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1.  A Novel Marker for Screening Paroxysmal Nocturnal Hemoglobinuria Using Routine Complete Blood Count and Cell Population Data 
Annals of Laboratory Medicine  2014;35(1):35-40.
Background
Final diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) may take years demanding a quick diagnosis measure. We used the facts that PNH cells are damaged in acid, and reagents for measuring reticulocytes in Coulter DxH800 (Beckman Coulter, USA) are weakly acidic and hypotonic, to create a new PNH screening marker.
Methods
We analyzed 979 complete blood counts (CBC) data from 963 patients including 57 data from 44 PNH patients. Standard criteria for PNH assay for population selection were followed: flow cytometry for CD55 and CD59 on red blood cells (RBCs) to a detection level of 1%; and fluorescent aerolysin, CD24 and CD15 in granulocytes to 0.1%. Twenty-four PNH minor clone-positive samples (minor-PNH+) were taken, in which the clone population was <5% of RBCs and/or granulocytes. Excluding PNH and minor-PNH+ patients, the population was divided into anemia, malignancy, infection, and normal groups. Parameters exhibiting a distinct demarcation between PNH and non-PNH groups were identified, and each parameter cutoff value was sought that includes the maximum [minimum] number of PNH [non-PNH] patients.
Results
Cutoff values for 5 selected CBC parameters (MRV, RDWR, MSCV, MN-AL2-NRET, and IRF) were determined. Positive rates were: PNH (86.0%), minor-PNH+ (33.3%), others (5.0%), anemia (13.4%), malignancy (5.3%), infection (3.7%), normal (0.0%); within anemia group, aplastic anemia (40.0%), immune hemolytic anemia (11.1%), iron deficiency anemia (1.6%). Sensitivity (86.0%), specificity (95.0%), PPV (52.1%), and NPV (99.1%) were achieved in PNH screening.
Conclusion
A new PNH screening marker is proposed with 95% specificity and 86% sensitivity. The flag identifies PNH patients, reducing time to final diagnosis by flow cytometry.
doi:10.3343/alm.2015.35.1.35
PMCID: PMC4272963  PMID: 25553278
Paroxysmal nocturnal hemoglobinuria; Screening marker; Complete blood count; Reticulocyte-related parameters
2.  A platelet and granulocyte membrane defect in paroxysmal nocturnal hemoglobinuria: usefulness for the detection of platelet antibodies 
Journal of Clinical Investigation  1969;48(7):1199-1210.
The tendency of platelets and leukocytes to lyse after their interaction with antibody and complement was studied by measuring the release of 51Cr from cells labeled with this isotope. Platelets from six patients with paroxysmal nocturnal hemoglobinuria (PNH) were 15-230 times more sensitive to antibodies and 10-32 times more sensitive to complement than normal platelets or platelets from patients with other types of thrombocytopenic or hemolytic disorders. Mixed white blood cell (WBC) preparations from patients with PNH were 3-20 times more sensitive to anti-WBC antibodies and 5-10 times more sensitive to C′ than were WBC preparations from normal subjects, but PNH lymphocytes showed normal immunologic reactivity. PNH platelets, like PNH erythrocytes, lysed more readily than normal platelets in acidified serum and in media of reduced ionic strength, but these characteristics were not demonstrable with PNH WBC's under the conditions of study. In PNH, platelets appear to comprise a single population with respect to their sensitivity to immune lysis, yet their survival time as measured with 51Cr falls within normal limits. PNH granulocytes likewise appear to consist of a single, uniformly sensitive population.
It is concluded that, in PNH, platelets and granulocytes share the membrane defect characteristic of erythrocytes in this disorder. These observations support the concept that PNH arises as the result of a somatic mutation in a primitive cell capable of differentiating into erythroblast, myeloblast, and megakaryoblast lines. PNH platelets or enzymatically treated normal platelets permit the detection of some types of platelet antibodies in dilutions up to 2000-fold greater than is possible with currently available methods, a finding suggesting that the immune lysis technique will prove useful for the study of platelet immunology.
PMCID: PMC322341  PMID: 5798625
3.  Improved Detection and Characterization of Paroxysmal Nocturnal Hemoglobinuria Using Fluorescent Aerolysin 
Paroxysmal nocturnal hemoglobinuria (PNH) is caused by a somatic mutation in the gene PIGA, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIGA mutation results in absence or marked deficiency of more than a dozen proteins on PNH blood cells. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of specific GPI anchor proteins, such as CD59. However, because no single GPI anchor protein is always expressed in all cell lineages, no one monoclonal antibody can be used with confidence to diagnose PNH. We describe a new diagnostic test for PNH, based on the ability of a fluorescently labeled inactive variant of the protein aerolysin (FLAER) to bind selectively to GPI anchors. We compared GPI anchor protein expression in 8 patients with PNH using FLAER and anti-CD59. In all cases, FLAER detected similar or higher proportions of PNH monocytes and granulocytes compared with anti-CD59. Because of the increased sensitivity of detection, FLAER could detect small abnormal granulocyte populations in patients to a level of about 0.5%; samples from healthy control subjects contained substantially fewer FLAER-negative cells. FLAER gives a more accurate assessment of the GPI anchor deficit in PNH.
PMCID: PMC4124633  PMID: 10989647
Paroxysmal nocturnal hemoglobinuria; Aerolysin; GPI anchor proteins; CD59; Aplastic anemia
4.  Paroxysmal nocturnal hemoglobinuria: pathophysiology, natural history and treatment options in the era of biological agents 
Biologics : Targets & Therapy  2008;2(2):205-222.
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal non-malignant hematological disease characterized by the expansion of hematopoietic stem cells (HSCs) and progeny mature cells, whose surfaces lack all the proteins linked through the glycosyl-phosphatidyl inositol anchor. This defect arises from an acquired somatic mutation in the X-linked phosphatidylinositol glycan class A gene, with subsequent clonal expansion of the mutated HSCs as a result of a concomitant, likely immune-mediated, selective pressure. The disease is characterized by complement-mediated chronic intravascular hemolysis, resulting in hemolytic anemia and hemosiderinuria; capricious exacerbations lead to recurrent gross hemoglobinuria. Additional cardinal manifestations of PNH are a variable degree of bone marrow failure and an intrinsic propensity to thromboembolic events. The disease is markedly invalidating, with chronic symptoms requiring supportive therapy – usually including periodical transfusions; possible life-threatening complications may also ensue. The biology of PNH has been progressively elucidated in the past few years, but therapeutic strategies remained unsatisfactory for decades, the only exception being stem cell transplantation, which is restricted to selected patients and retains significant morbidity and mortality. Recently, a biological agent to treat PNH has been developed – the terminal complement inhibitor eculizumab – which has been tested in a number of clinical trials, with exciting results. All the data from worldwide clinical trials confirm that eculizumab radically modifies the symptoms, the biology, and the natural history of PNH, strongly improving the quality of life of PNH patients.
PMCID: PMC2721357  PMID: 19707355
paroxysmal nocturnal hemoglobinuria; GPI-AP; PIG-A; complement; eculizumab
5.  Acute myeloblastic leukemia in paroxysmal nocturnal hemoglobinuria. Evidence of evolution from the abnormal paroxysmal nocturnal hemoglobinuria clone. 
Journal of Clinical Investigation  1987;79(1):314-317.
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder in which the blood cells demonstrate aberrant interactions with serum complement. In part, this is due to the absence of the complement regulatory protein, decay accelerating factor (DAF). A small number of patients with PNH have gone on to develop acute nonlymphocytic leukemia, which is thought to arise from the injured marrow as a second hematopoietic disorder. We have studied a patient with PNH who developed acute myeloblastic leukemia (AML); the blasts from this patient were found to lack DAF as measured by polyclonal antibody binding and fluorescence flow cytometry as well as by immunoblotting. The blasts from 11 other patients with AML bound anti-DAF antibody in amounts similar to normal mononuclear cells from healthy donors. Cells of the human leukemia cell lines HL-60, K562, U937, and HEL also bound anti-DAF antibody. In addition to DAF deficiency, blasts from the PNH patient had undetectable alkaline phosphatase activity, in contrast to human leukemia cell lines. These data suggest that the leukemic cells of the PNH patient arose out of the PNH clone and that AML in the setting of PNH is not a separate disorder.
Images
PMCID: PMC424052  PMID: 2432090
6.  Paroxysmal Nocturnal Hemoglobinuria from Bench to Bedside 
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare hematologic disease that presents with protean manifestations. Clinical and laboratory investigation over the past 25 years have uncovered most of the basic science underpinnings of PNH and have led to the development of a highly effective targeted therapy. PNH originates from a multipotent hematopoietic stem cell (HSC) that acquires a somatic mutation in a gene called phosphatidylinositol glycan anchor biosynthesis, class A (PIG-A). The PIG-A gene is required for the first step in glycosylphosphatidylinositol (GPI) anchor biosynthesis. Failure to synthesize GPI anchors leads to an absence of all proteins that utilize GPI to attach to the plasma membrane. Two GPI-anchor proteins, CD55 and CD59, are complement regulatory proteins; their absence on the surface of PNH cells leads to complement-mediated hemolysis. The release of free hemoglobin leads to scavenging of nitric oxide and contributes to many clinical manifestations, including esophageal spasm, fatigue, and possibly thrombosis. Aerolysin, a pore-forming toxin, binds GPI anchored proteins and kills normal cells, but not PNH cells. A fluorescinated aerolysin variant (FLAER) binds GPI-anchor and serves as a novel reagent diagnosing PNH. Eculizumab, a humanized monoclonal antibody against C5, is the first effective drug therapy for PNH.
doi:10.1111/j.1752-8062.2011.00262.x
PMCID: PMC3128433  PMID: 21707954
7.  The use of the complement inhibitor eculizumab (Soliris®) for treating Korean patients with paroxysmal nocturnal hemoglobinuria 
The Korean Journal of Hematology  2010;45(4):269-274.
Background
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disorder characterized by chronic complement-mediated hemolysis. Eculizumab, a humanized monoclonal antibody against the terminal complement protein C5, potently reduces chronic intravascular hemolysis. We tested the clinical efficacy and safety of a 24-week treatment with eculizumab in 6 Korean patients with PNH.
Methods
We enrolled 6 patients with PNH who had clinically significant hemolysis. Eculizumab was administered intravenously at 600 mg/week for the first 4 weeks followed by 900 mg at week 5 and 2nd weekly thereafter.
Results
Three men and 3 women with a median age of 39.5 years (24-61 years) were enrolled. The median duration of PNH was 11 years (6-25 years). Hemolysis occurred in all patients [median lactate dehydrogenase (LDH) level, 7.95 times the upper limit of the reference range of LDH]. All patients treated with eculizumab had a rapid and sustained reduction in the degree of hemolysis. RBC transfusion requirements for 3 months were decreased from 0-12 units (median requirement, 1.5 units) to 0-6 units (median requirement, 0 units). Improvement in fatigue was noted in 4 patients. Further, 5 patients who had been receiving corticosteroids either reduced the dose or discontinued therapy. No significant adverse events related to eculizumab therapy were observed.
Conclusion
These results show that eculizumab reduces the degree of intravascular hemolysis, reduces or eliminates the requirement of RBC transfusion, and improves anemia and fatigue in patients with PNH. Eculizumab is an effective and safe option for treating Korean patients with PNH.
doi:10.5045/kjh.2010.45.4.269
PMCID: PMC3023054  PMID: 21253430
Paroxysmal nocturnal hemoglobinuria; Eculizumab; Efficacy; Safety
8.  Mechanisms of Immune Lysis of the Red Cells in Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum Test and Paroxysmal Nocturnal Hemoglobinuria 
The red cells of patients with hereditary erythroblastic multinuclearity with a positive acidified serum test (HEMPAS), a form of congenital dyserythropoietic anemia, and the cells of patients with paroxysmal nocturnal hemoglobinuria (PNH) are lysed more readily than normal cells by certain antibodies, notably cold agglutinins (anti-I) and complement. With some but not other examples of anti-I, HEMPAS and PNH cells adsorbed more antibody than normal cells. Equal quantities of adsorbed antibody bound equal quantities of the first component of complement (C1) to normal, PNH, and HEMPAS cells. However, for a given quantity of bound antibody and C1, much more of the fourth component of complement (C4) was bound to HEMPAS cells than to normal cells. This resulted in the binding of proportionately larger quantities of the third component of complement (C3) to these cells. The same amount of bound C3 was found on the membranes of normal and HEMPAS cells for a given degree of lysis. Hence, the marked increase in lysis of HEMPAS cells is due to the increased adsorption of antibody and/or increased binding of C4.
PNH cells bound the same amount of C4 per bound C1 as normal cells but bound more C3 than normal cells. However, the mean concentration of C3 on the membrane of PNH cells was one-third to one-fifth that on normal cells for a given degree of lysis. Hence, the increased lysis of PNH cells is due to the increased binding of C3 and increased hemolytic effectiveness of the bound C3.
PMCID: PMC301435  PMID: 4855546
9.  Complement Lysis of Human Erythrocytes 
Journal of Clinical Investigation  1979;64(2):428-433.
Although enhanced sensitivity of erythrocytes to complement-mediated lysis is a hallmark of paroxysmal nocturnal hemoglobinuria (PNH), subpopulations of erythrocytes in such patients vary significantly in this respect. One PNH erythrocyte subpopulation (termed type III) comprises exquisitely sensitive cells, whereas type II PNH erythrocytes are intermediate in complement sensitivity between PNH type III and normal human erythrocytes. Differences in the action of the terminal complement components that would account for the differing lytic behavior of types II and III PNH erythrocytes have been proposed but not directly demonstrated.
The present studies, making use of carefully selected cases with pure populations of type II or type III erythrocytes, confirm a prior observation that antibody-coated PNH erythrocytes of both types II and III display comparably supranormal C3 binding in whole human serum. However, when lysis was induced by the isolated C5b-9 membrane attack mechanism, bypassing the requirement for C3 binding, only type III PNH cells exhibited greater than normal lysis. This finding suggests that type III PNH erythrocytes have an additional membrane abnormality not present in type II cells. Thus, the differing lytic behavior of these two cell types in whole serum may reflect the additive effects on type III cells of both exaggerated C3 binding and enhanced sensitivity to C5b-9, whereas the more moderate lysis of type II PNH cells may be determined mainly or entirely by the earlier-acting mechanism producing augmented C3 binding.
The failure of guinea pig C8 and C9, as opposed to human C8 and C9, to reveal the true lytic sensitivity of PNH-III E in our earlier study is illustrated, and its implications briefly discussed.
PMCID: PMC372136  PMID: 457861
10.  Potential link between MHC–self-peptide presentation and hematopoiesis; the analysis of HLA-DR expression in CD34-positive cells and self-peptide presentation repertoires of MHC molecules associated with paroxysmal nocturnal hemoglobinuria 
Cell Biochemistry and Biophysics  2012;65(3):321-333.
The mechanisms of MHC allele associations with paroxysmal nocturnal hemoglobinuria (PNH) and its aplastic anemia subtype (AA/PNH) remain unclear. It might be dependent on MHC molecule functional properties, such as a scope and frequency of antigen sampling and presentation. For documented PNH-associated MHC alleles we analyzed current reference databases on MHC molecule-eluted peptide presentation repertoires and searched for a range of presented peptides. MHC class II expression was measured on CD34+ cells and appeared to be increased in PNH patients. Two class I alleles (HLA-A*24:02 and B*18:01) have been previously confirmed to associate with protection and increased risk of AA/PNH, respectively. Their product molecules presented immunodominant epitopes derived from proapoptotic (serine/threonine–protein phosphatase) and antiapoptotic (phospholipase D), respectively, intracellular enzymes dependent on phosphoinositide (PI) content. For total PNH and non-aplastic PNH (n/PNH) subtype-associated DRB1*15:01 and DRB1*04:01 class II molecules presentation of exceptionally broad arrays of their own peptide fragments has been found. We conclude that self antigen peptides presented with high frequency in the context of MHC molecules of increased expression may be involved in the immune recognition and the regulation of HSC in the periphery. The block in the normal plasma membrane PI production due to the PIG-A mutation can help explain the differences in the activation of intracellular regulatory pathways observed between PNH and normal HSC. This is evident in the variation in MHC association patterns and peptide presentation repertoires between these two groups of patients.
Electronic supplementary material
The online version of this article (doi:10.1007/s12013-012-9435-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s12013-012-9435-1
PMCID: PMC3601265  PMID: 23076633
Paroxysmal nocturnal hemoglobinuria; Hematopoietic stem cell; MHC molecules; HLA-DR expression; Self-peptide presentation; Regulatory T cells; Apoptosis; PNH clone domination; PNH clone selection
11.  Enhanced reactive lysis of paroxysmal nocturnal hemoglobinuria erythrocytes. Studies on C9 binding and incorporation into high molecular weight complexes 
As part of a broader analysis of the mechanism(s) by which the most sensitive (type III) paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes are excessively sensitive to reactive lysis by isolated C5b6, C7, C8, and C9, we have compared type III PNH (PNH-III) and normal human E in respect to both total specific binding of 125I-C9 and the proportion of cell-bound C9 appearing in high molecular weight (HMW) complexes. In a previous report, we found that after exposure to purified C5b6 and 125I-C7, specific C7 binding and, by implication, EC5b-7 formation were equal for PNH-III E and normal E. In the present study, C8-dependent binding of 125I-C9 to PNH-III EC5b-7 and normal EC5b-7 was also similar, although lysis of the PNH-III E was up to five times greater; that is, PNH-III E required fewer bound C9 molecules to produce an effective lytic site than did normal E. To quantify radioactivity in monomeric and HMW forms of membrane-bound C9, lysed and unlysed E were subjected to low ionic strength buffers to convert all E to ghosts. These ghosts were solubilized in 0.1 or 2% SDS (without reduction) and electrophoresed on 2.4-11% polyacrylamide gradient gels followed by autoradiography and densitometric scanning. With 0.1% SDS, broad, heterodisperse zones of HMW C9 were recovered from both PNH and normal ghosts; the amounts of C9 incorporated into the HMW complexes were similar for PNH-III E and normal E. In selected experiments, 125I-C7 could be shown in these same HMW bands. When membranes were solubilized in 2% SDS, the overall proportion of HMW C9 complexes compared with dimer and monomer C9 was reduced on both PNH and normal membranes. In many, but not all experiments, more of the highest mol wt C9 complexes were detected from PNH-III E membranes solubilized in 2% SDS than from normal or PNH-II E membranes similarly treated. When antibody-sensitized E were lysed by purified C1-C9, PNH- III EA bound far more C9 than did normal EA, and both lysis and C9 incorporation into HMW complexes were markedly and proportionately increased over normal; however, lytic efficiency of 125I-C9 bound to PNH EA was equal to or less than that bound to normal EA.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC2188431  PMID: 3760783
12.  Deficient biosynthesis of N-acetylglucosaminyl-phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria 
Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic disorder caused by a deficiency of biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor, but the biochemical defect is not completely understood. In the present study, we have analyzed affected cell lines established recently from two Japanese patients with PNH. Two lines of evidence indicate that these cells do not synthesize N- acetylglucosaminyl-phosphatidylinositol, the first intermediate in the GPI anchor biosynthesis. First, somatic cell hybridization analysis using Thy-1-deficient murine thymoma cell lines with known biochemical defects as fusion partners showed that the PNH cell lines belong to complementation class A, which is known not to synthesize N- acetylglucosaminyl-phosphatidylinositol. Second, analysis of in vitro glycolipid biosynthesis demonstrated that cell lysates of these PNH cell lines in fact did not support biosynthesis of N-acetylglucosaminyl- phosphatidylinositol. Thus, we have characterized for the first time the exact biochemical defect leading to PNH.
PMCID: PMC2190897  PMID: 8426120
13.  Anti-Complement Treatment in Paroxysmal Nocturnal Hemoglobinuria: Where we Stand and Where we are Going 
Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal, non-malignant, hematological disorder characterized by the expansion of hematopoietic stem cells and progeny mature blood cells which are deficient in some surface proteins, including the two complement regulators CD55 and CD59. PNH is the paradigm of diseases implying complement dysregulation as main pathogenic mechanism; in fact, PNH erythrocytes are uncapable to modulate on their surface physiologic complement activation, which eventually leads to the typical clinical hallmark of PNH – the chronic complement-mediated intravascular anemia. Indeed, due to the lack of CD55 complement is continuously activated on erythrocyte surface, which subsequently enables the terminal lytic complement because of the lack of CD59, finally resulting in erythrocyte lysis. The availability of eculizumab as the first complement inhibitor for clinical use renewed the interest for this rare hematological disease. Indeed, in the last decad the anti-C5 monoclonal antibody has proven effective for the treatment of PNH, resulting in a sustained control of complement-mediated intravascular hemolysis, with a remarkable clinical benefit. Anti-complement treatment allowed transfusion independence in at least half of PNH patients receiving eculizumab, with adequate control of all hemolysis-associated symptoms even in almost all remaining patients. In addition, the risk of thromboembolic events – an other clinical hallmark of PNH, which significantly affects prognosis and survival – seems substantially reduced on eculizumab treatment, apparently resulting in improved survival. Even with all these remarkable effects, eculizumab treatment does not result in hemoglobin normalization, and most patients remain anemic. It has been demonstrated that this is due to persistent activation of the early phases of complement activation (upstream the C5), leading to complement-mediated extravascular hemolysis. Ongoing researches are focusing on possible strategies to improve current anti-complement therapies, aiming to develop second-generation complement therapeutics. Here we review PNH and its complement-mediated pathophysiology, summarizing available data on anti-complement treatment; we’ll also discuss recent pathogenic insights which drive the development of novel strategies of complement inhibition.
PMCID: PMC4000462  PMID: 24778997
Paroxysmal Nocturnal Hemoglobinuria; complement alternative pathway; complement component 3; complement component 5; eculizumab; complement therapeutics; C3-targeted therapy
14.  Serum-Red Cell Interactions at Low Ionic Strength: Erythrocyte Complement Coating and Hemolysis of Paroxysmal Nocturnal Hemoglobinuria Cells* 
Journal of Clinical Investigation  1967;46(5):753-761.
Complement coating and hemolysis were observed when erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) were incubated in isotonic sucrose solution in the presence of small amounts of serum. Normal cells were likewise coated with complement components but did not hemolyze. Both normal and PNH erythrocytes reduced the hemolytic complement activity of the serum used in this reaction.
Experience with other simple saccharides and related compounds suggests that the low ionic strength of the sucrose solution is the feature that permitted complement coating of red cells and hemolysis of PNH erythrocytes. Isotonic solutions of other sugars or sugar alcohols that do not readily enter human erythrocytes could be substituted for sucrose.
The mechanism for these reactions may possibly relate to the agglutination observed with erythrocytes tested in the serum-sucrose system. Even though PNH hemolytic activity could be removed by prior heating of serum or barium sulfate treatment of plasma, the agglutination phenomenon still persisted.
The in vitro conditions necessary for optimal sucrose hemolysis of PNH erythrocytes were described and compared with those of the classical acid hemolysis test. The requirement for less serum in the sucrose hemolysis system than needed in the standard acid hemolysis reaction makes certain experiments, especially those using large amounts of autologous PNH serum, much more feasible. Additional advantages of the sucrose hemolysis test are that it can be carried out at room temperature in the presence of oxalate and citrate and that critical pH control is not essential. To date, the sucrose hemolysis test has been a sensitive and specific one for PNH. A modified test used for screening purposes, the “sugar water” test, is very easy to perform.
PMCID: PMC297078  PMID: 6025481
15.  Deep sequencing reveals stepwise mutation acquisition in paroxysmal nocturnal hemoglobinuria 
The Journal of Clinical Investigation  2014;124(10):4529-4538.
Paroxysmal nocturnal hemoglobinuria (PNH) is a nonmalignant clonal disease of hematopoietic stem cells that is associated with hemolysis, marrow failure, and thrombophilia. PNH has been considered a monogenic disease that results from somatic mutations in the gene encoding PIGA, which is required for biosynthesis of glycosylphosphatidylinisotol-anchored (GPI-anchored) proteins. The loss of certain GPI-anchored proteins is hypothesized to provide the mutant clone with an extrinsic growth advantage, but some features of PNH argue that there are intrinsic drivers of clonal expansion. Here, we performed whole-exome sequencing of paired PNH+ and PNH– fractions on samples taken from 12 patients as well as targeted deep sequencing of an additional 36 PNH patients. We identified additional somatic mutations that resulted in a complex hierarchical clonal architecture, similar to that observed in myeloid neoplasms. In addition to mutations in PIGA, mutations were found in genes known to be involved in myeloid neoplasm pathogenesis, including TET2, SUZ12, U2AF1, and JAK2. Clonal analysis indicated that these additional mutations arose either as a subclone within the PIGA-mutant population, or prior to PIGA mutation. Together, our data indicate that in addition to PIGA mutations, accessory genetic events are frequent in PNH, suggesting a stepwise clonal evolution derived from a singular stem cell clone.
doi:10.1172/JCI74747
PMCID: PMC4191017  PMID: 25244093
16.  Expression of cryptantigen Th on paroxysmal nocturnal hemoglobinuria erythrocytes in association with a hemolytic exacerbation. 
Journal of Clinical Investigation  1995;96(1):201-206.
Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes lack complement regulatory membrane proteins and are susceptible to complement. Although the critical role of complement in intravascular hemolysis in PNH is accepted, the precise mechanism of complement activation in vivo is unknown. Accordingly, in a PNH patient who was suffering from a hemolytic precipitation soon after a common cold-like upper respiratory infection, we analyzed the erythrocytes with lectins and by flow cytometry to detect membrane alteration that lead to complement activation. The lectin reactivity of erythrocytes showed the expression of cryptantigen Th. The patient serum at the time of the hemolysis induced the expression of Th on erythrocytes from PNH patients and from healthy volunteers in vitro, whereas neither the patient serum after recovery from the hemolysis nor blood type-matched control serum from healthy donor showed this activity. Moreover, autologous serum selectively hemolyzed Th+ PNH erythrocytes, but not Th- PNH erythrocytes, or Th+ control erythrocytes. Hemolysis was not observed either in complement-inactivated serum or in blood type-matched cord blood serum, which lacks natural antibodies to cryptantigens. These findings indicate that the immunoreaction of infection-induced Th with natural antibody on PNH erythrocytes is a trigger of the complement activation, leading to intravascular hemolysis.
Images
PMCID: PMC185189  PMID: 7542278
17.  Response of Paroxysmal Nocturnal Hemoglobinuria Clone with Aplastic Anemia to Rituximab 
Case Reports in Hematology  2012;2012:106182.
Paroxysmal nocturnal hemoglobinuria is caused by expansion of a hematopoietic stem cell clone with an acquired somatic mutation in the PIG-A gene. This mutation aborts the synthesis and expression of the glycosylphosphatidylinositol anchor proteins CD55 and CD59 on the surface of blood cells, thereby making them more susceptible to complement-mediated damage. A spectrum of disorders occurs in PNH ranging from hemolytic anemia and thrombosis to myelodysplasia, aplastic anemia and, myeloid leukemias. Aplastic anemia is one of the most serious and life-threatening complications of PNH, and a PNH clone is found in almost a third of the cases of aplastic anemia. While allogeneic bone marrow transplantation and T cell immune suppression are effective treatments for aplastic anemia in PNH, these therapies have significant limitations. We report here the first case, to our knowledge, of PNH associated with aplastic anemia treated with the anti-CD20 monoclonal antibody rituximab, which was associated with a significant reduction in the size of the PNH clone and recovery of hematopoiesis. We suggest that this less toxic therapy may have a significant role to play in treatment of PNH associated with aplastic anemia.
doi:10.1155/2012/106182
PMCID: PMC3420688  PMID: 22937317
18.  Evaluation of Danazol, Cyclosporine, and Prednisolone as Single Agent or in Combination for Paroxysmal Nocturnal Hemoglobinuria 
Turkish Journal of Hematology  2013;30(4):366-370.
Objective: The responses of 32 patients with paroxysmal nocturnal hemoglobinuria (PNH) were assessed after the patients were put on various combinations of danazol, prednisolone, and cyclosporine.
Materials and Methods: Nineteen males and 13 females aged between 14 and 60 years with confirmed diagnosis of PNH were treated with danazol (4), danazol + cyclosporine (7), cyclosporine (1), and prednisolone + danazol (20). Response to these interventions was assessed regularly. Danazol was added to cyclosporine in patients with aplastic bone marrow after 3 months of cyclocporine use only unless the former therapy was successful. Four patients with aplastic marrow received only danazol because they had renal insufficiency at presentation. Patients were evaluated with regular complete blood count and routine liver and renal function tests.
Results: One patient responded to cyclosporine only. Thirteen of 32 patients (40%) had complete response, 12/32 patients (37%) had partial response leading to freedom from red cell transfusion, and 2/32 (7%) had no response. Five patients (16%) died due to thrombosis or hemorrhage within 3 months of therapy before their response to therapy could be assessed. The median period of review of the cases was 4 years and 6 months.
Conclusion: Danazol is a useful addition to PNH therapy both in combination with cyclosporine for hypoplastic PNH and with prednisolone for other forms of PNH, and this therapy could be a good alternative where eculizumab and anti-lymphocyte globulin cannot be used for various reasons.
Conflict of interest:None declared.
doi:10.4274/Tjh.2012.0199
PMCID: PMC3874974  PMID: 24385826
cyclosporine; Danazol; Hemoglobinuria; Paroxysmal; Immunosuppression; Prednisolone
19.  Eculizumab in paroxysmal nocturnal hemoglobinuria with Budd-Chiari syndrome progressing despite anticoagulation 
Paroxysmal nocturnal hemoglobinuria (PNH) is a progressive, life-threatening disorder characterized by chronic intravascular hemolysis caused by uncontrolled complement activation. Hepatic vein thrombosis (Budd-Chiari syndrome) is common in PNH patients. This case report describes the response to eculizumab (a humanized monoclonal antibody that inhibits terminal complement activation) in a 25-year-old male with progressive liver function deterioration despite standard anticoagulation therapy and transjugular intrahepatic porto-systemic shunt. The patient presented with anemia, severe thrombocytopenia, headache, abdominal pain, and distention. He was diagnosed with PNH, cerebral vein thrombosis, and Budd-Chiari syndrome. Despite adequate anticoagulation, diuretic administration, and placement of a transjugular shunt, additional thrombotic events and progressive liver damage were observed. Eculizumab therapy was initiated, resulting in rapid blockade of intravascular hemolysis, increased platelet counts, ascites resolution, and liver function recovery, all of which are presently sustained. Since starting eculizumab the patient has had no further thrombotic events and his quality of life has dramatically improved. This is the first report to confirm the role of complement-mediated injury in the progression of Budd-Chiari syndrome in a patient with PNH. This case shows that terminal complement blockade with eculizumab can reverse progressive thromboses and hepatic failure that is unresponsive to anticoagulation therapy and suggests that early initiation of eculizumab should be included in the therapeutic regimen of patients with PNH-related Budd-Chiari syndrome.
doi:10.1186/2162-3619-1-26
PMCID: PMC3514094  PMID: 23210433
Budd-Chiari syndrome; Complement inhibition; Eculizumab; Paroxysmal nocturnal hemoglobinuria
20.  Distribution of decay-accelerating factor in the peripheral blood of normal individuals and patients with paroxysmal nocturnal hemoglobinuria 
Decay-accelerating factor (DAF) is a 70,000 Mr protein that has been isolated from the membrane of red cells. The function of DAF is to inhibit the assembly of amplifying enzymes of the complement cascade on the cell surface, thereby protecting them from damage by autologous complement. We raised monoclonal antibodies to DAF and used them to study its distribution in cells from the peripheral blood of normal individuals and of patients with paroxysmal nocturnal hemoglobinuria (PNH), a disease characterized by the unusual susceptibility of red cells to the hemolytic activity of complement. The results of immunoradiometric assays and of fluorescence-activated cell sorter analysis showed that DAF was present not only on red cells but was widely distributed on the surface membrane of platelets, neutrophils, monocytes, and B and T lymphocytes. By Western blotting, we observed small but consistent differences in the Mr of DAF from the membranes of various cell types. Quantitative studies showed that phagocytes and B lymphocytes, which presumably enter more frequently in contact with immune complexes and other potential activators of complement, had the highest DAF levels. As previously reported by others, the red cells from PNH patients were DAF deficient. When the patients' red cells were incubated in acidified serum (Ham test), only the DAF-deficient cells were lysed. In addition, we detected defects in DAF expression on platelets and all types of leukocytes. The observed patterns of DAF deficiency in these patients were consistent with the concept that the PNH cells were of monoclonal origin. In one patient, abnormal and normal cells were found only in the erythroid, myeloid, and megakaryocytic lineages. In two other patients, the lymphocytes were also DAF deficient, suggesting that a mutation occurred in a totipotent stem cell. It appears, therefore, that the lesion leading to PNH can occur at various stages in the differentiation of hematopoietic cells.
PMCID: PMC2187705  PMID: 2409211
21.  Paroxysmal nocturnal hemoglobinuria presenting as cerebral venous sinus thrombosis: a case report 
Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare type of acquired hemolytic anemia that is frequently associated with thrombophilia. It may rarely present with cerebral venous sinus thrombosis, which manifests clinically with signs of raised intracranial pressure and requires lifelong anticoagulation therapy. One such rare presentation was seen in a 28 years old male who had history of recurrent episodes of passing red colored urine and this time presented with severe headache. He was diagnosed to have cerebral venous sinus thrombosis and on further workup was found to be suffering from PNH.
doi:10.1186/1755-7682-7-39
PMCID: PMC4138960  PMID: 25143784
22.  Paroxysmal nocturnal hemoglobinuria type III. Lack of an erythrocyte membrane protein restricting the lysis by C5b-9. 
The complement-mediated lysis is inefficient when complement and target cells are homologous with regard to the species. In erythrocytes from patients suffering from paroxysmal nocturnal hemoglobinuria (PNH), the species restriction is lost: PNH-erythrocytes (PNH-E) are susceptible to lysis by human complement. In human erythrocytes (huE) the species restriction is ascribed to an integral membrane protein, designated C8-binding protein (C8bp). In the present study, we tested membranes of PNH-E type III for the presence of C8bp. A protein with C8-binding capacity could not be detected. C8bp, which was isolated from the membrane of huE, inhibited the lysis of PNH-E by C5b-9 as well as the C9 polymerization. Thus, addition of C8bp restored the species restriction in PNH-E. In conclusion, we propose that lack of C8bp might represent the defect in PNH-E type III membranes, which is responsible for their enhanced lytic susceptibility towards lysis by the late complement components.
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PMCID: PMC442194  PMID: 3597779
23.  Decay-accelerating factor is present on paroxysmal nocturnal hemoglobinuria erythroid progenitors and lost during erythropoiesis in vitro 
The Journal of Experimental Medicine  1985;162(4):1182-1192.
A glycoprotein that regulates the deposition of C3b on the erythrocyte surface, called decay-accelerating factor or DAF, is absent from the red blood cells (RBC) of patients with paroxysmal nocturnal hemoglobinuria (PNH), explaining in part their abnormal sensitivity to complement. We used a specific antiserum to DAF, flow microfluorometry, and clonogenic assays for erythroid progenitor cells to study PNH erythropoiesis in vitro. By fluorescence-activated cell sorter analysis, all RBC from normal individuals are DAF+. In contrast, the RBC of six patients with PNH showed discrete populations of DAF- cells (10-44%; x +/- SEM = 31 +/- 6%). The DAF- RBC population was partly eliminated by prior acidified serum lysis. To determine whether erythropoietic progenitors expressed DAF, bone marrow cells were sorted by flow microfluorometry and the separated DAF+ and DAF- populations then cultured in vitro. In two normal individuals, but also in six patients with PNH, erythroid colonies formed only from cells in the DAF+ fraction. However, a variable proportion of the normoblast progeny of these DAF+ progenitor cells from patients with PNH was DAF-. Individual bursts removed from cultures of PNH bone marrow showed two discrete populations by fluorescence; the majority of normoblasts were DAF-, only 3 of 27 individual bursts had greater than 50% DAF+ cells, and in three patients, DAF- normoblasts averaged 79%. In contrast, the progeny of individual bursts from normal individuals comprised a unimodal DAF+ population. In each PNH patient, one normal burst (greater than 80% DAF+ normoblasts) was detected, possibly reflecting a normal residual population of erythroid progenitors. By the criterion of DAF expression, there was no evidence of separate populations of normal and PNH type progenitor cells. The phenotypically normal erythroid progenitors of PNH bone marrow acquire the PNH characteristics during differentiation in vitro.
PMCID: PMC2187865  PMID: 2413153
24.  Preferential expression of human Fc gamma RIIIPMN (CD16) in paroxysmal nocturnal hemoglobinuria. Discordant expression of glycosyl phosphatidylinositol-linked proteins. 
The isoform of Fc gamma RIII (CD16) expressed on PMN has a GPI membrane anchor, and in paroxysmal nocturnal hemoglobinuria (PNH) there is a deficiency in Fc gamma RIII expression on PMN. Contrary to expectation, however, CD16 expression is preserved (albeit at reduced levels) in all affected PNH PMN that completely lack the GPI-anchored proteins DAF (CD55) and CD59. Fc gamma RIII negative PMN are not observed in any of the six PNH patients examined in this study. Analysis of the molecular weight of both glycosylated and deglycosylated Fc gamma RIII from PMN with reduced Fc gamma RIII expression indicates no variations in size relative to normal donor Fc gamma RIIIPMN. Indeed, the Fc gamma RIII expressed at intermediate levels is phosphatidylinositol-specific phospholipase C (PI-PLC)-sensitive. Thus, there is no evidence suggestive of expression of a transmembrane isoform and all data indicate that Fc gamma RIIIPMN on affected cells in PNH is a GPI-linked isoform. With Fc gamma RIIIPMN expression preserved at reduced levels on affected cells in PNH, PMN from PNH patients retain the capacity to internalize the Fc gamma RIIIPMN-specific probe E-ConA (at reduced levels) as well as IgG-opsonized erythrocytes. Reduced expression of GPI-anchored molecules on PNH PMN is not restricted to Fc gamma RIIIPMN since intermediate levels of CD59 were observed in the PNH PMN that were decay-accelerating factor (DAF)-negative and Fc gamma RIIIPMN intermediate. In addition, discordant expression of GPI-linked molecules in individual cells is not restricted to PMN since DAF+/CD14- monocytes were observed in one PNH patient. These data suggest that, when analyzed on an individual cell level, the GPI anchor defect in PNH is not absolute and must involve either a hierarchy of access of different protein molecules to available GPI anchors, distinct anchor biochemistries for the different proteins, or differential regulation of protein-anchor assembly.
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PMCID: PMC294990  PMID: 1702101
25.  Relationship between decay accelerating factor deficiency, diminished acetylcholinesterase activity, and defective terminal complement pathway restriction in paroxysmal nocturnal hemoglobinuria erythrocytes. 
Journal of Clinical Investigation  1987;80(1):165-174.
Paroxysmal nocturnal hemoglobinuria (PNH) erythrocytes exhibit abnormalities in decay accelerating factor (DAF), acetylcholinesterase, and resistance to autologous C5b-9 attack. To investigate the nature of the lesion underlying PNH cells, we examined the relationship of these abnormalities to one another. Analyses of DAF in acetylcholinesterase-negative erythrocytes revealed that these two abnormalities involve functionally independent molecules, coincide precisely in the same cell populations, and are similarly expressed in PNH II and more complement-sensitive PNH III erythrocytes. The DAF and acetylcholinesterase deficiencies contrast with the C3b/C4b receptor (CR1) deficit, which is less profound and similarly distributed in complement-insensitive cell populations. Hemolytic studies showed that defective resistance to autologous C5b-9 attack is mediated by another mechanism. Whereas reconstitution of PNH II erythrocytes with DAF completely corrected their complement sensitivity, DAF reconstitution of PNH III erythrocytes restored their ability to circumvent C3b uptake but had no effect on their heightened susceptibility to reactive lysis. Assays of complement-insensitive (PNH I) erythrocytes surviving after reactive lysis disclosed partial DAF and acetylcholinesterase deficits. These findings indicate that the PNH lesion involves multiple membrane components and that PNH I erythrocytes are also abnormal.
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PMCID: PMC442215  PMID: 2439544

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