Platelet Immunology

July 18, 2002
13:00 to 17:00
Plaza Room
Boston Park Plaza Hotel

Chairman:  BH Chong, Australia
Co-Chairs: RH Aster, USA; JB Bussel, USA; M Ertem, Turkey; S Santoso, Germany; T Warkentin, Canada

This meeting was dedicated to Dr. Albert von dem Borne (Amsterdam, The Netherlands) for his substantial and significant contributions to the field of Platelet Immunology.  Of his 353 publications from 1969 to 2001, 76 dealt with platelet immunology. His leadership and contributions to the field are poignantly illustrated by the observation that 8 platelet alloantigens were discovered by Albert and his coworkers.
The meeting was divided into three sections: (1) autoimmune thrombocytopenia; (2) alloimmune thrombocytopenia; and (3) drug-induced immune thrombocytopenia. Specific tributes were made to Dr. von dem Borne’s contributions before each of these three segments.

Autoimmune thrombocytopenia

Dr. BH Chong. Critical review of laboratory testing for ITP.
In this presentation, the useful contribution of platelet antibody testing for diagnosis of ITP was summarized, but with the key caveat that positive laboratory testing is not essential for diagnosis given the moderate sensitivity of the tests for detecting platelet-reactive autoantibodies, even using the newest assays. The history of antibody testing was reviewed, including the recognition that the "classic" platelet-associated IgG assays are not specific for ITP. Brief descriptions of various platelet glycoprotein-specific assays were provided, particularly the glycoprotein immobilization assays (microtiter well assay, immunobead assay, MACE, MAIPA & PAICA). Technical problems of these assays were described. Prospective studies suggest that the sensitivity and specificity of the MAIPA for ITP (using anti-GPIIb/IIIa and anti-GPIb/IX monoclonal antibodies) are about 50-66% and about 90%, respectively. A concluding comment was that other laboratory information (eg, reticulated platelets, TPO levels, plasma glycocalicin) may also be helpful in defining ITP.

Dr. JB Bussel. H Pylori and ITP.
This presentation summarized published and non-published data indicating that about 42% of patients with ITP can be shown to have H pylori infection. Of the 66 patients in whom infection was successfully eradicated, 48% of patients showed platelet count improvement. Unfortunately, the validity of the treatment effect remains uncertain, as the five studies reviewed show considerable variability in response (0/13=0%, 6/14=43%, 6/12=50%, 12/19=63%, 8/8=100%).

Dr. D. Beardsley. Animal model for study of ITP.
Although dogs can spontaneously develop ITP, this does not offer a practical animal model for ITP. A strain of murine ITP-prone mice may have some value. Other models were described, including human Fcg RIIA transgenic mice, and comparisons of the effects of murine antiplatelet antibodies in murine FcgRIII knock-out vs human FcgRIIIA transgenic mice.

A new animal model was described: NOD/SCID immunodeficient mice were reconstituted with human stem cells, allowing for subsequent experiments examining the effects of anti-HPA-1a antibodies (in animals with significant numbers of circulating human platelets) or following transfusion with human platelets. Human macrophages and megakaryocytes are demonstrable in these "Harrington mice." Addition of high concentrations of IgG can inhibit alloantibody-mediate platelet clearance. A proposal was made that members of the Platelet Immunology Subcommittee interact with other groups having animal models and aim by 2003 to have a joint agreement with the Subcommittee on Animal Models.

Alloimmune Thrombocytopenia

Drs. EA Kolb, JB Bussel. A prospective randomized trial evaluating the safety and efficacy of risk-based therapy for neonatal alloimmune thrombocytopenia (NAIT).
The US group reviewed their experience performing randomized trials of prenatal therapy in various at-risk groups for NAIT, as defined by severity of thrombocytopenia at first fetal blood sampling (FBS) at 20-24 wk and history of intracranial hemorrhage (ICH) in previous siblings. Standard-risk fetuses (platelets >20x109/L, no siblings with ICH) were randomized to IVIG (1 g/kg/wk) or prednisone 0.5 mg/kg/wk, with salvage therapies (prednisone 1 mg/kg/d or IVIG 1 g/kg/wk, respectively) in non-responding fetuses (assessed at follow-up FBS every 3-6 wk). No significant differences in outcomes were seen. In high-risk fetuses (platelets <20x109/L or sibling with ICH), randomization to IVIG 1g/kg/wk plus prednisone (1 mg/kg/d) was compared with IVIG alone (salvage: add IVIG 1 g/kg/wk to total 2 g/kg/wk or add prednisone, respectively). Significantly better outcomes were observed in the group receiving IVIG plus prednisone.

Non-randomized studies were performed in very high-risk fetuses (sibling with ICH between 28-36 wk): IVIG (1 g/kg/wk) beginning at 12 wk, FBS beginning at 20-24 wk, with salvage prednisone (1 mg/kg/d). In this group, 8 out of 12 needed salvage therapy. Finally, an extremely high-risk group (sibling with ICH <28 wk) was upgraded to 2 g/kg/wk after the first fetus died (1 g/kg/wk), and both fetuses receiving the higher regimen later required salvage prednisone therapy.

Overall, in these studies, 6 patients had ICH (74 pregnancies); 211 FBS procedures in 91 patients were complicated by 3 fetal deaths, and 14 deliveries precipitated by FBS, among other complications. Taking these complications into account, the new protocols under current study emphasize early treatment with stratification according to risk and with initial FBS deferred until 30-33 wk. The standard-risk protocol (no sibling with ICH) now compares IVIG 2 g/kg/wk with IVIG 1 g/kg/wk plus prednisone (0.5 mg/kg/d). Therapy now starts at 20-24 wk and the first FBS is performed at 30-33 wk. Protocols for the high-risk (previously, very high-risk) and very high-risk fetuses were similarly modified.

Drs. MF Murphy, J Birchall, C Kaplan & H Kroll, on behalf of the European FMAIT Study Group. European collaborative study on the management of fetomaternal alloimmune thrombocytopenia (FMAIT).
Results of an observational study was reported. Fetuses with FMAIT due to HPA-1a incompatibility and a previously affected sibling, underwent initial FBS (generally, 20-24 wk) followed either by: (1) maternal therapy with IVIG (1 g/kg/wk) and/or prednisolone; (2) serial intrauterine platelet transfusions; or (3) fetal platelet monitoring by FBS with or without pre-delivery intrauterine platelet transfusion. There were 56 fetuses from 55 pregnancies in 50 women. Comparison of ICH in previous and present (treated) fetuses was: 15 vs 3 (suggesting a treatment effect). Other conclusions included: (1) there was a good correlation between the severity of FMAIT in siblings and severity of thrombocytopenia in fetuses; (2) FBS can have major complications (4 premature delivery, 2 deaths); (3) fetal platelet count rose to >50 in the 67% who received maternal therapy. In future studies, antenatal care will be based on sibling history. Antenatal therapy will commence prior to first FBS. Therapy with IVIG will be given with higher-dose IVIG or prednisolone or serial intrauterine transfusions added as salvage treatment if required.
 
Drs. JB Bussel, C Kaplan, M Murphy & H Kroll (presented by H Kroll). Guidelines for diagnosis and management of fetal/neonatal alloimmune thrombocytopenia (FNAIT).
The proposed new guidelines for diagnosis and management of FNAIT were presented (the last was developed in 1991 by Neonatal Hemostasis Committee of SSC/ISTH). This work is being developed as a joint ISTH/ISBT initiative. The topics to be discussed are when to suspect alloimmune thrombocytopenia in the fetus and neonate, lab testing, antenatal and postnatal management. The topic of screening for FNAIT will also be discussed. Specific technical aspects of testing, as well as test interpretation, will be reviewed. Emphasis on minimizing fetal blood sampling and using maternal therapy for "standard risk" fetuses will be made. For postnatal management, therapy with HPA-1a and -5b negative platelets is recommended, as >95% of neonates will respond, and there are logistical problems with providing washed and irradiated maternal platelets. In urgent situations, random donor platelets with IVIG can be given although response may be poor. Studies of antenatal screening are underway. The intention is to have the draft of this manuscript completed by September 2002, to receive comments by November 2002 with revisions made in December 2002, and to publish the report in Journal of Thrombosis and Haemostasis.

Drs. S Santoso, C Kaplan. Human platelet alloantigens: nomenclature and guidelines for new inclusions.
The history and terminology of the human platelet alloantigen (HPA) system for designating platelet alloantigens were reviewed and compared with "classical" and gene-based naming. Twenty-two platelet alloantigens were listed, with 4 not having been given HPA designations (Oea, Gova, Govb, Duva). The Platelet Nomenclature Committee was described, consisting of representative membership of scientists and physicians active in the field of platelet immunology (including members both from ISBT and ISTH), and officers, Chairman, Co-Chairman, and Secretary, elected from membership of committee, maximum two 3-year terms. Discussions would take place at ISBT and ISTH meetings, with acceptance of recommendations at ISBT meetings. Current representatives include C. Kaplan, Chairman, (representing ISBT), S. Santoso, Co-Chairman, (representing ISTH), and W. Ouwehand (Secretary). Other committee members are A. von dem Borne, R.H. Aster, V. Kiefel, Y. Shibata, J. Smith, and R. Kekomaki. Submissions should be made to the Nomenclature committee 3 months before ISTH and ISBT meetings. Procedures for acceptance of new alloantigen(s) were described in detail. Six reference labs were designated. Repository laboratories (Amsterdam & Giessen) would act as trustees for reference materials (sera, DNA, platelets).

Drs. P Metcalfe, WH Ouwehand, D Sands, TW Barrowcliffe (presented by BH Chong). Proposal for the adoption by WHO of a candidate preparation as an international reference reagent for human antibody against HPA-5b.
Plasma was obtained from two patients with well-characterized anti-HPA-5b alloantibodies. Stability studies showed minimal loss of reactivity when stored at -20oC or 4oC. Extensive collaborative studies were performed with multiple laboratories from several countries employing a wide variety of laboratory techniques: when plasma from the two patients was pooled (designated "99/666"), anti-HPA-5b reactivity of 1/64 and 1/16 in MAIPA and platelet immunofluorescence (flow cytometry) was shown, respectively. With few exceptions, laboratories readily detected anti-HPA-5b reactivity with 99/666, without detecting other HPA or HLA alloantibodies.
Dr Chong asked the members to vote on whether the Platelet Immunology subcommittee should recommend that 99/666 be adopted as an international reference reagent for human antibody against HPA-5b. The vote was 9 for, none against, remainder abstaining.  This recommendation will be presented to the SSC Voting members at this year's Business Meeting.
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Drug-induced Immune Thrombocytopenia (DIT)

Dr. BH Chong. Drug-induced thrombocytopenia. Mapping of the epitope of the quinine-induced platelet antibody.
The history of research into the pathogenesis of quinine-induced immune thrombocytopenia (first described in 1865) was presented. Immunoprecipitation of GP Ib by quinidine-dependent antibodies helped to support the view that this syndrome was not caused by an "innocent bystander" mechanism involving platelet Fc receptors. In general, GP Ib/IX is more frequently invoked as the target GP in DIT (particularly the GP IX component) compared with GP IIb/IIIa. A series of GP IX mouse-human chimeras were constructed to investigate the target region on GP IX more precisely. The epitope of the quinine-induced antibody was mapped to the C-terminus extra-cellular region of GP IX. Interestingly the epitope of SZ 1, an anti-GP IX monoclonal antibody which is known to block the binding of quinidine-, rifampicin- and ranitidine-dependent antibodies to GP IX, was also mapped to this region of the protein suggesting that this is a common binding site of several drug-dependent antibodies.

Dr. TE Warkentin. Clinical and laboratory diagnosis of HIT: refining the scoring system.
The concept of immune HIT as a clinicopathologic syndrome was emphasized. Thus, diagnosis requires both clinical (particularly, thrombocytopenia) and laboratory (detection of HIT antibodies) criteria. The availability of sensitive assays for clinically significant HIT antibodies and the fairly common occurrence of subclinical seroconversion in heparin-treated patients means that it is far easier to refute than to confirm a diagnosis of HIT. Indeed, a positive test result simply increases the pre-test probability of HIT to a higher post-test probability. A simple scoring system was proposed wherein a maximum of two points each (0, 1, 2) could be given for three criteria that help distinguish HIT from non-HIT disorders (timing of onset of thrombocytopenia; occurrence of new thrombosis; lack of another explanation for thrombocytopenia; maximum score, 6/6). Generation of sensitivity-specificity trade-off curves was shown for two assays: PF4/heparin-EIA and washed platelet activation assay (serotonin release test). Likelihood ratios for various test results were interpreted in relation to the estimated pre-test probability. It was concluded that a scoring system might help physicians arrive at appropriate estimations of pre-test probability for HIT, however, wide variation in the available laboratory assays and their performance characteristics means that it will be difficult to generate scoring systems that take into account both clinical and laboratory criteria applicable to all clinical settings. It was also suggested that quantitative results of laboratory testing can provide diagnostically useful information.

Dr. JM Walenga. Platelet-leukocyte activation in heparin-induced thrombocytopenia.
Evidence for a pro-inflammatory response in HIT was reviewed. Evidence was shown that neutrophils and monocytes (but not lymphocytes) can bind to platelets in the presence of platelet activation by HIT antibodies. Furthermore, this activation can be prevented by antibodies against P-selectin. Leukocyte activation leads to various pro-inflammatory and prothrombotic effects.