Small Paroxysmal Nocturnal Hemoglobinuria Clones Research Articles

Comparison of human leukocyte antigen in patients with paroxysmal nocturnal hemoglobinuria of different clone sizes.

Glycosylphosphatidylinositol-anchored protein-deficient hematopoietic stem and progenitor cell development caused by PIGA mutations cannot fully explain the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Herein, patients newly diagnosed with PNH at our hospital between April 2019 and April 2021 were recruited. The human leukocyte antigen (HLA) class I and II loci were analyzed, and patients were stratified by PNH clone sizes: small (< 50%) and large (≥ 50%). In 40 patients (29 males; 72.5%), the median PNH clone size was 72%. Thirteen (32.5%) and twenty-seven (67.5%) patients harbored small and large PNH clones, respectively. DRB1*15:01 and DQB1*06:02 had higher frequencies in patients with PNH than in healthy controls (adjusted P-value = 4.10 × 10-4 and 4.10 × 10-4, respectively). Whole HLA class I and II allele contributions differed (P = 0.046 and 0.065, not significant difference) when comparing patients with small and large PNH clones. B*13:01 and C*04:01 allelic frequencies were significantly higher in patients with small clones (P = 0.032 and P = 0.032, respectively). Patients with small clones had higher class II HLA evolutionary divergence (HED) (P = 0.041) and global class I and II HED (P = 0.019). In the entire cohort, 17 HLA aberrations were found in 11 (27.5%) patients. No significant differences in HLA aberrations were found between patients with small or large clones. In conclusion, patients with small clones tended to have a higher frequency of immune attack-associated alleles. A higher HED in patients with small clones may reflect a propensity for T cell-mediated autoimmunity. HLA aberrations were similar between patients with small and large clones.

ANALYSIS OF BASELINE CHARACTERISTICS, DISEASE BURDEN AND LONG-TERM FOLLOW-UP OF 167 PATIENTS WITH BRAZILIAN PAROXYSMAL NOCTURNAL HEMOGLOBINURIA - ANOTHER NATURAL HISTORY

Paroxysmal nocturnal hemoglobinuria (PNH) may occur as classical hemolytic disease or a small PNH clone found in a patient with bone marrow failure. To describe clinical features and long-term follow-up of 167 patients with PNH and demonstrate differences between disease categories. Multiparametric flow cytometry performed on 1025 patients referred from Jan/2000 to Dec/2019 found 167 (16.3%) confirmed PNH clone, 87M/70F. Clinical characteristics at first visit, and laboratory results, incidence of thrombosis, treatment and outcome during follow-up visits were considered for statistical analysis. Most patients (89.2%) had hypocellular bone marrow at diagnosis; 55(32.9%) developed hemoglobinuria, and 22(13.2%) developed thrombosis during monitoring. Mean age at aplasia diagnosis 28.4 years (range 5.6 - 71.2), and mean age at PNH clone detection 31.0 years (7.2 - 71.3 years). Clonal evolution occurred in 77 (46.1%) patients at a median of 4 years (range 6-281 months) after the onset of cytopenia. The cohort was divided into 15 classic PNH, 55 hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 definitive bone marrow disease with minor PNH clones - subclinical PNH (sc-PNH). Subclinical patients had lower erythrocyte (2.0% vs 24.0% vs 57.8%) and granulocyte PNH clones (11.7% vs 58.8% vs 81.2%) than PNH/AA and classic PNH, respectively. LDH, reticulocytes, absolute neutrophils and bone marrow cellularity were all lower in sc-PNH than in the hemolytic group. Pearson's analysis showed a statistically significant correlation (p < 0.001) between PNH granulocyte clone and RBC clone (0.697), LDH (0.602), absolute neutrophils (0.310), platelet count (0.430), absolute reticulocytes (0.546), total bilirubin (0.330), and bone marrow biopsy (0.435). PNH granulocyte clone greater than 50% was found in 16/22 thrombotic patients. Mean granulocyte clone was 89.1% (3.4%-99.2%) versus 12.3%(0.1%-34.0%), p < 0.001, and erythrocytes 28.4% (3.4-99.8) versus 0.7% (0.1-14.0) in thrombotic versus the remaining 145 patients. Treatment was highly heterogeneous during this 20-year follow-up: immunosuppressive therapy with CSA and/or thymoglobulin was given to 72 patients in the subclinical group (73.1%) and 45 (64.3%) in the hemolytic group; 52 patients received HSCT (31.1%) of which 40 in sc-PNH and 11 in PNH/AA; a total of 19 patients received Eculizumab after 2008 and 3 patients never received treatment. Incidentally, there were 17 thrombotic events in hemolytic patients (38.6%) diagnosed by 2007, compared to only five events (19.2%) in patients diagnosed after 2008. The average follow-up period was 74 months (1 - 330 months), with 31 deaths in total (80.4% OS). Sepsis was the leading cause of sc-PNH mortality (16/18, 88.8%), and thrombosis in both hemolytic groups (11/13, 84.6%). Log rank test shows similar survival when comparing patients with diagnosis until and after 2007 (p = 0.04). this study suggests clonal evolution in the long-term follow-up of aplastic patients and confirms the observation that high levels of PNH and LDH clones are associated with life-threatening hemolysis and thrombosis, while small PNH clones and young age are associated with the subclinical form of the disease. Although without statistical significance, the cumulative incidence of thrombosis in the group diagnosed after 2008 appears to be lower than the previous period, perhaps due to the routine use of Eculizumab.

Analysis of baseline characteristics, disease burden and long-term follow-up of 167 patients with Paroxysmal Nocturnal Hemoglobinuria at a single center in Brazil

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure. MethodsDescribe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up. Results167 patients, mean age at diagnosis 28.4 (7.1–71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%). ConclusionLarge PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

Platelet counts on peripheral blood and Mean Platelet Volume as markers of clinical severity in Sickle Cell Disease

Paroxysmal nocturnal hemoglobinuria (PNH) can occur as a hemolytic form or small PNH clone found in a patient with bone marrow failure.Describe Brazilian retrospective PNH cohort and identify the impact of disease burden on long-term follow-up.167 patients, mean age at diagnosis 28.4 (7.1–71.2 years), four years mean interval between onset of cytopenia/aplasia diagnosis and PNH clone detection. Patients were divided into 15 Classic PNH, 55 Hemolytic PNH with bone marrow hypoplasia (PNH/AA), and 97 Subclinical PNH (sc-PNH). Hypocellular bone marrow was found in 89.2%; 55 had hemoglobinuria and 22 thrombosis during monitoring. WBC PNH clone correlated with RBC PNH clone, LDH and cytopenia. Subclinical patients had lower median lower RBC clone (2.0% vs 24.0% vs 57.8%) and WBC clone (11.7% vs 58.8% vs 81.2%) than PNH/AA and Classic PNH, respectively. PNH granulocyte clone was 89.1% in thrombotic patients. Ten-year overall survival 80.4% and mortality in transplanted patients 9.6%. Sepsis was mortality cause in subclinical PNH (16/18, 88.8%), and thrombosis in hemolytic PNH (11/13, 84.6%).Large PNH clones and LDH burden were associated with increased hemolysis and thrombosis risks, while young patients were associated with small PNH clones and subclinical form of the disease. Knowledge of the patient profile, the low risk associated with HSCT, and the use of long-term IST may be instrumental in the clinical management of PNH in restricted-resources countries.

Clinical and prognostic significance of small paroxysmal nocturnal hemoglobinuria clones in myelodysplastic syndrome and aplastic anemia

In this large single-centre study, we report high prevalence (25%) of, small (<10%) and very small (<1%), paroxysmal nocturnal hemoglobinuria (PNH) clones by high-sensitive cytometry among 3085 patients tested. Given PNH association with bone marrow failures, we analyzed 869 myelodysplastic syndromes (MDS) and 531 aplastic anemia (AA) within the cohort. PNH clones were more frequent and larger in AA vs. MDS (p = 0.04). PNH clone, irrespective of size, was a good predictor of response to immunosuppressive therapy (IST) and to stem cell transplant (HSCT) (in MDS: 84% if PNH+ vs. 44.7% if PNH−, p = 0.01 for IST, and 71% if PNH+ vs. 56.6% if PNH− for HSCT; in AA: 78 vs. 50% for IST, p < 0.0001, and 97 vs. 77%, p = 0.01 for HSCT). PNH positivity had a favorable impact on disease progression (0.6% vs. 4.9% IPSS-progression in MDS, p < 0.005; and 2.1 vs. 6.9% progression to MDS in AA, p = 0.01), leukemic evolution (6.8 vs. 12.7%, p = 0.01 in MDS), and overall survival [73% (95% CI 68–77) vs. 51% (48–54), p < 0.0001], with a relative HR for mortality of 2.37 (95% CI 1.8–3.1; p < 0.0001) in PNH negative cases, both in univariate and multivariable analysis. Our data suggest systematic PNH testing in AA/MDS, as it might allow better prediction/prognostication and consequent clinical/laboratory follow-up timing.

Difficult Cases of Paroxysmal Nocturnal Hemoglobinuria: Diagnosis and Therapeutic Novelties.

Paroxysmal nocturnal hemoglobinuria (PNH) is an intriguing disease that can pose many difficulties to physicians, as well as to hematologists, who are unfamiliar with it. Research regarding its pathophysiologic, diagnostic, and therapeutic aspects is still ongoing. In the last ten years, new flow cytometry techniques with high sensitivity enabled us to detect PNH clones as small as <1% of a patient’s hematopoiesis, resulting in increasing incidence but more difficult data interpretation. Particularly, the clinical significance of small PNH clones in patients with bone marrow failures, including aplastic anemia and myelodysplastic syndromes, as well as in uncommon associations, such as myeloproliferative disorders, is still largely unknown. Besides current treatment with the anti-C5 eculizumab, which reduced PNH-related morbidity and mortality, new complement inhibitors will likely fulfill unmet clinical needs in terms of patients’ quality of life and better response rates (i.e., responses in subjects with C5 polymorphisms; reduction of extravascular hemolysis and breakthrough hemolysis episodes). Still, unanswered questions remain for these agents regarding their use in mono- or combination therapy, when to treat, and which drug is the best for which patient. Lastly, long-term safety needs to be assessed in real-life studies. In this review, we describe some clinical vignettes illustrating practical aspects of PNH diagnosis and management; moreover, we discuss recent advances in PNH diagnostic and therapeutic approaches.

Ultradeep Sequencing Analysis of Paroxysmal Nocturnal Hemoglobinuria Clones Detected by Flow Cytometry: PIG Mutation in Small PNH Clones.

We aimed to determine whether small paroxysmal nocturnal hemoglobinuria (PNH) clones detected by flow cytometry (FCM) harbor PIG gene mutations with quantitative correlation. We analyzed 89 specimens from 63 patients whose PNH clone size was ≥0.1% by FCM. We performed ultradeep sequencing for the PIGA, PIGM, PIGT, and PIGX genes in these specimens. A strong positive correlation between PNH clone size by FCM and variant allele frequency (VAF) of PIG gene mutation was identified (RBCs: r = 0.77, P < .001; granulocytes: r = 0.68, P < .001). Granulocyte clone size of 2.5% or greater and RBCs 0.4% or greater by FCM always harbored PIG gene mutations. Meanwhile, in patients with clone sizes of less than 2.5% in granulocytes or less than 0.4% in RBCs, PIG gene mutations were present in only 15.9% and 12.2% of cases, respectively. In addition, there was not a statistically significant positive correlation between FCM clone size and VAF or the presence or absence of a PIG mutation. Our results showed that in small PNH clones PIG gene mutations were present in only a small portion without significant correlation to VAF or the presence or absence of a PIG mutation.

Presence of paroxysmal nocturnal hemoglobinuria in patients with idiopathic portal vein thrombosis: a single-center study

Background/aimParoxysmal nocturnal hemoglobinuria (PNH) is a very rare clonal hematopoietic stem cell disease characterized by chronic hemolytic anemia and thrombosis. We report data from a study of the occurrence of PNH among patients with idiopathic portal vein thrombosis (PVT). Materials and methodsPatients who were followed up with the diagnosis of idiopathic PVT were enrolled into this study. Those with laboratory and/or imaging evidence of any local or systemic factor that could lead to PVT were excluded. PNH clone was examined in all patients using established FLAER methodology.ResultsA total of 112 patients (42 males and 70 females), none of them had a markedly PNH clone, but 4 patients (3.6%) with confirmed tests two times had small PNH clones (size between 3.02% and 4.62%). The median ages of PNH clone (-) and PNH clone (+) patients were 42 (range; 24–59) vs 39 (range; 36–42) years, respectively. The median hemoglobin concentration, platelet count and leukocyte count were lower in the PNH clone (+) group than the PNH clone (-) group. Anemia, thrombocytopenia, and leukopenia were detected in all PNH clone (+) patients. In addition, the PNH clone positivity size in monocytes was higher than erythrocytes in all of 4 patients.ConclusionsPNH should be considered during differential diagnosis among patients with idiopathic PVT. Small PNH clones can be detected in PVT patients that we cannot clearly determine its relationship with PVT. We need furthermore studies to explore the potential role of this finding.

Evolution of Moderate Aplastic Anemia into Subclinical PNH/AA and Successful Treatment with Reduced Intensity Haplo-HSCT: A Pediatric Case Report

Introduction: Aplastic Anemia (AA) is diagnosis made after the exclusion of other acquired or inherited disease that can present with pancytopenia. In pediatric AA, there is often overlap with many other diseases including Paroxysmal Nocturnal Hemoglobinuria (PNH) and hypocellular myelodysplastic syndrome (MDS). The diagnosis is often difficult and can evolve. There are new tests and methodologies to guide diagnosis and treatment decision making. Case report: The patient is a previously healthy, 18-year-old Caucasian female referred for pancytopenia. Patient reported progressive fatigue and easy bruising for a month prior to evaluation. Initial white blood count was 2.4 x109/L, hemoglobin 9.7g/dl, platelets 39 x109/L, absolute neutrophil count (ANC) of 590, and absolute retic count 68 x 109/L. A bone marrow exam revealed a hypocellular marrow with 30% cellularity with no dysplasia or blasts on flow cytometry. Viral studies, folate and vitamin B12 levels were normal. Chromosomal analysis, Fluorescent In situ Hybridization (FISH) study for MDS, and telomere length assay were normal. A bone marrow failure (BMF) panel for sixty targeted genes showed no mutations associated with BMF. PNH studies by flow cytometry showed 2.6% erythroid, 2% granulocytes, 2.7% monocytes failed to express GPI associated anchoring proteins (GPI-APs) representing a small PNH clone, not unusual in patients with aplastic anemia (AA). Studies were c/w with moderate AA (mAA). Standard management of mAA is careful observation and treatment if the patient requires transfusions or ANC falls below 500. However, since our patient was soon starting college out of state, we decided to treat with Immunosuppressive Therapy (IST), consisting of equine Anti-thymocyte globulin (eATG) and Cyclosporine. At 24 weeks post IST, no response was observed and the patient was requiring more frequent platelet and red blood cell transfusions. Repeat PNH testing showed 2% erythroid, 5% monocytes and 4% granulocytes that were deficient in GPI-Aps (Table1). BM exam showed 60% cellularity that was consistent with a recovering marrow but there was relative erythroid hyperplasia, without evidence of dysplasia or blasts. At this time, with increasing marrow cellularity but persistent pancytopenia we began to strongly suspect MDS. However, repeat chromosomal analysis and FISH studies were normal and a Foundation One myeloid panel was negative for mutations associated with MDS. At 28 weeks post-IST, repeat BM exam revealed 70% cellularity, again with relative erythroid hyperplasia. At the same time PNH testing, showed that 1.3% erythrocytes, 5.6% granulocytes and 5.4% monocytes were negative for GPI-APs. We concluded that our patient evolved had from mAA to subclinical PNH/AA which accounted for the normocellular marrow. High Disease Activity (HDA) is defined as lactate dehydrogenase (LDH) ≥ 1.5 times upper limit of normal and is associated with large PNH clones. HDA can guide the use of eculizumab as pre-transplant therapy. (DeZern et al, 2012) Throughout the course of the disease, her LDH was normal. She did not have HDA and therefore, was not treated with eculizumab. Because of the normocellular marrow she was not eligible for CTN 1502-CHAMP study. However, the patient underwent reduced intensity conditioning (RIC) and a haplo paternal BMT, followed by post-transplant cyclophosphamide, as per CTN 1502- CHAMP. She tolerated her BMT well without major complications. She is now five months post-transplant, alive and well, fully engrafted with 100% donor chimerism and no signs of GVHD. Post-transplant PNH testing revealed all PNH clones were eliminated. Conclusion: This case illustrates the diagnostic challenges of patients presenting with pancytopenia and the overlap and possible evolution of disease classifications; new diagnostic testing including gene mutation screens to aid in diagnosis; new concepts such as HDA in PNH to help guide treatment decision making; and the successful use of RIC and haplo BMT in a patient with PNH/AA and a normocellular BM Disclosures No relevant conflicts of interest to declare.

Prevalence of Paroxysmal Nocturnal Hemoglobinuria Clones in Myeloproliferative Neoplasm Patients: A Cross-Sectional Study

IntroductionThe myeloproliferative neoplasms (MPN) are clonal diseases that confer an increased risk of thrombohemorrhagic complications. Paroxysmal nocturnal hemoglobinuria (PNH) is a rare clonal disease associated with an increased thrombotic risk. Small PNH clones are prevalent in aplastic anemia and myelodysplastic syndrome patients, but their prevalence in MPN patients is unknown. Patients and MethodsConsecutive patients with MPN followed up at a single center were recruited. PNH clones were analyzed in erythrocytes and white blood cells by flow cytometry. ResultsPNH clones were detected in 2% of patients and were more common in JAK2 V617F positive patients. We could not detect any differences in clinical manifestations or complications in patients either with or without PNH clones because of the small patient numbers. ConclusionThe prevalence of PNH clones in MPN is similar to that described in myelodysplastic syndromes. Whether PNH clones influence MPN phenotype and complications should be studied prospectively in larger patient cohorts and over long-term follow-up.

Pathogenic Germline Variants in Acquired Aplastic Anemia (AA) and Paroxysmal Nocturnal Hemoglobinuria (PNH)

Next generation sequencing (NGS) methods, including whole exome sequencing (WES) and sequencing of large panels of genes have become standard tools facilitating the discovery of the genetic etiology of complex disorders, including the inherited bone marrow failure syndromes (IBMFS). This method has also revealed pathogenic variants in genes not previously associated with IBMFS. Our understanding of disease prevalence/incidence has also changed as exomes and genomes from healthy individuals or those with atypical presentations are being sequenced. We hypothesized that in a proportion of otherwise typical AA/PNH, known or new germ line variants (GLVs) may be present. These variants may also contribute to timing of onset and disease evolution of AA/PNH. We screened a cohort of 258 AA/PNH pts (median age 44 years, range 6-89, male vs female 115:143). In aggregate, 564 exonic variants with an average 2.18 variants per sample were found. We then sub-classified these candidate alterations into Tier-1 and Tier-2 lesions: Tier-1 were defined as known disease-prone sequence alterations/mutations, new nonsense/frameshift mutations and highly recurrent missense mutations with low frequency in the general population. Tier-2 were defined as missense mutations with a high general population frequency of ˃=0.01% and were not studied further. In the final analysis, we identified 80 GLVs of Tier-1 and 484 GLVs of Tier-2. In analyses focused on Tier-1 lesions, 61/258 pts (24%) with AA/PNH had pathogenic GLVs and 19 pts carried co-occurrent GLVs. High frequencies of mutations in DNA repair genes including BRCA2 (8 pts), ATM (6 pts), BRCA1 (5 pts), FANCC (3 pts), MSH2 (3pts), MLH1 (2 pts) were found in our cohort, all heterozygous, as well as telomere-associated genes including POT1 (5 pts), CTC1 (4 pts), DKC1 (3 pts), WRAP53 (2 pts) and NOP10 (1 pt) together with several other genes including NF1 (8 pts), CFTR (7 pts), APC (3 pts), PTEN (2 pts) and single carried GLVs in FH, GAR1, SBDS, SETBP1 and TP53. These GLVS are enriched in our cohort compared with the ExAC database controls (p Further exploration focused on the relationship between GLVs and disease evolution. 10/61 Tier-1 GLVs carriers (16%) and 15/197 without Tier-1 GLVs (7.6%) evolved to MDS or AML (p In our cohort, none of the carriers of Tier-1 GLVs showed any physical signs of inherited congenital BMF syndromes. We identified 6 cases with a family history (FH) of hematologic disease and none of them were Tier-1 GLVs carriers. We also did not see any difference in FH of other cancer in these two groups. In conclusion, our results indicate that important GL alterations in cancer predisposition genes can be found in acquired AA/PNH patients. Some of the genes seems to contribute to disease persistence and disease evolution/progression. Inherited factors likely play an important role in the pathogenesis of acquired AA/PNH and a clinical family history appears to be insufficient alone to determine/identify these predisposition states. Disclosures Carraway:Agios: Consultancy, Speakers Bureau; FibroGen: Consultancy; Novartis: Speakers Bureau; Jazz: Speakers Bureau; Celgene: Membership on an entity9s Board of Directors or advisory committees, Research Funding, Speakers Bureau; Balaxa: Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity9s Board of Directors or advisory committees. Sekeres:Celgene: Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees; Opsona: Membership on an entity9s Board of Directors or advisory committees; Opsona: Membership on an entity9s Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Ra Pharmaceuticals, Inc: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity9s Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy.

PNH Clone Size By Flow Cytometry and Its Correlation with PIG Gene Mutation

Background: Routine diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) is based on flow cytometric measurement of PNH clone in RBC and granulocyte with sensitivity of 1%, and recent introduction of high-sensitive flow cytometry (FCM) makes it possible to detect 0.01% clone. However, the clinical significance of small PNH clone has not been elucidated, so even the necessity to treat or not is controversial. We investigated whether quantitative results of PNH clone size measured by FCM correlate with mutant burden of PIG gene.Methods: A total of 44 specimens from 20 patients whose PNH clone size was >1% in either RBC or granulocyte by routine FCM were enrolled (classical PNH n=10, AA/PNH n=8, MDS/PNH n=2). To detect small quantity of cells with PIG gene mutation, we performed ultra-deep sequencing (average depth 3000X) for PIGA, PIGM, PIGT, and PIGX genes on these 44 consecutive specimens.Results: Sixteen patients (80.0%) were found to harbor PIG gene mutations: 15 patients had PIGA mutation and 1 patient had PIGM mutation. Granulocyte PNH clone size and variant allele frequency (VAF) of PIG gene mutation showed higher correlation (Spearman's r=0.73, p=0.0002) than that of RBC (Spearman's r=0.61, p=0.0073). All patients harboring PIG gene mutation showed more than 10% PNH clone by FCM. In contrast, 4 patients who did not have PIG gene mutation showed less than 10% PNH clone by FCM.Conclusion: Considering the mechanism of PNH development, the presence of PIG gene mutation is a definitive evidence supporting PNH diagnosis. PNH granulocyte clone size more than 10% by FCM seems to be clinically significant in relevance to PIG gene mutation. We suggest that neither high sensitivity FCM nor the detection of PIG gene mutation is a requisite for routine clinical diagnosis of PNH. DisclosuresNo relevant conflicts of interest to declare.

Paroxysmal Nocturnal Hemoglobinuria Testing by Flow Cytometry: Brief Overview for Clinicians

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare, acquired hematopoietic stem cell disorder, caused by a somatic mutation in the PIG-A gene. This mutation is responsible for the synthesis of glycophosphatidylinositol (GPI) anchor that attaches a number of proteins to the cell surface. The mutant gene leads to partial deficiency or absence of all proteins normally linked to the cell membrane by GPI anchor. The primary clinical manifestations of PNH are complement mediated hemolytic anemia, thrombosis in atypical locations and blood cytopenias. Flow cytometry has become the gold standard method for PNH clone detection due to its high sensitivity and specificity and due to the ability to examine multiple GPI-linked proteins on red and white blood cells surface. It is the method of choice for the detection of very small PNH clones in subclinical PNH that often accompanies aplastic anemia and other bone marrow disorders. PNH clone detection traditionally involves the analysis of CD55 and CD59 on red and white blood cells. Other markers such as CD14, CD16, CD24, CD66b and CD157 are suitable to detect GPI-linked proteins in the surface of granulocytes and monocytes. The most useful reagent to assess white blood cell PNH clones is Fluorescent Aerolysin (FLAER) which is a mutated form of proaerolysin conjugated with a fluorochrome. Its advantage in PNH clones detection is due to the ability to bind directly to the glycan portion of the GPI anchor. Flow cytometry is a sophisticated method and a useful tool for clinical cell analysis. However, PNH is a clinical diagnosis and flow cytometric results should always be interpreted with respect to clinical manifestations and other laboratory findings.

Auto-Reactive Glycosyl-Phosphatidyl-Inositol (GPI) Specific T Cells Are Present in Most Patients with Idiopathic Aplastic Anemia (IAA)

Idiopathic aplastic anemia (IAA) is an acquired bone marrow disease probably caused by an auto-immune attack against hematopoietic stem cells (HSCs), which leads to bone marrow failure. Many abnormalities have been observed in the T cell compartment, but the putative auto-antigen(s) remain elusive. A large body of evidence links paroxysmal nocturnal hemoglobinuria (PNH) to IAA, supporting the notion that autoimmunity is a key pathogenic mechanism in both diseases. In PNH, auto-reactive T cells may be the 'noxious agent' capable of killing GPI positive HSCs while sparing GPI negative HSCs. Recently, CD1d restricted, GPI-specific T-cells have been demonstrated in PNH patients. Here, we investigate whether CD1d restricted, GPI-specific T cells are also present in IAA patients.When peripheral blood mononuclear cells (PBMNCs) from 14 newly diagnosed IAA patients [12 of whom had a small percentage (between 0.003% and 5%) of GPI-negative granulocytes] were co-cultured with antigen presenting cells (APCs) expressing CD1d and competent for the synthesis of GPI, we detected GPI specific T cells (CD8+CD1d/GPI dimer+ T cells) in 10 out of 14 patients (71%) at a significantly higher abundance than in co-culture experiments performed with PBMNCs from healthy controls (Fig. 1). In fact, the frequency of CD8+CD1d/GPI dimer+ T cells was below the cutoff value of 0.35% in all the 15 healthy controls but only in 4 out 14 IAA patients (Fisher test, P <0.00005). The frequency of GPI specific T cells in IAA patients was similar to that previously found in PNH patients (Fig.1). In the same experimental setting, in IAA we found increased frequency of INFγ producing T cells (CD8+INFγ+ T cells) than in healthy controls: interestingly, in the 4 patients with no detectable CD8+CD1d/h-GPI dimer+ T cells, we found INFγ producing T cells regardless of the GPI stimulus. Furthermore, we have observed a trend towards the association of higher frequency of CD8+CD1d/GPI dimer+ T cells and severe IAA versus non-severe IAA.In 5 of the 10 IAA patients with detectable CD8+CD1d/GPI dimer+ T cells we tested sequential samples; we observed that response to immunosuppressive treatment (IST) was associated with significant decrement of both GPI specific and INFγ producing T cells. In keeping with these data, GPI specific T cells were absent in 2 AA patients in remission after engraftment of HSC transplants.We then similarly analyzed 27 IAA patients who were already receiving IST. 8 of these patients did not have any GPI-negative granulocyte population, 10 had a small PNH clone (between 0.003% and 5%) and 9 had more than 5% of GPI-negative granulocytes. GPI specific T cells were detected in 20 out of these 27 (74%) patients, regardless of the presence/absence of a PNH (GPI-negative) population; the frequency of GPI specific T cells was lower than in patients at diagnosis. In these 20 patients, INFγ producing T cells were detected only when co-cultured in presence of GPI on CD1d+ APC; in the remaining 7 patients with no detectable CD8+CD1d/h-GPI dimer+ T cells, INFγ producing T cells were present regardless of any GPI stimulus.In one patient with a congenital form of mild AA due to a TERC mutation, GPI specific T cells were present. This finding might signify that although congenital AA patients are innately predisposed to AA, an auto-immune attack may be still needed to produce full-blown AA.We conclude that GPI specific T cells are present in about 70% of IAA patients regardless of the presence/absence of a PNH (GPI-negative) population; and that the presence and size of a PNH cell population results from the existence of a PIG-A mutant HSC and from its degree of 'stemness'. The presence of GPI specific T cells in the large majority of AA patients suggests that in most of these cases, GPI is the target of the auto-immune attack potentially resulting in the suppression of hematopoiesis. In the remaining IAA patients, different and still unknown auto-immune targets are presumably involved in the T-cell attack. These data strongly support the hypothesis that AA and PNH could be different clinical expression of the same pathogenetic mechanism, namely the auto-immune suppression of the GPI+ (normal) HSCs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Flow Cytometry Screening for Paroxysmal Nocturnal Hemoglobinuria Abnormal Clones in Saudi Arabia

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease with insidious process, chronic course and life-threatening condition. PNH is clinically defined by the deficiency of the endogenous glycosyl phosphatidylinositol (GPI)-anchored complement inhibitory protein. It has always aroused interest in the medical profession rendering screening and proper diagnosis by flow cytometry (FCM) technology a priorityWe reviewed all samples submitted for PNH/ FCM screening for the past 2 years (2012-2013) at hematology section, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center (General Organization). We collected the positive cases and reviewed them for age, gender, indication for screening, sample type, size of the PNH clone and cell type affected. Immunophenotypic analysis was performed using gating antibodies CD45, CD15, CD33, CD235a GPI-linked antibodies, CD59, CD14, and CD24 as well as fluorescent Aerolysin (FLAER).In a total of 366 peripheral blood samples submitted for PNH/ FCM screening fifteen samples (4%) were positive for PNH clones but only 12 patients were available for analysis. The median age for our patients was 34 years with approximately equal male to female distribution.12 cases showed type II and III clones within the RBCs with clone size ranging between 0.04% and 56%. Analysis of granulocytes and monocytes revealed type III clone in 8 cases, type II and III clone in 3 cases and non in one case. The percentage of the clone varies between the granulocytes and monocytes and ranges from 1% up to 100%.Of 12 positive PNH cases, 8 (66.7%) patients were diagnosed as having aplastic anemia (AA), 1 (8.3%) patient with Budd-Chiari syndrome, 1 (8.3%) patient has chronic immune thrombocytopenia (ITP), and 2 (16.7%) patients presented with pancytopenia.This study confirms the rarity of the disease since only 4% of the submitted samples for analysis turned to be positive for PNH. The detection limit for a PNH clone by FCM in the RBC or WBC is 0.01%. Identification of small PNH clone is greater FCM sensitivity relative to old test used for the same purpose (Ham test). The use of the FLAER allowed us to detect granulocytic PNH clone, however, granulocytes PNH clone detection alone without RBCs clone detection is not recommended. This review confirms the previous percentage of positive cases (5.9%) reported from this center on a smaller number of cases during the past few years. 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.

Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria phenotype in patients with splanchnic vein thrombosis

IntroductionSplanchnic vein thrombosis (SVT) is a serious complication in patients with paroxysmal nocturnal hemoglobinuria (PNH). Mutant PNH clones can be associated with an increased risk of SVT even in the absence of overt disease, but their prevalence in non-selected SVT patients remains unknown. Materials and MethodsPatients with objective diagnosis of SVT and without known PNH were tested for the presence of PNH clone using high-sensitivity flow cytometric analysis. ResultsA total of 202 SVT patients were eligible, 58.4% were males, mean age was 54.6years (range 17–94), site of thrombosis was portal in 103 patients, mesenteric in 67, splenic in 37, and supra-hepatic in 10. SVT was associated with JAK2 V6167F in 28 of 126 (22.2%) screened patients, liver cirrhosis in 15.3% patients, recent surgery in 10.9%, and myeloproliferative neoplasm in 10.6%, whereas in 34.6% of patients neither permanent nor transient risk factors were detected. None of the patients had a clearly demonstrable PNH clone, but in two patients (0.99%, 95% CI 0.17-3.91) we observed very small PNH clones (size 0.014% and 0.16%) confirmed in two independent samples. One patient had portal vein thrombosis and no associated risk factors, the second had superior mesenteric vein thrombosis and inflammatory bowel disease. ConclusionsVery small PNH clones can be detected in patients with SVT and no clinical manifestations of disease. Future studies are needed to explore the potential role of this finding in the pathogenesis of SVT.

Presence Of CD55- and/Or CD59-Deficient Erythrocytes In Patients With Rheumatic Diseases: An Immune-Mediated Phenomenon?

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder characterized by the somatic mutation of X-linked gene PIG-A, required for glycosylphosphatidylinositol (GPI)-anchor biosynthesis. This results in absent or decreased expression of all membrane proteins normally anchored by GPI - including CD55 and CD59 - in all circulating cells, leading to an unusual sensitivity of red blood cells (RBCs) to complement lysis and subsequently intravascular hemolysis and hemoglobinuria. According to the “dual pathogenesis” model, there is an immunoregulatory selection in favor of PNH clones to proliferate preferentially over normal hemopoiesis on a microenvironment of bone marrow failure. The incidence of “PNH-like” defect has been also demonstrated in many hematological diseases and on peripheral blood cells (PBC) of normal individuals.Complement system is recognized as having the potential to provoke severe impairment to host tissues. This is extensively demonstrated in autoimmune disease setting. Multiple regulatory and inhibitory enzymes, such as CD55 and CD59, known as complement regulatory proteins, adjust the progression of complement cascade at all levels, protecting the autologous cells. Complement activation and cytopenias have been associated with diminished CD55 and/or CD59 expression on PBC membranes. The aim of this study was to evaluate the presence of “PNH-like” red-cell populations in patients with rheumatic diseases and investigate possible correlations with clinical or laboratory parameters.CD55 and CD59 expression was evaluated in erythrocytes of 113 patients (94 females, 19 males, median age: 64 years) with rheumatic diseases: 38 with rheumatoid arthritis, 25 with systemic lupus erythematosus, 17 with Sjögren's syndrome, 7 with systemic sclerosis, 12 with vasculitis, 2 with dermatomyositis, 1 with ankylosing spondylitis and 11 with mixed connective tissue diseas, using the sephacryl-gel microtyping system, a semi-quantitative, inexpensive and simple method useful in screening “PNH-like” red-cell defect, with sensitivity comparable with that of flow cytometry. One hundred and twenty-one (121) healthy blood donors of similar age and gender and 10 patients with PNH were also studied, as control groups. In all samples with CD55- and/or CD59- negative RBCs, Ham and sucrose tests were also performed.Interestingly, the majority of patients (104/113, 92%) demonstrated “PNH-like” erythrocytic populations: 47 (41.6%) with concomitant deficiency of CD55 and CD59, 50 (44.2%) with isolated deficiency of CD55 and 6 (6.2%) with isolated deficiency of CD59. In healthy donors, only 2 (1%) had red cells with concomitant CD55/CD59 negativity and 3 (2%) with isolated CD55 or CD59 deficiency. “PNH-like” erythrocytic clones never surpassed 25% of the total red-cell population, while the most common proportion of deficiency for both antigens was 10%. All PNH patients exhibited simultaneous CD55/CD59 deficiency. Moreover, it should be high-lightened that we found an unprecedented relation between patients' hemoglobin (Hb) and CD55 expression on RBCs (rs= -0.205, p=0.029), while there was a significant difference (δ) when the mean concentration of Hb was compared between patients with normal expression of CD55 and those with deficiency of this protein (δ=-1.4534 g/dl, p=0.0151). There was no clinical or laboratory evidence of hemolysis in our patients. There was no association between the presence of “PNH-like” red-cell populations and cytopenias or specific treatment for the autoimmune disorder. Positive Ham and sucrose tests were found only in PNH patients.In conclusion, this study provides evidence supporting the presence of erythrocytes with CD55- and/or CD59- deficiency in patients with rheumatic diseases. The pre-existence of small PNH clones in the bone marrow of these patients, that acquire a survival advantage to proliferate against normal hemopoietic tissue and become detectable with our methodology, may be the underlying cause for this phenomenon. Moreover, it was demonstrated that CD55- deficiency on RBCs influences the levels of Hb, in these patients. Further studies, using molecular techniques, will be required, to clarify the exact pathophysiologic mechanisms for this deficiency. Disclosures:No relevant conflicts of interest to declare.

Clinical and Genetic Heterogeneity of Telomere Diseases.

AbstractAbstract 2373Genetic defects in telomerase genes are associated with a variety of clinical syndromes that includes marrow failure, cirrhosis and pulmonary fibrosis. Mutations in the RNA template (TERC) and the catalytic subunit (TERT) have been associated to adult onset aplastic anemia without the characteristic physical abnormalities observed in inherited forms of marrow failure where telomerase genes can be mutated. Clinical presentations can vary and a careful personal and family history often provides clues to an underlying telomeropathy. The implications for therapy are still evolving and the best strategy in approaching these patients not yet defined. At NHLBI we have systematically tested patients evaluated in our bone marrow failure clinic for telomere length and often for mutational analysis of telomerase genes, and have observed a range of phenotypes associated with telomere disorders. In this analysis we describe some of these phenotypes.We reviewed the clinical data of all patients referred to the Hematology Branch bone marrow failure clinic since 2003, when we began routinely testing for telomere disorders. One hundred fourteen patients were diagnosed as having a telomere disease, either by the presence of a telomerase gene mutation and/or very short peripheral blood leukocyte’ telomere lengths (below the first percentile as compared to age-matched controls). Sixty-seven mutations or gene variants were identified: 40% in TERT (46/114), 15% in TERC (17/114), 2 cases in DKC1, 1 case in TERF2 and 1 case in TINF2. Disease presentation varied: 42% (n=48) of patients had severe aplastic anemia (SAA) (including 2 hepatitis-associated SAA), 30% (n=34) were diagnosed with moderate aplastic anemia (MAA), 6% (n=7) had large granular lymphocytosis (LGL), and 11% (n=12) myelodysplasia (MDS). Eleven of 114 cases had liver disease and 10/114 cases had pulmonary fibrosis. Only 45/114 had family histories suggestive of a telomere disorder, such as a family member affected with a hematologic disorder, liver disease or pulmonary fibrosis. Hematologic disorders were reported in the vast majority (37/45) of cases who had a positive family history. A history of multiple malignancies within the family was not prominent in the cohort. In addition, 14/114 cases had paroxysmal nocturnal hemoglobinuria (PNH) clones. 8/114 cases had very small PNH clones (<5% of granulocytes) and only 5/14 had expanded PNH clones that were clinically significant (>30% of granulocytes).In addition, we report treatment outcomes for patients in the cohort who received immunosuppression therapy (IST) with anti-thymocyte globulin, cyclophosphamide and/or alemtuzumab at our institution: 28/114 patients received immunosuppression for SAA, of whom 13 demonstrated a clinically meaningful hematologic response of which 5 later relapsed.This data describes the various clinical presentations of telomeropathies in our clinic. Important points that can be inferred from our data set: 1) a family history need not be present in patients who present with an underlying genetic defect in telomerase genes suggesting that screening based on family history alone may not be adequate; 2) the presence of PNH or an LGL clone does not rule out the presence of an underlying telomeropathy; 3) the presence of a mutation in telomere genes and PNH or LGL clones suggests that an immune disarray may coexist with an underlying genetic defect in some cases; 4) hematologic recovery after immunosuppression in some mutants suggests that the genetic defect may not be the dominant pathogenic determinant in some cases and does not rule out the possibility of an immunomodulatory responsive disease. Disclosures:No relevant conflicts of interest to declare.

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.