Hunting for the elusive target antigen in gestational alloimmune liver disease (GALD).

The prevailing concept is that gestational alloimmune liver disease (GALD) is caused by maternal antibodies targeting a currently unknown antigen on the liver of the fetus. This leads to deposition of complement on the fetal hepatocytes and death of the fetal hepatocytes and extensive liver injury. In many cases, the newborn dies.

Droplet digital PCR-based testing for donor-derived cell-free DNA in transplanted patients as noninvasive marker of allograft health: Methodological aspects.

In solid organ transplantation, donor-derived cell-free DNA (dd-cfDNA) is a promising universal noninvasive biomarker for allograft health, where high levels of dd-cfDNA indicate organ damage. Using Droplet Digital PCR (ddPCR), we aimed to develop an assay setup for monitoring organ health. We aimed to identify the least distinguishable percentage-point increase in the fraction of minute amounts of cfDNA in a large cfDNA background by using assays targeting single nucleotide polymorphisms (SNPs).

Pneumatic tube transport of blood samples affects global hemostasis and platelet function assays.

Introduction: Pneumatic tube systems (PTS) are frequently used for rapid and cost-effective transportation of blood samples to the clinical laboratory. The impact of PTS transport on platelet function measured by the Multiplate system and global hemostasis measured by the TEG 5000 was evaluated.

Methods: Paired samples from healthy adult individuals were obtained at two study sites: Rigshospitalet (RH) and Nordsjaellands Hospital (NOH).

The association of IgA deficiency on infection rate, self-perceived health, and levels of C-reactive protein in healthy blood donors.

The clinical importance of immunoglobulin A (IgA) deficiency in otherwise healthy individuals is not well described. We aimed to investigate the self-reported mental and physical health and the risk of infection in IgA-deficient blood donors compared to healthy control blood donors. Infectious events, recorded in public health registries either as prescriptions filled of any antimicrobial medicine or as hospital infections, were compared between 177 IgA-deficient blood donors and 1770 control blood donors.

How to evaluate PCR assays for the detection of low-level DNA.

High sensitivity of PCR-based detection of very low copy number DNA targets is crucial. Much focus has been on design of PCR primers and optimization of the amplification conditions. Very important are also the criteria used for determining the outcome of a PCR assay, e.

Uptake and presentation of myelin basic protein by normal human B cells.

B cells may play both pathogenic and protective roles in T-cell mediated autoimmune diseases such as multiple sclerosis (MS). These functions relate to the ability of B cells to bind and present antigens. Under serum-free conditions we observed that 3-4% of circulating B cells from healthy donors were capable of binding the MS-associated self-antigen myelin basic protein (MBP) and of presenting the immunodominant peptide MBP85-99, as determined by staining with the mAb MK16 recognising the peptide presented by HLA-DR15-positive cells.

Pre-analytical conditions in non-invasive prenatal testing of cell-free fetal RHD.

Background: Non-invasive prenatal testing of cell-free fetal DNA (cffDNA) in maternal plasma can predict the fetal RhD type in D negative pregnant women. In Denmark, routine antenatal screening for the fetal RhD gene (RHD) directs the administration of antenatal anti-D prophylaxis only to women who carry an RhD positive fetus. Prophylaxis reduces the risk of immunization that may lead to hemolytic disease of the fetus and the newborn.

Maternofetal transplacental transport of recombinant IgG antibodies lacking effector functions.

The neonatal Fc receptor (FcRn) directs the transfer of maternal immunoglobulin G (IgG) antibodies across the placenta and thus provides the fetus and newborn with passive protective humoral immunity. Pathogenic maternal IgG antibodies will also be delivered via the placenta and can cause alloimmunity, which may be lethal. A novel strategy to control pathogenic antibodies would be administration of a nondestructive IgG antibody blocking antigen binding while retaining binding to FcRn.

Next-generation sequencing: proof of concept for antenatal prediction of the fetal Kell blood group phenotype from cell-free fetal DNA in maternal plasma.

Background: Maternal immunization against KEL1 of the Kell blood group system can have serious adverse consequences for the fetus as well as the newborn baby. Therefore, it is important to determine the phenotype of the fetus to predict whether it is at risk. We present data that show the feasibility of predicting the fetal KEL1 phenotype using next-generation sequencing (NGS) technology.

Is current serologic RhD typing of blood donors sufficient for avoiding immunization of recipients?

Background: Avoiding immunization with clinically important antibodies is a primary objective in transfusion medicine. Therefore, it is central to identify the extent of D antigens that escape routine RhD typing of blood donors and to improve methodology if necessary.

Study Design And Methods: We screened 5058 D- donors for the presence of the RHD gene, targeting Exons 5, 7, and 10 with real-time polymerase chain reaction.

Fetomaternal hemorrhage in women undergoing elective cesarean section.

Objective: To investigate the degree of fetomaternal hemorrhage (FMH) caused by elective cesarean section.

Design: Descriptive study.

Settings: University Hospitals in Copenhagen, Denmark.

Quality assessment of a placental perfusion protocol.

Validation of in vitro test systems using the modular approach with steps addressing reliability and relevance is an important aim when developing in vitro tests in e.g. reproductive toxicology.

Recombinant human immunoglobulin (Ig)A1 and IgA2 anti-D used for detection of IgA deficiency and anti-IgA.

Background: To avoid anaphylactic reactions, immunoglobulin (Ig)A-deficient patients with anti-IgA should be transfused with IgA-deficient blood components. There is a need for fast and robust assays for demonstration of IgA deficiency and for detection of anti-IgA.

Study Design And Methods: Recombinant human IgA1 and IgA2 anti-D molecules were constructed, expressed in Chinese hamster ovary cells, and purified.

In vitro assessment of recombinant, mutant immunoglobulin G anti-D devoid of hemolytic activity for treatment of ongoing hemolytic disease of the fetus and newborn.

Background: A specific treatment for ongoing hemolytic disease of the fetus and newborn (HDFN) due to anti-D would be very attractive. One approach could be administration to the mother of nonhemolytic anti-D, which by crossing the placenta can block the binding of hemolytic maternal anti-D.

Study Design And Methods: Two anti-D immunoglobulin G3 (IgG3) heavy-chain mutants were expressed in Chinese hamster ovary cells.

A novel antibody-dependent cellular cytotoxicity mechanism involved in defense against malaria requires costimulation of monocytes FcgammaRII and FcgammaRIII.

Clinical experiments have shown that the Ab-dependent cell-mediated inhibition of Plasmodium falciparum is a major mechanism controlling malaria parasitemia and thereby symptoms. In this study, we demonstrate that a single merozoite per monocyte (MN) is sufficient to trigger optimal antiparasitic activity. Using particulate Ag as pseudomerozoites, we show that only Ags, and no other parasite-derived factor, are required to trigger MN activation and that a single Ag is as potent as the complex combination of Ags constituting the merozoite surface.

Functional in vitro studies of recombinant human immunoglobulin G and immunoglobulin A anti-D.

Background: The use of anti-D purified from human serum to prevent hemolytic disease of the fetus and newborn due to D is well established. Owing to supply and safety reasons, however, an unlimited and non-plasma-derived source of antibodies for Rhesus prophylaxis is needed.

Study Design And Methods: Recombinant human immunoglobulin G (IgG)1, IgG2, IgG3, IgG4, IgA1, and IgA2 anti-D with the same variable region were expressed in Chinese hamster ovary cells.

Detecting fetomaternal hemorrhage by flow cytometry.

Purpose Of Review: The aim of this review is to summarize the most recent developments in the area of detection of fetomaternal hemorrhage by flow cytometry.

Recent Findings: Maternal red blood cell chimerism is readily detectable by flow cytometry. Fetal and maternal red blood cells differ in their content of fetal hemoglobin (alpha2gamma2).

[Antenatal determination of fetal RhD-blood type based on fetal DNA in plasma from the RhD-negative mother--secondary publication].

We present a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD-women. This test is needed for a future antenatal RH prophylaxis. A new real time PCR based assay targeting RHD exon 7 and a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma from 56 pregnant women in 15th-36th week of gestation.

In vitro functional test of two subclasses of an anti-RhD antibody produced by transient expression in COS cells.

For over 35 years hemolytic disease of the fetus and newborn (HDFN) due to RhD has been effectively prevented by anti-RhD antibodies obtained from alloimmunized women or deliberately immunized men. However, due to the reduced number of immunized women and for ethical reasons it is foreseen that other sources of anti-RhD will be needed. One such source is recombinant human antibodies.

Human recombinant antibodies against Plasmodium falciparum merozoite surface protein 3 cloned from peripheral blood leukocytes of individuals with immunity to malaria demonstrate antiparasitic properties.

Immunoglobulins from individuals with immunity to malaria have a strong antiparasitic effect when transferred to Plasmodium falciparum malaria infected patients. One prominent target of antiparasitic antibodies is the merozoite surface antigen 3 (MSP-3). We have investigated the antibody response against MSP-3 residues 194 to 257 (MSP-3(194-257)) on the molecular level.

Reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women.

Objectives: The objective of this study was to establish a reliable test for prenatal prediction of fetal RhD type using maternal plasma from RhD negative women. This test is needed for future prenatal Rh prophylaxis.

Methods: A novel real-time PCR-based assay targeting RHD exon 7 combined with a published assay for RHD exon 10 were used to determine the fetal RHD status in DNA extracted from plasma, sampled from 56 pregnant RhD negative women in 15th-36th week of gestation.

Efficient purification of unique antibodies using peptide affinity-matrix columns.

Phage display technology was used to identify peptide ligands with unique specificity for a monoclonal model antibody, MK16, that recognises the human multiple sclerosis associated MHC class II molecule DR2 in complex with a myelin basic protein (MBP)-derived peptide corresponding to residue 85-99. Several peptide epitopes were identified and all of them recognised specifically MK16. One peptide, ER6.

Human anti-rhesus D IgG1 antibody produced in transgenic plants.

Transgenic plants represent an alternative to cell culture systems for producing cheap and safe antibodies for diagnostic and therapeutic use. To evaluate the functional properties of a 'plantibody', we generated transgenic Arabidopsis plants expressing full-length human IgG1 against the Rhesus D antigen, which is responsible for alloimmunization of RhD- mothers carrying an RhD+ fetus. Anti-RhD extracted from plants specifically reacted with RhD+ cells in antiglobulin technique, and elicited a respiratory burst in human peripheral blood mononuclear cells.