Detection of platelet autoantibodies to identify immune thrombocytopenia: state of the art

Abstract Introduction: Immune thrombocytopenia (ITP) is a bleeding disorder caused by an autoimmune response against platelets. In the majority of cases, ITP is thought to be caused by the presence of autoreactive B cells that produce anti-platelet autoantibodies and target platelets for destruction by phagocytic cells. However, in about 40% of ITP patients platelet autoantibodies cannot be detected and there is some evidence that cytotoxic cells might also be responsible for platelet death. Indeed, many patients repeatedly fail to respond to current immunosuppressive therapies that target B cells and their autoantibodies. As a consequence, these patients retain very low platelet counts with increased bleeding diathesis. In this study we have immunophenotyped a group of adult chronic ITP patients that have not responded to traditional immunosuppressive therapies and we identified 2 subgroups of patients with either an increase or decrease in the frequency of CD8+ T effector memory CD45RA+ cells (CD8TEMRA) compared to healthy controls. Methods: PBMCs were isolated from blood samples of 14 ITP patients with platelet counts <100x109/L and 14 matched healthy controls. The cells were phenotyped using a variety of antibodies including: CD3, CD4, CD8, CD45RA, CCR7, CD127, CD25, CD14, CD16 and CD19. In addition, at least 5x106 PBMCs were stimulated with PMA (50ng/ml) and ionomycin (1µg/ml) for 5 hours at 37°C, 5% CO2 and stained with antibodies against CD3 and CD8, then fixed and permeabilised before staining with antibodies specific to Granzyme B and Interferon-γ. Results and discussion: In our cohort of ITP patients we were able to identify two subgroups of patients based on their frequency of CD8TEMRA cells, identified as CD45RA+ CCR7- cells, gated on CD3+ CD8+ cells. Compared to healthy controls (mean=16.33%), 6/14 patients had significantly lower frequencies of CD8TEMRA cells (mean=11.31%) and 8/14 patients showed a significant increase (mean=31.50%). Interestingly, these two groups of patients also show significant differences between them in the frequency of CD19+ B cells (gated on CD3- cells), as the group with the lowest CD8TEMRA frequency showed a significant increase in B cells compared to the high CD8TEMRA group. Considering that CD8TEMRA cells are described as highly differentiated cytotoxic T cells, these results suggest that in patients with active ITP in which the CD8TEMRA population is more prevalent and the frequency of B cells is reduced, cytotoxic T cells might play an important role in platelet destruction. Although an increase in the frequency of CD8TEMRA with age has been described we did not find a correlation between these two variables in our cohort of patients. In the low CD8TEMRA group we also observed a significant increase in the frequency of T regulatory cells (Tregs) and monocytes when compared to healthy controls, whereas the trend in the high CD8TEMRA group was for frequencies closer to controls. In addition, when analysing the production of Granzyme B and Interferon-γ after a short in vitro stimulation, we found that the trend was for the CD8+ T cells in the high CD8TEMRA group to produce higher levels of both Granzyme B and Interferon-γ when compared to the patients in the low CD8TEMRA group. This would support the hypothesis that in patients with increased frequency of CD8TEMRA there has been an expansion of cells with cytotoxic properties. Further work will be required to confirm that in this cohort of patients there is a CD8+ T cell population that can specifically target and lyse platelets, thus contributing to ITP pathogenesis. Disclosures Provan: UCB: Consultancy; GSK: Equity Ownership, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Medimmune: Consultancy.

Download Full-text

The ITP syndrome: pathogenic and clinical diversity

Blood ◽

10.1182/blood-2009-01-129155 ◽

2009 ◽

Vol 113 (26) ◽

pp. 6511-6521 ◽

Cited By ~ 398

Author(s):

Douglas B. Cines ◽

James B. Bussel ◽

Howard A. Liebman ◽

Eline T. Luning Prak

Keyword(s):

Natural History ◽

Immune Thrombocytopenia ◽

Genetic Factors ◽

Response To Therapy ◽

Patient Specific ◽

Platelet Destruction ◽

Autoimmune Syndrome ◽

Immune Deficiencies ◽

Platelet Autoantibodies ◽

Autoantibody Formation

Abstract Immune thrombocytopenia (ITP) is mediated by platelet autoantibodies that accelerate platelet destruction and inhibit their production. Most cases are considered idiopathic, whereas others are secondary to coexisting conditions. Insights from secondary forms suggest that the proclivity to develop platelet-reactive antibodies arises through diverse mechanisms. Variability in natural history and response to therapy suggests that primary ITP is also heterogeneous. Certain cases may be secondary to persistent, sometimes inapparent, infections, accompanied by coexisting antibodies that influence outcome. Alternatively, underlying immune deficiencies may emerge. In addition, environmental and genetic factors may impact platelet turnover, propensity to bleed, and response to ITP-directed therapy. We review the pathophysiology of several common secondary forms of ITP. We suggest that primary ITP is also best thought of as an autoimmune syndrome. Better understanding of pathogenesis and tolerance checkpoint defects leading to autoantibody formation may facilitate patient-specific approaches to diagnosis and management.

Download Full-text

Flow cytometric immunobead assay for quantitative detection of platelet autoantibodies in immune thrombocytopenia patients

Journal of Translational Medicine ◽

10.1186/s12967-017-1317-2 ◽

2017 ◽

Vol 15 (1) ◽

Cited By ~ 2

Author(s):

Juping Zhai ◽

Mengyuan Ding ◽

Tianjie Yang ◽

Bin Zuo ◽

Zhen Weng ◽

...

Keyword(s):

Immune Thrombocytopenia ◽

Quantitative Detection ◽

Flow Cytometric ◽

Platelet Autoantibodies

Download Full-text

Platelet antibodies in immune thrombocytopenia and related conditions

Journal of Laboratory Medicine ◽

10.1515/labmed-2020-0012 ◽

2020 ◽

Vol 0 (0) ◽

Author(s):

Volker Kiefel

Keyword(s):

Immune Thrombocytopenia ◽

Common Finding ◽

Blood Products ◽

Clinical Value ◽

Drug Induced ◽

Primary Hemostasis ◽

Platelet Antibodies ◽

Alloimmune Thrombocytopenia ◽

Transfusion Reactions ◽

Platelet Autoantibodies

AbstractPlatelet autoantibodies are a common finding in immune thrombocytopenia (ITP) and in rare cases of antibody-mediated platelet function (“acquired thrombasthenia”). In drug-induced immune thrombocytopenia, antibodies react with platelets only in the presence of the offending drug. Alloantibodies reacting with platelets are induced by transfusion of cellular blood products or during pregnancy. They are responsible for fetal/neonatal alloimmune thrombocytopenia (FNAIT), they are able to cause febrile, nonhemolytic transfusion reactions and they give rise to insufficient platelet increments following platelet transfusions. Two rare transfusion reactions: post-transfusion purpura (PTP) and passive alloimmune thrombocytopenia (PAT) are triggered by platelet alloantibodies. This review discusses the clinical value of tests for platelet antibodies in various clinical situations related to insufficient primary hemostasis.

Download Full-text

Detection of Platelet Autoantibodies Revisited to Identify Immune Thrombocytopenia

Blood ◽

10.1182/blood.v126.23.1142.1142 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1142-1142 ◽

Cited By ~ 3

Author(s):

Leendert Porcelijn ◽

Elly Huiskes ◽

Gonda Oldert ◽

Rob Fijnheer ◽

Martin R. Schipperus ◽

...

Keyword(s):

Sensitivity And Specificity ◽

Clinical Evaluation ◽

Predictive Value ◽

Immune Thrombocytopenia ◽

Response To Therapy ◽

Platelet Glycoprotein ◽

Control Group ◽

Control Groups ◽

Platelet Counts ◽

Platelet Autoantibodies

Abstract Introduction: Immune Thrombocytopenia (ITP) is still diagnosed by exclusion of many other causes for thrombocytopenia. In order to prevent misdiagnosis, an ITP-specific diagnostic test would be very helpful. In addition, characterization of glycoprotein specificity of platelet autoantibodies may explain (the severity of) bleeding symptoms and response to therapy. In this regard, we optimized the cut-off value of the direct monoclonal antibody immobilization of platelet antigens (MAIPA) assay for detection of platelet glycoprotein directed autoantibodies and re-evaluated its sensitivity and specificity for the diagnosis of ITP. Materials and Methods: The MAIPA was performed as part of our routine protocol, described by Kiefel et al. (1985). For the determination of a new cut-off value, and to calculate the sensitivity and specificity, blood samples were tested from 462 healthy blood donors and 43 non-immune-mediated thrombocytopenic patients, suffering from either hematological malignancies or aplastic anemia (n=20), hepato-splenomegalic pooling (n=3), drug-induced thrombocytopenias (n=4), viral infections (n=6), pregnancy related thrombocytopenia (n=7), pseudothrombocytopenias (n=2) and microangiopathy (n=1) and from 60 known ITP patients. We then have tested 120 prospectively collected samples from thrombocytopenic patients, sent for diagnostic tests to our laboratory, and categorized these samples based on subsequently obtained clinical evaluation into 'most likely ITP' (n=64) or 'most likely non-ITP' (n=56). Results: The calculated direct MAIPA sensitivity and specificity, using a cut-off value of E=0.130, in the ITP and non-ITP control groups (n=103) were 85% (95% CI, 73-93%) and 100% (95% CI, 92-100%), respectively (see Figure). The platelet auto-antibodies in the ITP control group (n=60) were directed against glycoprotein (GP)IIb/IIIa (66.7%), GPIb/IX (60%), GPV (51.7%), GPIa/IIa (40.6%) and/or GPIV (26.9%). The calculated sensitivity and specificity for detection of platelet auto-antibodies in the prospective diagnosed ITP and non-ITP patient control groups (n=120) were 75% (95% CI, 63-85%) and 96% (95% CI, 88-100%), respectively (see Figure). For this group of patients, the direct MAIPA showed, for diagnosis of ITP, a negative predictive value (NPV) of 77% (95% CI, 66-86%) and a positive predictive value (PPV) of 96% (95% CI, 86-100%). Furthermore, in 23 ITP patients the sequential sampling in a rituximab-treatment protocol showed platelet counts that were significantly and inversely correlated with the direct-MAIPA extinctions (p=0.006). In this respect, we excluded that higher platelet counts impaired the detection of platelet autoantibodies - e.g. by diluting them over an higher platelet mass-since autoantibodies were successfully detected in samples from ITP patients (n=4) with, as a result of splenectomy, platelet counts above 100 x 109/L and in untreated ITP patients with platelet counts between 75 and 100 x 109/L. These findings may implicate that response to rituximab as reflected by a rise in platelet counts is dependent on antibody presence, but the mechanism of effective lowering of platelet autoantibody levels by rituximab is still unclear. In conclusion, the revisited direct MAIPA showed to be a valuable technique for the detection of platelet autoantibodies both at diagnosis and during treatment and can possibly become a guide for optimizing therapy towards a more personalized treatment of ITP. Direct MAIPA O.D. above 0.13 is considered positive. Control samples: historically well characterized ITP patients. Prospective study: requests for serological ITP diagnostics, after final clinical evaluation classified as ITP or non-ITP. Figure 1. Figure 1. Disclosures Schipperus: Novartis: Consultancy.

Download Full-text

Platelet autoantibodies in the bone marrow of patients with immune thrombocytopenia

Blood Advances ◽

10.1182/bloodadvances.2020001846 ◽

2020 ◽

Vol 4 (13) ◽

pp. 2962-2966 ◽

Cited By ~ 3

Author(s):

Sabrina Shrestha ◽

Ishac Nazy ◽

James W. Smith ◽

John G. Kelton ◽

Donald M. Arnold

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Immune Thrombocytopenia ◽

Bone Marrow Cells ◽

Bone Marrow Aspiration ◽

Platelet Glycoprotein ◽

Platelet Destruction ◽

Immune Reactions ◽

Healthy Donors ◽

Platelet Autoantibodies

Abstract Autoantibodies cause platelet destruction in patients with immune thrombocytopenia (ITP); yet only 50% to 60% of patients have detectable platelet autoantibodies in peripheral blood. We hypothesized that in some ITP patients, platelet autoantibodies are sequestered in the bone marrow where pathological immune reactions target megakaryocytes or newly formed platelets. In this study, we modified the platelet glycoprotein-specific assay to test bone marrow aspiration samples for free platelet autoantibodies or antibodies bound to bone marrow cells in aspirate fluid from patients with ITP (n = 18), patients with nonimmune thrombocytopenia (n = 3), and healthy donors (n = 6). We found that 10 (56%) of 18 patients with ITP had autoantibodies in the bone marrow, including 5 (50%) of 10 with autoantibodies in bone marrow only, and 5 (50%) of 10 with autoantibodies in bone marrow and peripheral blood. In comparison, 6 (33%) of 18 ITP patients had autoantibodies in peripheral blood, most of whom (5 [83%] of 6) also had autoantibodies in bone marrow. Bone marrow autoantibodies were not detected in patients with nonimmune thrombocytopenia or healthy donors; however, peripheral blood autoantibodies were detectable in 1 (33%) of 3 patients with nonimmune thrombocytopenia. The sensitivity of platelet autoantibodies for the diagnosis of ITP increased from 60% (peripheral blood testing) to 72% (peripheral blood and bone marrow testing). Immune reactions limited to the bone marrow may be characteristic of certain subsets of ITP patients.

Download Full-text

Practical Picture Processing

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051700 ◽

1974 ◽

Vol 32 ◽

pp. 338-339

Author(s):

T. A. Welton

Keyword(s):

Radiation Damage ◽

Coherence Length ◽

Spatial Information ◽

State Of The Art ◽

Coherent Radiation ◽

The State ◽

Energy Spread ◽

Electron Micrograph ◽

Picture Processing ◽

Molecular Skeleton

Various authors have emphasized the spatial information resident in an electron micrograph taken with adequately coherent radiation. In view of the completion of at least one such instrument, this opportunity is taken to summarize the state of the art of processing such micrographs. We use the usual symbols for the aberration coefficients, and supplement these with £ and 6 for the transverse coherence length and the fractional energy spread respectively. He also assume a weak, biologically interesting sample, with principal interest lying in the molecular skeleton remaining after obvious hydrogen loss and other radiation damage has occurred.

Download Full-text

A Macintosh Interface for the Cameca Camebax-Micro Electron Microprobe

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010018094x ◽

1990 ◽

Vol 48 (1) ◽

pp. 438-439

Author(s):

Carl E. Henderson

Keyword(s):

Electron Microprobe ◽

Processing Speed ◽

Word Processing ◽

State Of The Art ◽

Computer Control ◽

Third Party ◽

Disk Storage ◽

The Past ◽

User Facility ◽

Instrument Control

Over the past few years it has become apparent in our multi-user facility that the computer system and software supplied in 1985 with our CAMECA CAMEBAX-MICRO electron microprobe analyzer has the greatest potential for improvement and updating of any component of the instrument. While the standard CAMECA software running on a DEC PDP-11/23+ computer under the RSX-11M operating system can perform almost any task required of the instrument, the commands are not always intuitive and can be difficult to remember for the casual user (of which our laboratory has many). Given the widespread and growing use of other microcomputers (such as PC’s and Macintoshes) by users of the microprobe, the PDP has become the “oddball” and has also fallen behind the state-of-the-art in terms of processing speed and disk storage capabilities. Upgrade paths within products available from DEC are considered to be too expensive for the benefits received. After using a Macintosh for other tasks in the laboratory, such as instrument use and billing records, word processing, and graphics display, its unique and “friendly” user interface suggested an easier-to-use system for computer control of the electron microprobe automation. Specifically a Macintosh IIx was chosen for its capacity for third-party add-on cards used in instrument control.

Download Full-text

Current Issues: Advanced Digital Technology for Supervising Graduate Clinicians

Perspectives on Administration and Supervision ◽

10.1044/aas20.1.9 ◽

2010 ◽

Vol 20 (1) ◽

pp. 9-13 ◽

Cited By ~ 2

Author(s):

Glenn Tellis ◽

Lori Cimino ◽

Jennifer Alberti

Keyword(s):

Real Time ◽

Digital Technology ◽

Clinical Training ◽

State Of The Art ◽

Clinical Skills ◽

Slow Motion ◽

Video Capture ◽

Microsoft Excel ◽

Clinical Supervisors ◽

Training And Supervision

Abstract The purpose of this article is to provide clinical supervisors with information pertaining to state-of-the-art clinic observation technology. We use a novel video-capture technology, the Landro Play Analyzer, to supervise clinical sessions as well as to train students to improve their clinical skills. We can observe four clinical sessions simultaneously from a central observation center. In addition, speech samples can be analyzed in real-time; saved on a CD, DVD, or flash/jump drive; viewed in slow motion; paused; and analyzed with Microsoft Excel. Procedures for applying the technology for clinical training and supervision will be discussed.

Download Full-text