Diagnosis of Paroxysmal Nocturnal Hemoglobinuria (PNH) by the REDQUANT and the CELLQUANT kits
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Background : Paroximal nocturnal hemoglobinuria (PNH) is a disorder of the pluripotent stem cells resulting in a deficient expression of membrane-bound GPI-anchored proteins in different cell types. We evaluated REDQUANT and CELLQUANT kits (Biocytex, Marseille, France) for PNH test. Methods : Seventy patients with peripheral blood cytopenia and 16 healthy controls were studied. RBCs and granulocytes were tested for CD55 and CD59 expression using the REDQUANT and CELLQUANT kits and an Epics XL flow cytometer. According to the manufacturer’s instruction, results were interpreted abnormal when more than 3% of cells were deficient in the expression of CD55 or CD59, and a test was considered positive for PNH if three of the four markers tested were abnormal. Results : The percentage of CD55/CD59 deficient RBCs and granulocytes was 0.3/3.1 and 3.5/ 10.0, respectively, in the patient group, and 0.1/1.0 and 0.3/9.7, respectively, in the control group. PNH was diagnosed in three patients who had a deficiency in the expression of three or four antigens; two other patients showed a deficiency in two antigens. There were many who had CD59 deficiency only: on granulocytes in 30 patients and 11 controls, and on RBCs in 6 patients and 2 controls. One patient had CD55 deficient granulocytes. Conclusion : The REDQUANT/CELLQUANT kit is a standardized method and does not require normal samples as the control, but one should be cautious in interpreting the results showing CD59 expression on granulocytes.Keywords:
Paroxysmal nocturnal hemoglobinuria
CD59
Hemoglobinuria
Aplastic anemia
Cytopenia
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Summary Introduction Paroxysmal nocturnal hemoglobinuria ( PNH ) is a hemolytic, clonal and acquired disorder of the hematopoietic stem cell with a deficiency of all glycophosphatidyl‐inositol ( GPI ) linked proteins. The aim of this retrospective study was to analyse haematological and biochemical data from 152 patients referred to our laboratory for diagnosis of PNH by flow cytometry ( FC ). Methods Patients and healthy donor (152 and 99 respectively) were studied. Ham, sucrose, lactate dehydrogenase ( LDH ), Iron, haptoglobin (Hp), blood cell morphology and Kaplow cytochemical stain for leukocyte alkaline phosphatase ( LAP ) were carried out. GPI ‐proteins anti‐ CD 55 and CD 59 in erythrocytes and the former, plus anti CD 16b and CD 66b on neutrophils were evaluated by FC . Results Anemia and/or leukopenia and/or thrombocytopenia, increased reticulocyte count and LDH were observed in patients with PNH clone. Some of them had dacriocytes, schistocytes. LAP was low. On average, we detected 50% CD 59 (−) erythrocytes and 29, 83, 78% CD 55/59 (−), CD 16b (−), CD 66b (−) neutrophils, respectively. Conclusion Paroxysmal nocturnal hemoglobinuria clone was detected in 20/152 patients. Negative population's percentages were high in patients with classic PNH , Hematimetry, LAP and adequate use of CF contribute to PNH clone detection in the laboratory.
Paroxysmal nocturnal hemoglobinuria
Hemoglobinuria
clone (Java method)
Haemolysis
Schistocyte
Haptoglobin
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Paroxysmal nocturnal hemoglobinuria
Aplastic anemia
CD59
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Paroxysmal nocturnal hemoglobinuria
Aplastic anemia
clone (Java method)
Hemoglobinuria
Thrombomodulin
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Citations (133)
INTRODUCTION: Paroxysmal nocturnal hemoglobinuria (PNH) is an uncommon disease. Many cases go undiagnosed as high index of clinical suspicion is required for its detection. This study was performed to detect the presence of PNH defect by flow cytometric immunophenotyping (FCMI) in patients with suspected PNH disease and evaluate their clinical and laboratory profile.MATERIALS AND METHODS: In this retrospective study, a total of 136 patients with suspected PNH who fulfilled the inclusion criteria for the study were evaluated for PNH defect by FCMI using monoclonal antibodies against CD55 and CD59 on red blood cell, granulocytes, and monocytes.RESULTS: Forty-eight (35.3%) of 136 patients evaluated had a PNH defect. Nineteen (39.5%) of these 48 patients had classical PNH (hemolytic type). The remaining 29 patients had PNH Clone in association with aplastic anemia. The clinical and laboratory data of these 19 patients with classical PNH were analyzed in this retrospective study. The median age was 34 years (range: 19–65 years). Thrombotic events were observed in 3 (16%) of the 19 cases (one each with Budd–Chiari syndrome, cerebral venous thrombosis, and abdominal vein thrombosis). The flow cytometric data of these patients were further analyzed for the presence of and size of PNH clone on erythrocytes, granulocytes, and monocytes. PNH clone was detected in 84% of erythrocytes, 76.9 % of monocytes and in 100% granulocytes.CONCLUSION: Classical PNH is not rare in India as previously thought. A high index of clinical suspicions and evaluation by FCMI is necessary for its detection. CD59 is a better marker for identification of PNH clone than CD55 in all three cell types.
Paroxysmal nocturnal hemoglobinuria
Eculizumab
Hemoglobinuria
Aplastic anemia
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Objective To establish an efficient way for the diagnosis of paroxysmal nocturnal hemoglobinuria(PNH) through the combination of the hemolytic anemia(HA) associated routine tests with flow cytometry (FCM) of the red cell CD55/CD59 expression. Methods The peripheral erythrocytes and granulocytes were analyzed with flow cytometry for CD55/CD59 expression while the deficient red blood cells were further studied for forward scatter (FS), FS/CD55 and FS/CD59. Other hemolytic anemia associated tests were also employed as routine. Results PNH could be diagnosed based on the results of FCM and routine tests for peripheral erythrocytes and granulocytes, which showed the characteristic defects when the HA broke out. During remission , PNH deficient red blood cells were mainly destroyed and the peripheral blood was negative for Ham's test. FCM analysis revealed those PNH cells with CD55/CD59 deficiency and the changes of FS/CD59, which was helpful in the diagnosis and the differentiation from those PNH patients with aplastic anemia whose PNH cells also exist. Conclusions FCM analysis of the peripheral red blood cells' CD55/CD59 expression in combination with HA-related routine tests was efficient in the diagnosis of PNH.
Paroxysmal nocturnal hemoglobinuria
CD59
Aplastic anemia
Hemoglobinuria
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Background. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal haematopoietic stem cell disorder characterised by intravascular haemolysis, bone marrow failure and increased tendency to thrombosis. It is caused by a somatic mutation in the PIG-A gene, which encodes an enzyme essential for the synthesis of glycosylphosphatidylinositol (GPI) anchors. The PIG-A mutation results in a clone of blood cells with total or partial deficiency of membrane proteins anchored to the cell surface through GPI anchor. For many years, an increased susceptibility of the PNH red cells to complement lysis in acidified serum (Ham’s test) has been essential for diagnosis of the PNH. Current flow cytometric assays for PNH rely on the use of labeled antibodies to detect deficiencies of the specific GPI anchor proteins on blood cells. We evaluated a three-colour flow cytometry method for detection and quantification of CD55/59 negative netrophils. Patients and methods. Nine patients (6 with PNH and 3 with aplastic anaemia) who were previously diagnosed as having PNH or aplastic anaemia were evaluated. In the period of three years the patients were serially evaluated for the extent of PNH clone by quantification of CD55/59 negative neutrophils. Three-colour flow cytometry method was used for quantification of the CD55/CD59 negative neutrophils. We used directly conjugated monoclonal antibodies anti-DC15PC5 for identification of neutrophils and anti-CD59-FITC and anti-CD55-PE for the GPI-linked antigens. Results. Patients with haemolytic PNH had > 50% CD59/CD55 negative granulocytes. The proportion of the PNH granulocytes was higher in the patient with frequent and serious haemolytic attacks. Over the period of three years slow growth of the PNH clone was seen in two cases. Two patients with PNH diagnosed 19 years ago and in remission at the time of flow cytometric analysis was devoid of the PNH clone. Three patients with aplastic anaemia (hypoplastic PNH) had the proportion of CD59/CD55 negative granulocytes < 40%. In one of them PNH clone increased. Conclusions. Three colour flow cytometry of granulocytes using combination of anti-CD15/55/59 provides the accurate technique for detection and quantification of the PNH clone. Monitoring the PNH clone size has clinical and prognostic value. The size of the PNH clone is an important determinant for thrombotic risk. Serial studies allow prediction of remission in some cases or progress from hypoplastic to hemolytic PNH in others.
CD59
Haemolysis
Paroxysmal nocturnal hemoglobinuria
clone (Java method)
Eculizumab
Aplastic anemia
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To evaluate the significance of glycophosphatidylinositol deficient cells in the diagnosis of paroxysmal nocturnal hemoglobinuria (PNH).RBC, and PMN from peripheral blood and mononuclear cells (MNCs) from bone marrow of 29 PNH patients were treated with FITC labelled anti-CD59 monoclonal antibody. Percentages of labelled and unlabelled cells were discriminated by flow cytometry.Normally CD9(-) cells constituted less than 5% in RBCs, PMNs and marrow MNCs, while in each of the patients more than 10% of RBCs were CD59(-) (ranging from 12.8% to 98.0%). More than 10% of PMNs were CD59(-) as well in each of the patients (ranging from 12.0% to 90.1%). Marrow MNCs from the patients contained even more CD59(-) cells (ranging from 17.9% to 73.5%). Ten patients with atypical manifestations were confirmed to be PNH, whereas 7 suspected cases were excluded by this measurement.Determination of CD59(-) cells in RBCs, PMNs and more preferably in marrow MNCs could be considered a direct, specific and reliable method for diagnosis of PNH.
Paroxysmal nocturnal hemoglobinuria
CD59
Hemoglobinuria
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Objective To investigate the expression of glycosyl-phosphatidyl inositol(GPI) anchored protein on peripheral blood cells from patients with paroxysmal nocturnal hemoglobinuria (PNH) and provide a basis for diagnosis and differential diagnosis of PNH.Methods The percentages of CD55,CD59,CD16 and CD14 expression on the peripheral blood cells from 32 patients with PNH were determined by flow cytometry. Results The expression levels of CD55 and CD59 were decreased significantly on the erythrocytes and granulocytes from patients with PNH compared with control group (P0.001),the expression levels of CD55 and CD59 on granulocytes were lower than those on erythrocytes (P0.05).The expression levels of CD55 and CD59 on the thrombocytes of PNH patients were lower than those in control group(P0.001).The expression levels of CD55 and CD59 on the lymphocytes from 27 PNH patients were lower than those on granulocytes and erythrocytes,the deficiency rate was 30%.The percentage of CD16 expression varied greatly from 34% to 83% on the granulocytes,there was significant difference compared with control group (P0.001). The deficiency of CD14 clones was detected on the monocytes from PNH patients.Conclusion The diagnosis reliability of patients with PNH is increased by the assay of CD55 and CD59 on all kinds of cells of peripheral blood.The detection of CD16 and CD14 is also an optimal and helpful index to diagnose PNH.
Paroxysmal nocturnal hemoglobinuria
CD59
Hemoglobinuria
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Aplastic anemia
Paroxysmal nocturnal hemoglobinuria
CD59
Eculizumab
Ineffective erythropoiesis
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To detect paroxysmal nocturnal hemoglobinuria (PNH) clone in aplastic anemia (AA) patients.Flow cytometric technique along with antibodies against CD59 was used to estimate the amount of GPI-P deficient cells, characteristic of PNH abnormalities.Among 23 cases of AA studied, 13 possessed no excessive percentage of CD59(-) cells in peripheral PMNs and RBCs. Eight of these 13 cases underwent bone marrow examination, and no increase of CD59(-) mononuclear cell (MNC) was found either. Three other AA patients had slightly increased amount (> 5%) of CD59(-) cells in both peripheral blood and bone marrow. In seven cases, percentages of CD59(-) cells were normal in peripheral RBCs and PMNs but increased in bone marrow MNCs.Measurement of CD59(-) cells in periperal blood and bone marrow by flow cytometry may serve as the best way of detecting PNH clone in AA, and thus could be used for the early diagnosis of AA-PNH syndrome.
Paroxysmal nocturnal hemoglobinuria
CD59
Aplastic anemia
clone (Java method)
Hemoglobinuria
Bone marrow failure
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