Paroxysmal Nocturnal Hemoglobinuria Phenotype Research Articles

Unraveling the Clinical Spectrum: A Case Study of Classic and Aplasia-Related Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare nonmalignant clonal disorder of hematopoietic stem cells characterized by a phosphatidylinositol glycan protein A (PIGA) gene mutation. This mutation results in the deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins, CD55 and CD59, on the cell surface, rendering red blood cells (RBC) susceptible to complement-mediated destruction. This leads to hemolytic anemia, thromboses, and bone marrow failure. Here we describe two cases of PNH presenting to a tertiary care center. Case 1 A 29-year-old male with a history of polysubstance use presented to the Emergency Department with a 2-day history of progressively worsening left lower quadrant abdominal pain. CT abdomen pelvis showed multiple renal infarcts and findings suggestive of pneumatosis intestinalis. A complete blood count (CBC) revealed pancytopenia with a leukocyte count (WBC) of 3 x 10 9/L, hemoglobin (Hb) of 94 g/L, and platelet count (Plt) of 48 x 10 9/L. Lactate dehydrogenase (LDH) was elevated (900 IU/L), haptoglobin level was low ( <8mg/dL), and direct antiglobulin test (DAT) was negative. Urine was positive for hemosiderin, and flow cytometry showed RBCs (6.7%), granulocytes (97.97%), and monocytes (89.02%) with PNH phenotype. Bone marrow biopsy (BMB) was negative for dysplasia and fibrosis with normal hematopoiesis but showed increased erythroid precursors. Enoxaparin was started for thromboses and levofloxacin for neutropenia. Treatment for PNH was initiated with a loading dose of ravulizumab and meningococcal vaccination. The patient was lost to follow-up after receiving the first maintenance dose. Case 2 An 81-year-old female with a medical history of hypertension and coronary artery disease was hospitalized for gradually worsening shortness of breath and fatigue ongoing for 2 months. She reported easy bruising but denied a history of obvious bleeding from any orifice. Physical exam was significant for petechial rash in her lower extremities. Labs revealed pancytopenia (WBC 1.44 x 10 9/L, Hb 68 g/L, Plt 8 x 10 9/L), low reticulocyte count, LDH of 276 IU/L, and negative DAT. BMB showed marked aplasia, and she became transfusion dependent. Subsequently, flow cytometry revealed PNH phenotype in 1.45% of RBCs, 54% granulocytes, and 54% monocytes. Eculizumab therapy was started for the diagnosis of PNH. However, her transfusion requirements did not decrease. She developed iron overload and showed positive conversion on DAT. Darbepoetin alfa and romiplostim were added. Her Hb stabilized at >70g/L in 3 years, following which she only required romiplostim to maintain Plt >20,000 ( Graph 1). After 5 years, she opted to discontinue all treatment. She is being followed up regularly and has since remained stable. Discussion The two cases above are reflective of how PNH can present in different ways, ranging from the classic triad of symptoms to those with subclinical findings associated with other underlying conditions, such as aplastic anemia or myelodysplastic syndrome. The introduction of complement inhibitors completely changed the natural history of PNH, as patients' median survival shifted from 15-20 years to align with age-matched controls. Indications for treatment include severe anemia, thrombosis, or PNH symptoms like dyspnea, fatigue, and painful paroxysms. Currently, eculizumab and ravulizumab are the only FDA-approved drugs for PNH treatment. Ravulizumab is non-inferior to eculizumab, with the added advantage of less frequent dosing (every 8 weeks versus 2 weeks). Regular monitoring for hemolysis is warranted with treatment. Many patients require long-term therapy, which continues to be a significant challenge, even with the spaced dosing of ravulizumab. Eculizumab protects from terminal complement-mediated damage preventing intravascular hemolysis. However, these cells are still deficient in CD55, making them vulnerable to opsonization by C3 and premature removal in the spleen. As a result, >50% of PNH patients receiving treatment show positive conversion on DAT, as seen in case 2. Heparin is preferred in acute thrombotic states. Most hematologists recommend anticoagulation till a few months after starting complement inhibition.

Paroxysmal Nocturnal Haemoglobinuria (PNH) Arising from Non-Canonical Mutations

Introduction Acquired somatic mutations in the X-linked PIGA gene are the most commonly described pathological mutations found in the serious haematopoietic disorder paroxysmal nocturnal haemoglobinuria (PNH), though incidental cases of mutations in PIGT, PIGB and PIGV have been reported. Mutations in the family of 20 PIG (phosphatidylinositol glycan) genes can disrupt biosynthesis of glycosylphosphatidylinositol (GPI) anchors, critical for tethering many important proteins, including the vital complement regulators CD55 and CD59, to the cell membrane. Their absence in PNH leads to complement dysregulation and complications including potentially life-threatening haemolysis and thrombosis, which may be mitigated by lifelong anti-complement therapy. From the cohort of 273 PNH patients currently on complement inhibitor therapy at the Leeds National PNH Centre we have identified 37 patients whose diagnostic flow cytometry report or clinical course differs significantly from expectation, particularly with evidence of inflammatory presentations, which may be indicative of an alternative, non- PIGA, aetiology. Methods Patients consented to the Leeds PNH Research Tissue Bank with unusual clinical and laboratory presentations were identified, genomic DNA was extracted from peripheral blood and initially screened by targeted high sensitivity NGS of the PIGA gene. From the first ten patient samples sequenced, three lacked any evidence of PIGA mutations and so whole exome sequencing (WES) was carried out. Results Patient 1:An 80 year old man, diagnosed with MDS with rearrangement of chromosomes 3 and 7 resulting in a dicentric chromosome and loss of chromosome 20q, was diagnosed with PNH following an increase in transfusion requirement. In addition to classical symptoms of PNH he was diagnosed with seronegative rheumatoid arthritis. Flow cytometry showed an unusual PNH phenotype with a large population of partially GPI-anchor deficient cells only (Table 1). Symptoms improved on eculizumab but he remained transfusion dependent and the disease later transformed to acute myeloid leukaemia. WES: exonic frameshift insertion in PIGT (figure1), pathogenic variant rs760395465. Patient 2:A 34 year old woman presented with a 13 year history of intermittent episodes of generalized urticaria associated with nausea, vomiting, general malaise and myalgia, increasing in intensity and latterly being associated with haemoglobinuria, lethargy and jaundice leading to a PNH diagnosis. Episodes were triggered by pregnancy or infections, but she remained asymptomatic between attacks. Flow cytometry revealed a PNH clone with a majority of cells being partially GPI deficient. She responds well to C5 inhibition with no further symptoms, including urticaria. WES: exonic frameshift insertion in PIGT (figure 1), pathogenic variant rs760395465. Patient 3: A 14 year old girl presented with intermittent episodes of urticaria and severe lethargy and was diagnosed with symptomatic significant transfusion-dependent PNH, with completely GPI-deficient cells. Diagnosis followed an episode of massive haemolysis resulting in transient acute renal failure requiring haemodialysis. She commenced anticoagulation initially until eculizumab was approved 2 years later; despite occasional episodes of breakthrough haemolysis she displays no further urticaria. She has also had symptoms of diffuse arthralgia. WES: stopgain mutation in PIGV (figure1), pathogenic variant rs145160045. Conclusions These cases add to the emerging picture of non- PIGA associated PNH cases and suggest that the presence of joint problems and episodic urticaria may be sufficient indication to prompt screening for alternative causative mutations. Similar to the previously reported PIGB mutated case, the patients with PIGT mutations only had partially deficient cells. It is important to fully characterise these emerging non-canonical variants in order to improve our understanding of the pathophysiology of this disorder and to provide more individualised care.

Ahemolytic PNH: Clinical Features of a Distinct Phenotype of Paroxysmal Nocturnal Hemoglobinuria

Introduction: Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem/progenitor cell disorder caused by PIGA mutations. All hematopoietic lineages derived from the affected clone exhibit the PNH phenotype [deficiency of glycosyl phosphatidylinositol linked membrane proteins (GPI-APs)]. Diagnosis is made by flow cytometric analysis of red blood cells (RBC), granulocytes, and monocytes. The percentage of granulocytes and monocytes with absent expression of GPI-APs is a measure of the size of the PNH clone. Typically, the percentage of GPI-AP deficient RBCs is somewhat less than that of granulocytes and monocytes due to selective destruction by complement-mediated intravascular hemolysis, manifested by high LDH, bilirubin, reticulocyte count and low haptoglobin. Herein, we report five patients with large PNH clones based on percentage of GPI-AP deficient neutrophils and monocytes, but with absent or near absent PNH RBCs and no biochemical evidence of hemolysis. We call this unique disease subtype, ahemolytic PNH. Methods: The medical records of PNH patients (174 patients with PNH granulocytes >20%) at the University of Texas Southwestern, Cleveland Clinic Foundation, and National Institutes of Health from 2000 to 2022 were examined. Information pertaining to their clinical, laboratory, and molecular attributes (NGS sequencing) was collected. Five patients with granulocyte clones >50% and RBC clones <5% were identified (Table). Results: Patient 1 is a 63-year-old male with a remote history of non-severe aplastic anemia (AA) diagnosed at 11 years old. The first PNH flow cytometry, obtained at age 61 years, showed 0.02% type II PNH RBC, 0.5% type III PNH RBC, 96% PNH monocyte and 97% PNH granulocytes (Table). There was no biochemical evidence of hemolysis at the time of diagnosis or in the preceding 5 years. Patient 2 is a 50-year-old male who presented with pancytopenia and was diagnosed with acute myeloid leukemia with myelodysplastic-related mutations. Flow cytometry showed 85% PNH monocytes and 59% PNH with no detectable PNH RBCs and no biochemical evidence of hemolysis at diagnosis or in 3 years of follow-up (Table). Patient 3 is a 73-year-old female who presented with pancytopenia and marrow aplasia with unremarkable morphology, and a PNH granulocyte clone of 93.6% with 1.3% type III PNH RBCs and 1.3% and 0.5% type II PNH RBCs, with no evidence of hemolysis (Table). Patient 4 is a 21-year-old female who presented with Budd-Chiari syndrome. Flow cytometry showed 73% PNH granulocytes with 3.6% type III PNH RBCs and 1.8% type II PNH RBCs. There was no biochemical evidence of hemolysis, but because of thrombosis, she was treated complement inhibitor therapy (Table). Patient 5 is a 61-year-old female with history of severe aplastic anemia treated with immunosuppression (IST) and eltrombopag. PNH flow cytometry showed PNH granulocytes of 92.4% and 2.1% PNH type II RBCs 2.1% post IST treatment with no biochemical evidence of hemolysis (Table). Conclusion: Herein, we present five patients with a distinct PNH phenotype that we call ahemolytic PNH. Except for the absence or near absence of PNH erythrocytes, this entity shares clinical and pathophysiological features with classic PNH, including thrombophilia (Budd-Chiari Syndrome, Table). The basis of this phenotype is speculative but may be determined by the genotype of the affected HSPC in which the somatic mutation of PIGA first occurred. According to this hypothesis, somatic mutations other than PIGA skew differentiation toward granulocyte/monocyte lineages. The International PNH Interest Group recognizes the following three categories of PNH: classic PNH, PNH in the setting of another define bone marrow abnormality, and subclinical PNH. Like ahemolytic PNH, subclinical PNH has no biochemical evidence of hemolysis. In this case, however, the absence of hemolysis is due to the small size of the PNH clone (median clone size <1%) as determined by flow cytometry analysis of peripheral blood granulocytes and monocytes. We propose that ahemolytic PNH, defined as patients having less than <5% PNH red cells and >50% PNH granulocytes/monocytes, be recognized as a distinct category of PNH.

Multi-Center Analysis of Clinical Characteristics, Outcomes and Overall Survival of an Under-Studied Entity: Paroxysmal Nocturnal Hemoglobinuria Associated with Bone Marrow Disorder

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease. Parker described three phenotypes: Classic PNH (C-PNH), PNH associated with another bone marrow disorder (BMD-PNH), and subclinical PNH associated with another bone marrow disorder (BMD-SCPNH). Virtually all research of complement inhibitors is focused on C-PNH, while BMD-PNH remains a poorly studied phenotype. The main objective of this study was to analyze survival of BMD-PNH patients compared to that in C-PNH. Methods: This is a multi-center retrospective study. We analyzed the outcomes of patients diagnosed with PNH from March 1990 to January 2022 in three hospitals in Mexico. PNH was classified according to Parker´s criteria. We compared clinical characteristics between patients with C-PNH and BMD-PNH. Univariate and multivariate analyses were performed to assess the impact of PNH phenotype on overall survival and Kaplan Meier curves were plotted to compare survival between groups. Results: We found 106 patients diagnosed with PNH, of which 6 patients with the BMD-SCPNH phenotype were excluded. We analyzed 100 patients, median age 49 years (37.5-59), 50% female, C-PNH 72% (n=72), BMD-PNH 28% (n=28). Relevant differences between groups (C-PNH vs BMD-PNH) include serum LDH median 1,147 vs 256 U/L, p=0.001, LDH ≥1.5 SLN 72.2% vs 26.08%, p=0.001, clone size 78.7% vs 18.8%, and platelets median 120x109 U/L vs 13x109U/L, p=0.0001. In the BMD-PNH group, 46.42% (n=13), had a clone size ≥50%, 7.14% (n=2) presented thrombotic events, and 21.42% (n=6) had high disease burden. Regarding treatment agents (C-PNH vs BMD-PNH), complement inhibitor use was reported in 30.55% (n=22) vs only 3.57% (n=1), p=0.003, and anticoagulation treatment was also more frequent in C-NPH, 47.22% vs 7.14%, p=0.001. Use of bone marrow failure treatment (immunosuppressants and/or androgens) was frequent in the BMD-PNH group, 96.43%, n=27. Table 1 describes pronostic factors found to influence survival. Of note, overall survival of BMD-PNH was considerably lower: median 1y (CI95%; 0-2.4h) vs 10y (CI95%;6.6-13.3y), p=0.012, as presented in Fig 1. In the multivariate analysis, lack of either complement inhibitor (HR 4.64; 95% CI, 1.66-12.94, p=0.003) or lack anticoagulation treatment was independently associated with increased mortality (HR 2.00; 95%CI 1.24-3.25, p=0.005). Conclusions: We found that BMD-PNH patients had a notably inferior survival, and infrequently received agents considered to be standard in C-PNH treatment. Interestingly, 21.42% of BMD-PNH had a high disease burden, a frequent indication of complement inhibitor use in C-PNH. Since specific treatment of BMD-PNH hasn't been defined (that is, beyond immunosuppression and/or androgens), use of complement inhibitor therapy could be a promising option. To our knowledge, clinical trial information of complement inhibitors in BMD-PNH patients is currently lacking. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Immune Checkpoint Molecules Regulate Paroxysmal Nocturnal Hemoglobinuria Clonal Evolution

In paroxysmal nocturnal hemoglobinuria (PNH), the acquisition of somatic PIGA mutations provides a selection advantage to hematopoietic stem cells from aplastic anemia (AA) immune attack. In addition to this immune evasion mechanism, HLA mutations, deletions, and somatic UPD involving 6p21 locus have been also described in AA, all of which implicated in such adaptive clonal escape. It is well-known that blood cells derived from a PIGA-mutant clone lack membrane-bound complement regulators (e.g., CD55, CD59) and exhibit an increased vulnerability to complement attack. Nevertheless, PIGA-mutant cells may still be susceptible targets for AA T cell-mediated immune attack. In conjunction with mutational analysis, HLA protein expression may be investigated by flow cytometry to identify a haploinsufficient (clonal) vs. possible adaptive down-modulation. Indeed, the absence of these otherwise constitutionally expressed proteins represents an excellent marker of clonality. Additionally, in an attempt to further hinder the creation of a successful immunological synapsis, other mechanisms, including modulation of immune checkpoints expression, may be also involved in such AA/PNH intertwine. Whether these immune escape mechanisms are convergent or instead represent alternative pathways is a subject of intense research. Here, we comprehensively explore the dynamics governing PNH immune ontogenesis by combining HLA/PIGA genomics and flow cytometric measurements of HLA (A, B, C, DQ, DR, DP) and the immune checkpoint B7/CD28H family (PD1, PDL1, PDL2, VISTA, B7-H6, HHLA2, CD28H), stimulatory (GITR, CD30L) and inhibitory immune checkpoints (CD47) in CD45+CD15+CD59+ and CD59- fractions. A deep targeted sequencing panel covering HLA classical loci along with an in-house developed pipeline was also applied to assess fine HLA aberrations (mutational and copy number events), whereas multi-amplicon deep sequencing was deployed to identify PIGA mutational status. A total of 27 PNH patients classified as fully blown hemolytic PNH (n=15) or PNH/AA overlap (n=12) were included in this study with additional 4 healthy controls (HC). Overall, 41% of our patients harboured concomitant myeloid clones (most common hits were in DNMT3A, TET2, ASXL1, BCOR), while 58% of cases carried somatic HLA alterations, involving allelic losses or mutations. The latter were nonsense (33%), missense (11%), or 5’ or 3’ UTR (56%) hits with a median VAF of 11% (3-95%). Somatic lesions (including losses) were found in HLA-A (10%), B (30%), DQA1 (10%), or DQB1 (50%) loci. Since HLA aberrations may constitute the extreme pole of immune evasion and a marker of enhanced immune selection, we performed sorting/sequencing experiments on CD59+vs CD59- cells. A similar distribution and clonal burden of HLA-mutant subclones was found between the 2 fractions, indicating that PNH cells do not provide absolute immune privilege.1 Next, we compared the expression of HLA and other immunomodulatory molecules across HC, CD59+ cells from PNH patients and their corresponding CD59- cells (GPI+ and GPI-, respectively). Using MFI as a readout, GPI- cells showed similar expression levels of HLA-DP, -DR, PDL2, HHLA2 and GITR as compared to HC cells, while these molecules were significantly 1.5 to 5-fold upregulated on corresponding GPI+ cells consistent with their trigger/target function. This conclusion was also supported by the higher levels of CD28H found on GPI+ cells vs. GPI-(median MFI, 13.3 vs. 6.0; p<0.0001). Of note is that GPI- cells exhibited lower levels of HLA-DQ (0 vs. 7.0, p=0.01), B7H6 (0 vs. 39, p<0.0001) and CD30L (0 vs. 41, p <0.0001) vs. HC cells and upregulation of VISTA (460 vs. 227, p=0.006), suggesting that PNH clones may exhibit additional (to PNH phenotype) immune deterrence pathways. HLA alterations resulted in downregulation of HLA-DQ (0 vs. 29, p=.03), -DR (20 vs. 136, p=.03), B7H6 (0 vs. 45, p=.02), and CD30L (7.5 vs. 52, p=.02), as well as upregulation of HLA-B (2671 vs. 1909, p=.03) and VISTA (561 vs. 291, p=.02) in GPI- vs. GPI+, while no difference was noted in HLAWT cases. In conclusion, differential expression of immunomodulatory molecules on PNH vs. residual phenotypically normal cells in PNH patients and HC highlights various stimulatory and immune escape mechanisms operating in this disease. Understanding these processes may enable the identification of therapeutically relevant immune pathways.

Immunodynamics of Paroxysmal Nocturnal Hemoglobinuria

In the context of immune-mediated attack in aplastic anemia (AA), PIGA mutations produce a clonally-restricted escape phenotype in an attempt to restore hematopoiesis. Along with PIGA lesions, HLA class I/II mutations may represent an alternative way to render cytotoxic T cell recognition less effective. Microdeletions of 6p21, uniparental disomy [UPD] of 6p and mutations of HLA genes have been previously identified in AA patients. One could stipulate that paroxysmal nocturnal hemoglobinuria (PNH) may be an alternative mode of clonal evolution as opposed to HLA mutation acquisition or instead be a redundant pathway of immune escape co-existing with such lesions. Here, we took advantage of a large cohort (n=92) of patients with hemolytic PNH (granulocytes clone size >20% and LDH>x2.5 ULN) to elucidate the immunodynamics of PIGA/HLA clone(s) (Fig1A).We first used a deep targeted sequencing panel covering HLA classical loci and applied an in-house newly developed pipeline on 62 patients with PNH. Overall, 35% of cases had HLA alterations, which were found at a similar rate in patients studied at onset (33%) and during the course of the disease (32%). These alterations were classified as allelic losses in 15% of cases while majority of mutations were missense (57%) followed by 5‘ or 3‘ UTR (20%), nonsense (15%) and frameshifts (8%) with a median variant allelic frequency (VAF) of 5% (range, 2-95). Of note, the canonical exon 1 HLA B14:02 mutation (c.19C>T, p.R7X) 1,2was found in 2 patients who both had de novo PNH with a granulocyte clone size of 60% at disease onset (Fig1B).To clarify whether the PNH phenotype conveys an absolute resistance from immune attack, one should determine whether in these patients HLA mutations arise indeed within non-PNH cells. Consequently, we also performed HLA sequencing on 9 patients whose samples (n=18) were previously sorted by flow cytometry to enrich for CD59+ and CD59- fractions. HLA mutations were identified in both CD59+ and CD59- fractions at similar rates (44% each) and VAF (5% vs 3%, p=0.3) while no “canonical” exon 1 mutation was found.When looking at associations with disease-specific characteristics, we did not identify any significant differences in terms of burden of HLA events and disease ontogeny (primary vs secondary to AA cases) as well as baseline demographics characteristics (gender, age at presentation), dominant erythrocytes type (II vs III), and thrombotic events. No correlations with specific types of PIGA mutations nor their burden were found in HLA-mutated vs non-mutated groups as also confirmed by the absence of relationship with PNH granulocyte clone size (62% vs 72%, p=0.5). Of note, in HLA mutants the VAFs of HLA lesions were significantly lower than those of PIGA (5% vs 18%, p<0.001) underlying their possible less powerful role in driving immune escape within the context of AA overshooting reactions. To validate these results, we have designed a highly quantitative flow cytometric method for detection of HLA expression (A, B, C for class I; DQ, DR, DP for class II) on CD59+ vs CD59- fractions of CD34+ and CD15+ cells obtained from PNH patients. Preliminary results in 7 patients showed a general downregulation of HLA expression within PNH clones (delta HLA MFI of CD59+ vs CD59- fractions), consistent with our ability to detect HLA mutations also in PNH cells (Fig1C). This preferential down-modulation on PNH cells suggests a possible selection mechanism operating agonistically and in a non-mutually exclusive fashion with the PNH-intrinsic privilege. Moreover, these findings may also represent a byproduct of antigen processing machinery impairment due to proteasomal accumulation of proteins, which would have been GPI-linked and exported to the membrane.In sum, our study indicates that PNH phenotype conveyed by PIGA mutations operates independently from HLA mutations as means of immune escape. In addition, based on these results we stipulate that HLA down-modulation via various mechanisms is operative in the context of the PNH phenotype and the immune privilege of PIGA clones is not absolutely protective. Patients with PNH may have elements of immune suppression. In fact, their responses in the context of reduced-intensity transplant do eliminate hosts PNH clones following transplantation. 3 [Display omitted] DisclosuresPatel: Alexion: Consultancy, Other: educational talks, Speakers Bureau; Apellis: Consultancy, Other: educational talks, Speakers Bureau. Maciejewski: Bristol Myers Squibb/Celgene: Consultancy; Novartis: Consultancy; Alexion: Consultancy; Regeneron: Consultancy.

The importance of flow cytometry in the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria

ABSTRACT Introduction: The paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal disease of the hematopoietic stem cells, and it is clinically characterized by chronic intravascular hemolysis, bone marrow failure and hypercoagulability leading to thrombosis. It is a rare disorder of the hematopoietic stem cells that occurs due to a somatic mutation in the gene phosphatidylinositol glycan class A (PIG-A). Objective: Here we reviewed the importance of screening and monitoring of individuals with high risk of developing PNH, since the early diagnosis of the disease is essential for better prognostic and treatment choice for the patient. Method: A review was carried out with great focus on the pathophysiology and diagnosis of PNH, mainly with the use of flow cytometry technique to detect the disease. Results: This gene codifies an enzyme essential to the formation of glycosylphosphatidylinositol (GPI), which acts as a molecular anchor for many membrane proteins. The alteration of GPI synthesis promotes a partial or complete loss of proteins that needs this molecular anchor to bind to the cell surface. Among these proteins are the CD55 and the CD59, which control the activation of the complement cascade. Conclusion: The immunophenotyping exam with flow cytometry is considered the reference test for PNH diagnosis, since the technique is highly sensitive and specific, presenting advantages as the quantitative identification of small populations of cells with PNH phenotype and the capacity to distinguish cells with partial or total deficiency of GPI-anchored proteins.

Acute kidney injury with dark urine: the case of paroxysmal nocturnal hemoglobinuria

In this case report; we describe a patient with severe attack of paroxysmal nocturnal hemoglobinuria (PNH) following Ciprofloxacin therapy. He presented with recurrent abdominal pain, repeated vomiting and dark urine. Physical examination revealed severe pallor and jaundice. Laboratory investigations showed severe intravascular hemolysis with negative Coomb’s test, progressive acute kidney injury (AKI), sterile blood cultures and negative serology for autoimmune diseases. Subsequently, he developed pancytopenia. Ham test was positive and flow cytometry, later on, confirmed PNH. He was supported with multiple transfusions of packed blood cells and hemodialysis. Ciprofloxacin was discontinued and his PNH was treated with Solumedrol 1 g daily for 3 days followed by Prednisone 60 mg/day for 2 months. Two weeks later; his hemolysis abated and his AKI improved. Up to 5 years later, he still has minor PNH clone yet without disease activity. In conclusion; our patient had acute drug-induced hemolytic crisis associated with minor PNH clone. With drug-vigilance; no further relapses were reported and his PNH clone remained stable for 5 years. The case expands the spectrum of PNH phenotypes and its triggering factors.&#x0D; Keywords: AKI, anemia, autoimmune, flow cytometry, hemolysis, PNH.

CD71 improves delineation of PNH type III, PNH type II, and normal immature RBCS in patients with paroxysmal nocturnal hemoglobinuria.

The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

Paroxysmal Nocturnal Hemoglobinuria in Patients with Aplastic Anemia: Challenges, Characteristics, and Analysis of Clinical Experience

Background &amp; Aims. Paroxysmal nocturnal hemoglobinuria (PNH) is a disease caused by an acquired clonal disorder of hematopoietic stem cells with clone cell membrane hypersensitivity to the complement. PNH can exist as an independent disease and can also be associated with other pathological conditions characterized by bone marrow deficiency, first of all with aplastic anemia (AA). In PNH-associated AA (AA/PNH) pathological clones may be initially of different size. In some patients a gradual growth of PNH clone is observed together with occurring signs of intravascular hemolysis and transformation into classical hemolytic PNH. In this case it is important to assess the clinical situation and determine eligibility for complement inhibitor therapy. During targeted therapy it is necessary to assess the efficacy of treatment based on monitoring of complement-mediated hemolysis and to identify probable reasons for insufficient effect. Materials &amp; Methods. The paper deals with 1 clinical case. A female patient born in 1964, with initial diagnosis of AA was followed-up from 1989 till present at the Russian Research Institute of Hematology and Transfusiology. Her treatment included blood-component therapy, the use of antilymphocyte immunoglobulin, cyclosporine, plasmapheresis, eculizumab, and symptom-relieving drugs. Results. The study deals with the case of transformation of non-severe AA with remission after immune-suppressive therapy into classical hemolytic PNH. The case report describes the characteristic features, AA/PNH diagnosis and treatment issues at different stages of the disease, and the reasons for incomplete effect of targeted therapy. Conclusion. The case under discussion confirms the relevance of current methods of detecting PNH clone at early stages of AA diagnosis and dynamic follow-up with respect to a probable growth of clone with PNH phenotype, especially at the stage of hematopoietic recovery. Determination of PNH clone size and lactate dehydrogenase serum level is required for timely amendment of treatment strategy with a switch to long-term targeted monitoring of hemolysis which allows to prevent irreversible visceral changes and severe complications. In case of insufficient effect of targeted therapy with ongoing anemia Coombs test is recommended because of probability of C3-mediated extravascular hemolysis.

A brief, but comprehensive, guide to clonal evolution in aplastic anemia.

Acquired aplastic anemia (AA) is an immune-mediated bone marrow aplasia that is strongly associated with clonal hematopoiesis upon marrow recovery. More than 70% of AA patients develop somatic mutations in their hematopoietic cells. In contrast to other conditions linked to clonal hematopoiesis, such as myelodysplastic syndrome (MDS) or clonal hematopoiesis of indeterminate potential in the elderly, the top alterations in AA are closely related to its immune pathogenesis. Nearly 40% of AA patients carry somatic mutations in the PIGA gene manifested as clonal populations of cells with the paroxysmal nocturnal hemoglobinuria phenotype, and 17% of AA patients have loss of HLA class I alleles. It is estimated that between 20% and 35% of AA patients have somatic mutations associated with hematologic malignancies, most characteristically in the ASXL1, BCOR, and BCORL1 genes. Risk factors for evolution to MDS in AA include the duration of disease, acquisition of high-risk somatic mutations, and age at AA onset. Emerging data suggest that several HLA class I alleles not only predispose to the development of AA but may also predispose to clonal evolution in AA patients. Long-term prospective studies are needed to determine the true prognostic implications of clonal hematopoiesis in AA. This article provides a brief, but comprehensive, review of our current understanding of clonal evolution in AA and concludes with 3 cases that illustrate a practical approach for integrating results of next-generation molecular studies into the clinical care of AA patients in 2018.

Issue Highlight - July 2018.

Issue Highlight - July 2018.

High-sensitivity 5-, 6-, and 7-color PNH WBC assays for both Canto II and Navios platforms.

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a rare acquired hematopoietic stem cell disorder characterized by an inability to make Glyco-Phosphatidyl-Inositol (GPI)-linked cell surface structures. Fluorescent proaerolysin (FLAER-Alexa488) is increasingly used to detect GPI-deficient WBCs by flow cytometry. However, FLAER is not available in all countries and is expensive to obtain in others. An earlier study to compare FLAER-based and non-FLAER assays confirmed very good agreement between the two tubes suggesting a cost effective simultaneous evaluation of PNH neutrophils and monocytes is possible without FLAER. We have used a single tube approach with a 7-color assay comprising FLAER-CD157-CD15-CD64-CD24-CD14-CD45. Conjugates were carefully selected and validated so that stained samples could be analyzed on either 10-color Navios or 8-color FACSCanto II platforms. The 6-color (minus CD14) and 5-color (minus CD24 and CD14) versions were also developed and compared with our predicate clinical lab 5-color assay comprising FLAER-CD157PE-CD64ECD-CD15PC5-CD45PC7. CD15-gated PNH neutrophil clone size was quantified using either FLAER and CD157, FLAER and CD24, or CD157 and CD24. CD64-gated PNH monocyte clone size was quantified using either FLAER and CD157, FLAER and CD14, or CD157 and CD14. Analysis of >40 PNH samples showed that the FLAER-based plots derive virtually identical data to the non-FLAER plot for neutrophils (R2 = 1) and monocytes (R2 = 0.9999) and that closely similar data can be acquired using both Canto II and Navios platforms with 7-, 6-, and 5-color versions of the assay. Assessment of non-PNH samples confirmed extremely low background rate of PNH phenotypes (neutrophils and monocytes) with all three approaches. © 2018 International Clinical Cytometry Society.

ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in paroxysmal nocturnal hemoglobinuria (PNH) and related disorders part 2 - reagent selection and assay optimization for high-sensitivity testing.

Since publication in 2010 of the International Clinical Cytometry Society (ICCS) Consensus Guidelines for detection of Paroxysmal nocturnal hemoglobinuria (PNH) by flow cytometery, a great deal of work has been performed to develop, optimize, and validate a number of high-sensitivity assays to detect PNH phenotypes in both red blood cells (RBCs) and white blood cells (WBCs, neutrophils, and monocytes). This section (Part 2) of the updated ICCS PNH Consensus Guidelines will focus on specific instrument setup for these PNH assays, the identification and proper testing of appropriate antibody conjugates and combinations therof, and basic assay design. © 2017 International Clinical Cytometry Society.

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.

161 - Analysis of PNH Clones in Bone Marrow in Myelodysplastic Syndromes at Diagnosis and Upon Treatment with the Immunomodulatory Agent Lenalidomide

Paroxysmal nocturnal hemoglobinuria (PNH), an acquired clonal disorder is manifested by failure of hematopoietic cells to express phosphatidylinositol glycan-anchored proteins (PIG-AP). Since the PIG-A mutation is present at the stem cell level, all cell lines may be affected. Although the pathogenesis of hemolytic anemia in PNH is related to the absence of CD55 and CD59 molecules on the membrane of red cells, the mechanism responsible for the increased incidence of thrombotic events in PNH is not clear. In this study we measured two glycosylphosphatidylinositol (GPI)-linked molecules on platelets (CD55 and CD59) and two GPI-linked proteins on neutrophils (CD14 and CD16), comparing their expression on normal and PNH patients. Using two-color flow cytometric analysis with antibodies directed against CD42b and CD41a, we found that CD55 and CD59 were constitutively expressed by normal fresh platelets, but that the expression levels decreased during the five day storage of platelets. A substantial population of platelets lacking the GPI-linked proteins were detected in most cases. We demonstrated varying degrees of deficiency in the expression of GPI-anchored molecules with neutrophils, monocytes and platelets with the highest proportion of deficient cells found within monocytic lineage. Similar numbers of platelets with the PNH phenotype and normal platelets expressed activation markers before and after exposure to platelet agonists. Flow cytometry is more sensitive than Ham's test in monitoring expression of PNH in platelets. Differences in the numbers of circulating GPI-deficient platelets and myeloid cells suggest that either the survival of platelets and mature myeloid cells differs or megakaryocytopoeisis is abnormal within the GPI-deficient clone.

High-Sensitivity Detection of PNH Red Blood Cells, Red Cell Precursors, and White Blood Cells.

Flow cytometry is the method of choice to 'diagnose' paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub-clinical PNH samples. A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four-color FLAER-based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single-tube 5-color FLAER/CD157-based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra- and inter-laboratory performance characteristics even in laboratories with little prior experience performing PNH testing.

Complement-mediated haemolysis and the role of blood transfusion in paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) is a rare form of acquired chronic haemolytic anaemia with unique characteristics. From a clinical point of view the classical triad is that of intravascular haemolysis, thrombosis and cytopenias1,2. From the point of view of pathogenesis, PNH can be regarded as a non-malignant clonal disorder, in which a mutation of the X-linked PIG-A gene in a haematopoietic stem cell affects the its mature progeny in the peripheral blood, including erythrocytes, leucocytes and platelets3: this progeny is, therefore, referred to as a PNH clone4. In PNH patients the PNH clone co-exists with qualitatively normal blood cells, although in many cases these are a minority5. PNH has no gender predilection and an estimated population frequency of between 1 and 10 per million. Molecular basis of paroxysmal nocturnal haemoglobinuria The first biochemical abnormality to be demonstrated in red cells from patients with PNH was a deficiency of the membrane protein acetylcholinesterase6. Subsequent studies showed that several more membrane proteins were missing or markedly decreased in the blood cells belonging to the PNH clone. From the serological point of view it is interesting that one of these (decay accelerating factor, see below) bears the Cromer blood groups7. In the 1980s it emerged that these deficient membrane proteins have one important feature in common: they are anchored to the cell surface through a glycosyl phosphatidyl inositol (GPI) molecule8,9. This suggested that the underlying biochemical abnormality would be in the GPI biosynthetic pathway, and it was indeed pinpointed at the step at which N-acetylglucosamine (NAcGlcN) is transferred onto phoshatidyl inositol10. Through expression cloning in a GPI-deficient cell line Taroh Kinoshita’s group identified the PIG-A (phosphatidyl inositol glycan complementation group A) gene as being defective in PNH cells11, and PIG-A somatic mutations were indeed identified in PNH patients12. PIG-A maps to the short arm of the X chromosome (band p22.1)13. The PIG-A product is a 484 amino acid protein which, with three other proteins, forms NAcGlcN transferase, the enzyme that catalyses the first step of the biosynthesis of the GPI anchor. Almost all the somatic mutations of the PIG-A gene identified so far in PNH patients are either frame-shift or nonsense mutations, only two are large deletions14,15. It should be noted that one of these inactivating mutations is sufficient to give rise on its own to the PNH phenotype because PIG-A is X-linked: as a result, only one allele is present in men, and only one allele is active in somatic cells in women. Very recently a PNH patient has been reported who had no PIG-A mutation: instead, she had a germ-line mutation of another gene (PIG-T) encoding a GPI biosynthetic enzyme, and a somatic mutation of the other PIG-T allele16. This patient provides a powerful confirmation of the notion that the critical biochemical failure in the PNH clone is in biosynthesis of the GPI anchor; in terms of frequency, this is likely to remain an exceptional situation17.

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.

Paroxysmal nocturnal haemoglobinuria phenotype cells and leucocyte subset telomere length in childhood acquired aplastic anaemia

The significance of paroxysmal nocturnal haemoglobinuria (PNH(pos) ) cells and leucocyte subset telomere lengths in paediatric aplastic anaemia (AA) is unknown. Among 22 children receiving immunosuppressive therapy (IST) for AA, 73% (16/22) were PNH(pos) , of whom 94% achieved at least a partial response (PR) to IST; 11/16 (69%) achieved complete response (CR). Only 2/6 (33%) PNH(neg) patients achieved PR. PNH(pos) patients were less likely to fail IST compared to PNH(neg) patients (odds ratio 0·033; 95% confidence interval 0·002-0·468; P=0·012). Children with AA had short granulocyte (P=7·8×10(-9) ), natural killer cell (P=6·0×10(-4) ), naïve T lymphocyte (P=0·002) and B lymphocyte (P=0·005) telomeres compared to age-matched normative data.