization of lymphocytes from nine patients with PNH. By labeling cells with myo-[3H]inositol we have found that PNH LCLs produce phosphatidylinositol normally. By contrast, PNH LCLs fail to incorporate [3H]mannose into GPI anchor precursors. When cell-free extracts of PNH LCLs and normal LCLs obtained from the same patients (and expected therefore to be isogeneic except for the PNH mutation) were incubated with uridine diphospho-N-acetyl[3H]glucosamine (UDP-[3H]GlcNAc), we observed complete failure or marked reduction in the production of N-acetylglucosaminyl(alpha-1,6)phosphatidylinositol and glucosaminyl(alpha-1,6)phosphatidylinositol by the PNH LCLs in all cases. These findings pinpoint the block in PNH at an early stage in the biosynthesis of the GPI anchor, suggesting that the defective enzyme is UDP-GlcNAc:phosphatidylinositol-alpha-1,6-N- acetylglucosaminyltransferase. The existence of PNH type III cells and type II cells is probably explained by the transferase deficiency being total or partial, respectively.