Paroxysmal Nocturnal Hemoglobinuria Testing Research Articles

FLAER as a standalone reagent for paroxysmal nocturnal hemoglobinuria: Do we need to reconsider the guidelines for testing?

Flow cytometry-based paroxysmal nocturnal hemoglobinuria (PNH) testing involves utilization of monoclonal antibodies against GPI-linked proteins and FLAER. The ability of FLAER to bind to a wide variety of GPI-linked structures and to be utilized across different leukocyte subsets is remarkable. We hypothesize that FLAER as a standalone reagent may be equally effective for detecting PNH clones. The present study intends to compare the results of a FLAER alone-based strategy to the recommended FLAER+GPI-linked protein-based approach for applicability in clinical settings. EDTA-anticoagulated blood samples from patients for PNH workup were tested for PNH by multiparametric flow cytometry. A conventional panel comprising gating markers (CD45 for WBC, CD15 for granulocytes, and CD64 for monocytes) and a combination of FLAER and GPI-linked markers, such as CD24 and CD14, henceforth referred to as the "routine panel," was employed. Second, a "FLAER-only panel" comprising the gating markers and FLAER alone (excluding the GPI-linked markers CD24 and CD14) was set up. The samples were processed using the lyse-wash-stain-wash technique, and events were acquired on BC Navios Ex flow cytometer (Beckman Coulter, Inc., USA) and analyzed on Kaluza Software 2.1. The presence of a PNH clone was reported at a value of ≥0.01%. A total of 209 patients were tested. Both panels found a PNH clone in 20.1% of patients (n = 42/209) with a 100% concordance rate. The PNH clone range for granulocytes was 0.01%-89.68%, and for monocyte was 0.04%-96.09% in the routine panel. The range in the FLAER-only panel for granulocytes was 0.01%-89.61%, and for monocytes, it was 0.01%-96.05%. Pearson correlation statistics revealed a significant correlation between the size of the PNH clone of granulocytes and monocytes among the two panels tested (granulocytes r = 0.9999, p < 0.0001, 95% CI = 0.9999 to 1.000; monocytes r = 0.9974, p < 0.0001, 95% CI = 0.9966-0.9980). Based on our results, FLAER as a standalone marker is specific and sensitive for identifying PNH clones in granulocytes and monocytes, even for high-sensitivity PNH assay. The proposed "FLAER-only panel" panel is efficient and cost-effective for highly sensitive PNH testing in two different cell lineages, especially in resource-limited clinical settings.

Pediatric-Specific Patterns of Clonal Evolution Arising from Acquired Aplastic Anemia

Introduction Acquired aplastic anemia (aAA) occurs in both children and adults, though pathophysiology and clinical outcomes differ significantly by age. While clinical testing for paroxysmal nocturnal hemoglobinuria (PNH) clones is routine, targeted next generation sequencing (NGS) for myelodysplastic syndrome (MDS) associated variants has only recently been implemented. Furthermore, single nucleotide polymorphism array (SNP-A) assessing for copy neutral loss of heterozygosity in the human leukocyte antigen (HLA) region on chromosome 6p (6p CN-LOH) and HLA NGS assessing for allele specific loss of function (LoF) mutations, which together are seen in up to 25% of aAA cases, are only now being considered for clinical use. Recent studies have reported on the frequency and overlap of these 3 types of clonal evolution (PNH, MDS-associated, HLA-targeting) in adult aAA cohorts, but none focus specifically on pediatric patients, whose MDS-associated clonal evolution patterns are distinct. Delineating patterns of clonal hematopoiesis in pediatric aAA to determine whether evolution driven by immune escape (PNH and HLA loss) affects likelihood of MDS development is critical for personalizing therapeutic options. Thus, we aimed to describe the frequency, clinical significance, and overlap of clonal evolution including PNH, HLA loss, and MDS-associated gene mutations in a pediatric cohort of aAA patients. Methods This study identified 85 pediatric aAA patients treated at Children's Hospital of Philadelphia (CHOP) and enrolled on the IRB-approved CHOP/Penn BMF Registry and Repository in whom clinical testing for all patterns of clonal hematopoiesis was available or could be performed with biorepository samples. PNH flow cytometry was performed clinically in all cases; subjects with PNH > 0.05% in the granulocyte fraction were considered as having a PNH clone. Peripheral blood and bone marrow samples were used for SNP-A, HLA-NGS, and an institutional somatic mutation NGS panel for hematological malignancies encompassing 118 genes. Descriptive and contingency statistical analyses along with Fisher's Exact Test were performed using SAS (Cary, NC). Results Demographic composition of the 85 pediatric subjects with aAA included a median age of 10.7 years (1.7-20.8), male/female ratio of 59%/41%, and a representative race/ethnicity distribution (Table 1). In total, 66% displayed at least one clonal event. As expected, PNH clones were most common, present in 45% (38/85) of the cohort. An additional 16 (19%) exhibited acquired HLA loss by 6p CN-LOH (n=6), allele specific LoF mutations (n=6), or both (n=4). Fifteen (18%) had clonal hematopoiesis associated with MDS, of whom 3 had cytogenetic lesions that progressed to clinical MDS (3.5%), and 12 developed no morphologic features of MDS but demonstrated a total of 14 mutations in MDS-associated genes (BCOR/BCORL1 n=6, KMT2C/KMT2D n=3, RUNX1 n=1, ASXL1 n=1, SRSF2 n=1, PRPF8 n=1, CALR n=1). Critically, pediatric subjects with aAA who developed HLA loss did not develop MDS-associated mutations, with no patient demonstrating both subtypes of clonal events (Figure 1). In contrast, PNH and MDS-associated clonal events overlapped in 11% of the overall cohort. In total, 60% of patients with MDS-associated clonal hematopoiesis also possessed PNH clones, and 23% of patients with detectable PNH clones demonstrated at least one MDS-associated clonal event. Only 3.53% (n=3) of the cohort demonstrated both PNH and HLA loss, and PNH clone size was < 1% in all 3 patients. Furthermore, patients with HLA loss were significantly less likely to have PNH clones than those without HLA somatic alterations (19% vs 51%, p = 0.026). Correlations between patterns of clonal hematopoiesis and clinical outcomes will be presented. Concalusions Pediatric aAA patients treated with IST are at risk for developing MDS and hemolytic PNH. Presence and evolution of clonal hematopoiesis influenced decisions to pursue IST versus alternative donor transplant. Importantly, our study demonstrates that occurrence of clonal hematopoiesis by HLA loss does not increase and may even decrease the risk of developing MDS-associated or PNH clones. This finding strongly suggests that treatment decisions for aAA patients whose only clonal event is HLA loss need not be considered distinctly from patients with no clonal changes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

Small Paroxysmal Nocturnal Hemoglobinuria Clones in Autoimmune Hemolytic Anemia: Clinical Implications and Different Cytokine Patterns in Positive and Negative Patients.

Autoimmune hemolytic anemia (AIHA) is characterized by immune mediated erythrocytes destruction by autoantibodies with or without complement activation. Additional pathologic mechanisms include cellular cytotoxicity, cytokline dysregulation, and inadequate bone marrow compensation with fibrosis/dyserythropoiesis. The latter resembles that of bone marrow failures, namely aplastic anemia and myelodysplastic syndromes. Paroxysmal nocturnal hemoglobinuria (PNH) clones are increasingly recognized in bone marrow failure syndromes, and their selection and expansion are thought to be mediated by immune mechanisms. In this study, we aimed to evaluate the prevalence of PNH clones in 99 patients with primary AIHA, and their correlations with disease features and outcomes. Moreover, in the attempt to disclose the physiopathology of PNH positivity in AIHA, serum levels of several immunomodulatory cytokines were tested. A PNH clone was found in 37 AIHA patients (37,4%), with a median size of 0.2% on granulocytes (range 0.03–85). Two patients showed a large clone (16 and 85%) and were therefore considered as AIHA/PNH association and not included in further analysis. Compared to PNH negative, PNH positive cases displayed a higher hemolytic pattern with adequate bone marrow compensation. AIHA type, response to therapy, complications and outcome were comparable between the two groups. Regarding cytokine levels, IFN-γ and IL-17 were lower in PNH positive vs. PNH negative AIHAs (0.3 ± 0.2 vs. 1.33 ± 2.5; 0.15 ± 0.3 vs. 3,7 ± 9.1, respectively, p = 0.07 for both). In PNH positive AIHAs, IFN-γ positively correlated with reticulocytes (r = 0.52, p = 0.01) and with the bone marrow responsiveness index (r = 0.69, p = 0.002). Conversely, IL-6 and IL-10 showed the same pattern in PNH positive and PNH negative AIHAs. IL-6 levels and TGF-β positively correlated with clone size (r = 0.35, p = 0.007, and r = 0.38, p = 0.05, respectively), as well as with LDH values (r = 0.69, p = 0.0003, and r = 0.34, p = 0.07, respectively). These data suggest testing PNH clones in AIHA since their prevalence is not negligible, and may correlate with a prominent hemolytic pattern, a higher thrombotic risk, and a different therapy indication. PNH testing is particularly advisable in complex cases with inadequate response to AIHA-specific therapy. Cytokine patterns of PNH positive and negative AIHAs may give hints about the pathogenesis of highly hemolytic AIHA.

FLAER‐negative CD15+ neutrophils can be used for the simplified screening of suspected PNH cases

The flow cytometry analysis of GPI-linked proteins on red blood cells and leukocytes is crucial for paroxysmal nocturnal hemoglobinuria (PNH) diagnostics. However, the commonly used multicolor panels cannot be implemented in low-resourced hematology laboratories. In order to develop a simple prediagnostic test for PNH screening, we analyzed the diagnostic accuracy of the two-color (FLAER/CD15) detection of GPI-deficient neutrophils. We reanalyzed multicolor data set of 1594 peripheral blood samples of patients screened for PNH applying only two markers (FLAER/CD15). The quantitative positivity/negativity was reported. Then, these results were compared in a blinded manner with previously obtained multicolor data from the same samples. Among the 1594 samples included in the study, 507 samples were PNH-positive by the multicolor assay. The two-color method revealed 510 PNH-positive samples. The detailed examination of this discrepancy revealed 12 false-positives and 9 false-negatives. Therefore, FLAER/CD15 screening method displayed 98.90% of the diagnostic specificity and 98.22% of the sensitivity. This simple two-color evaluation of FLAER-negative neutrophils is a highly effective screening test for PNH. Although this approach is not intended to replace the multicolor diagnostic procedure, it could minimize the number of patients requiring a conventional multicolor flow cytometric assay.

The utility of thrombophilia testing in patients with newly diagnosed portal vein thrombosis.

: Thrombophilia testing is frequently performed in both seemingly provoked and unprovoked portal vein thrombosis (PVT), yet the clinical implications of these expensive laboratory tests are unknown. We investigated the frequency of clinical management changes in patients with newly diagnosed PVT. This is a retrospective analysis of adult patients with a newly diagnosed PVT at a single institution. The primary outcome is change in clinical management, defined as documented change in choice, dose, or duration of anticoagulation, future thromboprophylaxis, or counseling of asymptomatic family members. Five-hundred and forty-four patients with PVT were identified, 438 (80.5%) of whom had an identifiable pretesting provoking factor, most commonly cirrhosis (39.2%). Two-hundred ninety-one patients (53.5%) had at least one hypercoagulable laboratory test performed. The most frequently positive test was PAI-1 polymorphism, followed by elevated homocysteine and MTHFR mutational analysis. However, the only test that was frequently positive and consistently altered management was JAK2 mutational analysis (15.3%). Factor V Leiden was commonly positive but rarely changed clinical decision-making (1.5%), as was flow cytometric testing for paroxysmal nocturnal hemoglobinuria (0.8%), and antiphospholipid antibodies (0.7%). Patients with cirrhosis rarely had thrombophilia testing results that were clinically significant. A rough cost estimate was dramatically reduced from $231 000 to $76 000 if only clinically meaningful tests were employed in the hypercoagulable work-up. These results highlight the need for focused thrombophilia testing in patients with PVT.

Study for the Diagnostic Screening of Paroxysmal Nocturnal Hemoglobinuria in Older Patients with Unexplained Anemia and/or Cytopenia.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disease that may lead to weakness and death of patients, if unrecognized and untreated. Although consensus guidelines were reviewed recently for the diagnostic screening of PNH with multi-parameter flow cytometry (FCM), until now, no study has investigated the efficiency of such clinical indications in older patients. Overall, 20 centers participated in the study and a total of 1,689 patients were included, 313 of whom were at geriatric age and 1,376 were aged 18 - 64 years. We evaluated the efficiency of consensus clinical indications for PNH testing using FCM in peripheral blood samples and compared the results of older patients and patients aged 18 - 64 years. PNH clones were detected positive in 7/313 (2.2%) of the older patients. Five (74.4%) of the patients with PNH clones had aplastic anemia, 1 had unexplained cytopenia, and 1 patient had myelodysplastic syndrome (MDS) with refractory anemia. PNH clones were not detected in any older patients who were screened for unexplained thrombosis, Coombs (-) hemolytic anemia, hemoglobinuria, and others (e.g., elevated lactate dehydrogenase (LDH), splenomegaly). We detected PNH clones in 55/1376 (4%) samples of the patients aged under 65 years. Forty-two (76.4%) patients with PNH clones had aplastic anemia, 2 patients had Coombs (-) hemolytic anemia, 3 patients had unexplained cytopenia, 1 patient had MDS with refractory anemia, 1 patient had hemoglobinuria, and 6 (10.9%) had others (e.g., elevated LDH, splenomegaly). PNH clones were not detected in any patients who were screened for unexplained thrombosis. There was no statistical difference between the geriatric population and patients aged 18 - 64 years in terms of clinical indications for PNH screening with FCM (p = 0.49). Our results showed that the current clinical indications for PNH screening with FCM were also efficient in older patients. We suggest that older patients with unexplained anemia, myelodysplastic syndrome with refractory anemia, and unexplained cytopenia should be screened for PNH with FCM to identify patients who would benefit from treatment.

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.

Screening for paroxysmal nocturnal hemoglobinuria (PNH) in patients presenting with cerebral sinovenous thrombosis (CSVT): Results of a FLAER based flowcytometry study in Indian patients.

Patients with paroxysmal nocturnal hemoglobinuria (PNH) may present with thrombosis at unusual sites, of which cerebral sinovenous thrombosis (CSVT) is one and screening for PNH is recommended in this condition. Though many patients diagnosed with PNH develop CSVT, it is unclear how many patients with PNH would present for the first time with thrombosis. We analysed the results of screening for PNH by flowcytometry in our patients with CSVT. The laboratory data of patients referred for thrombophilia and PNH testing in CSVT was examined to assess the frequency of PNH at presentation in these patients. FLAER and CD24 on granulocytes and FLAER and CD14 on monocytes respectively were used to screen the leucocytes for PNH by flowcytometry. The data for Protein C, S and Antithrombin deficiency, antiphospholipid antibodies and the Factor V Leiden mutation was examined and circumstantial risk factors were also assessed. Of the 180 cases of CSVT screened by flowcytometry for PNH, not a single case tested positive. Positivity for anti-phospholipid antibodies was the most common thrombophilic risk factor (5%). Pregnancy was the most common circumstantial risk factor. Our data on FLAER based flowcytometry in the North Indian population with CSVT suggests that PNH is not a common risk factor in our patients with thrombosis at this unusual site.

Paroxysmal nocturnal hemoglobinuria testing in patients with myelodysplastic syndrome in clinical practice-frequency and indications.

Myelodysplastic syndrome (mds) is characterized by peripheral blood cytopenias, with most patients developing significant anemia and dependence on red blood cell (rbc) transfusion. In paroxysmal nocturnal hemoglobinuria (pnh), mutations in the PIGA gene lead to lack of cell-surface glycosylphosphatidylinositol, allowing complement-mediated lysis to occur. Paroxysmal nocturnal hemoglobinuria results in direct antiglobulin test-negative hemolysis and cytopenias, and up to 50% of patients with mds test positive for pnh cells. We wanted to determine whether pnh is considered to be a contributor to anemia in mds. Patients with a diagnosis of mds confirmed by bone-marrow biopsy since 2009 were reviewed. High-resolution pnh testing by flow cytometry examined flaer (fluorescein-labeled proaerolysin) binding and expression of CD14, CD15, CD24, CD45, CD59, CD64, and CD235 on neutrophils, monocytes, and rbcs. In 152 patients with mds diagnosed in 2009 or later, the mds diagnosis included subtypes associated with pnh positivity (refractory anemia, n = 7, and hypoplastic mds, n = 4). Of 11 patients who underwent pnh testing, 1 was positive (9.0%). Reasons for pnh testing were anemia (n = 3), new mds diagnosis (n = 2), hypoplastic mds (n = 2), decreased haptoglobin (n= 1), increased rbc transfusion requirement (n= 1), and unexplained iron deficiency (n= 1). Testing for pnh was infrequent in mds patients, and the criteria for testing were heterogeneous. Clinical indicators prompted pnh testing in 6 of 11 patients. Given that effective treatment is now available for pnh and that patients with pnh-positive mds can respond to immunosuppressive therapy, pnh testing in mds should be considered. Prospective analyses to clarify the clinical significance of pnh positivity in mds are warranted.

Issue Highlight - July 2018.

Issue Highlight - July 2018.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 1 - clinical utility.

Paroxysmal nocturnal hemoglobinuria (PNH) arises as a consequence of the non-malignant clonal expansion of one or more hematopoietic stem cells with an acquired somatic mutation of the PIGA gene (Brodsky RA. Blood 113 (2009) 6522-6527). Progeny of affected stem cells are deficient in glycosyl phosphatidylinositol-anchored proteins (GPI-APs). This deficiency is readily detected by flow cytometry. Though this seems straightforward, the clinical utility of this testing requires that the ordering clinician understand not only the characteristics of the test, but also the biology of the underlying disease, and the clinical and laboratory manifestations in the individual patient. When interpreted correctly, the results from PNH flow cytometry testing, including presence and size of the clonal populations and the cell types involved, can allow the clinician to classify the disease appropriately; evaluate the risk of disease progression; and subsequently monitor response to therapy. In these guidelines, we discuss the evaluation of a patient with suspected PNH or other bone marrow failure disorders, with specific emphasis on the contribution of this testing to the diagnosis, classification, and monitoring of patients. For convenience we will commonly refer to these flow cytometry studies as "PNH testing" recognizing that an abnormal result is not diagnostic of PNH; rather both laboratory and clinical features are used to establish this diagnosis. © 2017 International Clinical Cytometry Society.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 3 - data analysis, reporting and case studies.

Over the past several years, a diverse group of physicians and other laboratory scientists have developed various recommendations and guidelines regarding best practices for PNH testing. This manuscript is based on these previous recommendations as well as various other relevant publications of experts in the area of PNH testing. The goal is to provide flow cytometry laboratories with an updated consensus approach to analysis and reporting of PNH results and to address the most common analytical challenges for accurate reporting of this rare disease. A comprehensive case library is included in this section. © 2017 International Clinical Cytometry Society.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 4 - assay validation and quality assurance.

Over the past six years, a diverse group of stakeholders have put forth recommendations regarding the analytical validation of flow cytometric methods and described in detail the differences between cell-based and traditional soluble analyte assay validations. This manuscript is based on these general recommendations as well as the published experience of experts in the area of PNH testing. The goal is to provide practical assay-specific guidelines for the validation of high-sensitivity flow cytometric PNH assays. Examples of the reports and validation data described herein are provided in Supporting Information. © 2017 International Clinical Cytometry Society.

Diagnosis of Paroxysmal Nocturnal Hemoglobinuria: Recent Advances.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder with its protean clinical manifestations. This is due to partial or complete absence of 'glycophosphatidyl-inositol-anchor proteins' (GPI-AP). The main aim of this review is to highlight various diagnostic modalities available, basic principle of each test and recent advances in the diagnosis of PNH. Recently among various tests available, the flow cytometry has become 'the gold standard' for PNH testing. In order to overcome the difficulties encountered by the testing and research laboratories throughout the world, International Clinical Cytometry Society has come up with guidelines regarding the indications for testing, protocol for sample collection, processing, panel of antibodies as well as gating strategies to be used, how to interpret the test and reporting format to be used. It is essential to test at least two GPI-linked markers on at least two different lineages particularly on red cells and granulocytes/monocytes. The fluorescent aerolysin combined with other monoclonal antibodies in multicolour flow cytometry offered an improved assay not only for diagnosis but also for monitoring of PNH clones. It is equally important to diagnose this rare entity with high index of suspicion.

Standardized high-sensitivity flow cytometry testing for paroxysmal nocturnal hemoglobinuria in children with acquired bone marrow failure disorders: A single center US study.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired hematopoietic stem cell disorder that has not been well-documented in children, particularly those with acquired bone marrow failure disorders (ABMFD)-acquired aplastic anemia (AAA) and myelodysplastic syndrome (MDS). Therefore, we sought to determine the prevalence of PNH populations in children with ABMFD. PNH testing was performed in children with an ABMFD diagnosis using high sensitivity (≥0.01%) fluorescent aerolysin (FLAER)-based assay according to 2010 International Clinical Cytometry Society (ICCS) PNH Consensus Guidelines and 2012 Practical PNH Guidelines. FLAER/CD64/CD15/CD24/CD14/CD45 and CD235a/CD59 panels were used for white blood cell and red blood cell testing, respectively. Thirty-seven patients with ABMFD (34 AAA, 3 MDS) were included (17M/20F, age 2-18years, median 9years). PNH populations were identified in 17 of 37 (46%) patients. Of the 17 patients with PNH populations identified, 7 were PNH clones (>1% PNH population), and 10 had minor PNH population or rare cells with PNH phenotype (≤1% PNH population). This is the first study to use a standardized high-sensitivity FLAER-based flow cytometry assay and the recommended cutoff of 0.01% to identify cells with PNH phenotype in pediatric patients with ABMFD in the United States. The identification of a PNH population in 46% of ABMFD supports the recommendation for high sensitivity PNH testing in children with these disorders. As a less sensitive assay using a cutoff of ≥ 1% PNH population would have missed 10 (27%) patients with minor PNH population or rare cells with PNH phenotype. © 2017 International Clinical Cytometry Society.

Diagnosing nocturnal paroxysmal hemoglobinuria: a single-center 4-year experience.

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease and can present as a wide range of signs and symptoms. As such, the indication for diagnostic testing for PNH is not always straightforward. Therefore, we analyzed all first-time samples tested over a 56-month period to determine the clinical settings with a high probability of detecting a PNH clone. We retrospectively analyzed 323 first-time PNH flow cytometry tests, including LDH, cytopenias, direct antiglobulin test (DAT), and clinical indication for testing as available at the time of testing. The probability of finding a PNH clone was 47% in patients tested because of aplastic/hypoplastic bone marrow disorders, 10% in DAT-negative hemolytic anemia (HA), 5% in myelodysplastic syndromes (MDS), 3% in cytopenias other than HA, and 2% in thrombosis. When testing for another reason than the indications described before, there were no positive samples. Our findings reinforce guidelines from the International PNH Interest Group which suggest testing for PNH in the setting of unusual thrombosis, HA, aplastic/hypoplastic bone marrow disorders, or MDS, as these have a higher pretest probability. This probability drops to zero in our study in nonrecommended indications. This reflects the need for better education of clinicians about the disease PNH and the indications for diagnostic testing.

Diagnostic screening of paroxysmal nocturnal hemoglobinuria: Prospective multicentric evaluation of the current medical indications.

Although consensus guidelines have been proposed in 2010 for the diagnostic screening of paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometry (FCM), so far no study has investigated the efficiency of such medical indications in multicentric vs. reference laboratory settings. Here we evaluate the efficiency of consensus medical indications for PNH testing in 3,938 peripheral blood samples submitted to FCM testing in 24 laboratories in Spain and one reference center in Brazil. Overall, diagnostic screening based on consensus medical indications was highly efficient (14% of PNH+ samples) both in the multicenter setting in Spain (10%) and the reference laboratory in Brazil (16%). The highest frequency of PNH+ cases was observed among patients screened because of bone marrow (BM) failure syndrome (33%), particularly among those with aplastic anemia (AA; 45%) and to a less extent also a myelodysplastic syndrome (MDS; 10%). Among the other individuals studied, the most efficient medical indications for PNH screening included: hemolytic anemia (19%), hemoglobinuria (48%) and unexplained cytopenias (9%). In contrast, only a minor fraction of the patients who had been submitted for PNH testing because of unexplained thrombosis in the absence of cytopenia, were positive (0.4%). In summary, our results demonstrate that the current medical indications for PNH screening by FCM are highly efficient, although improved screening algorithms are needed for patients presenting with thrombosis and normal blood cell counts. © 2016 International Clinical Cytometry Society.

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.

Significance of the detection of paroxysmal nocturnal hemoglobinuria clones in patients with multiple myeloma undergoing autologous stem cell transplantation

There are reports about the presence of paroxysmal nocturnal hemoglobinuria (PNH) clones in multiple myeloma (MM), but these have been demonstrated only in red blood cells (RBCs) and the previous reports utilized an obsolete diagnostic method. We carried out a study to identify the clones by flow cytometry (FC) and to understand their clinical significance. A prospective study on consecutive patients with newly diagnosed MM who were candidates for autologous stem cell transplantation (ASCT) from 2008 to 2012. We screened peripheral blood samples by FC for CD55- and/or CD59-deficient RBC, neutrophils, and monocytes. PNH testing was carried out at diagnosis, before ASCT and 3 months after ASCT, as well as sporadically during MM remission and at disease relapse. A total of 31 patients were included in the study. PNH clones reaching a median size of 10.8% (range 4.0-18.7%) were found in 10 patients (32.3%). Clones were detected at diagnosis in nine patients and 3 months after ASCT in one patient. A correlation between the presence of the clones and subclinical hemolysis was observed. Nevertheless, the presence of the clones did not influence the overall management and prognosis of the patients. We confirmed findings of previous reports with current diagnostic guidelines and showed that although the size of the clones may be relatively large, their presence is probably not detrimental. The clinical significance of these clones and the possible mechanisms underlying their expansion in MM must be a subject of further investigation.