Paroxysmal Nocturnal Hemoglobinuria Clone Size Research Articles

A New Approach of PNH Clone Detection

IntroductionThe presence of the CD157 GPI-protein on both monocytes and granulocytes only gives opportunity to use it as a target for paroxysmal nocturnal hemoglobinuria (PNH) phenotype cells detection instead of CD24 and CD14, thus improving the technique of PNH-cells evaluation. In present study we used standard technique (International Clinical Cytometry Society (ICCS) guideline proposal) together with 5 color combination of mAbs for detection of PNH-clone on the monocytes and granulocytes simultaneously in one test-tube.Objectives: improvement of PNH cells detection technique.Materials and methods: Blood samples were collected from 37 patients (pts) with bone marrow failure syndrome (aplastic anemia – 34 pts., PNH - 2 pts., myelodysplastic syndrome – 1 pt.; 23 female and 14 men; median age 24 (15 to 66). PNH clone was detected by flow cytometry in all 37 pts. Three healthy donors were in control group.The comparison of the standard 4-color technique of PNH clone size detection on monocytes (FLAER, CD14, CD64, CD45 reagents) and on granulocytes (FLAER, CD24, CD15, CD45 reagents) with the 5-color technique using CD157 GPI-protein antibodies for the both cells populations in one test-tube (FLAER, CD157, CD15, CD64, CD45) were performed.Results: Both methods showed the same high sensitivity for determining of PNH clone. The correlation coefficient was 0,9994 for granulocytes and 0,9924 for monocytes (Figure 1). [Display omitted] In group of patients with minor PNH-clone (range from 0,01% to 0,99%) the clone size was twice times bigger on monocytes compared to granulocytes using the standard method, whereas with antibodies against CD157 this difference disappeared. In patients with PNH-clone between 1% and 10% the clone was nearly three times less on granulocytes than monocytes using both methods; in the group with clone more than 10% there was no differences between cells populations.Table 1Average value of PNH clone on leukocytes in groups of patients both techniquesPNH cloneGr, % (mean)Mon, % (mean)CD24-/FLAER-CD157-/FLAER-CD14-/FLAER-CD157-/FLAER-0%-0,99%0,1780,3490,3230,3781%-9,99%3,983,769,549,98>10%71,5271,5271,8571,34Conclusion: The results of the PNH clone detection obtained with CD157 mAbs are comparable with the standard technique proposed by ISSC. However, the use of CD157 antibodies has important advantage: the PNH clone size detection in one test-tube with 5-color combination reduces time and expenses. Additionally, using of 5-color CD157 antibodies kit would be preferable for the monitoring and detection of minor PNH clone. DisclosuresNo relevant conflicts of interest to declare.

Allo-HSCT from MUD/MRD As a Curative Treatment in Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired clonal abnormality of the hematopoietic stem cell caused by somatic mutation in the phosphatidylinositol glycan-class A (PIG-A) gene located on the short arm of the X chromosome. Cells with lack phosphatidylinositol glycoproteins are more sensitive to complement-mediated lysis. Despite the efficient symptomatic treatment of hemolytic PNH with eculizumab, allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only curative treatment of the disease, although outcomes presented in the past were controversial.Material and methods: We report 32 allo-HSCTs: 31 from MUD and 1 from MRD performed for PNH in 2004-2014. Median age of recipients was 28 years (range 20-55) and donors 33(19-53), median time from diagnosis to allo-HSCT was 18(2-307) months. Median size of PNH clone was 80% granulocytes (0.41%-98%). Indication for allo-HSCT was aplastic/hypoplastic bone marrow (15 pts), overlapping MDS (2 pts), severe course of PNH with hemolytic crises and transfusion-dependency without access to eculizumab (15 pts). Additional risk factors were Budd-Chiari syndrome and hepatosplenomegaly (1 pt), history of renal insufficiency requiring hemodialyses (2 pts) and chronic hepatitis B (1 pt). The preparative regimen consisted of treosulfan 3x14 g/m2 plus fludarabine 5x30 mg/m2 (25 pts) or treosulfan 2x10 g/m2 plus cyclophosphamide 4x40 mg/kg (7 pts). Standard GVHD prophylaxis consisted of cyclosporine-A, methotrexate and pre-transplant ATG or thymoglobulin in MUD-HSCT. 2 pts instead cyclosporine-A received mycophenolate mofetil and tacrolimus. Source of cells was bone marrow (12 pts) or peripheral blood (20 pts) with median 7.7x10(8)NC/kg, 5.3x10(6)CD34+cells/kg, 24.2x10(6)CD3+cells/kg. Myeloablation was complete in all pts with median 9 days (6-13) of absolute agranulocytosis <0.1 G/l. Median number of transfused RBC and platelets units was 8.5(1-16) and 8(3-18).Results All pts engrafted, median counts of granulocytes 0.5 G/l, platelets 50 G/l and Hb 10 g/dl were achieved on days 17.5(13-33), 17.5(11-39) and 19.5(11-34). Acute GVHD grade I,II and III was present in 14, 6 and 1 pt, limited chronic GVHD in 11 pts. LDH decreased by 77%(5%-91%) in first 30 days indicating disappearance of hemolysis. 100% donor chimerism was achieved in all pts. In 1 patient donor chimerism decreased to 83% what was treated with donor lymphocytes infusion (DLI). 2 patients died, 1 previously hemodialysed pt died on day +102 in a consequence of nephrotoxicity complicating adenoviral/CMV hemorrhagic cystitis and second on day +56 because of severe pulmonary infection. Complications in survivors were FUO (7 pts), CMV reactivation (8), VOD (1), neurotoxicity (1), venal thrombosis (1), hemorrhagic cystitis (1) and mucositis (8). 30 pts (93.7%) are alive 42 months (1-85) post-transplant and are doing well without treatment. Complete disappearance of PNH clone was confirmed by flow cytometry in all surviving pts.Conclusions: Our results indicate, that allo-HSCT with treosulfan-based conditioning is effective and well tolerated curative therapy in PNH. DisclosuresNo relevant conflicts of interest to declare.

Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia.

Flow cytometry is the gold standard methodology for screening of paroxysmal nocturnal hemoglobinuria. In the last few years, proaerolysin conjugated with fluorescein (FLAER) has become an important component of antibody panel used for the detection of paroxysmal nocturnal hemoglobinuria (PNH) clone. This study aimed to compare PNH clone detection by flow cytometry in the pre-FLAER era versus the FLAER era. This was a retrospective analysis of 4 years and included 1004 individuals screened for PNH clone, either presenting as hemolytic anemia or as aplastic anemia. In the pre-FLAER time period, the RBCs and neutrophils were screened with antibodies against CD55 and CD59. With the introduction of FLAER, neutrophils were screened with FLAER/CD24/CD15 and monocytes with FLAER/CD14/CD33 combination. A comparative analysis was done for detection of PNH clone in aplastic anemia patients versus non-aplastic anemia patients, as well as between pre-FLAER and FLAER era. Out of a total of 1004 individuals, 59 (5.8%) were detected to have PNH clone positivity. The frequency of PNH clone detected in aplastic anemia and non-aplastic anemia groups was 12.02 and 3.36%, respectively. The detection rate of PNH clone increased from 4.5% (32/711) in the pre-FLAER era to 9.2% (27/293) with the introduction of FLAER. However, this increase could be attributed to increased detection of PNH clone in the aplastic anemia group, which showed a significant increase from 8.3 to 18.2% after use of FLAER. In the non-aplastic group, PNH clone was detected with similar frequencies before and after use of FLAER (3.2 versus 3.8%, respectively). Mean PNH clone size was lower in the aplastic anemia group when compared with the non-aplastic group. RBCs always showed a lower clone size than neutrophils. PNH clone on neutrophils and monocytes was however similar. Inclusion of FLAER increases the sensitivity of the test which is especially useful in picking up small PNH clones in patients of aplastic anemia.

Rapidly evolving skin manifestations due to progressive thrombosis in a patient with paroxysmal nocturnal haemoglobinuria resolved with prompt initiation of eculizumab

Funding sources: none. Conflicts of interests: P.M. has sat on advisory boards for Alexion. Dear Editor, Paroxysmal nocturnal haemoglobinuria (PNH) is a rare acquired clonal disorder of the haematopoietic stem cell. PNH arises from mutations in the PIGA gene, which encodes glycosylphosphatidylinositol (GPI)‐anchored proteins. Consequently, these mutations result in a deficiency of GPI‐anchored proteins in cell membranes.1 This deficiency causes PNH red blood cells to be susceptible to complement‐induced intravascular haemolysis.2 However, PNH is also characterized by a high rate of thrombosis.3 4 Venous and arterial thrombosis can be the first symptom of PNH. PNH‐induced thrombosis often occurs at unusual sites and may progress in days to weeks.5 Moreover, thromboembolic complications and consequences are the leading cause of morbidity and death in PNH.4 The pathogenesis of thrombosis in PNH is complex and not completely elucidated at this time.3 The clinical picture of PNH is highly variable and depends on the PNH clone size, factors of complement activation and whether or not there is an underlying bone marrow disease. Complications of PNH occur spontaneously or can be triggered by bacterial or viral infections, for example by highly contagious parvovirus B19.6 7 Cutaneous manifestations of PNH comprise rapidly evolving purpura, haemorrhagic bullae and ulcerations presumably due to necrosis, secondary to intravascular clotting.8 9 10

Analysis of abnormal clones by the fluorescent aerolysin method in paroxysmal nocturnal haemoglobinuria and other marrow disorders

Flow cytometry is the most sensitive and specific diagnostic modality for the assessment of clone size in paroxysmal nocturnal haemoglobinuria (PNH) and other bone marrow failure states. In this study, we attempt to distinguish PNH from aplastic anaemia (AA) and myelodysplastic syndromes (MDS) associated with PNH clones at diagnosis by clone size, clinical and laboratory features. A total of 29 samples included 19 PNH cases and 10 AA/MDS cases with PNH clones. Flow cytometry was performed using fluorescent aerolysin (FLAER)-based assay and comparison of clinical features, laboratory parameters and PNH clone size was carried out at diagnosis. The PNH clone size on granulocytes varied from 0.4% to 99.2% and correlated with the clone size on monocytes (r=0.966; P<0.001). Paroxysmal nocturnal haemoglobinuria clone size on granulocytes (median=34.6%) and monocytes (median=49.9%) was always larger than erythrocytes (median=10.9%). The median clone size in PNH (median granulocytes=74.9%, monocytes=71.8%) was significantly greater than in AA/MDS associated with PNH clone (median granulocytes=2.9%, monocytes=6%). In PNH patients, a significant negative correlation was seen between PNH clone on monocytes and the haemoglobin concentration. In our small study using the FLAER method, the clone size was >70% in majority of PNH cases. In other marrow disorders like AA/MDS, the clone size was usually <10%.

Baseline characteristics and disease burden in patients in the International Paroxysmal Nocturnal Hemoglobinuria Registry.

Paroxysmal nocturnal hemoglobinuria is a rare, acquired disease associated with hemolytic anemia, bone marrow failure, thrombosis, and, frequently, poor quality of life. The International PNH Registry is a worldwide, observational, non-interventional study collecting safety, effectiveness, and quality-of-life data from patients with a confirmed paroxysmal nocturnal hemoglobinuria diagnosis or detectable paroxysmal nocturnal hemoglobinuria clone, irrespective of treatment. In addition to evaluating the long-term safety and effectiveness of eculizumab in a global population, the registry aims to improve diagnosis, optimize patient management and outcomes, and enhance the understanding of the natural history of paroxysmal nocturnal hemoglobinuria. Here we report the characteristics of the first 1610 patients enrolled. Median disease duration was 4.6 years. Median granulocyte paroxysmal nocturnal hemoglobinuria clone size was 68.1% (range 0.01-100%). Overall, 16% of patients had a history of thrombotic events and 14% a history of impaired renal function. Therapies included anticoagulation (31%), immunosuppression (19%), and eculizumab (25%). Frequently reported symptoms included fatigue (80%), dyspnea (64%), hemoglobinuria (62%), abdominal pain (44%), and chest pain (33%). Patients suffered from poor quality of life; 23% of patients had been hospitalized due to paroxysmal nocturnal hemoglobinuria-related complications and 17% stated that paroxysmal nocturnal hemoglobinuria was the reason they were not working or were working less. This international registry will provide an ongoing, valuable resource to further the clinical understanding of paroxysmal nocturnal hemoglobinuria.

Paroxysmal Nocturnal Haemoglobinuria Clone In Aplastic Anaemia Patients At The Beginning Of Disease and In Remission

IntroductionAplastic Anaemia (AA) and Paroxysmal Nocturnal Haemoglobinuria (PNH) are severe hematological diseases accompanied by bone marrow failure syndromes. The high-sensitivity flow cytometry standartised methods helped us to detect the PNH clone incedence at AA patients at the different stages of disease and of treatement and to reveal its' influence on the immunosuppressive therapy (IST) effectiveness. Objectiveto detect the PNH clone at AA patients at different stages of disease and to reveal its' influence on the IST effectiveness. Methods63 patients with severe AA (SAA) who received combined IST with antithymocytic globulin (hATG) and cyclosporin A (CsA) have been included into the study. Mediane age – 26 years (16-65).All 63 patients were divided into 2 groups. The 1st one included de novo AA patients (n=28); the 2nd group – AA patients in complete remission (CR) after IST (n=35). The median remission duration was 3 years (2-6 y). The results of the de novo AA treatment (1stgroup) were evaluated at 3, 6 and 12 months from the start of IST.We used the flow cytometry (Becton Dickinson (BD) FACS Canto II and Beckman Coulter (BC) FC 500) to evaluate the PNH clone.Peripheral blood samples were analyzed with antibodies CD45(BD), CD15(BD), CD64(BD), CD235a(BC), GPI-tying antibodies CD59 (Invitrogen), CD14(BC), CD24(BC) and FLAER (Cedarlane). Minor PNH clone was detected when the count of GPI- deficient cells did not exceed 1%. ResultsThe PNH clone was found in 18 patients among 28 (64%) from the 1st group. The minor clone was found in 4 patients, in 3 patients the clone size exceeded 50%. Median (Me) clone size on the Red Blood Cells (RBC: type II + type III) was 0,25% (0,03-25,3%), Granulocytes (GR) – 1,7% (0,02- 93,92%), Monocytes (Mon)- 23,2% (0,05-95,66%).7/18 SAA patients (38,9%) with the PNH clone, showed a haematological response at 3 months from the treatment start, including 3 patients with the minor PNH clone. 2/18 patients (11.1%) underwent allogenic bone marrow transplantation (alloBMT). 9/18 did not get the remission by the 3d month.4/18 patients (22,2%), without response at 6 months, received the second course of IST; 5/18 patients (27.7%) are being followed up and can‘t be analyzed at 6 months.It worth to note, that PNH clone disappeared after allo-BMT (n=2) and in 1 patient with CR after IST.None of 10 patients without PNH clone attained response at 3 months (p=0,02). 4 out of 10 (40%) achieved only partial remission at 6 months. In these cases minor PNH clone appeared after hematological response and persisting from 6 till 18 months. 6 other patients are still on the treatment (ATG).In the 2nd group the PNH clone was detected in 26 of 35 cases (74,3%), only 11 of them had a minor clone. The Me of PNH clone size on RBC - 1,4% (0,02 to 3,76%), Gr – 25,2% (0,01-93,73%), Mon - 23,52% (0,01-54,32%) ConclusionThe PNH clone has been detected in more than 60 % of de novo SAA patients. The disease was characterized by pancytopenia and aplasia of the bone marrow without clinical signs of intravascular hemolysis. In our study we observed the quick IST response at 3 months in SAA patients with the PNH clone (38,9%), in patients without PNH clone at time of diagnosis achievement of partial remission at 6 months was followed by PNH clone appearance and persistence. Disclosures:No relevant conflicts of interest to declare.

Value Of Magnetic Resonance Imaging In The Study and Follow-Up Of Paroxysmal Nocturnal Hemoglobinuria

IntroductionRenal damage is relatively frequent in Paroxysmal Nocturnal Hemoglobinuria (PNH). Iron accumulation from hemolysis in the renal tubules has been found to be one of the triggering factors. Hemosiderin accumulates in the epithelial cells of the proximal renal tubules localized in the renal cortex, and they can be measured (and monitoring) using magnetic resonance imaging (MRI). MethodsWe studied 7 PNH patients with MR using T2-weighted gradient echo and multiecho sequences. We quantified renal, hepatic and myocardial iron with T2* relaxometry model. Four of these patients were studied at diagnosis and after 1 year of treatment with eculizumab. In 2 of them the MRI was done after 2 and 3 years respectively of treatment with eculizumab. One patient was studied after 8 months on eculizumab, and MRI was repeated at 12 and 24 months. Liver and myocardial iron deposition was quantified in all cases. ResultsAt diagnosis the median hemoglobin of the 7 patients was 8.9 g/dl (8.2-12-4), the PNH median PNH clone size was 77% (60-90) and the median LDH level was 2340 U/L (1100-3600). Three patients presented an important accumulation of iron in the renal cortex, with much lower signal intensity than the medulla in the T2-weighted MR images. One patient with mild hemolysis (Hb 12.4 g/dl, 20% reticulocytes and 1,100 LDH) did not present iron accumulation in the renal cortex, and neither did the 2 patients studied after 2 a 3 years of eculizumab treatment. All the patients improved, presenting median of Hb of 11,2 g/dl ( 9,5-12,9) and of LDH of 520 U/L ( 430- 721). The patient studied after 8 months showed an increase of iron in the renal cortex that persisted, despite the improvement with the treatment with eculizumab, with increase of the Hb and decrease of the LDH levels; this patients had a C hepatitis and positivity on the hemochromatosis genes C282Y/H63D, with important iron hepatic overload (11-17 mgFe/g). In the rest of the patients the hepatic iron was normal, with values from 0.5 to 2 mgFe/g, except in one case where the levels were higher (10 mgFe/g) due to previous transfusions used to treat an aplastic anemia pre-PNH. Myocardial iron was normal in the 7 patients, with values ranging from 33 to 60 ms in T2. Only one of the patients presented several episodes of acute renal insufficiency, related with hemolytic crisis, with residual proteinuria, that disappeared when treated with eculizumab. ConclusionsA diffuse signal loss in the renal cortex without liver and spleen alteration is indicative of intravascular hemolysis, suggesting PNH. Serial MRI studies of iron overload in the renal cortex can be used for the diagnosis, and for monitoring the effects of treatment. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A prospective multicenter study of paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure

BackgroundParoxysmal nocturnal hemoglobinuria (PNH), a rare clonal hematopoietic stem cell disorder, is characterized by chronic, uncontrolled complement activation leading to intravascular hemolysis and an inflammatory prothrombotic state. The EXPLORE study aimed to determine the prevalence of undiagnosed PNH in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS), and/or other bone marrow failure (BMF) syndromes and the effect of PNH clone size on hemolysis.MethodsPatients, selected from medical office chart reviews, had blood samples collected for hematologic panel testing and for flow cytometry detection of PNH clones.ResultsGranulocyte PNH clones ≥ 1% were detected in 199 of all 5,398 patients (3.7%), 93 of 503 AA patients (18.5%), 50 of 4,401 MDS patients (1.1%), and 3 of 130 other BMF patients (2.3%). Higher-sensitivity analyses detected PNH clones ≥ 0.01% in 167 of 1,746 patients from all groups (9.6%) and in 22 of 1,225 MDS patients (1.8%), 116 of 294 AA patients (39.5%), and four of 54 other BMF patients (7.8%). Among patients with PNH clones ≥ 1%, median clone size was smaller in patients with AA (5.1%) than in those with MDS (17.6%) or other BMF (24.4%), and the percentage of patients with lactate dehydrogenase levels (a marker for intravascular hemolysis) ≥ 1.5 × upper limit of normal was smaller in patients with AA (18.3%) than in those with MDS (42.0%).ConclusionsThese results confirm the presence of PNH clones in high-risk patient groups and suggest that screening of such patients may facilitate patient management and care.

A Prospective Multicenter Study of Paroxysmal Nocturnal Hemoglobinuria Cells in Patients with Bone Marrow Failure.

Background: Paroxysmal nocturnal hemoglobinuria (PNH), a rare clonal hematopoietic stem cell disorder, is characterized by chronic, uncontrolled complement activation leading to intravascular hemolysis and an inflammatory prothrombotic state. The EXPLORE study aimed to determine the prevalence of undiagnosed PNH in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS), and/or other bone marrow failure (BMF) syndromes and the effect of PNH clone size on hemolysis. Methods: Patients, selected from medical office chart reviews, had blood samples collected for hematologic panel testing and for flow cytometry detection of PNH clones. Results: Granulocyte PNH clones ≥ 1% were detected in 199 of all 5398 patients (3.7%), 93 of 503 AA patients (18.5%), 50 of 4401 MDS patients (1.1%), and 3 of 130 other BMF patients (2.3%). Higher-sensitivity analyses detected PNH clones ≥ 0.01% in 167 of 1746 patients from all groups (9.6%) and in 22 of 1225 MDS patients (1.8%), 116 of 294 AA patients (39.5%), and 4 of 54 other BMF patients (7.8%). Among patients with PNH clones ≥ 1%, median clone size was smaller in patients with AA (5.1%) than in those with MDS (17.6%) or other BMF (24.4%), and the percentage of patients with lactate dehydrogenase levels (a marker for intravascular hemolysis) ≥ 1.5 × upper limit of normal was smaller in patients with AA (18.3%) than in those with MDS (42.0%). Conclusions: These results confirm the presence of PNH clones in high-risk patient groups and suggest that screening of such patients may facilitate patient management and care. © 2013 Clinical Cytometry Society.

Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection

Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH.CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages.Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R(2) >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples.While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs.

Intra‐ and interlaboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high‐sensitivity paroxysmal nocturnal hemoglobinuria testing

Sutherland et al. recently published the Practical Guidelines for high-sensitivity detection of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM), containing concise protocols for PNH testing. Using this approach, we studied the intra- and interlaboratory variability observed in a multicenter study in which fresh blood samples containing three clinically relevant PNH clone sizes within the granulocytic, monocytic, and red blood cell (RBC) populations were shipped to each participating center. Coefficients of variation (CVs) for precision/reproducibility analysis ranged from 0.01%/0.02% to 0.48%/0.45% (big clone), from 0.69%/1.52% to 4.24%/5.80% (small-intermediate clone), from 1.47%/3.91% to 15.01% /17.83% (minor clone) for PNH white blood cells (WBCs) and from 0.24%/0.48% to 1.76%/1.83% (big clone), from 0.80%/1.14% to 2.39%/4.45% (small-intermediate clone), from 1.09%/3.36% to 10.54%/10.23% (minor clone) for PNH RBCs, respectively. Linear regression analysis showed excellent performance correlation between centers (r > 0.99), Wilcoxon rank test revealed no statistically significant differences for PNH granulocytes, monocytes, and RBCs (P > 0.05%), Bland-Altman analysis demonstrated good performance agreement for all target PNH clones (mean bias ranging from -1.47 to 0.71). Our results demonstrate very good intra- and interlaboratory performance characteristics for both precision and reproducibility analyses and excellent correlation and agreement between centers for all target PNH clone sizes. Our data confirm the reliability and robustness of the recently published Practical Guidelines approach for high sensitivity PNH testing by flow cytometry and suggest that such an approach represents an excellent basis for standardization of PNH testing by flow cytometry.

Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection.

Background: Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH. Methods: CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15 and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages. Results: Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R2 >0.99) with predicate values over a range (0.06% - 99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples. Conclusion: While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs. © 2013 Clinical Cytometry Society.

Bone marrow histology in patients with a paroxysmal nocturnal hemoglobinuria clone correlated with clinical parameters

Paroxysmal nocturnal hemoglobinuria (PNH) is a disease with variable presentation, including classical PNH (cPNH) and PNH with aplastic anemia (AA-PNH). Here, we describe bone marrow (BM) histology related to clinical findings in 67 patients with a PNH clone. Patients were divided in AA-PNH (n = 39) and cPNH (n = 28) based on clinical criteria and were compared to 17 AA patients without PNH clone. Median PNH clone size was higher in cPNH (75 %) than in AA-PNH (10 %) (p < 0.0001). BM cellularity was normal or increased in 65 % of cPNH, while it was decreased in AA-PNH and AA (95 % and 100 %) (p < 0.0001). Myelopoiesis and megakaryopoiesis were decreased in 85 % and 100 % of AA-PNH and 100 % of AA patients, but also in 86 % and 46 % of cPNH patients, even when peripheral blood values were normal. The percentage of CD59-deficient late-stage myeloid cells determined immunohistochemically correlated to PNH granulocyte clone size. Lymphoid nodules and increased mast cell numbers were present in all groups but more frequently in AA-PNH (38 % and 73 %) than in cPNH (20 % and 43 %). BM iron was decreased in 88 % of cPNH, while it was increased in AA-PNH (64 %) and AA (100 %) (p < 0.0001). Hemolysis was present in all cPNH but also in AA-PNH patients (67 %). In conclusion, cPNH patients had more cellular BM and more prominent erythropoiesis than AA-PNH patients. Nonetheless, cPNH patients also show AA features such as myeloid and megakaryocyte hypoplasia and inflammatory infiltrates, although more subtle than in AA-PNH. No significant differences were found between BM of AA patients with and without PNH clone.

The clinical study of myelodysplastic syndromes with PNH clones

To analyze the clinical characteristics and risk factors on responses and survival of myelodysplastic syndromes (MDS) patients with paroxysmal nocturnal hemoglobinuria (PNH) clones. The clinical data of 31 MDS cases with PNH clones from October 2004 to June 2012 were retrospectively analyzed to reveal the influence of PNH clone size on responses and survival. ①The chromosome karyotypes were analyzed in all patients, 23 patients with normal karyotype, 7 patients with abnormal karyotype [including 3 patients with +8, 2 -Y, 1 del(7q) and 1 Xp+] and 1 patient with no mitosis. 1 patient belonged to low-risk, 27 intermediate-1 risk, 2 intermediate-2 risk and 1 high-risk groups, respectively, according to IPSS. There were significantly statistical differences between responders and nonresponders in terms of infection, ANC, Reticulocyte count and IPSS (P values were 0.049, 0.006, 0.031 and 0.043, respectively). ②The overall responsive rate was 67.7%, no patients progressed to acute leukemia (AL) during median follow-up of 19 months after immunosuppressive therapy (IST). The 3-year and 5-year overall survival rates were 82.7% and 55.1%,respectively. ③According to univariate analysis,age, infection and ANC had significant influence on survival (P values were 0.050, 0.031 and 0.026, respectively). ④The PNH clone size had no significant influence on survival through univariate and COX analyses (P=0.393). MDS patients with PNH clone had less cytogenetic abnormalities, higher probability of response to IST and lower probability of progression to AL; Furthermore, the PNH clone size had no significant influence on response and survival.

Role of Paroxysmal Nocturnal Hemoglobinuria (PNH) Clones in Refractory Anemias

AbstractAbstract 4406 Background:The presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in the setting of aplastic anemia (AA) and myelodysplastic syndrome (MDS) highlights the pathogenetic link between these disorders. The awareness that small clones of PNH cells could be detected by flow cytometry in aplastic anaemia patients who had negative Ham test has led to classifying PNH into two broad groups such as Haemolytic PNH which can have 50% presenting with thrombosis and a hypo plastic group which behaves like aplastic anaemia. Even if there is a small PNH clone in the setting of aplastic anaemia, then the patients respond better to immunosuppressive therapy.Flowcytometry is a relatively new advanced technique in many parts of India and there is no widespread consensus among methodology. Further, the clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is yet to be established here. Objective:To validate the role of flowcytometry in the diagnosis of PNH, in the setting of aplastic anaemia, myelodysplastic syndrome or abdominal or cerebral vein thrombosis in the setting of PNH which may help to prognosticate the course and treatment. Material and methods:The study was both retrospective and prospective. The prospective arm of the study was conducted from September 2009 to April 2010. Ham's test and Sucrose lysis test were done as screening tests and were reported as positive or negative. Other laboratory studies including hemogram and bone marrow analysis were done as part of standard care. In the retrospective group there were 32 patients who were diagnosed as PNH based on Ham's and Sucrose lysis test followed up from 1990.Four color flowcytometry immunophenotypic analysis with CD55 and CD59 was used to detect PNH clones in RBCs. EDTA-anticoagulated whole blood was stained with anti–CD 55-Phycoerythrin (PE) and anti–CD 59-PE.A minimum of 10,000 events were acquired. For analysis, RBCs were identified by light-scatter properties. For the purpose of the study, 3% of type II and/or type III cells were considered as threshold to diagnose PNH clones in both CD55 and CD 59 gated population. The results of laboratory studies including transfusion history and flow cytometry were correlated. Institutional ethical committee approval was obtained for the conduct of the study. Results:33 patients were included as part of the prospective group. The number of males was more compared to females (19:14). 78% of patients had hemoglobin of less than 7gm%. The presentation varied from unexplained anemia to thrombosis. Based on flow cytometry, 3 patients were found to have PNH clones in the RBC lineage by both CD 55 and CD 59. CD 55 alone picked up one more patient. In the retrospective group there were 32 patients who were followed up from 1990. Interestingly, two of them who had earlier presented with aplastic anemia had transformed into PNH in a span of 3years and 6 months. All patients were treated with Stanazolol and cyclosporine. The median survival was 12.14. months with Kaplan-Meier survival estimates of 30 and 20 percent at 121 months, and 143 months after diagnosis, respectively. The patients in the prospective group are being followed up. Discussion:The Ham test, sucrose lysis test, and modified Ham tests rely on the differential sensitivity of PNH red cells to haemolysis. These tests, although suitable for haemolytic PNH, cannot reliably detect small populations of PNH red cells nor differentiate partially and completely deficient cells. One patient who presented with DVT in whom increase in clone size was demonstrated by serial flowcytometry. Testing for the presence of PNH RBC clones provides additional diagnostic information and the thrombotic event may probably directly related to the size of the PNH clone.Conclusions: Our data demonstrate the utility of RBC assay in flow cytometry in the primary screening of PNH, AA, and MDS patients. The clinical and prognostic significance of monitoring PNH clone size by serial flow cytometry is relatively new and could be demonstrated in one patient. Disclosures:No relevant conflicts of interest to declare.

Clinical features and prognostic factors of Asian patients with paroxysmal nocturnal hemoglobinuria: results from a single center in China

Although all patients with paroxysmal nocturnal hemoglobinuria (PNH) have acquired mutations in the phosphatidylinositol glycan class-A(PIG-A)gene, their clinical courses are highly variable. We reviewed 280 PNH cases referred to our hospital from January 1990 through June 2010 to assess clinical presentations, prognostic factors influencing survival, difference among subcategories, and clinical significance of PNH clone size. The overall survival at 10years after diagnosis estimated by Kaplan-Meier was 77.6%. Both univariate and multivariate analyses identified risk factors affecting survival, including age >40years, absolute neutrophil count<0.5 × 10(9) cells/L, development of thrombotic events, evolution to myelodysplastic syndrome or acute myelogenous leukemia, and recurrent infections. The cohort of patients were divided into subcategories of classic PNH, PNH/AA, and PNH-sc/AA based on the recent proposed PNH working clinical classification, hemoglobinuria as the initial symptomatic manifestation was high up to 82.0% in classic PNH subcategory, whereas only as low as 1.4% in PNH-sc/AA subcategory; the frequencies of infectious (26.0%) and bleeding symptoms (14.0%) in classic PNH subcategory were significantly less than those in PNH/AA (25.3% and 51.7%, respectively) and PNH-sc/AA (48.3% and 83.2%, respectively) subcategories. Our results revealed that large PNH clone was associated with increased risks for hemoglobinuria and thrombosis, whereas small PNH clone was associated with bone marrow failure. Thus, this study may shed insights into Chinese PNH patients to set up individually therapeutic regimens.

Screening Patients with Myelodysplastic Syndrome and Aplastic Anemia for Paroxysmal Nocturnal Hemoglobinuria Clones: A Retrospective Study,

Abstract 3426 Introduction:Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare disorder due to a somatic mutation in the hematopoietic stem cell. The introduction of highly sensitive flow cytometric and aerolysin testing have shown the presence of PNH clones in patients with a variety of other hematological disorders such as aplastic anemia (AA) and myelodysplasic syndrome (MDS). It is hypothesized that patients with these disorders and PNH clones may share an immunologic basis for marrow failure with relative protection of the PNH clone, due to their lack of cell surface expression of immune accessory proteins. This is supported by the literature showing responsiveness in AA and MDS to immunosuppressive treatments. Preliminary results from a recent multicenter trial, EXPLORE, notes that PNH clones can be seen in 70% of AA and 55% of MDS patients, and therefore there may be utility in the general screening of all patients with bone marrow failure (BMF) syndromes. Furthermore, it has been suggested that the presence of PNH cells in MDS is a predictive biomarker that is clinically important for response to immunosuppressive therapy. Methods:Our retrospective cohort study in a tertiary care center used a high sensitivity RBC and FLAER assay to detect PNH clones as small as 0.01%. Of all patients screened with this method, those with bone marrow biopsy and aspirate proven MDS, AA, or other BMF syndromes (defined as unexplained cytopenias) were analysed. Results from PNH assays were compared to other clinical and laboratory parameters such as LDH. Results:Overall, 102 patients were initially screened over a 12 month period at our center. 30 patients were excluded as they did not have biopsy or aspirate proven MDS, AA, or other BMF syndromes. Of the remaining 72 patients, four patients were found to have PNH clones, where 2/51 had MDS (both RCMD, IPSS 0) [3.92%] and 2/4 had AA [50%]. The PNH clone sizes of these four patients were 0.01%, 0.01%, 0.02%, and 1.7%. None of the MDS patients with known recurrent karyotypic abnormalities had PNH clones present. Only one of the four patients had a markedly increased serum LDH level. Conclusions:Our retrospective study indicates much lower incidence of PNH clones in MDS patients or any patients with BMF syndromes when compared to the preliminary data from the EXPLORE trial. There is also significant disagreement in other smaller cohorts in regards to the incidence of PNH in AA and MDS. Screening for PNH clones in patients with bone marrow failure needs further study before adoption of widespread use. Disclosures:Keeney:Alexion Pharmaceuticals Canada Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Wells:Alexion Pharmaceuticals Canada Inc: Honoraria. Sutherland:Alexion Pharmaceuticals Canada Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Natural history of paroxysmal nocturnal hemoglobinuria clones in patients presenting as aplastic anemia

To investigate the natural history of paroxysmal nocturnal hemoglobinuria (PNH) clones in patients with acquired aplastic anemia (AA). Twenty-seven patients with AA and a detectable PNH clone were monitored for a median of 5.7 years (range 1.5-11.5 years). Twenty-two patients received high-dose cyclophosphamide (HiCy) therapy. The erythrocyte and granulocyte PNH clone sizes were measured using flow cytometry and analyzed via CellQuest software. PE-conjugated anti-glycophorin A, anti-CD15, FITC-conjugated anti-CD59, and FLAER staining were used to define glycosylphosphatidylinositol-AP-deficient cells. We found a linear relationship between PNH clone size and the development of intravascular hemolysis, assessed by lactate dehydrogenase (LDH) values (Pearson correlation coefficient = 0.80, P < 0.001 for erythrocyte PNH clones; and Pearson correlation coefficient = 0.73, P < 0.0001 for granulocyte PNH clones). An erythrocyte PNH size of 3-5% and granulocyte PNH size of 23% were the thresholds to predict hemolysis as measured by an elevated LDH (receiver operating characteristic analyses with AUC = 0.96 for erythrocyte PNH clone sizes and AUC = 0.88 for granulocyte PNH clone sizes). Patients with small (≤15%) initial PNH clone sizes were less likely to develop an elevated LDH (mean ± SD: 236.9 ± 109.9 vs. 423.1 ± 248.8; P = 0.02). Over time, the PNH clone sizes remained stable in 25.9% of patients; 48.1% experienced a rise in the PNH clone size; and 25.9% experienced a decrease. The risk of developing clinically significant PNH after HiCy therapy appears to be low in AA patients with PNH clones, especially for those with small initial PNH clones and for those who respond to HiCy therapy.

PNH clone assessment by flow cytometry and its clinical correlation in PNH and aplastic anemia

Flow cytometry is the most sensitive and specific diagnostic modality for paroxysmal nocturnal hemoglobinuria (PNH) clone assessment in PNH and other bone marrow failure states. A total of 101 samples included 23 PNH, 46 aplastic anemia (AA), seven myelodysplastic syndrome (MDS) cases, and 25 normal controls. Flow cytometry was performed using CD55, CD59, and stain–lyse–wash method, and the PNH clone size was correlated with the severity of disease. The PNH clone size on granulocytes in PNH patients varied from 7% to 97% detected with a sensitivity of 0.2% and correlated with the clone on monocytes (r = 0.563; p 0.05). PNH clone (0.4–38.7%) was detected in 23 (50%) AA patients. Three (42.9%) of the seven MDS patients showed PNH clone (<1%). CD55 and CD59 have the advantage of simultaneously analyzing granulocytes and monocytes for PNH clone which correlated with degree of anemia in PNH. A larger patient data is required to assess the clinical impact of PNH clone of variable size in AA and MDS patients.