Low anti‐RhD IgG‐Fc‐fucosylation in pregnancy: a new variable predicting severity in haemolytic disease of the fetus and newborn

Haemolytic disease of the fetus and newborn (HDFN) may occur when maternal IgG antibodies against red blood cells (RBCs), often anti‐RhD (anti‐D) antibodies, cross the placenta and mediate the destruction of RBCs via phagocytic IgG‐Fc‐receptors (FcγR). Clinical severity is not strictly related to titre and is more accurately predicted by the diagnostically‐applied monocyte‐based antibody‐dependent cellular cytotoxicity (ADCC), a sensitive test with relatively low specificity. This suggests that other factors are involved in the pathogenesis of HDFN. Binding of IgG to FcγR requires the N‐linked glycan at position 297 in the IgG‐Fc‐region, consisting of several different glycoforms. We therefore systematically analysed IgG‐derived glycopeptides by mass spectrometry from 70 anti‐D IgG1 antibodies purified from the plasma of alloimmunized pregnant women. This revealed a variable decrease in Fc‐fucosylation in the majority of anti‐D IgG1 (even down to 12%), whereas the total IgG of these patients remained highly fucosylated, like in healthy individuals (>90%). The degree of anti‐D fucosylation correlated significantly with CD16 (FcγRIIIa)‐mediated ADCC, in agreement with increased affinity of defucosylated IgG to human FcγRIIIa. Additionally, low anti‐D fucosylation correlated significantly with low fetal‐neonatal haemoglobin levels, thus with increased haemolysis, suggesting IgG‐fucosylation to be an important pathological feature in HDFN with diagnostic potential.     

Provision of platelet support for fetuses and neonates affected by severe fetomaternal alloimmune thrombocytopenia

Severe fetomaternal alloimmune thrombocytopenia requires urgent treatment with compatible platelet concentrates. As prompt treatment is sometimes delayed owing to the unavailability of compatible platelets, we established an accredited platelet donor panel to provide effective and timely transfusion support for fetal and neonatal therapy. After a mass screening programme of over 60,000 blood donations, 45 HPA-1a-negative donors with no antibodies to HPA, HLA, red cell antigens and granulocytes/lymphocytes, and with low titre anti-A and/or -B were accredited. All accredited donors were fully genotyped for HPA-1, -2, -3 and -5 by PCR-SSP. Ninety-one per cent of the accredited donors were also negative for HPA-5b.     

Frequencies of maternal platelet alloantibodies and autoantibodies in suspected fetal/neonatal alloimmune thrombocytopenia, with emphasis on human platelet antigen-15 alloimmunization

BACKGROUND AND OBJECTIVES: Serological evaluation of maternal sera for platelet antibodies in suspected fetal/neonatal alloimmune thrombocytopenia (FNAITP) discloses in only approximately 30% of individuals a platelet-specific antibody. Transfusion-induced alloimmunization against human platelet antigen-15 (HPA-15) has been reported to be about as common as against HPA-5, the second most common platelet antibody. Thus, anti-HPA-15 may also contribute significantly to yet-unclear cases of FNAITP. MATERIALS AND METHODS: In this retrospective analysis, we provide data on maternal platelet antibodies from 309 mothers who delivered an offspring with suspected FNAITP. RESULTS: Genotyping maternal and paternal samples (together n = 573) revealed a gene frequency of 0.496 for HPA-15a and a gene frequency of 0.504 for HPA-15b. HPA-15 antibodies were detected in 2% of all samples. Anti-HPA-15a and -15b were detected in two and three samples, respectively. One serum reacted equally with HPA-15a and -15b platelets. The most frequent platelet-specific antibodies were anti-HPA-1a (22%), but anti-HPA-5b (8.4%) were more frequent than anti-HPA-15. In addition, panreactive (5.5%) or autoreactive (5.2%) anti-GPIIb/IIIa or anti-GPIb/IX were detectable in maternal samples. CONCLUSIONS: These data indicate that HPA-15 alloimmunization needs only to be considered in subjects with suspected FNAITP if no other platelet-specific antibody is detectable. The presence of panreactive or autoreactive antibodies should also be considered in neonatal thrombocytopenia.     

The effect of rituximab on anti‐platelet autoantibody levels in patients with immune thrombocytopenia

Rituximab is an effective therapy resulting in a platelet count improvement in 60% of patients with immune thrombocytopenia (ITP). Rituximab depletes B cells; thus, a reduction in platelet autoantibody levels would be anticipated in patients who achieve a clinical response to this treatment. The objectives of this study were to determine whether rituximab was associated with a reduction in platelet autoantibody levels, and to correlate the loss of autoantibodies with the achievement of a treatment response. We performed a case‐control study nested within a previous randomized controlled trial of standard therapy plus adjuvant rituximab or placebo. We measured platelet‐bound anti‐glycoprotein (GP) IIbIIIa and anti‐GPIbIX using the antigen capture test. Of 55 evaluable patients, 25 (45%) had a detectable platelet autoantibody at baseline. Rituximab was associated with a significant reduction in anti‐GPIIbIIIa levels (P = 0·02) but not anti‐GPIbIX levels (P = 0·51) compared with placebo. Neither the presence of an autoantibody at baseline nor the loss of the autoantibody after treatment was associated with a response to rituximab. The subset of patients with persistent autoantibodies after treatment failed to achieve a platelet count response, suggesting that persistence of platelet autoantibodies can be a marker of disease severity.     

Maternal HLA genotyping is not useful for predicting severity of fetal and neonatal alloimmune thrombocytopenia

Lack of reliable laboratory parameters is the main challenge in the management of fetal and neonatal alloimmune thrombocytopenia (FNAIT ). Despite the long‐known association between the HLA ‐DRB 3* 01:01 allele and human platelet antigen 1a (HPA ‐1a) alloimmunisation, maternal human leucocyte antigen (HLA ) typing has been of little clinical value. Recently, other DRB 3 allele variants have been suggested to predict the severity of FNAIT . In this nationwide population‐based retrospective cohort study, we performed extensive HLA typing of 96 women, accounting for 87% of our cohort of 110 families with confirmed or possible HPA ‐1a‐immunisation. The HLA type was compared with anti‐HPA ‐1a levels, severity of neonatal disease and responsiveness to maternally administrated intravenous gammaglobulin (IVIG ). HLA haplotypes were constructed to investigate further HLA associations. Despite significantly lower anti‐HPA ‐1a levels in DRB 3* 01:01‐negative women, the carrier status of this particular allele could not be used to confirm or rule out FNAIT in the absence of detectable antibodies. In the haplotype analysis, the DRB 3* 01:01 allele was the actual factor associated with FNAIT . No other HLA allele was shown to be of additional value as a predictor of severe FNAIT or non‐responsiveness to IVIG treatment. Thus, HLA genotyping was not found useful in differentiating high‐ and low‐risk pregnancies or in guiding antenatal treatment in affected families.     

Antigen specificity determines anti‐red blood cell IgG‐Fc alloantibody glycosylation and thereby severity of haemolytic disease of the fetus and newborn

Haemolytic disease of the fetus and newborn (HDFN) is a severe disease in which fetal red blood cells (RBC) are destroyed by maternal anti‐RBC IgG alloantibodies. HDFN is most often caused by anti‐D but may also occur due to anti‐K, ‐c‐ or ‐E. We recently found N‐linked glycosylation of anti‐D to be skewed towards low fucosylation, thereby increasing the affinity to IgG‐Fc receptor IIIa and IIIb, which correlated with HDFN disease severity. Here, we analysed 230 pregnant women with anti‐c, ‐E or –K alloantibodies from a prospective screening cohort and investigated the type of Fc‐tail glycosylation of these antibodies in relation to the trigger of immunisation and pregnancy outcome. Anti‐c, ‐E and –K show – independent of the event that had led to immunisation – a different kind of Fc‐glycosylation compared to that of the total IgG fraction, but with less pronounced differences compared to anti‐D. High Fc‐galactosylation and sialylation of anti‐c correlated with HDFN disease severity, while low anti‐K Fc‐fucosylation correlated with severe fetal anaemia. IgG‐Fc glycosylation of anti‐RBC antibodies is shaped depending on the antigen. These features influence their clinical potency and may therefore be used to predict severity and identify those needing treatment.     

Effects of rituximab and dexamethasone on regulatory and proinflammatory B‐cell subsets in patients with primary immune thrombocytopenia

Objective

To investigate the cytokine production and surface marker composition of B cells in adult patients with newly diagnosed primary immune thrombocytopenia (ITP) before and 12 months after treatment with rituximab + dexamethasone (RTX+DXM) or dexamethasone (DXM).

Methods

Peripheral blood mononuclear cells were isolated from nine patients treated with RTX+DXM, seven patients treated with DXM, and seven healthy donors. Expression of the cell‐surface markers CD5, CD27, CD25, and CD19, and intracellular content of IL‐6 and IL‐10 were measured by flow cytometry.

Results

PBMCs from ITP patients at baseline contained a lower proportion of IL‐10⁺ B cells (P < .01) and IL‐6⁺ B cells (P < .01) than healthy controls. All patients responded to therapy and levels were normalized at 12 months. The proportion of CD5⁺ B cells increased (P < .01) and CD27⁺ memory B cells decreased (P < .05) 12 months after treatment with RTX+DXM compared to baseline, with an inverse correlation between platelet numbers and the proportion of CD27⁺ B cells (R = ?0.71; P < .05).

Conclusion

Both treatment regimens normalized the frequencies of cytokine‐producing B cells. The additional increase in CD5⁺ B cells after RTX+DXM is compatible with induction of Bregs.     

Incidence and outcome of lung involvement in IgG4‐related autoimmune pancreatitis

We evaluated the incidence and outcome of lung involvement in 35 patients with autoimmune pancreatitis (AIP). Our results indicate that lung involvement is commonly observed in AIP (40%). In addition, corticosteroid treatment improved the lung lesions and appeared to reduce the probability of relapse compared with pancreatic lesions (0% vs 36%). This is the first report to assess the long‐term outcome of lung involvement in AIP (52 ± 33 months).     

Response loss and development of neutralizing antibodies during long‐term treatment with romiplostim in patients with immune thrombocytopenia: a case series

Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by low platelet counts resulting from both immune‐mediated platelet destruction and inappropriate bone marrow platelet production. Therefore, in patients with ITP failing immunosuppressants/splenectomy, an alternative approach is to enhance platelet production stimulating thrombopoiesis. Studies on the development of recombinant thrombopoietins (rhTPO) were halted as a minority of patients developed an autoantibody that neutralized pegylated rhTPO and also cross‐reacted with and neutralized endogenous TPO resulting in thrombocytopenia. Clinical use of romiplostim, a second‐generation TPO‐RAs, has shown that during long‐term treatment, it may elicit the development of neutralizing antibodies to this agent resulting in acute thrombocytopenia. In our case series of 47 primary adult patients with ITP treated with romiplostim, 28 of 47 are evaluable for response loss. Among these, we observed eight patients who either progressively (3 of 8) or abruptly (5 of 8) lost response which accounts for a prevalence of 28.5%. Neutralizing antibody testing could be performed in 4 of 8 patients and 3 of 4 tested positive. These antibodies did not cross‐react with endogenous TPO and retesting of 2 patients at 9 and 7 months yielded a negative result. At follow‐up, 5 of 8 patients – including the 3 patients with neutralizing antibodies – went into long‐term complete response when switched to a different therapy while 3 of 8 patients never regained a response on subsequent lines of therapy. Response loss does not seem to be so rare an event during romiplostim administration (28.5% in our series) and in a minority of patients, it can be associated with development of drug neutralizing antibodies. Although recognized by the manufacturer as a possible adverse event ensuing during romiplostim administration, development of neutralizing antibody in everyday clinical practice has so far not been specifically addressed in reports on romiplostim use outside controlled studies. Unfortunately, testing for these antibodies requires adhesion to strict procedures which is not easily accomplished in everyday clinical practice. This complexity represents a significant drawback in extending antibody testing to all patients who lose response to romiplostim.