MONITORING OF SMALL PAROXYSMAL NOCTURNAL HEMOGLOBINURIA CLONE IN PATIENTS AFFECTED BY SEVERE APLASTIC ANEMIA OR APLASTIC ANEMIA
(Abstract release date: 05/19/16)
EHA Library. Milani R. 06/09/16; 134668; PB1768
Dr. Raffaella Milani
Contributions
Contributions
Abstract
Abstract: PB1768
Type: Publication Only
Background
Flow cytometry is the gold standard method for screening of paroxysmal nocturnal hemoglobinuria (PNH). The most reliable marker to detect and monitor small granulocyte or monocyte PNH clones is FLAER (fluorescent aerolysin), especially in conditions such as myelodysplastic syndromes or bone marrow failure, when traditional GPI-linked surface marker expression can be significantly altered. More than 50 % of aplastic anemia patients have a PNH clone detectable by highly sensitive assays, although the underlying mechanism is yet to be unveiled.
Aims
Monitoring of small PNH clone in patients followed at our Institute for severe aplastic anemia or aplastic anemia.
Methods
Between May, 2011 and Feb, 2016, sixteen patients with severe aplastic anemia (sAA)/ aplastic anemia (AA) provided 52 blood samples for PNH flow cytometry at different time points in the course of their evaluation and treatment. We performed serial quantification of the PNH clone size by multiparameter flow cytometry using a Navios cytometer (Beckman Coulter) and Navios software. The immunophenotype evaluation is performed on EDTA whole blood samples. Granulocytes, monocytes and red blood cells (RBCs) were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers FLAER and CD59 were comparatively evaluated. Neutrophils and monocytes were screened with FLAER/CD24/CD14/CD33/CD45 combination. The sensitivity of detection allowing identification of PNH clone sizes as small as 0.01% for RBCs and 0.03% for granulocytes.
Results
Eight of 16 sAA/AA patients were detected to have PNH clone positivity. All of them were treated with standard immunosuppressive therapy such as antithymocyte globulin and cyclosporine (ATG/CsA) or CsA alone and two non responder patients received allogeneic stem cell transplantation (alloSCT) after myeloablative conditioning. Granulocyte PNH clone was <1% in 4 patients, 1-5% in 3 patients and >30% in one patient. Lactate dehydrogenase (LDH) values were normal in all patients except the one with PNH clone >30%, who had clinical evidence of hemolysis. Haptoglobin level was normal in 5 patients. Two patients with PNH clone 1-5% had very low concentration of haptoglobin, without altered LDH values. These were patients who did not respond to ATG/CsA and received alloSCT. Direct Antiglobulin Test (DAT) were negative in all patients. PNH clone sizes were stable during immunosuppressive therapy courses with a median follow up of 42 months (3-60), while PNH clone became undetectable after alloSCT and haptoglobin levels returned to normal values (respectively 6 month and 36 month follow up).
Conclusion
Whether the presence of a small PNH clone in the setting of hypocellular marrow failure has clinical significance or predicts response to treatment and outcomes is still controversial, an accurate monitoring of PNH clone with high sensitive assays and hemolysis parameters might be useful to clarify the pathophysiology of these complex and rare diseases.
Session topic: E-poster
Keyword(s): Bone marrow failure, Flow cytometry, PNH
Type: Publication Only
Background
Flow cytometry is the gold standard method for screening of paroxysmal nocturnal hemoglobinuria (PNH). The most reliable marker to detect and monitor small granulocyte or monocyte PNH clones is FLAER (fluorescent aerolysin), especially in conditions such as myelodysplastic syndromes or bone marrow failure, when traditional GPI-linked surface marker expression can be significantly altered. More than 50 % of aplastic anemia patients have a PNH clone detectable by highly sensitive assays, although the underlying mechanism is yet to be unveiled.
Aims
Monitoring of small PNH clone in patients followed at our Institute for severe aplastic anemia or aplastic anemia.
Methods
Between May, 2011 and Feb, 2016, sixteen patients with severe aplastic anemia (sAA)/ aplastic anemia (AA) provided 52 blood samples for PNH flow cytometry at different time points in the course of their evaluation and treatment. We performed serial quantification of the PNH clone size by multiparameter flow cytometry using a Navios cytometer (Beckman Coulter) and Navios software. The immunophenotype evaluation is performed on EDTA whole blood samples. Granulocytes, monocytes and red blood cells (RBCs) were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers FLAER and CD59 were comparatively evaluated. Neutrophils and monocytes were screened with FLAER/CD24/CD14/CD33/CD45 combination. The sensitivity of detection allowing identification of PNH clone sizes as small as 0.01% for RBCs and 0.03% for granulocytes.
Results
Eight of 16 sAA/AA patients were detected to have PNH clone positivity. All of them were treated with standard immunosuppressive therapy such as antithymocyte globulin and cyclosporine (ATG/CsA) or CsA alone and two non responder patients received allogeneic stem cell transplantation (alloSCT) after myeloablative conditioning. Granulocyte PNH clone was <1% in 4 patients, 1-5% in 3 patients and >30% in one patient. Lactate dehydrogenase (LDH) values were normal in all patients except the one with PNH clone >30%, who had clinical evidence of hemolysis. Haptoglobin level was normal in 5 patients. Two patients with PNH clone 1-5% had very low concentration of haptoglobin, without altered LDH values. These were patients who did not respond to ATG/CsA and received alloSCT. Direct Antiglobulin Test (DAT) were negative in all patients. PNH clone sizes were stable during immunosuppressive therapy courses with a median follow up of 42 months (3-60), while PNH clone became undetectable after alloSCT and haptoglobin levels returned to normal values (respectively 6 month and 36 month follow up).
Conclusion
Whether the presence of a small PNH clone in the setting of hypocellular marrow failure has clinical significance or predicts response to treatment and outcomes is still controversial, an accurate monitoring of PNH clone with high sensitive assays and hemolysis parameters might be useful to clarify the pathophysiology of these complex and rare diseases.
Session topic: E-poster
Keyword(s): Bone marrow failure, Flow cytometry, PNH
Abstract: PB1768
Type: Publication Only
Background
Flow cytometry is the gold standard method for screening of paroxysmal nocturnal hemoglobinuria (PNH). The most reliable marker to detect and monitor small granulocyte or monocyte PNH clones is FLAER (fluorescent aerolysin), especially in conditions such as myelodysplastic syndromes or bone marrow failure, when traditional GPI-linked surface marker expression can be significantly altered. More than 50 % of aplastic anemia patients have a PNH clone detectable by highly sensitive assays, although the underlying mechanism is yet to be unveiled.
Aims
Monitoring of small PNH clone in patients followed at our Institute for severe aplastic anemia or aplastic anemia.
Methods
Between May, 2011 and Feb, 2016, sixteen patients with severe aplastic anemia (sAA)/ aplastic anemia (AA) provided 52 blood samples for PNH flow cytometry at different time points in the course of their evaluation and treatment. We performed serial quantification of the PNH clone size by multiparameter flow cytometry using a Navios cytometer (Beckman Coulter) and Navios software. The immunophenotype evaluation is performed on EDTA whole blood samples. Granulocytes, monocytes and red blood cells (RBCs) were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers FLAER and CD59 were comparatively evaluated. Neutrophils and monocytes were screened with FLAER/CD24/CD14/CD33/CD45 combination. The sensitivity of detection allowing identification of PNH clone sizes as small as 0.01% for RBCs and 0.03% for granulocytes.
Results
Eight of 16 sAA/AA patients were detected to have PNH clone positivity. All of them were treated with standard immunosuppressive therapy such as antithymocyte globulin and cyclosporine (ATG/CsA) or CsA alone and two non responder patients received allogeneic stem cell transplantation (alloSCT) after myeloablative conditioning. Granulocyte PNH clone was <1% in 4 patients, 1-5% in 3 patients and >30% in one patient. Lactate dehydrogenase (LDH) values were normal in all patients except the one with PNH clone >30%, who had clinical evidence of hemolysis. Haptoglobin level was normal in 5 patients. Two patients with PNH clone 1-5% had very low concentration of haptoglobin, without altered LDH values. These were patients who did not respond to ATG/CsA and received alloSCT. Direct Antiglobulin Test (DAT) were negative in all patients. PNH clone sizes were stable during immunosuppressive therapy courses with a median follow up of 42 months (3-60), while PNH clone became undetectable after alloSCT and haptoglobin levels returned to normal values (respectively 6 month and 36 month follow up).
Conclusion
Whether the presence of a small PNH clone in the setting of hypocellular marrow failure has clinical significance or predicts response to treatment and outcomes is still controversial, an accurate monitoring of PNH clone with high sensitive assays and hemolysis parameters might be useful to clarify the pathophysiology of these complex and rare diseases.
Session topic: E-poster
Keyword(s): Bone marrow failure, Flow cytometry, PNH
Type: Publication Only
Background
Flow cytometry is the gold standard method for screening of paroxysmal nocturnal hemoglobinuria (PNH). The most reliable marker to detect and monitor small granulocyte or monocyte PNH clones is FLAER (fluorescent aerolysin), especially in conditions such as myelodysplastic syndromes or bone marrow failure, when traditional GPI-linked surface marker expression can be significantly altered. More than 50 % of aplastic anemia patients have a PNH clone detectable by highly sensitive assays, although the underlying mechanism is yet to be unveiled.
Aims
Monitoring of small PNH clone in patients followed at our Institute for severe aplastic anemia or aplastic anemia.
Methods
Between May, 2011 and Feb, 2016, sixteen patients with severe aplastic anemia (sAA)/ aplastic anemia (AA) provided 52 blood samples for PNH flow cytometry at different time points in the course of their evaluation and treatment. We performed serial quantification of the PNH clone size by multiparameter flow cytometry using a Navios cytometer (Beckman Coulter) and Navios software. The immunophenotype evaluation is performed on EDTA whole blood samples. Granulocytes, monocytes and red blood cells (RBCs) were gated using forward and side scatter as well as lineage-specific markers. The GPI-linked markers FLAER and CD59 were comparatively evaluated. Neutrophils and monocytes were screened with FLAER/CD24/CD14/CD33/CD45 combination. The sensitivity of detection allowing identification of PNH clone sizes as small as 0.01% for RBCs and 0.03% for granulocytes.
Results
Eight of 16 sAA/AA patients were detected to have PNH clone positivity. All of them were treated with standard immunosuppressive therapy such as antithymocyte globulin and cyclosporine (ATG/CsA) or CsA alone and two non responder patients received allogeneic stem cell transplantation (alloSCT) after myeloablative conditioning. Granulocyte PNH clone was <1% in 4 patients, 1-5% in 3 patients and >30% in one patient. Lactate dehydrogenase (LDH) values were normal in all patients except the one with PNH clone >30%, who had clinical evidence of hemolysis. Haptoglobin level was normal in 5 patients. Two patients with PNH clone 1-5% had very low concentration of haptoglobin, without altered LDH values. These were patients who did not respond to ATG/CsA and received alloSCT. Direct Antiglobulin Test (DAT) were negative in all patients. PNH clone sizes were stable during immunosuppressive therapy courses with a median follow up of 42 months (3-60), while PNH clone became undetectable after alloSCT and haptoglobin levels returned to normal values (respectively 6 month and 36 month follow up).
Conclusion
Whether the presence of a small PNH clone in the setting of hypocellular marrow failure has clinical significance or predicts response to treatment and outcomes is still controversial, an accurate monitoring of PNH clone with high sensitive assays and hemolysis parameters might be useful to clarify the pathophysiology of these complex and rare diseases.
Session topic: E-poster
Keyword(s): Bone marrow failure, Flow cytometry, PNH
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