The complement-mediated prozone effect in the Luminex single-antigen bead assay and its impact on HLA antibody determination in patient sera.

The Luminex xMAP system has become an important tool for HLA antibody screening and identification in sera of transplant patients. Recently, the Luminex single antigen bead assay was shown to be prone to an artefact, the so called prozone phenomenon: Sera with high titer HLA antibodies gave negative results when tested neat, but reacted strongly positive after 1:10 dilution. We also observed such a phenomenon and found that it was most likely caused by the complement component 1 (C1) by competitively displacing the detection antibodies. In this article we review the complement-mediated prozone effect and other mechanisms of interference with solid phase assays, and we discuss possible consequences for HLA antibody testing with the Luminex SAB assay.

Successful haematopoietic stem cell transplantation from a matched unrelated donor following three graft failures from HLA-mismatched related donors.

Graft rejection and graft failure are serious complications after allogeneic stem cell transplantation (SCT). We report a patient with CML in first chronic phase who finally engrafted with a transplant from an HLA-identical unrelated donor after graft failures from two related HLA-mismatched sibling donors. After failure of one BM and two PBSC grafts from two 1 HLA-antigen mismatched related donors, the patient was finally successfully transplanted from a subsequently identified HLA-identical unrelated donor (donor 3). At 5 years post transplant, the patient is in complete cytogenetic and molecular remission.

Successful transplantation of highly selected CD34+ peripheral blood stem cells in a HLA-sensitized patient treated with immunoadsorption onto protein A.

BACKGROUND: Haploidentical bone marrow transplantation with preexisting anti-HLA antibodies is associated with a high risk of graft failure. METHODS: A 27-year-old female patient with chronic myeloid leukemia and evidence of several osseous chloromas had no suitable matched bone marrow donor, and fluorescence cytometric cross-match (FCXM) revealed antibodies against donor-specific HLA-molecules. Immunoadsorption onto staphylococcal protein A was applied to remove these antibodies, and peripheral stem cell transplantation was performed from her haploidentical sister after a negative FCXM was documented after immunoadsorption and conditioning treatment. RESULTS: FCXM for donor lymphocytes and stem cells remained negative throughout the posttransplant period, and engraftment of donor cells was documented on day +69. CONCLUSION: Immunoadsorption onto protein A should be considered in stem cell transplantation even from an haploidentical donor where anti-HLA antibodies and a positive FCXM are documented.

Platelet-specific antibodies in female blood donors after pregnancy.

Sera of 500 female blood donors after pregnancy were tested for platelet-specific antibodies (HPA-1, 3, 5) using the MAIPA assay. Twenty-one sera (4.2%) were found to be positive: four anti-HPA-1a, one anti-HPA-5a and 16 anti-HPA-5b. Sera were positive up to 30 years after the last pregnancy. All women with antibodies were HLA-DR52 positive and 79% of the women with anti-HPA-5b were HLA-DR6 positive.

Platelet antibodies in patients with migraine.

The reported decrease of platelet serotonin receptors in patients with migraine could be due to an autoimmune reaction. We therefore, examined sera from 42 migraineurs without aura, 26 migraineurs with aura, and 107 headache-free blood donors for platelet-reactive antibodies using the platelet adhesion immunofluorescence test, the NIH-lymphocytotoxicity test, and the monoclonal antibody-specific immobilization of platelet antigens test. IgG antibodies against non-HLA class I platelet antigens were found in 9.5% of patients with migraine without aura, 7.6% of patients with migraine with aura, and in 7.5% of controls; IgM antibodies were found in 11.9% of patients with migraine without aura, in 30.8% of patients with migraine with aura, and in 13.1% of controls. Most antibodies were directed against glycoprotein complexes II-III (fibrinogen receptor) or Ib-IX (thrombin receptor). Two patients with migraine without aura but no patient with migraine with aura nor any control subject had IgG antibodies of unknown specificity. One patient (2.4%) with migraine without aura and two patients (7.7%) with migraine with aura, as well as 2 controls (1.9%) had IgM antibodies not further specified. The differences in frequency of platelet antibodies of antibodies of known or unknown specificity in patients with migraine without aura and migraine with aura and controls were not statistically significant. Therefore, our data do not support the hypothesis of a pathophysiologically relevant autoimmune reaction against platelet serotonin receptors in th majority of patients with migraine. We can not exclude the occurrence of antibodies against neuron-specific serotonin receptors.

Limits of the MAIPA assay when differentiating high-titered platelet-reactive antibodies.

Experience with the MAIPA assay for the diagnosis of platelet-reactive antibodies has shown that high-titered antibodies falsify the test results. We here demonstrate 2 cases: i) A serum with high-titered HLA antibodies (100% panel reactivity in the LCT, titer between 4,000 and 12,000), and ii) Serum with a high-titered anti-HPA-1a (titer in the MAIPA assay 1,000). In both cases, it can be demonstrated that these antibodies led to unspecific reactions. In the 1st case, they interfered with the diagnosis of additional platelet-specific antibodies. Only the use of HLA-compatible platelets allowed a correct identification. On the other hand, in the high-titered anti-HPA-1a unspecific reactions were seen with the glycoproteins Ib/IX, Ia/IIa, and beta 2-microglobulin, leading to misinterpretations. These examples demonstrate that, in the test conditions as described, a correct diagnosis of high-titered sera might only be achieved by using compatible HLA or HPA cells.

Frequency and specificity of platelet-specific alloantibodies in HLA-immunized haematologic-oncologic patients.

The frequency and specificity of platelet-alloantibodies to human platelet antigens (HPA) -1, -3 and -5 was investigated in 59 multitransfused, HLA-immunized patients. Using the MAIPA test (monoclonal antibody specific immobilization of platelet antigens) platelet alloantibodies could be demonstrated in 10 (17%) patients. In one patient the antibody was present prior to any transfusions and probably induced by multiple previous pregnancies. This antibody was directed to HPA-5b. The remaining nine antibodies were found in patients (n = 36) with HLA-antibodies reacting with over 95% of unselected lymphocytes. In these patients the target antigens were HPA-1b in six, HPA-3a in one and both antigens in two patients. Our findings demonstrate platelet alloimmunization induced by transfusions to be restricted to patients with high HLA-immunization. 25% of these patients (9/36) show platelet-specific antibodies, primarily HPA-1b.

[Severe neonatal alloimmune thrombocytopenia with delayed antibody detection]. / Schwere neonatale Alloimmunthrombozytopenie mit verzögertem Antikörpernachweis.

Neonatal alloimmune thrombocytopenia (NAIT) is caused by maternal immunisation against a paternal antigen on fetal platelets. The antigen involved in the majority of cases is HPA-1 a (PIA1). Usually circulating platelet alloantibodies are detectable in the mother. In this report, we present a thrombocytopenic newborn with severe hemorrhagic diathesis due to materno-fetal HPA-1 a (PIA1) incompatibility. Platelet antibodies could initially not be demonstrated in the mother's serum but became detectable after four weeks. Because of the severe and protracted course of the disease, repeated platelet substitution was necessary throughout the first two months of life.

Reactivation of recipient antibody to blood cell antigens soon after allogeneic bone marrow transplantation.

Reactivation of recipient antibody to HLA and red blood cell antigens is described in 8 patients after allogeneic bone marrow transplantation. These IgG antibodies can be detected between day 10 and day 40 after transplantation and, in 1 patient, can be shown to be antigen-independent. We hypothesize that, induced by graft recognition of recipient antigens, antigen-independent activation of sensitized recipient B cells takes place leading to transient antibody production.

[Diagnosis and therapy of fetal alloimmune thrombocytopenia]. / Diagnostik und Therapie der fetalen Alloimmunthrombozytopenie.

In fetal alloimmune thrombocytopenia, maternal IgG antibodies directed against platelets pass into the fetal circulation and lead to the destruction of fetal platelets. Fetal thrombocytopenia is usually first noted postnatally, but the maternal alloantibodies may lead to severe fetal haemorrhage in utero in the second or, more commonly, third trimester. Platelet-specific antibodies appear to play the major role in the pathogenesis of fetal alloimmune thrombocytopenia. Antenatal serological diagnosis is complicated by the fact that no platelet-specific alloantibodies are detectable in untreated maternal serum in about 20% of cases. Even when antibodies are detected, repeated estimation of the titre in the mother gives no indication of the severity of the fetal thrombocytopenia. Therefore, when the diagnosis of fetal alloimmune thrombocytopenia is suspected, it should be confirmed and subsequently monitored by cordocentesis. The intrauterine transfusion of compatible platelets and the administration of high dose IgG infusions to the mother and/or fetus are currently being used as approaches to treatment. Despite advances in the antenatal diagnosis and therapy of fetal alloimmune thrombocytopenia, the clinical effects on the fetus remain unpredictable.

Reactivation of antibodies of donor and recipient origin to platelet antigens early after allogeneic bone marrow transplantation: a case report.

Reactivation of platelet-reactive antibodies of donor and recipient origin is described in a patient following allogeneic BMT (donor: anti-HPA-5b; recipient: anti-HLA, anti-HPA-1b). The antibodies were detected around day 15 after BMT, peaked around day 25, and then decreased. These antibodies are interpreted as an antigen-independent reactivation of secondary B-cell responses, activated in the context of recognition of host antigens by the graft.

[Demonstration of platelet-specific alloantibodies in HLA-sensitized hematologic oncologic patients]. / Nachweis thrombozytenspezifischer Alloantikörper bei HLA-sensibilisierten hämatologisch-onkologischen Patienten.

Alloimmunization to the platelet-specific antigen systems HPA-1, -3 and -5 was studied in 59 multitransfused patients (31 females, 16 males, 12 children) with haematologic-oncologic disorders. All patients tested had broad-reacting or multispecific HLA antibodies. Of these, 10 (17%) were found to have additional platelet-specific alloantibodies. With respect to HLA sensitization, 9 out of 10 platelet-specific alloantibodies were found in sera which showed HLA antibodies reacting with more than 95% of unselected panel cells. Determination of antibody specificity revealed 6 patients with anti-HPA-1b, one patient with anti-HPA-3a, and 2 patients with anti-HPA-1b combined with anti-HPA-3a. One patient had anti-HPA-5b; in retrospect, only the HPA-5b antibody was demonstrable before the beginning of transfusion therapy and before HLA sensitization.

Difficulties in the antenatal assessment of neonatal alloimmune thrombocytopenia.

In this paper we present a patient with an initially questionable history of neonatal alloimmune thrombocytopenia (NAT) due to materno-fetal HPA-1a (PLA1) incompatibility. No circulating antibodies were detectable in untreated maternal serum, but an adsorption/elution technique enabled the demonstration of the platelet-specific anti-HPA-1a (anti-PLA1) in maternal serum. Cordocentesis at 35 weeks of gestation revealed a fetal platelet count of 18 x 10(9)/l. Intrauterine platelet transfusion with HPA-1a (PLA1)-negative donor platelets was performed prior to cesarean section.

Serological screening, using three different test systems of platelet-transfused patients with hematologic-oncologic disorders.

Sera of hematologic-oncologic patients were tested regularly after platelet transfusions in three test systems: lymphocytotoxicity test, platelet adhesion immunofluorescence test, and--only selected sera--in the monoclonal antibody-specific immobilization of platelet antigen test. Of 388 patients 53 (14%) had HLA antibodies 5 of these in combination with platelet-specific alloantibodies. Lymphocyte-restricted (non-HLA) reactions were observed in 20 patients, the majority of which was attributed to lymphocyte-specific auto- or alloantibodies. Sera of 27 patients showed platelet-specific reactions, usually cold-reacting autoantibodies which have no effect in vivo.