The prospective Hemophilia Inhibitor PUP Study reveals distinct antibody signatures prior to FVIII inhibitor development.

Preventing factor VIII (FVIII) inhibitors following replacement therapies with FVIII products in patients with hemophilia A remains an unmet medical need. Better understanding of the early events of evolving FVIII inhibitors is essential for risk identification and the design of novel strategies to prevent inhibitor development. The Hemophilia Inhibitor Previously Untreated Patients (PUPs) Study (HIPS; www.clinicaltrials.gov #NCT01652027) is the first prospective cohort study to evaluate comprehensive changes in the immune system during the first 50 exposure days (EDs) to FVIII in patients with severe hemophilia A. HIPS participants were enrolled prior to their first exposure to FVIII or blood products ("true PUPs") and were evaluated for different immunological and clinical parameters at specified time points during their first 50 EDs to a single source of recombinant FVIII. Longitudinal antibody data resulting from this study indicate that there are 4 subgroups of patients expressing distinct signatures of FVIII-binding antibodies. Subgroup 1 did not develop any detectable FVIII-binding immunoglobulin G (IgG) antibodies. Subgroup 2 developed nonneutralizing, FVIII-binding IgG1 antibodies, but other FVIII-binding IgG subclasses were not observed. Subgroup 3 developed transient FVIII inhibitors associated with FVIII-binding IgG1 antibodies, similar to subgroup 2. Subgroup 4 developed persistent FVIII inhibitors associated with an initial development of high-affinity, FVIII-binding IgG1 antibodies, followed by IgG3 and IgG4 antibodies. Appearance of FVIII-binding IgG3 was always associated with persistent FVIII inhibitors and the subsequent development of FVIII-binding IgG4. Some of the antibody signatures identified in HIPS could serve as candidates for early biomarkers of FVIII inhibitor development.

In-depth comparison of N-glycosylation of human plasma-derived factor VIII and different recombinant products: from structure to clinical implications.

Essentials Glycosylation heterogeneity of recombinant proteins affects pharmacokinetics and immunogenicity. N-glycomics/glycoproteomics of plasma-derived Factor VIII and 6 recombinant FVIIIs were compared. Depending on cell line, significant differences to plasma-derived FVIII were observed. Recombinant FVIIIs expressed distinct and immunologically relevant epitopes. SUMMARY: Background/Objective Human factor VIII (FVIII) is a plasma glycoprotein, defects of which result in hemophilia A. Current substitution therapy uses FVIII products purified from human plasma or from various cell lines (recombinant FVIII) with different levels of B-domain deletion. Glycosylation is a post-translational protein modification in FVIII that has a substantial influence on its physical, functional and antigenic properties. Variation in glycosylation is likely to be the reason that FVIII products differ in their pharmacokinetics, pharmacodynamics and immunogenicity. However, the literature on FVIII glycosylation is inconsistent, preventing assembly into a coherent model. Seeking to better understand the glycosylation mechanisms underlying FVIII biology, we studied the N-glycosylation of human plasma-derived (pd)FVIII and six rFVIII products expressed in CHO, BHK or HEK cell lines. Methods FVIII samples were subjected to head-to-head detailed glycomic and glycoproteomic characterization using a combination of MALDI-MS and MS/MS, GC-MS and UPLC-UV-MSE technologies. Results/Conclusion The results of our study detail the N-glycan repertoire of pdFVIII to an unprecedented level, and for the first time, provide evidence of N-glycolylneuraminic acid (NeuGc) found on pdFVIII. Although site-specific glycosylation of rFVIII proved consistent with pdFVIII regardless of the expression system, the entire N-glycan content of each sample appeared significantly different. Although the proportion of biologically important epitopes common to all samples (i.e. sialylation and high-mannose) varied between samples, some recombinant products expressed distinct and immunologically relevant epitopes, such as LacdiNAc (LDN), fucosylated LacdiNAc (FucLDN), NeuGc, LewisX/Y and Galα1,3 Gal epitopes. rFVIII expressed in HEK cells showed the greatest glycomic differences to human pdFVIII.

Comparative analysis of marketed factor VIII products: recombinant products are not alike vis-a-vis soluble protein aggregates and subvisible particles.

Essentials Aggregation is a critical quality attribute of protein therapeutics influencing immunogenicity. Aggregates and subvisible particles in 9 recombinant factor VIII (rFVIII) products were analyzed. Major differences in aggregate and particle concentrations were detected after reconstitution. rFVIII product quality determined aggregation propensity under use-relevant stress. SUMMARY: Background Recombinant protein technologies have facilitated the development of novel factor VIII (FVIII) therapeutics with improved production efficiency, potency and half-live, and a low risk of viral transmission. The increasing number of recombinant FVIII (rFVIII) products and information on their efficacy, safety and cost allow patients and healthcare professionals to adjust treatment to individual needs. Nonetheless, 20-32% of previously untreated patients with severe hemophilia A develop inhibitory antibodies to rFVIII following treatment. The root cause of the immunogenicity of rFVIII products is not well understood. Data for human interferon and human insulin products suggest that critical quality parameters such as soluble protein aggregates (SPAs) and subvisible particles (SVPs) influence the immunogenicity of protein therapeutics. Therefore, we analyzed SPA and SVP concentrations in commercially available rFVIII products and determined how these parameters change upon exposure of rFVIII products to relevant stress conditions. Objectives Compare critical quality parameters such as SPA and SVP concentrations in rFVIII products under intended use and use-relevant stress conditions. Methods Nine rFVIII products (≥ 3 lots each) were analyzed by high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) and flow cytometry-based particle analysis. Results/conclusions SPAs and SVPs were present at different concentrations in all freshly reconstituted rFVIII products: SPA concentrations ranged from 0.2% to 11.6%; SVPs were 0.7 × 106 to 114.0 × 106 / 1000 IU. Under use-relevant stress conditions (agitation and shear stress) the products formed additional SPAs and SVPs to different degrees. The collected data indicate that product quality determines its propensity to form SVPs and SPAs, and highlights differences between marketed rFVIII products.

Dangerous liaisons: how the immune system deals with factor VIII.

Only a fraction of patients with hemophilia A develop a neutralizing antibody (inhibitor) response to therapeutic infusions of factor VIII. Our present understanding of the underlying causes of the immunogenicity of this protein is limited. In the past few years, insights into the uptake and processing of FVIII by antigen-presenting cells (APCs) have expanded significantly. Although the mechanism of endocytosis remains unclear, current data indicate that FVIII enters APCs via its C1 domain. Its subsequent processing within endolysosomes allows for presentation of a heterogeneous collection of FVIII-derived peptides on major histocompatibility complex (MHC) class II, and this peptide-MHC class II complex may then be recognized by cognate effector CD4(+) T cells, leading to anti-FVIII antibody production. Here we aim to summarize recent knowledge gained about FVIII processing and presentation by APCs, as well as the diversity of the FVIII-specific T-cell repertoire in mice and humans. Moreover, we discuss possible factors that can drive FVIII immunogenicity. We believe that increasing understanding of the immune recognition of FVIII and the cellular mechanisms of anti-FVIII antibody production will lead to novel therapeutic approaches to prevent inhibitor formation in patients with hemophilia A.

Taking immunogenicity assessment of therapeutic proteins to the next level.

Therapeutic proteins provide innovative and effective therapies for numerous diseases. However, some of these products are associated with unwanted immunogenicity that may lead to clinical consequences such as reduced or loss of efficacy, altered pharmacokinetics (PK), general immune and hypersensitivity reactions, and neutralisation of the natural counterpart (e.g. the physiological hormone). Regulatory guidance on immunogenicity assessment needs to take into consideration a great diversity of products, indications and patient populations as well as constantly advancing manufacturing technologies. Such guidance needs to be sufficiently specific while, at the same time, allowing interactive discussion and adjusted benefit-risk weighing of each product on a case-by-case basis, e.g. for a unique treatment of a life threatening disease acceptable treatment risks may differ considerably from the ones in case of less serious disease. This theme was the focus of the international conference "Taking immunogenicity assessment of therapeutic proteins to the next level", held at the Paul-Ehrlich-Institut in Langen, Germany, on the 10-11. June 2010. The objectives of the conference were to highlight how the field could move from that of a mere description of risk factors to a system of risk assessment and mitigation, as well as an understanding of the impact of unwanted immunogenicity on the overall benefit/risk consideration for a medicinal product. More than 150 experts from industry, academia and regulatory authorities worldwide discussed the phenomenon of undesired immunogenicity from different perspectives. The conference focussed on issues relevant to three areas: (1) new European guidelines that are currently the subject of discussion; (2) testing strategies for immunogenicity assessment; and (3) scientific progress on the product-related factors that may contribute to the development of pathogenesis of immunogenicity, in particular in the field of protein aggregation and post-translational modifications. This report provides an overview of issues, insights, and conclusions that were discussed and achieved during the meeting.

Animal models of inhibitors.

SUMMARY: Antibody responses to clotting factor concentrates remain a major treatment limitation. In conjucation with ongoing clinical studies, the pathogenesis and potential treatment of clotting factor immune responses is being evaluated in a variety of animal models.

Modulation of factor VIII-specific memory B cells.

The development of inhibitory antibodies against factor VIII (FVIII) is the major complication in patients with haemophilia A who are treated with FVIII products. Memory B cells play an essential role in maintaining established antibody responses. Upon re-exposure to the same antigen, they are rapidly re-stimulated to proliferate and differentiate into antibody-secreting plasma cells (ASC) that secrete high-affinity antibodies. It is, therefore, reasonable to believe that memory B cells have to be eradicated or inactivated for immune tolerance induction therapy to be successful in patients with haemophilia A and FVIII inhibitors. The aim of our studies was the development of strategies to prevent FVIII-specific memory B cells from becoming re-stimulated. We established a 6-day in vitro culture system that enabled us to study the regulation of FVIII-specific murine memory-B-cell re-stimulation. We tested the impact of the blockade of co-stimulatory interactions, of different concentrations of FVIII and of ligands for toll-like receptors (TLR). The blockade of B7-CD28 and CD40-CD40 ligand interactions prevented FVIII-specific murine memory B cells from becoming re-stimulated by FVIII in vitro and in vivo. Furthermore, high concentrations of FVIII blocked re-stimulation of FVIII-specific murine memory B cells. Triggering of TLR7 amplified re-stimulation by low concentrations of FVIII and prevented blockade by high concentrations of FVIII. We conclude that we defined modulators that either amplify or inhibit the re-stimulation of FVIII-specific murine memory B cells. Currently, we are investigating whether the same modulators operate in patients with haemophilia A and FVIII inhibitors.

Non-genetic risk factors and the development of inhibitors in haemophilia: a comprehensive review and consensus report.

SUMMARY: The development of inhibitors to the infused factor in patients with haemophilia is a serious clinical problem. Recent evidence suggests that alongside the strong genetic contribution to inhibitor formation, there are a number of non-genetic factors--perceived by the immune system as danger signals--which promote formation of inhibitors. This study provides a comprehensive review of clinical studies relating to these factors and also presents a survey of opinion concerning their importance and clinical influence, conducted among the members of the European Haemophilia Treatment Standardisation Board (EHTSB). Taken together, this information highlights the lack of robust data concerning the influence of several non-genetic risk factors on inhibitor development, and an urgent need for prospective, well-conducted studies that adhere to recommendations made by the European Medicines Agency (EMEA) for studying inhibitors. Based on current literature, the EHTSB formulated consensus recommendations. It is desirable to minimize intensive treatment wherever possible, given the clinical situation. Prophylaxis should be offered to all children, although we still need to determine optimal dosing with respect to inhibitor development, and age for starting treatment. Vaccinations should be given subcutaneously and concomitant factor concentrate infusions avoided. According to the board, there is no evidence in the literature supporting suggestions that the type of concentrate influences inhibitor risk; but all patients should be monitored during their first exposures. Furthermore, there is no evidence to support an association between pregnancy-related issues, breast feeding and treatment-related factors (e.g. route of administration, or use of blood components) and inhibitor development.

New early prophylaxis regimen that avoids immunological danger signals can reduce FVIII inhibitor development.

SUMMARY: The most problematic complication of haemophilia A treatment is the development of inhibitors to FVIII. The highest risk of developing inhibitors is during the first 20 exposure days (EDs). If the patient can be brought through this high risk period without inhibitor development, the subsequent risk is low. Therefore, as a pilot project, we developed a prophylaxis regimen for the first 20-50 EDs specifically designed to induce tolerance to the administered FVIII and to minimize inhibitor development by avoiding immunological danger signals. Twenty-six consecutive previously untreated patients (PUPs) with severe haemophilia A were treated with the new prophylaxis regimen and the incidence of inhibitor development in this group was compared with that in a historical control group of 30 consecutive PUPs treated with a standard joint protection prophylaxis regimen (40-50 IU kg(-1), three times a week). There were no significant differences between the study and control groups in patient-related inhibitor risk factors such as ethnicity (all Caucasian), severity of haemophilia (all <1% FVIII), severity of FVIII gene mutation (P < 0.0006) nor in some treatment-related factors such as product type, age at first exposure, vaccination regimen or the need for surgery. 14 of 30 subjects given standard prophylaxis but only one of the 26 subjects given the new regimen developed an inhibitor (P = 0.0003, odds ratio 0.048, 95% CI: 0.001-0.372). Our results indicate that minimizing danger signals during the first 20 EDs with FVIII may reduce the risk of inhibitor formation. These results should be confirmed in a larger prospective clinical study.

Opportunities and limitations of mouse models humanized for HLA class II antigens.

MHC class II molecules are essential for shaping the CD4+ T-cell repertoire in the thymus and for selecting antigenic peptides that are presented to CD4+ T cells in the periphery. A range of different mouse models humanized for HLA class II antigens have been developed to study the regulation of MHC-class II restricted immune responses. These mouse models have been used to identify immunodominant peptides that trigger diseases and to characterize the interactions of T-cell receptors with disease-associated peptides and MHC class II molecules. Peptides presented to CD4+ T cells in these mouse models were shown to be similar to peptides presented to CD4+ T cells in patients who carry the same MHC class II haplotype. Opportunities and limitations associated with these mouse models will be discussed and the potential application of these models for understanding the regulation of antibody responses against factor VIII in hemophilia A will be indicated.

Variation of anti-Fas antibodies in different lots of intravenous immunoglobulin.

BACKGROUND AND OBJECTIVES: Previous studies have presented evidence that human immunoglobulin G preparations for intravenous use contain antibodies directed against the death receptor Fas (CD95). The function of these antibodies was described as either antagonistic or agonistic; therefore, inhibiting or stimulating Fas-dependent apoptosis. Based on these reports, we asked whether the proportion of antagonistic and agonistic anti-Fas activities differs between different lots of intravenous immunoglobulin (IVIG). Variations between lots would open the possibility to preselect suitable lots of IVIG for different therapeutic purposes. MATERIALS AND METHODS: Eleven lots of IVIG were tested for their ability to induce or inhibit Fas-dependent apoptosis. The biological significance of anti-Fas antibodies was confirmed by including anti-Fas antibodies purified from IVIG and IVIG depleted of anti-Fas antibodies in the study. RESULTS: All 11 lots inhibited FasL-induced apoptosis. In addition, five lots stimulated apoptosis in the absence of FasL. Depletion of anti-Fas antibodies from IVIG abolished the capacity of IVIG to inhibit FasL-induced apoptosis, but reduced the ability to induce apoptosis only slightly. CONCLUSION: The inhibition of FasL-induced apoptosis by IVIG is because of the presence of antagonistic anti-Fas antibodies. The activity of these antibodies differs considerably between different lots. On the other hand, the induction of apoptosis by IVIG is probably because of the concerted action of a range of different antibodies. The variation in the proportion of stimulating and inhibiting anti-Fas activities between different lots of IVIG opens the possibility to preselect suitable lots for different therapeutic purposes.

Fc function of a new intravenous immunoglobulin product: IGIV 10% triple virally inactivated solution.

BACKGROUND AND OBJECTIVES: Baxter AG has developed a new liquid intravenous immunoglobulin product [Immune Globulin Intravenous (IGIV) 10%] using a new manufacturing procedure. A modified Cohn fractionation and ion exchange chromatography is used to produce an IgG solution with no alterations to the Fc region. Three dedicated virus reduction steps are included: solvent-detergent treatment, nanofiltration, and incubation at low pH and elevated temperature in final formulation. We applied the reference method of the European Pharmacopoeia (EP) together with a flow-cytometric binding assay for the evaluation of the Fc function of the new product. MATERIALS AND METHODS: The EP reference method was done as described in the EP. The flow-cytometric method measured binding of IgG to Fc receptors of human monocytic THP-1 cells after exclusion of apoptotic cells. RESULTS: Sixteen lots of the new product expressed Fc functions between 84% and 110% when analysed with the EP reference method and Fc-binding activities between 82% and 121% when determined by the flow-cytometric method. CONCLUSION: All tested lots of the new product demonstrated a high level of Fc activity and met the requirements of the EP for Fc function.

Immunomodulatory properties of human serum immunoglobulin A: anti-inflammatory and pro-inflammatory activities in human monocytes and peripheral blood mononuclear cells.

Our study investigated the immunomodulatory activities of human plasma-derived serum immunoglobulin (Ig)A. Previous findings seem contradictory indicating either pro- or anti-inflammatory activities. We used serum IgA purified from large plasma pools and studied the modulation of the release of cytokines and chemokines from resting and lipopolysaccharide (LPS, endotoxin)-stimulated human adherent monocytes and human peripheral blood mononuclear cells (PBMC). Our results indicate that IgA down-modulates the release of the pro-inflammatory chemokines monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1alpha and MIP1beta from LPS-stimulated PBMC and the release of MCP1, MIP1alpha and MIP1beta from LPS-stimulated monocytes. Furthermore, we confirmed previous reports that plasma-derived serum IgA down-modulates the release of the pro-inflammatory cytokines, interleukin (IL)-6 and tumour necrosis factor (TNF)-alpha, from LPS-stimulated monocytes and PBMC, and up-regulates the release of IL-1 receptor antagonist (IL-1RA) from resting and LPS-stimulated monocytes and resting PBMC. This IgA-mediated up-regulation of IL-1RA is independent of the simultaneous up-regulation of IL-1beta release, as shown by blocking the biological activity of IL-1beta with a neutralizing antibody. On the other hand, we also found an IgA-induced pro-inflammatory activity, namely IgA-mediated up-regulation of the release of pro-inflammatory IL-1beta as well as down-regulation of the anti-inflammatory cytokines IL-10 and IL-12p40 from LPS-stimulated monocytes and PBMC and a down-regulation of transforming growth factor (TGF)-beta from resting and LPS-stimulated PBMC. We conclude that human serum IgA has both an anti-inflammatory and a pro-inflammatory capacity and this dual capacity might contribute to the feedback mechanisms maintaining a balance between pro-inflammatory and anti-inflammatory activities.

Increase in autoantibodies against Fas (CD95) during carcinogenesis in the human colon: a hope for the immunoprevention of cancer?

A thorough understanding of the naturally occurring events in the immune system in response to carcinogenesis will facilitate the development of strategies for the immunoprevention of cancer. The adenoma-carcinoma sequence in the human colon is a well-established clinical example of multi-step carcinogenesis and can be used for immunological studies. Based on previous observations that both apoptosis and the expression of Fas (Apo-1, CD95) are altered during carcinogenesis in the human colon, we asked the question whether serum titers of autoantibodies against Fas show any modification during the adenoma-carcinoma sequence. Healthy controls (38), patients with colorectal adenomas (38) and patients with colorectal adenocarcinomas (21) were investigated. Anti-Fas antibody titers were found to be significantly higher in patients with colorectal adenomas than in healthy controls and higher still in patients with colorectal adenocarcinomas. This increase in anti-Fas autoantibody titers during carcinogenesis might reflect the activation of natural defense mechanisms by the immune system.

Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance.

Patients with severe hemophilia A frequently develop neutralizing anti-factor VIII antibodies after replacement therapy with factor VIII (FVIII). In a search for new strategies to induce immune tolerance against FVIII in these patients, we used a murine model of hemophilia A to investigate the importance of CD40/CD40 ligand (CD40L) interactions for the initiation of the anti-FVIII immune response. We focused our attention in particular on the induction of neutralizing anti-FVIII antibodies and the Th1/Th2 polarization of FVIII-specific T cells. The development of anti-FVIII antibodies was analyzed by ELISA systems (detection of total anti-FVIII antibodies) and Bethesda assays (determination of neutralizing anti-FVIII antibodies). Factor VIII-specific T cells were characterized by multiparameter flow cytometry and cytokine ELISAs for the detection of cytokine production in splenic CD4+ T cells after in vitro restimulation with FVIII. Hemophilic mice received four doses of FVIII and anti-CD40L antibody MR1 (24 h before FVIII). Subsequently mice received four doses of FVIII only. The induction of neutralizing anti-FVIII antibodies in hemophilic mice after treatment with human FVIII could be prevented completely by a blockade of CD40/CD40L interactions using MR1. Furthermore, FVIII-specific T-cell responses that included both Th1 and Th2 cells were suppressed when mice were treated with FVIII and MR1. The initial blockade of CD40/CD40L interactions was, however, not sufficient to induce a lasting immune tolerance against FVIII. The immune suppression was abolished and both neutralizing anti-FVIII antibodies and FVIII-specific T cells developed when treatment with FVIII was continued after the omission of MR1. In addition, there were no alterations in the Th1/Th2 polarization induced by the initial blockade of CD40/CD40L interactions.

Characterization of antibodies induced by human factor VIII in a murine knockout model of hemophilia A.

To investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy. knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.

Apoptosis in haemopoietic progenitor cells exposed to extremely low-frequency magnetic fields.

Epidemiological studies have indicated a modestly increased risk for the development of acute myeloid leukaemia in children who live close to high-voltage power-lines. Recent evidence has suggested that a common property shared by a number of known and suspected tumour promoters is their ability to block the process of apoptosis. Therefore, one possible mechanistic explanation for the apparent leukaemogenic effect of weak, low-frequency magnetic fields, such as emitted by power-lines and electrical appliances, would be their expression of tumour-promoting activity by interfering with the regulation of apoptosis in multipotent haemopoietic progenitor cells. In order to test this hypothesis, we have employed the well-characterized multipotential haemopoietic progenitor cell line FDCP-mix(A4). These cells are non-leukaemic and undergo apoptosis when deprived of appropriate growth factors such as Interleukin-3. We have tested a series of different regimes of weak, low-frequency magnetic fields: nulled fields, Ca2+-ion cyclotron resonance conditions at 50 Hz, and vertical 50 Hz fields of 6 microT(RMS), 1 mT(RMS) and 2 mT(RMS), exposing the cells for 2 hours, 24 hours, 4 days or 7 days under various culture conditions. We have not seen any significant alteration in apoptosis induced by any of the exposure regimes tested. We therefore conclude that the regulation of viability and apoptosis in FDCP-mix(A4) cells is not disturbed by weak magnetic fields of the magnitude and type indicated.

Detection of phosphatidylinositol glycan class A gene transcripts by RT in situ PCR hybridization. A comparative study using fluorescein, Texas Red, and digoxigenin-11 dUTP for color detection.

Gene-specific probes labeled with fluorescein, Texas Red, and digoxigenin-11 dUTP (DIG) were used for RT in situ PCR hybridization to detect PIG-A gene (phosphatidylinositol glycan class A) transcripts. The PIG-A gene is responsible for biosynthesis of the glycosylphosphatidyl-inositol (GPI) anchor. Lack of GPI anchor expression due to mutations can cause an acquired clonal hematologic disorder called paroxysmal nocturnal hemoglobinuria (PNH). In this RT in situ PCR study, two types of labeling methods, a direct method (using fluorescein and Texas Red) and an indirect method (using DIG-11 dUTP) were compared. Both were successfully applied to detect and localize the PIG-A gene transcripts within single cells of the cell lines AA2, H9, and JY. Furthermore, similar results for sensitivity and reproducibility were obtained. Advantages and disadvantages of the different labeling techniques are discussed. In addition, peripheral blood mononuclear cells from PNH patients were also included in this study.