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). We mimicked a clinical sample from a recipient in a number of spike-in experiments, where cfDNA from healthy volunteers were mixed. A total of 40 assays were tested and approved by qPCR and ddPCR. Limit of detection (LOD) was demonstrated to be approximately 3 copies per reaction, observed at a fraction of 0.002%, and which would equal 6 copies per mL plasma. Limit of quantification (LOQ) was 35 copies per reaction, estimated to 0.038%. The lowest detectable increase in percentage point of dd-cfDNA was approximately 0.04%. Our results demonstrated that ddPCR has great sensitivity, high precision, and exceptional ability to quantify low levels of cfDNA. The ability to distinguish small differences in mimicking dd-cfDNA was far beyond the desired capability. While these methodological data are promising, further prospective studies are needed to determine the clinical utility of the proposed method.
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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. In subsequent pregnancies early treatment of the woman with intravenous immunoglobulin can be instituted, and the prognosis for the fetus will be excellent. Without treatment the prognosis can be severe. Crucial improvements of diagnosis require identification of the target antigen. For this identification, this work was based on two hypotheses: 1. The GALD antigen is exclusively expressed in the fetal liver during normal fetal life in all pregnancies; 2. The GALD antigen is an alloantigen expressed in the fetal liver with the woman being homozygous for the minor allele and the father being, most frequently, homozygous for the major allele. We used three different experimental approaches to identify the liver target antigen of maternal antibodies from women who had given birth to a baby with the clinical GALD diagnosis: 1. Immunoprecipitation of antigens from either a human liver cell line or human fetal livers by immunoprecipitation with maternal antibodies followed by mass spectrometry analysis of captured antigens; 2. Construction of a cDNA expression library from human fetal liver mRNA and screening about 1.3 million recombinants in Escherichia coli using antibodies from mothers of babies diagnosed with GALD; 3. Exome/genome sequencing of DNA from 26 presumably unrelated women who had previously given birth to a child with GALD with husband controls and supplementary HLA typing. In conclusion, using the three experimental approaches we did not identify the GALD target antigen and the exome/genome sequencing results did not support the hypothesis that the GALD antigen is an alloantigen, but the results do not yield basis for excluding that the antigen is exclusively expressed during fetal life., which is the hypothesis we favor.
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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. The reliability of predicting the fetal RhD type depends on pre-analytical factors and assay sensitivity. We evaluated the testing setup in the Capital Region of Denmark, based on data from routine antenatal RHD screening.
Blood samples were drawn at gestational age 25 weeks. DNA extracted from 1 mL of plasma was analyzed for fetal RHD using a duplex method for exon 7/10. We investigated the effect of blood sample transportation time (n = 110) and ambient outdoor temperatures (n = 1539) on the levels of cffDNA and total DNA. We compared two different quantification methods, the delta Ct method and a universal standard curve. PCR pipetting was compared on two systems (n = 104).
The cffDNA level was unaffected by blood sample transportation for up to 9 days and by ambient outdoor temperatures ranging from -10 °C to 28 °C during transport. The universal standard curve was applicable for cffDNA quantification. Identical levels of cffDNA were observed using the two automated PCR pipetting systems. We detected a mean of 100 fetal DNA copies/mL at a median gestational age of 25 weeks (range 10-39, n = 1317).
The setup for real-time PCR-based, non-invasive prenatal testing of cffDNA in the Capital Region of Denmark is very robust. Our findings regarding the transportation of blood samples demonstrate the high stability of cffDNA. The applicability of a universal standard curve facilitates easy cffDNA quantification.
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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.
Study Design and Methods
The KEL1/2 single‐nucleotide polymorphism was polymerase chain reaction (PCR) amplified with one adjoining base, and the PCR product was sequenced using a genome analyzer (GAIIx, Illumina); several millions of PCR sequences were analyzed.
Results
The results demonstrated the feasibility of diagnosing the fetal KEL1 or KEL2 blood group from cell‐free DNA purified from maternal plasma.
Conclusion
This method requires only one primer pair, and the large amount of sequence information obtained allows well for statistical analysis of the data. This general approach can be integrated into current laboratory practice and has numerous applications. Besides DNA‐based predictions of blood group phenotypes, platelet phenotypes, or sickle cell anemia, and the determination of zygosity, various conditions of chimerism could also be examined using this approach. To our knowledge, this is the first report focused on antenatal blood group determination using NGS.
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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. In the presence of serum, however, the majority of B cells bound MBP in a complement-dependent manner, and almost half of the B cells became engaged in presentation of MBP85-99. Even though complement receptor 1 (CR1, CD35) and CR2 (CD21) both contributed to binding of MBP to B cells, only CR2 was important for the subsequent presentation of MBP85-99. A high proportion of MBP85-99 presenting B cells expressed CD27, and showed increased expression of CD86 compared to non-presenting B cells. MBP-pulsed B cells induced a low frequency of IL-10-producing CD4+ T cells in 3 out of 6 donors, indicating an immunoregulatory role of B cells presenting MBP-derived peptides. The mechanisms described here refute the general assumption that B-cell presentation of self-antigens requires uptake via specific B-cell receptors, and may be important for maintenance of tolerance as well as for driving T-cell responses in autoimmune diseases.
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How to evaluate PCR assays for the detection of low-level DNA Clausen, Frederik Banch; Urhammer, Emil; Rieneck, Klaus ...
APMIS : acta pathologica, microbiologica et immunologica Scandinavica,
September 2015, Volume:
123, Issue:
9
Journal Article
Peer reviewed
Open access
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.g. how many replicates are needed and how many of these should be positive or what amount of template should be used? We developed a mathematical model to obtain a simple tool for quick PCR assay evaluation before laboratory optimization and validation procedures. The model was based on the Poisson distribution and the Binomial distribution describing parameters for singleplex real‐time PCR‐based detection of low‐level DNA. The model was tested against experimental data of diluted cell‐free foetal DNA. Also, the model was compared with a simplified formula to enable easy predictions. The model predicted outcomes that were not significantly different from experimental data generated by testing of cell‐free foetal DNA. Also, the simplified formula was applicable for fast and accurate assay evaluation. In conclusion, the model can be applied for evaluation of sensitivity of real‐time PCR‐based detection of low‐level DNA, and may also assist in design of new assays before standard laboratory optimization and validation is initiated.
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Immunoglobulinsfrom individuals with immunity to malaria have a strong antiparasiticeffect when transferred to Plasmodium falciparum malariainfected patients. One prominent target of antiparasitic ...antibodies isthe merozoite surface antigen 3 (MSP-3). We have investigated theantibody response against MSP-3 residues 194 to 257(MSP-3₁₉₄₋₂₅₇) on the molecular level. mRNA fromperipheral blood leukocytes from clinically immune individuals was usedas a source of Fab (fragment antibody) genes. A Fab-phage displaylibrary was made, and three distinct antibodies designated RAM1, RAM2,and RAM3 were isolated by panning. Immunoglobulin G1 (IgG1) and IgG3full-length antibodies have been produced in CHO cells. Reactivity withthe native parasite protein was demonstrated by immunofluorescencemicroscopy, flow cytometry, and immunoblotting. Furthermore, theantiparasitic effect of RAM1 has been tested in vitro in anantibody-dependent cellular inhibition (ADCI) assay. Both the IgG1 andthe IgG3 versions of the antibody show an inhibitory effect on parasitegrowth.
PURPOSE OF REVIEWThe aim of this review is to summarize the most recent developments in the area of detection of fetomaternal hemorrhage by flow cytometry.
RECENT FINDINGSMaternal red blood cell ...chimerism is readily detectable by flow cytometry. Fetal and maternal red blood cells differ in their content of fetal hemoglobin (α2γ2). Fetal red blood cells contain fetal hemoglobin, and normal maternal red blood cells contain some percentage of fetal hemoglobin in a background of normal adult hemoglobin. All blood group systems with allelic differences between mother and fetus are readily applicable for detection of fetomaternal hemorrhage by fetal hemoglobin.
SUMMARYFetal hemoglobin for detection of fetomaternal hemorrhage is an accurate clinical diagnostic procedure for investigation of anemia in fetus and newborn.
Objective. To investigate the degree of fetomaternal hemorrhage (FMH) caused by elective cesarean section. Design. Descriptive study. Settings. University Hospitals in Copenhagen, Denmark. ...Population. Women scheduled for elective cesarean section, in the period September 2007 to January 2009, at the Department of Gynecology and Obstetrics, Hvidovre Hospital, University of Copenhagen, Denmark. Methods. Two maternal blood samples were taken, the first before cesarean section and the second immediately after. Both samples were analyzed at the Blood Bank, Rigshospitalet, Copenhagen, for the presence of fetal red blood cells (fRBCs) using flow cytometry. FMH associated with cesarean section was defined as the difference between the volumes of fRBCs in the two samples. Main Outcome Measures. The frequency and volume of FMH caused by elective cesarean section. Results. 207 women were included in the study. FMH was detected in 38 cases (18.4%). Of these, 22 women (10.6%) had FMH of less than 1 ml fRBCs, 13 women (6.3%) had FMH between 1 and 4 ml fRBCs, and three women (1.4%) had FMH above 4 ml fRBCs. Conclusions. We found no evidence for recommending general screening for FMH in connection with elective cesarean section, provided guidelines such as the current Danish guidelines for Rhesus prophylaxis are followed.
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