Abstract
Twenty-five B-cell–depleted patients (24 following anti-CD19/20 therapy) diagnosed with coronavirus disease 2019 had been symptomatic for a median of 26 days but remained antibody negative. ...All were treated with convalescent plasma with high neutralizing antibody titers. Twenty-one (84%) recovered, indicating the potential therapeutic effects of this therapy in this particular population.
The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR-4 contribute to stem cell homing and may play a role in the trafficking of leukemic cells. Therefore, we analyzed migration ...across Transwell filters of cells derived from bone marrow (BM) or peripheral blood (PB) from 26 acute myeloid leukemia (AML) patients. The presence of the extracellular matrix protein fibronectin (FN) strongly enhanced the spontaneous and SDF-1-induced migration of leukemic PB and BM cells. No differences in spontaneous, SDF-1-induced migration or CXCR-4 expression were observed between the different AML subtypes. Subsequently, it was determined whether SDF-1 preferentially promoted migration of subsets of leukemic cells. Leukemic cells expressing CD34, CD38 and HLA-DR were preferentially migrating, whereas cells expressing CD14 and CD36 showed diminished migration. Analysis of paired PB and BM samples indicated that significantly higher SDF-1-induced migration was observed in AML for CD34(+) BM-derived cells compared to CD34(+) PB-derived cells, suggesting a role for SDF-1 in the anchoring of leukemic cells in the BM or other organs. The lower percentage of circulating leukemic blasts in patients with a relatively high level of SDF-1-induced migration also supports this hypothesis. In conclusion, we have shown that primary AML cells are migrating towards SDF-1 independent of the AML subtypes.
Background and Objectives
Several comprehensive genotyping platforms for determining red blood cell (RBC) antigens have been established and validated for use in the Caucasian and Black populations, ...but not for the Chinese. The multiplex ligation‐dependent probe amplification (MLPA) assay was validated for RHD genotyping in the Chinese.
Materials and Methods
The blood samples of 200 D+, 200 D− and 62 D variant Chinese donors were collected. RhD antigen was routinely typed by serological method. D variant phenotype was determined by an anti‐D panel (D‐Screen), when RBCs were available. The RHD genotype and its zygosity were analysed with the RH‐MLPA technique. When the MLPA was unable to identify a RHD variant, direct sequencing of all exons of the RHD gene was performed.
Results
In 200 D+ donors, DD (168/200, 84%), D (12/200, 6%), DDD genotype (1/200) and D variant allele carriers (19/200, 9·5%) were found. In 200 D− donors, six reported RHD alleles, RHD*01EL.01, RHD*01N.03, RHD*01N.05, RHD*01N.16, RHD*DFR2 and RHD*weak partial 15 and one novel RHD*1154T allele were identified in 36·5% (73/200) of them. In 62 D variant donors, three novel RHD alleles, RHD*79_81delCTC, RHD*710T and RHD*689A, and twelve reported alleles, RHD*DVI.3, RHD*weak partial 15, RHD*DVI.4, RHD*01EL.01, RHD*01N.03, RHD*DLO, RHD*DV.5, RHD*D‐CE(2‐10), RHD*730C, RHD*weak D type 25, 33 and 72, were identified, either alone or in combination.
Conclusion
The RH‐MLPA assay correctly identified the common RHD variant alleles in the Chinese population. However, DNA sequencing was required to identify certain alleles; probes to detect these alleles should be added into the assay.
Background
Anti‐platelet antibody testing may be useful for the diagnosis and management of childhood immune thrombocytopenia (ITP).
Objectives
Here we aimed to assess the prevalence and prognostic ...significance of anti‐platelet glycoprotein‐specific IgM and IgG antibodies.
Methods
Children with newly diagnosed ITP were included at diagnosis and randomized to an intravenous immunoglobulins (IVIg) or careful observation group (TIKI trial). In this well‐defined and longitudinally followed cohort (N = 179), anti‐platelet glycoprotein‐specific IgM and IgG antibodies were determined by monoclonal antibody‐immobilization of platelet antigens.
Results
The dominant circulating anti‐platelet antibody class in childhood ITP was IgM (62% of patients); but IgG antibodies were also found (10%). Children without IgM platelet antibodies were older and more often female. There was weak evidence for an association between IgM anti‐GP IIb/IIIa antibodies and an increased bleeding severity (P = .03). The IgM and IgG anti‐platelet responses partially overlapped, and reactivity was frequently directed against multiple glycoproteins. During 1‐year follow‐up, children with IgM antibodies in the observation group displayed a faster platelet recovery compared to children without, also after adjustment for age and preceding infections (P = 7.1 × 10−5). The small group of patients with detectable IgG anti‐platelet antibodies exhibited an almost complete response to IVIg treatment (N = 12; P = .02), suggesting that IVIg was particularly efficacious in these children.
Conclusions
Testing for circulating anti‐platelet antibodies may be helpful for the clinical prognostication and the guidance of treatment decisions in newly diagnosed childhood ITP. Our data suggest that the development of even more sensitive tests may further improve the clinical value of antibody testing.
Please cite this paper as: Scheffer P, Ait Soussan A, Verhagen O, Page‐Christiaens G, Oepkes D, de Haas M, van der Schoot C. Noninvasive fetal genotyping of human platelet antigen‐1a. BJOG ...2011;118:1392–1395.
We describe a reliable noninvasive fetal human platelet antigen (HPA)‐1a genotyping assay on a real‐time polymerase chain reaction (PCR) platform using cell‐free fetal DNA isolated from maternal blood. Nonspecific amplification of maternal cell‐free DNA is overcome by pre‐PCR digestion of the cell‐free DNA with the Msp1 restriction enzyme. Noninvasive fetal HPA‐1a genotyping offers a safe method for alloimmunised pregnant women to determine whether their fetus is at risk of fetal or neonatal alloimmune thrombocytopenia (FNAIT) and whether interventions to prevent intracranial haemorrhage are required. The availability of this test is relevant to the ongoing debate on screening pregnancies for HPA‐1a‐mediated FNAIT.
Essentials
There is a need for improved tools to predict persistent and chronic immune thrombocytopenia (ITP).
We developed and validated a clinical prediction model for recovery from newly diagnosed ...ITP.
The Childhood ITP Recovery Score predicts transient vs. persistent ITP and response to intravenous immunoglobulins.
The score may serve as a useful tool for clinicians to individualize patient care.
Background
Childhood immune thrombocytopenia (ITP) is an autoimmune bleeding disorder. The prognosis (transient, persistent, or chronic ITP) remains difficult to predict. The morbidity is most pronounced in children with persistent and chronic ITP. Clinical characteristics are associated with ITP outcomes, but there are no validated multivariate prediction models.
Objective
Development and external validatation of the Childhood ITP Recovery Score to predict transient versus persistent ITP in children with newly diagnosed ITP.
Methods
Patients with a diagnosis platelet count ≤ 20 × 109/L and age below 16 years were included from two prospective multicenter studies (NOPHO ITP study, N = 377 development cohort; TIKI trial, N = 194 external validation). The primary outcome was transient ITP (complete recovery with platelets ≥100 × 109/L 3 months after diagnosis) versus persistent ITP. Age, sex, mucosal bleeding, preceding infection/vaccination, insidious onset, and diagnosis platelet count were used as predictors.
Results
In external validation, the score predicted transient versus persistent ITP at 3 months follow‐up with an area under the receiver operating characteristic curve of 0.71. In patients predicted to have a high chance of recovery, we observed 85%, 90%, and 95% recovered 3, 6, and 12 months after the diagnosis. For patients predicted to have a low chance of recovery, this was 32%, 46%, and 71%. The score also predicted cessation of bleeding symptoms and the response to intravenous immunoglobulins (IVIg).
Conclusion
The Childhood ITP Recovery Score predicts prognosis and may be useful to individualize clinical management. In future research, the additional predictive value of biomarkers can be compared to this score. A risk calculator is available (http://www.itprecoveryscore.org).
Background
Cell‐free DNA (cfDNA) and nucleosomes, consisting of cfDNA and histones, are markers of cell activation and damage. In systemic inflammation these markers predict severity and fatality. ...However, the role of cfDNA in acute Graft‐versus‐Host Disease (aGvHD), a major complication of allogeneic hematopoietic stem cell transplantation (HSCT), is unknown.
Objective
The aim of this study is to investigate the role of cfDNA as a marker of aGvHD.
Methods
We followed nucleosome levels in 37 allogeneic HSCT patients and an established xenotransplantation mouse model. We determined the origin of cfDNA with a species‐specific polymerase chain reaction.
Results
In the plasma of aGvHD patients, nucleosome levels significantly increased around the time of aGvHD diagnosis compared to pretransplant, concurrently with a significant increase of known aGvHD markers ST2 and REG3α. In mice, we confirmed that nucleosomes were elevated during clinically detectable aGvHD. We found cfDNA to be mainly of human origin and to a lesser extent of mouse origin, indicating that cfDNA is released by (proliferating) human xeno‐reactive PBMC and damaged mouse cells.
Conclusion
We show increased cfDNA both in an aGvHD mouse model and in aGvHD patients. We also demonstrate that donor hematopoietic cells and to a lesser degree (damaged) host cells are the cellular source of cfDNA in aGvHD. We propose that nucleosomes and cfDNA might be an additive marker for aGvHD.
Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease ...manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.
The interactions of antibodies with myeloid Fcγ receptors and the complement system are regulated by an Asn297-linked glycan in the Fc portion of IgG. Alterations of serum IgG-Fc glycosylation have ...been reported in various autoimmune diseases, and correlate with treatment response and disease activity. We hypothesized that IgG-Fc glycosylation is altered in immune thrombocytopenia (ITP) and associates with response to anti-CD20 monoclonal antibody treatment (rituximab). IgG-Fc glycosylation was analyzed by liquid chromatography-mass spectrometry. We found that IgG-Fc glycosylation was identical between refractory ITP patients (HOVON64 trial; N = 108) and healthy controls (N = 120). Two months after rituximab treatment, we observed a shift in Fc glycosylation, with a mean 1.7% reduction in galactosylation for IgG1 and IgG4 and a mean 1.5% increase for bisection in IgG1, IgG2/3 and IgG4 (adjusted p < 1.7 × 10
and p < 2 × 10
, respectively). Neither baseline nor longitudinal changes in IgG-Fc glycosylation after rituximab were associated with clinical treatment response. We conclude that IgG-Fc glycosylation in refractory ITP is similar to healthy controls and does not predict treatment responses to rituximab. The observed changes two months after treatment suggest that rituximab may influence total serum IgG-Fc glycosylation. Overall, our study suggests that the pathophysiology of refractory ITP may differ from other autoimmune diseases.
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