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

Blood ◽  
2002 ◽  
Vol 100 (12) ◽  
pp. 3897-3902 ◽  
Author(s):  
Hongbo Wang ◽  
Tatsuya Chuhjo ◽  
Shizuka Yasue ◽  
Mitsuhiro Omine ◽  
Shinji Nakao
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Clinical Significance ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Refractory Anemia ◽  
Severe Thrombocytopenia ◽  
Minor Population ◽  
A Minor

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.

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

Blood ◽  
2003 ◽  
Vol 102 (4) ◽  
pp. 1211-1216 ◽  
Author(s):  
Ken Ishiyama ◽  
Tatsuya Chuhjo ◽  
Hongbo Wang ◽  
Akihiro Yachie ◽  
Mitsuhiro Omine ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Positive Response ◽  
Bone Marrow Failure ◽  
Refractory Anemia ◽  
Clonal Population ◽  
Inactivation Pattern ◽  
Minor Population ◽  
A Minor

Abstract Although 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.

NKG2D-Mediated Marrow Injury in Paroxysmal Nocturnal Hemoglobinuria and Its Related Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3674-3674
Author(s):  
Nobuyoshi Hanaoka ◽  
Tatsuya Kawaguchi ◽  
Kentaro Horikawa ◽  
Shoichi Nagakura ◽  
Sonoko Ishihara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Cells ◽  
Close Association ◽  
Bone Marrow Failure ◽  
Γδ T Cells ◽  
Refractory Anemia ◽  
Nkg2d Ligands ◽  
Marrow Cells

Abstract Immune mechanism is considered to exert in the pathogenesis of marrow failure in paroxysmal nocturnal hemoglobinuria (PNH), idiopathic aplastic anemia (AA) and myelodysplastic syndromes (MDS); however, the molecular events are unknown. We have currently reported the appearance of NKG2D ligands such as cytomegalovirus glycoprotein UL16 binding proteins (ULBPs) and MHC class I-related chains A and B (MICA/B) on granulocytes and CD34+ marrow cells of some patients with PNH and its related diseases (Hanaoka N, et al. Blood. 2006;107:1184–1191). ULBP and MICA/B are stress-inducible membrane proteins that appear in infection and transformation. The ligands share NKG2D receptor on lymphocytes such as NK, CD8+ T, and γδ T-cells and promote activation of the lymphocytes. Cells expressing the ligands are then deadly injured by NKG2D+ lymphocytes (Groh, PNAS 1996; Cosman, Immunity 2001). Indeed, cells expressing NKG2D ligands were killed in vitro by autologous NKG2D+ lymphocytes of our patients (Hanaoka N, et al. Blood. 2005;106:304a; Blood. 2006;108:295a). In further analysis, ligands were detected on granulocytes in 47 (53%) of 88 patients: 11 (58%) of 19 PNH, 28 (60%) of 47 AA, and 8 (36%) of 22 refractory anemia. Ligands were also detected on immature bone marrow cells in all 11 patients (3 PNH, 5 AA, and 3 refractory anemia) who permitted analysis of their marrow cells. In the patients, it is conceivable that blood cells were exposed to a certain stress to induce NKG2D ligands, leading to NKG2D-mediated marrow injury. We also observed a close association of the ligand expression with pancytopenia and favorable response to immunosuppressive therapy by prospective analysis of 5 patients (3 AA-PNH syndrome and 2 AA) for more than one year up to 5 years. Thus, we here propose that NKG2D-mediated immunity, which drives both NK and T-cells, is critically implicated in the pathogenesis of bone marrow failure of PNH and its related disorders.

Hematopoietic Stem Cell Differentiation Pathway in Humans Deduced From the Lineage Diversity of PNH-Type Cells in Patients with Bone Marrow Failure.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1489-1489
Author(s):  
Takamasa Katagiri ◽  
Zhirong Qi ◽  
Yu Kiyu ◽  
Naomi Sugimori ◽  
J. Luis Espinoza ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Bone Marrow Failure ◽  
Stem Cell Differentiation ◽  
Refractory Anemia ◽  
Hematopoietic Stem

Abstract Abstract 1489 Poster Board I-512 The hematopoietic stem cell (HSC) differentiation pathway in humans remains largely unknown due to the lack of an appropriate in vivo assay allowing the growth of HSCs as well as of clonal markers that enable the tracing of their progenies. Small populations of blood cells deficient in glycosylphosphatidylinositol-anchored proteins (GPI-APs) such as CD55 and CD59 are detectable in approximately 50% of patients with aplastic anemia (AA) and 15% of patients with refractory anemia (RA) of myelodysplastic syndrome defined by the FAB classification. Such blood cells with the paroxysmal nocturnal hemoglobinuria (PNH) phenotype (PNH-type cells) are derived from single PIGA mutant HSCs and their fate depends on the proliferation and self-maintenance properties of the individual HSCs that undergo PIG-A mutation by chance (Blood 2008;112:2160, Br J Haematol 2009 in press) Analyses of the PNH-type cells from a large number of patients on the diversity of lineage combination may help clarify the HSC differentiation pathway in humans because PIG-A mutant HSCs in patients with bone marrow failure appear to reflect the kinetics of healthy HSCs. Therefore, different lineages of peripheral blood cells were examined including glycophorin A+ erythrocytes (E), CD11b+ granulocytes (G), CD33+ monocytes (M), CD3+ T cells (T), CD19+ B cells (B), and NKp46+ NK cells (Nk) from 527 patients with AA or RA for the presence of CD55−CD59− cells in E and G, and CD55−CD59−CD48− cells in M,T, B, Nk with high sensitivity flow cytometry. Two hundred and twenty-eight patients (43%) displayed 0.003% to 99.1% PNH-type cells in at least one lineage of cells. The lineage combination patterns of PNH-type cells in these patients included EGM in 71 patients (31%), EGMTBNk in 43 (19%), EG in 37 (16%), T alone 14 (6%), EGMBNk in 11 (5%), G alone in 10 (4%), GM in 10 (4%), EGMNk in 7 (3%), EGMT in 7 (3%), EGMB in 6 (3%), EM in 5 (2%), EGMTB in 3 (1%), EGNk in 1 (0.4%), EGMTNk in 1 (0.4%), GMTB in 1 (0.4%), and GT in 1 (0.4%) (Table). All patterns included G or M, except for 14 patients displaying PNH-type T cells alone. No patients showed TB or TBNk patterns suggestive of the presence of common lymphoid progenitor cells. Peripheral blood specimens from 123 patients of the 228 patients possessing PNH-type cells were examined again after 3 to 10 months and all patients showed the same combination patterns as those revealed by the first examination. PIG-A gene analyses using sorted PNH-type cells from 3 patients revealed the same mutation in G and Nk for 1 patient and in G and T for 2 patients. These findings indicate that human HSCs may take a similar differentiation pathway to that of murine HSCs, the ‘myeloid-based model’ that was recently proposed by Kawamoto et al. (Nature 2008; 10:452), though the cases with PNH-type T cells alone remain to be elucidated. Table. Lineages of cells containing PNH-type cells in patients with AA or RA. The number in the parenthesis denotes the proportion of patients showing each combination pattern in the total patients possessing PNH-type cells. (+ ; presence of PNH-type cells) Disclosures No relevant conflicts of interest to declare.

Establishment of a flow cytometry assay for detecting paroxysmal nocturnal hemoglobinuria-type cells specific to patients with bone marrow failure

2018 ◽  
Vol 97 (12) ◽  
pp. 2289-2297
Author(s):  
Kohei Hosokawa ◽  
Chiharu Sugimori ◽  
Ken Ishiyama ◽  
Hiroyuki Takamatsu ◽  
Hideyoshi Noji ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Flow Cytometry Assay

Paroxysmal Nocturnal Hemoglobinuria: An Atypical Clinical Case Report and Proof of the Disease in Blood and BONE Marrow with FLOW Cytometry

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5166-5166
Author(s):  
Fabienne Pineau-Vincent ◽  
Pierre Lemaire ◽  
Habib Ghnaya ◽  
Guillaume Direz ◽  
Mohamed Kaabar ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Case Report ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Blood Smear ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Chromatin Condensation ◽  
Clinical Signs ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired disease, associated with hemolytic anemia and bone marrow failure. The cellular abnormality is a mutation in the phosphatidylinositol glycan class (PIG A) resulting in a deficiency of glycosylphosphadityl-inositol (GPI)-anchored complement regulatory proteins, including CD 55 and CD59, on the surface of blood cells. Case report We report the case of a French, 81 year-old-man, who was admitted to our institution with an unusual clinical presentation. He had a rheumatologic monitoring in the context of polyarthritis associated with anemia (98g/L). No hemolytic events were noticed and there was no notion of either transfusion. Biological results showed hemolytic regenerative anemia (98g/L) with 136G/L of reticulocytes, neutrophil polynuclears (4.2G/L) without degranulation and nevertheless rare degranulation cells, no blasts, normal level of platelets (258G/l), increase of LDH (Nx3), low haptoglobin (0.07g/L), negative direct Coombs test. The cytology aspect of medullar cells associated dysgranulopoiesis with degranulation of myeloid lineage and abnormal chromatin condensation, dyserythropoiesis, dysmegacaryopoiesis, in favor of a multilineage dysplasia without blasts. The marrow karyotype was normal. Due to the morphological results observed on the blood smear and their dissociation with the medullary cytology, flow cytometry (FC500) for GPI‘s expression study was performed. The used antisera were: CD55, CD59, CD14, CD16, CD24, CD66b, CD157, no FLEAR was tested. Results TableBloodBone marrowMononuclear cells CD14 FL378% intermediar cells70% negative cellsNeutrophil cells CD16 PE56% intermediar cells56% negative cellsNeutrophil cells CD66b FITC57% negative cells70% negative cellsGranular cells CD24 PE49% negative cells62% negative cellsRed cells CD55 FITC10% negative cells11% negative cellsRed cells CD59 FITC12% negative cells12% negative cells Figure 1 Blood Figure 1. Blood Figure 2 Bone Marrow Figure 2. Bone Marrow The confirmation was obtained by using CD157PE antisera on bone marrow with 70% negative mononuclear and granular cells. The results confirmed the PNH clone’s presence in the blood and also in bone marrow, and the results of flow cytometry could explain the cytological aspect of neutrophil polynuclear cells. It is rare to explore the expression of GPI molecules in bone marrow and there is no publication about the PNH clone whose identification required bone marrow cells for the confirmation of abnormalities in blood. Thus, the apoptosis in the bone marrow of the defective myeloid cells would explain the difference of granularity of polynuclear cells between bone marrow and blood smear. Conclusion The significance of this observation is related to the search of a PNH clone when cytological dissociation is observed between the peripheral blood and bone marrow, associated with biological hemolysis arguments (increased LDH and decreased haptoglobin). It is well known that 6 at 8% of myelodysplasia had PNH clone; the originality of this case report is the initial clinical signs and the laboratory proof of PNH in the blood and the bone marrow. This observation was submitted at the national reference center of PNH in France (St Louis Hospital - Hematology Department - Professor SOCIE) and the treatment by eculizumab was introduced. Disclosures No relevant conflicts of interest to declare.

The Pathophysiology of Disease in Patients with Paroxysmal Nocturnal Hemoglobinuria

Hematology ◽  
2008 ◽  
Vol 2008 (1) ◽  
pp. 104-110 ◽  
Author(s):  
Monica Bessler ◽  
Jeffrey Hiken
Keyword(s):  
Bone Marrow ◽  
Red Blood Cells ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Progenitor Cell ◽  
Clinical Phenotype ◽  
Bone Marrow Failure ◽  
Hematopoietic Progenitor Cell ◽  
Glycosyl Phosphatidylinositol ◽  
Abnormal Cells

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia caused by the expansion of a hematopoietic progenitor cell that has acquired a mutation in the X-linked PIGA gene. PNH occurs on the background of bone marrow failure. Bone marrow failure and the presence of the abnormal cells account for the clinical phenotype of patients with PNH including hemolysis, cytopenia, and thrombophilia. PIGA is essential for the synthesis of glycosyl phosphatidylinositol (GPI) anchor molecules. PNH blood cells are therefore deficient in all proteins that use such an anchor molecule for attachment to the cell membrane. Two of these proteins regulate complement activation on the cell surface. Their deficiency therefore explains the exquisite sensitivity of PNH red blood cells to complement-mediated lysis. Complement-mediated lysis of red blood cells is intravascular, and intravascular hemolysis contributes significantly to the morbidity and mortality in patients with this condition. PNH is an outstanding example of how an increased understanding of pathophysiology may directly improve the diagnosis, care, and treatment of disease.

A Minor Population of CD55−CD59− Blood Cells Detected by Two-Color Flow Cytometry in Aplastic Anemia Patients: A Reliable Marker for Good Response to Immunosuppressive Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1593-1593
Author(s):  
Chiharu Sugimori ◽  
Tatsuya Chuhjo ◽  
Xuzhang Lu ◽  
Xingmin Feng ◽  
Ken Ishiyama ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Aplastic Anemia ◽  
Blood Cells ◽  
Blood Collection ◽  
Glycophorin A ◽  
Newly Diagnosed ◽  
Color Flow ◽  
Minor Population ◽  
Reliable Marker ◽  
A Minor

Abstract Modern flow cytometry techniques using monoclonal antibodies (mAbs) specific to glycosylphosphatidylinositol-anchored membrane proteins have revealed an increase in the proportion of paroxysmal nocturnal hemoglobinuria (PNH)-type cells in a varying (30 to 50) percentage of acquired aplastic anemia (AA) patients who do not manifest any signs of hemolysis. However, the clinical significance of such a small proportion of PNH-type cells remains uncertain because previous studies have used a variety of flow cytometric assays with different mAbs and different definitions of an abnormal increase in the proportion of PNH-type cells, and also, few studies have focused on the correlation of presence of increased PNH-type cells with response to immunosuppressive therapy (IST) based on a large number of newly diagnosed AA patients. We previously demonstrated, using two-color flow cytometry capable of precisely determining the proportion of PNH-type cells less than 0.1%, that such a minor population of PNH-type cells represents a reliable marker for presence of immune pathophysiology in refractory anemia patients (Blood,100: 3897–3902, 2002; Blood,102:1211–1216, 2003). To determine if the PNH-type cells serve as a marker for immune pathogenesis in AA as well, we examined peripheral blood in 99 newly diagnosed AA patients (67 severe AA and 32 moderate AA) for presence of CD55−59−CD11b+ granulocytes and CD55−59−glycophorin A+ RBCs and studied correlation of response to ATG + cyclosporine (CsA) therapy with presence of increased PNH-type cells. When the influence of time from blood collection until treatments of cells with mAbs on the outcome of PNH-type-cell detection were examined, the percentages of PNH-type cells remained unchanged for 2 days for granulocytes and for 6 days for RBCs from the time of blood collection. Minor (>0.003% for CD11b+ granulocytes and >0.005% for glycophorin A+ RBCs) population of PNH-type cells were detected in 66 (66.7%) patients. The proportions of PNH-type cells in patients with increased PNH-type cells (PNH+ patients) were 0.005–0.01% in 10 (15.2%) patients, 0.01–0.1% in 20 (30.3%), 0.1–1.0% in 25 (37.9%), and 1.0–8.67% in 10 (15.2%). When clinical characteristics of PNH+ patients were compared with that of PNH− patients, PNH+ patients (mean, 54 year-old) were significantly older than PNH− patients (mean, 42 year-old, [Italic]P[/Italic]=0.042). 55 of 66 (83.3%) PNH+ patients improved with IST and achieved PR or CR whereas 17 of 33 (52.9%) PNH− patients responded. Kaplan-Meier analysis showed that PNH+ patients had a significantly better chance of response to IST than PNH- patients (Figure). These results indicate that a minor population of PNH-type cells defined by the two-color flow cytometry represents a good marker for response to IST in AA patients. Since CD55−59−glycophorin A+ RBCs less than 0.1% remain constant and detectable in blood stored at 4 ?C for up to 6 days, testing blood cells, particularly RBCs, using this sensitive flow cytometry is useful in identifying high responders to IST in AA patients. Figure Figure

scholarly journals Paroxysmal nocturnal hemoglobinuria clone in bone marrow of patients with pancytopenia

Blood ◽  
1995 ◽  
Vol 85 (5) ◽  
pp. 1371-1376 ◽  
Author(s):  
H Nakakuma ◽  
S Nagakura ◽  
N Iwamoto ◽  
T Kawaguchi ◽  
M Hidaka ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Blood Cells ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Unknown Origin ◽  
Diagnostic Value ◽  
Follow Up Study ◽  
Early Appearance ◽  
Pnh Clone

The lack of glycosylphosphatidylinositol (GPI)-anchored membrane proteins such as decay-accelerating factor (DAF) and CD59 on blood cells has a diagnostic value in paroxysmal nocturnal hemoglobinuria (PNH). Because PNH often develops in patients with aplastic anemia (AA), we attempted to detect a PNH clone in the bone marrow (BM) of patients with AA and pancytopenia before affected cells were evident in the peripheral blood (PB). We used flow cytometry with monoclonal antibodies against DAF and CD59 for the detection of the clone. Affected cells were observed in the BM of 3 of 7 patients with AA and 1 of 3 patients with pancytopenia of unknown origin, but not in their PB. All 8 patients with apparent PNH had affected cells in their BM and PB. On the basis of the early appearance of the PNH clone in the BM, a prospective 4-month follow-up study of the PB cells was performed. The study showed the release of affected mature cells first in granulocytes, then in monocytes, and finally in lymphocytes. Ham's test was positive before affected erythrocytes were detected by flow cytometry. Our findings indicate that detection of the PNH clone in BM could be predictive of the development of PNH in patients with AA and pancytopenia.

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