David Barth

Diagnosing PNH with FLAER and multiparameter flow cytometry.

PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl‐inositol (GPI)‐linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI‐linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement‐mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI‐linked molecules and causes lysis of normal but not GPI‐deficient PNH cells. FLAER is an Alexa488‐labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER‐based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders.

Use of a FLAER-Based WBC Assay in the Primary Screening of PNH Clones

Diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) with flow cytometry traditionally involves the analysis of CD55 and CD59 on RBCs and neutrophils. However, the ability to accurately detect PNH RBCs is compromised by prior hemolysis and/or transfused RBCs. Patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS) can also produce PNH clones. We recently described a multiparameter fluorescent aerolysin (FLAER)-based flow assay using CD45, CD33, and CD14 that accurately identified PNH monocyte and neutrophil clones in PNH, AA, and MDS. Here, we compared the efficiency of this WBC assay with a CD59-based assay on RBCs during a 3-year period. PNH clones were detected with the FLAER assay in 63 (11.8%) of 536 samples tested, whereas PNH RBCs were detected in only 33 (6.2%), and always with a smaller clone size. The FLAER assay on WBCs is a more sensitive and robust primary screening assay for detecting PNH clones in clinical samples.

Survival in Sensitized Lung Transplant Recipients With Perioperative Desensitization

Donor‐specific HLA antibodies (DSA) have an adverse effect on short‐term and long‐term lung transplant outcomes. We implemented a perioperative strategy to treat DSA‐positive recipients, leading to equivalent rejection and graft survival outcomes. Pretransplant DSA were identified to HLA‐A, B, C, DR and DQ antigens. DSA‐positive patients were transplanted if panel reactive antibody (PRA) ≥30% or medically urgent and desensitized with perioperative plasma exchange, intravenous immune globulin, antithymocyte globulin (ATG), and mycophenolic acid (MPA). PRA‐positive/DSA‐negative recipients received MPA. Unsensitized patients received routine cyclosporine, azathioprine and prednisone without ATG. From 2008–2011, 340 lung‐only first transplants were performed: 53 DSA‐positive, 93 PRA‐positive/DSA‐negative and 194 unsensitized. Thirty‐day survival was 96 %/99%/96% in the three groups, respectively. One‐year graft survival was 89%/88%/86% (p = 0.47). DSA‐positive and PRA‐positive/DSA‐negative patients were less likely to experience any ≥ grade 2 acute rejection (9% and 9% vs. 18% unsensitized p = 0.04). Maximum predicted forced expiratory volume (1 s) (81%/74%/76%, p = NS) and predicted forced vital capacity (81%/77%/78%, respectively, p = NS) were equivalent between groups. With the application of this perioperative treatment protocol, lung transplantation can be safely performed in DSA/PRA‐positive patients, with similar outcomes to unsensitized recipients.

A Canadian phase II study evaluating the efficacy of rituximab in the management of patients with relapsed/refractory thrombotic thrombocytopenic purpura

Rituximab is a chimeric monoclonal antibody that targets the human CD-20 antigen present on malignant and normal B lymphocytes. Recent clinical studies have shown a significant response rate when this drug is given to selected patients with thrombotic thrombocytopenic purpura (TTP). Given that the clinical manifestations of TTP may be the direct result of an auto-antibody against a regulatory Von Willebrand factor enzyme (ADAMTS13), it makes biological sense to consider a therapy that has the ability to diminish or eradicate antibody-producing B cells. Despite initial positive results, there is a need to identify which patients derive durable benefit from this agent. As in other conditions that utilize therapeutic immunosuppression, there is a risk that the addition of rituximab may also lead to serious opportunistic infections.

A phase‐II sequential case‐series study of all patients presenting to four plasma exchange centres with presumed relapsed/refractory thrombotic thrombocytopenic purpura treated with rituximab

The primary objective of this phase II study was to evaluate the efficacy of rituximab in the management of adult patients with physician‐diagnosed presumed thrombotic thrombocytopenic purpura (TTP); relapsed or refractory. We conducted a multicentre study in four Canadian hospital‐based apheresis units. Forty patients with presumed TTP (20 refractory and 20 relapsing) were sequentially enrolled and all received rituximab in a standardized manner. A complete response was documented in 14 of 19 refractory patients by week 8 and 15/16 were alive and in remission at 52 weeks (one patient was lost to follow‐up, one was a non‐responder, and three died). Among relapsing patients, 16/18 had a complete response at week 8 and 18/18 at week 52 (one patient lost to follow‐up and one withdrew). At 1 year, all relapsing and 85% of refractory patients survived. Of 38/40 patients who had ADMATS13 testing at study entry, 13/19 refractory and 10/19 relapsing patients had ADAMTS13 < 10% (typical TTP); whereas 6/19 refractory and 9/19 relapsing cases had ADAMTS13 > 10% (other thrombotic microangiopathy; TMA). Refractory‐typical TTP in contrast to refractory‐other TMA and all relapsing patients treated with plasma exchange and rituximab, were less likely to be responsive and more likely to die or relapse.

Long-term survival in two patients with hepatosplenic T cell lymphoma treated with interferon-alpha.

Hepatosplenic T cell lymphoma (HSTCL) is a distinct subset of post-thymic mature lymphoid malignancies which is mainly gd restricted (HSgdTCL). HSgdTCL typically presents with constitutional symptoms, hepatosplenomegaly without lymphadenopathy, anemia and thrombocytopenia. Histology reveals preferential invasion of hepatic sinusoids, splenic red pulp and bone marrow interstitium, with or without peripheral blood involvement [1]. Isochromosone 7q is the prototypic chromosomal abnormality observed [2]. While many cases arise de novo, one fourth of patients are immunocompromised and there is an association with underlying conditions involving chronic antigenic stimulation [3]. While most mature T lymphocytes express an alpha/beta (ab) T-cell receptor (TCR) less than 5% of the normal population of cells express the gamma/ delta (gd) receptor which is found mostly in extranodal sites including the splenic red pulp, gastrointestinal tract and skin. Common T-cell functions such as cytotoxicity, helper and suppressor actions are attributed to CD4 and CD8 cells expressing ab T-cells, whereas the natural role of gd T-cells is less well known. Studies into the function of gd T-cells have demonstrated involvement in regulating immune responses, modulating tumors and immune defence through mechanisms independent from ab T-cells [3]. HSgdTCL portends a poor prognosis with median survivals between 6 and 10 months. First line treatment is anthracycline-based chemotherapy or platinum-based chemotherapy. Long-term survival is rare regardless of treatment regimen (Table I). We describe two case reports of patients with HSgdTCL achieving complete remission and long-term survival with interferon-a (IFN-a). A 63-year-old male presented with direct antibody negative hemolytic anemia. Past medical history included Churg–Strauss vasculitis treated numerous times with corticosteroids. Transfusion requirements gradually increased and extensive investigations for an etiology of hemolysis were negative. Initial bone marrow biopsy was negative. No hepatosplenomegaly or adenopathy was seen on CT scan. Hemolysis was resistant to corticosteroids and intravenous immunoglobulin. Red cell survival was reduced to 5 days on RBC survival study. Repeat CT scan 3 months later demonstrated hepatosplenomegaly and a splenectomy for diagnostic and therapeutic indications was performed with no resolution of hemolysis. Pathology revealed red pulp expansion and infiltration of the splenic cords by lymphocytes staining positive for CD3, CD7, TIA-1, CD 43 and MIB-1. The cells were non-immunoreactive for CD5, CD4, CD8, CD 30, ALK-1, EMA, CD 56, CD57 and CD25. A monoclonal population of T-cell lineage was identified. Ebstein bar virus was absent on PCR. Bone marrow demonstrated sinusoidal infiltration by abnormal lymphocytes again exhibiting CD3 and CD7 expression and lacking CD4, CD8 and CD5.

Safety of plasma exchange therapy in patients with myasthenia gravis.

Introduction: Plasma exchange (PLEX) is effective in myasthenia gravis (MG), but there are concerns about its safety. Methods: We collected data prospectively from 42 patients randomized to PLEX treatment in a comparison study with intravenous immunoglobulin (IVIg). Detailed information on the PLEX treatment methodology and adverse events are reported. Results: Forty of 42 patients completed PLEX. Ninety percent were treated in an outpatient setting. Fifty‐five percent had no complications, and 45% had mild–moderate reactions that did not require stopping treatment; the majority were citrate reactions and peripheral vascular issues that were easily treated. Fifty‐seven percent of patients responded to treatment, and 83% completed PLEX via peripheral venous access. Two patients had severe adverse events: 1 related and 1 unrelated to PLEX. Comorbid disease and age did not predict reactions. Conclusion: PLEX is safe, effective, and well tolerated in patients with MG. Our results do not raise concerns about the safety of PLEX in patients with moderate–severe MG. Muscle Nerve 47: 510–514, 2013

Changes in quality of life scores with intravenous immunoglobulin or plasmapheresis in patients with myasthenia gravis

Background Intravenous immunoglobulin (IVIG) and plasmapheresis (plasma exchange (PLEX)) have comparable efficacy in reducing the Quantitative Myasthenia Gravis Score for disease severity (QMGS) in patients with moderate to severe myasthenia gravis (MG). Objective To determine if the improvement in the quality of life (QOL) after immunomodulation is comparable with either IVIG or PLEX. Methods 62 patients participated in the MG-QOL-60 study, completing the questionnaire at baseline and at day 14 after treatment. The MG-QOL-15 scores were computed from the MG-QOL-60 questionnaire responses. We analysed the change in the QOL scores from baseline to day 14 in both treatment groups. Results The scores in both QOL scales decreased at day 14 in the IVIG and PLEX groups, without significant difference between groups (QOL-15: IVIG −5.7±8.5, PLEX: −7.0±7.6, p=0.52; QOL-60: IVIG −13.3±16.9, PLEX −18.5±22.0, p=0.41). The improvement in QOL showed a good correlation with the decrease in QMGS. There was an excellent correlation between the MG-QOL-15 and MG-QOL-60 scores at baseline and at day 14. Conclusions This study of MG-QOL changes supports recent findings that IVIG and PLEX are comparable in the treatment of patients with moderate to severe MG and worsening symptoms. Furthermore, our study supports the use of the MG-QOL-15 as a secondary outcome measure in future clinical trials in MG.

IVIG and PLEX in the treatment of myasthenia gravis

Intravenous immunoglobulin (IVIG) and plasma exchange (PLEX) are used to treat myasthenia gravis (MG) but with little trial evidence. While a class I study provided evidence for the efficacy of IVIG treatment, the empirical support for PLEX has been less convincing until recently. In a randomized controlled single‐masked study of 84 MG patients with moderate to severe disease, IVIG and PLEX had comparable efficacy as demonstrated by reduction in the Quantitative Myasthenia Gravis Score (QMGS) for disease severity, percentage of responders, persistence of treatment effect, and tolerability, which were similar in both treatment arms. The change in QMGS was accompanied by improved disease‐specific quality of life. The only factor predicting response to treatment was baseline severity. FcR polymorphisms did not predict response to IVIG therapy, but an inhibitory polymorphism was associated with baseline disease severity. These studies support the choice of either IVIG or PLEX as comparable treatments in adult patients with moderate to severe MG.

Blastic plasmacytoid dendritic cell neoplasm with leukemic presentation: 10‐Color flow cytometry diagnosis and HyperCVAD therapy

Few studies describe the comprehensive immunophenotypic pattern of blastic plasmacytoid dendritic cell neoplasm (BPDCN) in the bone marrow and its treatment. This retrospective analysis evaluates the diagnostic flow cytometry (FCM) pattern and outcome of nine patients diagnosed with BPDCN. A four‐tube 10‐color FCM panel used for diagnosis of acute leukemia (AL), showed cells in the blast gate (CD45dim/low SSC) and were positive for CD4(bright), CD33(dim), CD56(heterogenous), CD123(bright), CD36, CD38, HLA‐DR, CD71. Seven patients received front‐line induction therapy with HyperCVAD with an overall response rate of 86%. Five of six responders underwent planned allogeneic hematopoietic cell transplantation (allo‐HCT). For a median follow up of 13.3 months, the 1‐year disease free survival and overall survival were 56 and 67%, respectively. An accurate diagnosis of BPDCN can be made by 10‐color FCM using a four‐tube AL panel demonstrating a characteristic pattern of antigen expression. Front‐line induction chemotherapy with HyperCVAD can yield high remission rates, but allo‐HCT is required for long‐term durable remissions. Am. J. Hematol. 91:283–286, 2016.