High sensitivity flow cytometry to detect small population of PNH clone in bone marrow failure syndrome in Japan

Immunobiology ◽  
2016 ◽  
Vol 221 (10) ◽  
pp. 1186
Author(s):  
Yasutaka Ueda ◽  
Jun-ichi Nishimura ◽  
Chiharu Sugimori ◽  
Kohei Hosokawa ◽  
Yuji Yonemura ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Bone Marrow Failure ◽  
High Sensitivity ◽  
Small Population ◽  
Bone Marrow Failure Syndrome ◽  
Pnh Clone

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.

Fluorescence Activated Cell Sorting (FACS) Followed by Fluorescence In Situ Hybridization (FISH) To Determine Clonal Origins of Cells in Myelodysplastic Syndrome (MDS) with Paroxysmal Nocturnal Hemoglobinuria (PNH).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4623-4623
Author(s):  
Minoo Battiwalla ◽  
Sheila Sait ◽  
AnneMarie W. Block ◽  
Ibrahim Kebbewar ◽  
Manmeet S. Ahluwalia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Bone Marrow Failure ◽  
Macrocytic Anemia ◽  
Negative Control ◽  
Fish Analysis ◽  
Fish Family ◽  
Immune Evasion Mechanism ◽  
Software House ◽  
Pnh Clone

Abstract The combination of FACS and FISH has been used to identify clonal cytogenetic abnormalities in small populations of cells in diseases such as multiple myeloma. We describe our refinement and application of this technique in a patient with known MDS/PNH to dissect the clonal origins of the disorder. The subject is a 45-year old woman with MDS diagnosed five years ago when she presented with macrocytic anemia and thrombocytopenia. Bone marrow was hypocellular (20% cellularity), with 4% blasts, FAB-RA, WHO- RCMD, Intermediate-1 by IPSS, del 20q clone by FISH. Family history is significant for a familial cytopenia with two sisters developing bone marrow failure, but negative for carriage of the hTERC mutation. HLA DRB1 type was 0101;1301. There was no evidence for T-LGL by flow cytometry or by TCR gene rearrangement assay. Management has been observational only without the need for transfusions. A new PNH clone was detected for the first time by flow cytometry six months ago. Heparinized blood was processed by the laboratory on the same day as collected. WBCs were separated from RBCs by a combination of dextran centrifugation and lysis of RBCs with ammonium chloride. Normal mouse IgG (3mg/mL) was added to block Fc receptors. The cells were stained with mAb cocktails containing CD16 PE (Clone 3G8, Invitrogen), CD55 PC5 (clone IA10, BD Bioscience), and CD15 APC (clone HI98, BD Bioscience) for granulocytes; or CD14 PE (clone TUK4, Invitrogen), CD55 PC5, and CD64 (clone M22, Trillium) for monocytes. Concurrent with mAb staining proaerolysin (FLAER Ax488, Protox Biotech) was added to each tube. The cells were incubated with mAbs and FLAER reagent at saturating concentrations in the dark, on ice, for 30 minutes. They were then washed once with PBS, fixed in 0.5% methanol free formaldehyde (Polysciences, Warrington, PA) for 20 minutes and finally resuspended in PBS. Cytofluorometric analysis and sorting was performed using a FACSAria (BD BioSciences) flow cytometer. Granulocytes were defined using a Boolean combination of forward, side scatter and CD15; monocytes were defined with forward, side scatter and CD64. Granulocytes and monocytes were sorted into PNH positive and negative cells based on FLAER, CD55 and either CD16 (granulocytes) or CD14 (monocytes) expression. Data was analyzed using WinList (Verity Software House, Topsham, ME). FISH analysis for del(20q) was performed on these cells using the Vysis LSI D20S108 SpectrumOrange Probe (Abbott Molecular Inc.). Our results (table 1) show that the PNH clone (FLAER negative) is a distinct non-overlapping population from the del(20q) dysplastic population. Conclusions: Our technique of flow-sorting followed by FISH is feasible for enriching and characterizing PNH positive and negative cellular fractions in bone marrow failure states. The PNH clone in this patient, contrary to other reports in the literature, does not overlap with the MDS clone, rather arising from normal non-dysplastic cells consistent with a putative immune evasion mechanism. This technique is a powerful clinical or research tool to elucidate clonal origins in PNH-associated bone marrow failure states. Table 1 Pre-sort Post-sort 20q− by FISH Unsorted WBCs 13/200 Monocytes PNH− 67% 76% 34/200 PNH+ 245 81% 5/200 = negative control Granulocytes PNH− 72% 94% 34/200 PNH+ 26% 94% 5/200 = negative control

scholarly journals Evaluation of Patients with PNH Treated By Eculizumab: Real World Data from Turkey

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4805-4805
Author(s):  
Fatma Keklik Karadag ◽  
Mustafa Nuri Yenerel ◽  
Yılmaz Mehmet ◽  
Hava uskudar Teke ◽  
Vildan Ozkocaman ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Retrospective Analysis ◽  
Bone Marrow Failure ◽  
Thrombotic Event ◽  
Specific Information ◽  
Intravascular Hemolysis ◽  
Clone Size ◽  
Cell Counts ◽  
Pnh Clone

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease which is characterized with complement mediated intravascular hemolysis, bone marrow failure and thrombosis. The prevelance of PNH is estimated 1 to 16 cases per million in USA. X-linked somatic mutation of phosphatidylinositol glycan‐A (PIGA) gene in hematopoetic stem cells causes an impairment of glycosylphosphatidylinositol (GPI) anchored proteins. The absence of GPI‐dependent molecules that are CD55 (decay accelerating factor; DAF) and CD59 (membrane inhibitor of reactive lysis; MIRL), normally protect the cell from complement‐mediated hemolysis by preventing the formation of the membrane attack complex. The diagnosis is based on flow cytometry which can detect the deficiency of these two complement regulary proteins. We report a retrospective analysis of demographic and clinical characteristics of PNH patients from different centers. Material and methods: We conducted a retrospective analysis of the patients' recorded data. Patients' demographics, medical and treatment history, comorbid conditions, PNH clone size, disease characteristics and outcomes, symptoms, PNH-specific treatments, PNH-related events, morbidity (including myeloproliferative disease, other malignancies, and infections), mortality. Clinical data captured include lactate dehydrogenase (LDH) levels, PNH clone size, hemoglobin levels, thrombotic events, renal functional tests at the time of diagnosis, and other laboratory data. Specific information collected for eculizumab-treated patients includes dosage and dose adjustments and blood cell counts, reticulocyte count and LDH level after eculizumab treatment. Results: 138 patients were included from 28 different centers. All patients were diagnosed by flow cytometry for GPI-linked antigens on red cells and neutrophils. The number of male (69/138) and female (69/138) patient was equal and the median age was 41 years. Median hemoglobin (hb) level was 8.75±2.13 gr/dL; Platelet (plt) level was 131× 109/L at the time of diagnosis. Overall, 49(35,5%) of the patients had been diagnosed with bone marrow failure, including aplastic anemia or hypoplastic anemia (n=31; 22,5%), myelodysplastic syndromes (n=18; 13%). A history of any prior thrombotic event was reported in 45 patients (32,6%). At the time of analysis, 12 (8,7%) patients had pulmonary hypertension. The median granulocyte and monocyte clone size was 63,6% (±32.26) and 66.76 ±28.75 respectively. Fatigue (58%) is the most commonly reported symptom and abdominal pain was seen in 8% of patients. After the eculizumab therapy, the median time for normalization of Hb and LDH level were 7 and 14,6 months, respectively. There was no correlation between thrombosis and clone size, hb, plt, LDH level at the time of diagnosis. LDH level was higher in fatigue patients compared with the patients who were not fatigue (p=0.021). Discussion: PNH is a clonal but non malignant disease that is very rare and knowledge on large case series is really limited. The clinical findings and symptoms could be variable and unfortunately it takes very long time to diagnose because of unawareness of physicians. Eculizumab is a good option in treatment of PNH for improving symptoms of intravascular hemolysis but we still need better understanding of thrombosis mechanism of PNH to better management. In the future, novel inhibitors of the alternative pathway of complement will be used to improve survival and quality of life for PNH patients. Disclosures No relevant conflicts of interest to declare.

Incidence of PNH Clones by Diagnostic Code Utilizing High Sensitivity Flow Cytometry

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1033-1033 ◽  
Author(s):  
Mayur K Movalia ◽  
Ilene c Weitz ◽  
Seah H Lim ◽  
Andrea Illingworth
Keyword(s):  
Bone Marrow ◽  
Hemolytic Anemia ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
High Sensitivity ◽  
Diagnostic Code ◽  
Follow Up Study ◽  
Follow Up Studies ◽  
Pnh Clone

Abstract Abstract 1033 Paroxysmal nocturnal hemoglobinuria (PNH) is a chronic and life-threatening hematopoietic stem cell disorder characterized by deficiency of the GPI-anchored complement inhibitory proteins CD55/59. Chronic hemolysis from this deficiency leads to serious clinical morbidities including thromboembolism, chronic kidney disease, and increased mortality. The International Clinical Cytometry Society (ICCS) recommends multiparameter high sensitivity flow cytometry (HSFC) as the method of choice for diagnosing PNH. The ICCS also provides guidance on the clinical indications for testing for PNH, including patients (pts) with bone marrow failure (BMF), unexplained cytopenias, unexplained thrombosis, hemoglobinuria and hemolysis. The aim of this study is to use HSFC with sensitivity up to 0.01% to analyze 6,897 pts who were screened for PNH clones utilizing CD235a/CD59 for RBCs, FLAER/CD24/CD15/CD45 for neutrophils and FLAER/CD14/CD64/CD45 for monocytes. We evaluated the clinical indications for PNH testing with the provided ICD-9 diagnostic (DX) codes and examined the change in PNH clone sizes among pts who had follow-up studies in 3–12 months. Based on a sensitivity of at least 0.01%, 6.1% of all pts (421/6897) were found to be PNH positive. Of those pts, 5,545 pts (80.1%) had ICD-9 DX codes provided. The distribution of PNH clone sizes in these PNH+ pts is shown in Figure 1. Aplastic anemia (AA) and hemolytic anemia comprised the most common reasons for testing. In bone marrow failure syndromes, AA pts had the highest incidence of PNH+ clones, 26.3%, followed by pts with unexplained cytopenia, 5.7%, myelodysplastic syndrome (MDS), 5.5%, and anemia (unspecified or in chronic illness), 3.6% (Table 1). The incidence of PNH+ clones for symptoms such as hemolytic anemia was 22.7%, followed by hemoglobinuria 18.9%, and unspecified hemolysis, 7.9%, unspecified iron deficiency, 2.5%, and thrombosis, 1.4%. Of the 421 PNH positive pts, 89 pts (22%) were identified as having follow-up studies in 3–12 months. These pts were categorized into PNH clone sizes of 0.01% – 0.1% (27 pts, 30%), 0.11% – 1% (7 pts, 8%), 1.1% – 10% (18 pts, 20%) and 10.1% – 100% (37 pts, 42%). Of the 64 pts who had PNH clone sizes of 0.01% – 0.1% or 10.1 – 100%, one patient (0.02%) had a follow-up study that resulted in a change of category. Of the 25 pts with PNH clones sizes between 0.11% – 1% and 1.1% – 10%, 10 pts (40%) had a follow-up study resulting in an increase in category, 6 pts (24%) had a follow-up study resulting in a decrease in category and 9 pts (36%) had a follow-up study resulting in no change in category.Figure 1.Distribution of PNH Clone Sizes based on 421 PNH+ PatientsFigure 1. Distribution of PNH Clone Sizes based on 421 PNH+ PatientsTable 1:Incidence of PNH Clones in Patients with ICD-9 Diagnostic Code at Dahl-Chase Diagnostic ServicesICD-9 Diagnostic CodeGeneral DescriptionIncidence of PNH Clone284, 284.01, 284.8, 284.81, 284.89, 284.9Aplastic Anemia26.3% (94/357)238.7, 238.72, 238.73, 238.74, 238.75, 238.76Myelodysplastic Syndrome (MDS)5.5% (32/585)287.5Unexplained Cytopenia5.7% (13/230)284.1Pancytopenia6.0% (63/1058)285.2, 285.21, 285.29, 285.9Anemia Unspecified3.6% (40/1122)283, 283.1, 283.10, 283.11, 283.19, 283.2, 283.9Hemolytic Anemia22.7% (147/647)791, 791.2Hemoglobinuria18.9% (14/74)790.6, 790.99, 790.4Hemolysis7.9% (18/227)325, 415.1, 415.11, 434, 434.01, 444.22, 451.11, 451.19, 452, 453, 453.0, 453.2, 453.4, 453.41, 453.89, 453.9, 557, 557.1Thrombosis1.4% (14/967)280.9Unspecified Iron Deficiency2.5% (7/278)Other ICD-9 diagnostic codes2.1% (26/1232)Not Provided4.8% (51/1065)Note: Table reflects patients who had more than one ICD9 code associated with their laboratory tests. In this single-laboratory experience, we evaluated the incidence of PNH in these high risk groups. In this study, 26.3% of pts with the diagnosis of BMF had PNH+ clones detected, underscoring the need to test this group of pts. The study confirmed the utility of testing pts with unexplained hemolytic anemia, hemolysis and hemoglobinuria where the combined rate of positivity was 48%. In addition, this study highlights the need to monitor pts with small PNH clones by HSFC analysis as these pts may show significant variation over time. This examination of ICD-9 DX code association with presence of PNH+ clones confirms the need to actively test high risk populations for PNH based on the ICCS recommendations to ensure accurate diagnosis and early intervention. Disclosures: Weitz: Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Illingworth:Dahl-Chase: Employment; Alexion: Consultancy, Honoraria, Research Funding.

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

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4868-4868
Author(s):  
Zalina Fidarova ◽  
Elena Mikhailova ◽  
Svetlana Lugovskaia ◽  
Elena Naumova ◽  
Vera Troitskaia ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Aplastic Anaemia ◽  
Partial Remission ◽  
De Novo ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Paroxysmal Nocturnal Haemoglobinuria ◽  
Clone Size ◽  
Pnh Clone

Abstract Introduction Aplastic 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. Objective to detect the PNH clone at AA patients at different stages of disease and to reveal its’ influence on the IST effectiveness. Methods 63 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%. Results The 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%) Conclusion The 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.

An Interim 4-Year Analysis of Prospective Multicenter Observational Study of PNH-Type Cells in Japanese Patients with Bone Marrow Failure Syndrome (OPTIMA study)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4781-4781
Author(s):  
Yukari Shirasugi ◽  
Hideyoshi Noji ◽  
Tsutomu Shichishima ◽  
Chiharu Sugimori ◽  
Naoshi Obara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
High Resolution ◽  
Clinical Significance ◽  
Interim Analysis ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
High Response Rate ◽  
Bone Marrow Failure Syndrome ◽  
Hematopoietic Stem

Abstract Background: Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder caused by the clonal expansion of the phosphatidylinositol glycan class A (PIGA) mutant hematopoietic stem cells, which results in a deficiency in glycosylphosphatidylinositol-anchored proteins (GPI-APs). Through the high-resolution flow cytometry-based method, GPI-APs deficient blood cells (i.e., PNH-type cells) are often detectable in patients with bone marrow failure syndromes (BMF), such as aplastic anemia (AA) and low-risk types of myelodysplastic syndromes (MDS). Sugimori et al reported that when BMF patients possessed increased PNH-type cells, the patients had a good prognosis and showed a high response rate to immunosuppressive therapies, suggesting that detection of PNH-type cells is potentially useful in determining an optimal treatment for BMF patients. Thus, we conducted a nationwide, multi-center prospective observational investigation, the OPTIMA study. Methods: From July 2011, we start recruiting the patients with BMF that were diagnosed at various hematology clinics throughout Japan to the OPTIMA study. The primary endpoint of this study was to determine the prevalence of BMF patients with PNH-type cells and to clarify the clinical significance of the presence and quantitative changes of these cells with regard to the clinical features. Six different university laboratories were assigned as regional analyzing centers. The percentage of PNH-type cells was measured by the high-resolution flow cytometry-based method, originally established in Kanazawa University. At six individual laboratories, cross validations were conducted twice a year to minimize the inter-laboratory variations in the detection sensitivities, cutoff values, etc. The liquid FLAER method (≥0.003%) and cocktail method (≥0.005%) with CD55 and CD59 antibodies were used for the detection of PNH-type granulocytes and erythrocytes, respectively. Results Between July 2011 and May 2015, a total of 2328 patients were enrolled to this study, and we analyzed 2212 patients who were eligible for the interim analysis. Of these patients, 74 (3.3%) were diagnosed with PNH, 690 (31.2%) with AA, 592 (26.8%) with MDS, and 856 (38.7%) with undiagnosed BMF. Using high-resolution flow cytometry-based method, 755 (34.1%; 95.9% in PNH, 52.8% in AA, 18.2% in MDS, and 24.8% in undiagnosed BMT) patients had ≥0.005% PNH-type erythrocytes and ≥0.003% PNH-type granulocytes. Overall, 181(8.2%) patients had ≥1% of both PNH-type erythrocytes and granulocytes; the prevalence in each disease subset was 68/74 (91.9%) in PNH, 67/690 (9.7%) in AA, 22/592 (3.7%) in MDS, and 24/856 (2.8%) in undiagnosed BMF. Regarding FAB and WHO classifications of MDS subtype, no patients with RARS (0/22), RAEB-1 (0/37) or RAEB-2 (0/23) had PNH-type cells. In contrast, 20.4% (56/275) patients with RCMD, 18.3% (26/153) patients with RCUD and 50% (2/4) patients with del (5q) MDS possessed increased PNH-type cells. Blood samples from 75 (65 with and 10 without PNH-type cells) patients were analyzed three years after the first examination. Of 65 PNH (+) patients, PNH-type cells disappeared in 4 (6.2%), while the percentage remained stable in 61 (93.8%). All of the 10 PNH (-) at the enrollment were also negative for PNH-type cells in 3 years. Conclusions: A high-resolution flow cytometry-based method that enables the detection of minimal PNH-type cells below 0.01% was successfully transferred from Kanazawa University to other laboratories in Japan. Our interim analysis confirmed previous findings that PNH-type cells were detectable in patients with 52.8% of AA and 18.2% of MDS patients. Regarding FAB and WHO classifications of MDS subtype, PNH-type cells were not detected in any of MDS RARS, RAEB-1 or RAEB-2 patients. Further analysis are required to determine the clinical significance of the minimal level of PNH-type cells as well as chronological changes in the PNH-type cell percentage, especially in terms of their relation to response to immunosuppressive therapy. Disclosures Ninomiya: Alexion Pharmaceuticals: Honoraria. Ando:Eisai Co., Ltd.: Honoraria, Research Funding. Yonemura:1. Chugai Pharma, 2. Alexion Pharma, 3. Japan Blood Products Organization, 4. OHARA Pharma: Research Funding. Kawaguchi:Alexion Pharmaceuticals: Honoraria. Ueda:Alexion Pharma: Research Funding. Nishimura:Alexion Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Prevalence of paroxysmal nocturnal hemoglobinuria (PNH) cells in patients with myelodysplastic syndromes (MDS), aplastic anemia (AA), or other bone marrow failure (BMF) syndromes: Interim results from the EXPLORE trial

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7082-7082 ◽  
Author(s):  
N. Galili ◽  
F. Ravandi ◽  
G. Palermo ◽  
J. Bubis ◽  
A. Illingworth ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Interim Analysis ◽  
Bone Marrow Failure ◽  
Treatment Options ◽  
White Blood Cells ◽  
High Sensitivity ◽  
Clone Size ◽  
Hematopoietic Stem ◽  
Point Prevalence Study ◽  
Pnh Clone

7082 Background: In patients with PNH, lack of the glycophosphatidylinositol (GPI)-anchored terminal complement inhibitor CD59 on hematopoietic stem cells results in chronic intravascular hemolysis, kidney and pulmonary disorders, thrombosis, and shortened life span. Presence of even minor populations of PNH cells in AA or MDS patients is medically important as it may indicate a higher likelihood of response to immunosuppressive therapy. We conducted the first large multicenter, point-prevalence study (EXamination of PNH, by Level Of CD59 on REd and white blood cells [EXPLORE]) of PNH cells in patients with AA, MDS, or other BMF syndromes. Here we report an interim analysis. Methods: A central laboratory conducted high-sensitivity flow cytometry utilizing a combination of GPI-linked antibodies (CD59, CD24, and CD14) and fluorescent aerolysin (FLAER) to identify GPI anchor-deficient PNH cells in RBC and WBC resulting in 0.01% sensitivity. The primary endpoint was percentage of patients who had a PNH WBC clone ≥1%. Results: Among 5,212 patients screened, 4,500 (86.3%) were MDS patients, 413 (7.9%) were AA patients, and 356 (6.8%) had other BMF syndromes. Approximately 1/4 (24.5%) of patients with AA, 1.2% with MDS, and 4.6% with other BMF were newly found to have a significant PNH clone ≥ 1%. Many of the newly identified clones were of clinical significance as the median PNH clone size was 11.1% in AA patients, 16.3% in MDS patients, and 32.6% in patients with other BMF. Presence of PNH cells (≥ 0.01%) was common in all examined BMF types: 70% of AA patients, 55% of MDS patients and 55% of patients with other BMF. PNH cells were identified in all MDS subtypes represented in the trial. Conclusions: Interim analysis from this first large multicenter study demonstrates that PNH cells are present in a majority of patients with AA, MDS, and other BMF. A spectrum of PNH clone sizes was noted in patients with each form of BMF. Screening patients with BMF with high-sensitivity flow cytometry for PNH cells may guide treatment options for the underlying BMF and/or PNH. The EXPLORE trial continues to enroll patients with AA. (EXPLORE Clinical Study Abstract 1/6/2009) [Table: see text]

scholarly journals Lactic Acidosis in a Congenital Bone Marrow Failure Syndrome

2021 ◽  
pp. 1-4
Author(s):  
Fatima Farid Mir ◽  
Anjan Madasu ◽  
Hani Humad ◽  
Asim Noor Rana
Keyword(s):  
Bone Marrow ◽  
Mitochondrial Dna ◽  
Metabolic Acidosis ◽  
Lactic Acidosis ◽  
Liver Failure ◽  
Bone Marrow Failure ◽  
Disease Course ◽  
Bone Marrow Failure Syndrome ◽  
Severe Lactic Acidosis ◽  
End Stage

Fifteen-month-old male child, known to have a congenital bone marrow failure syndrome, presented in a state of shock with severe lactic acidosis following a brief episode of vomiting. Hospital stay was complicated by recurrent bouts of metabolic acidosis and progressive hepatic failure. Blood mitochondrial DNA sequencing revealed a large heteroplasmic 4,977 bp mitochondrial deletion (approximately 40% of all mitochondrial copies) suggestive of Pearson marrow-pancreas syndrome. By virtue of natural disease course, within a month of admission child succumbed to end-stage liver failure with multi-organ failure and died.

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