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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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Macrophages possess widespread pro-inflammatory, destructive, and remodelling capabilities that can critically contribute to acute and chronic diseases, such as rheumatoid arthritis (RA). Continuous ...monitoring and measurement of selective counteraction of macrophage activity in patients require a sensitivity and non-invasive marker. We characterised the VICM (citrullinated and MMP degraded vimentin fragment) biomarker by investigating the release from in vitro activated macrophages and by monitoring the change in serum levels after treatment with the anti-GM-CSFRα-mAb (mavrilimumab).
Peripheral blood mononuclear cells were isolated, and lipopolysaccharide (LPS) was used to activate the macrophages and calcium chloride (CaCl2) was used to facilitate the citrullination process of vimentin. Supernatants, cell lysates, was collected and analysed by ELISA, and western blotting. RA patients were treated with mavrilimumab+methotrexate or methotrexate alone in a phase 2b study (NCT01706926) once every two weeks for 24 weeks. Serum levels of VICM were measured at baseline and multiple time points post-treatment. In addition, whole blood expression of peptidylarginine deiminase-2 (PAD-2) and matrix metalloproteinase-9 (MMP-9) transcripts were tested by quantitative reverse transcriptase PCR assays at day 0 and day 169 post-treatment.
VICM levels were significantly higher at day 5 and 8 in supernatants of activated macrophages compared to controls (p<0.01), which was confirmed by Western blot. In RA patients, VICM correlated with disease activity (DAS28), modified total sharp score (mTSS), joint space narrowing (JSN), joint erosions and CRP at baseline. VICM was dose-dependently and significantly (p<0.01) inhibited by mavrilimumab. This suppression of VICM serum levels was supported by a decreased expression of PAD2 and MMP9 transcripts in patients treated with mavrilimumab.
These data verified that VICM is released by activated macrophages. Treatment of RA patients with mavrilimumab significantly reduced release of VICM and peptidylarginine deiminases-2 (PAD-2) gene expression indicating that mavrilimumab indeed is targeting activated macrophages and that VICM may be a novel blood-based marker of anti-GM-CSF response.
Label‐free blood typing by Raman spectroscopy (RS) is demonstrated by training an artificial intelligence (AI) model on 271 blood typed donor whole blood samples. A fused silica micro‐capillary flow ...cell enables fast generation of a large dataset of Raman spectra of individual donors. A combination of resampling methods, machine learning and deep learning is used to classify the ABO blood group, 27 erythrocyte antigens, 4 platelet antigens, regular anti‐B titers of blood group A donors, regular anti‐A,‐B titers of blood group O donors, and ABH‐secretor status, from a single Raman spectrum. The average area under the curve value of the ABO classification is 0.91 ± 0.03 and 0.72 ± 0.09, respectively, for the remaining traits. The classification performance of all parameters is discussed in the context of dataset balance and antigen concentration. Post‐hoc scalability analysis of the models shows the potential of RS and AI for future applications in transfusion medicine and blood banking.
The ABO blood group and 33 additional blood group traits are determined label‐free by Raman spectroscopy (RS) and Artificial Intelligence (AI). Whole blood is analyzed in a fused silica micro‐capillary with no sample preparation steps ensuring fast generation of Raman spectra. The performance scalability of the trained AI models is discussed in the context of future applications in transfusion medicine.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
To evaluate two different multiplex real-time PCR assays detecting fetal RHD for screening of RhD negative women in relation to antenatal RhD prophylaxis.
We designed a duplex assay for the detection ...of RHD exon 7 and 10 and a triplex assay for the detection of RHD exon 7, 10 and 5. We used the same fluorescent dye for the exon 7 and 10 probes to increase sensitivity; exon 5 was VIC labeled. We evaluated possible inhibition of DNA amplification with dilution experiments. We then tested the two multiplex assays with DNA extracted from 97 plasma samples from 38 RhD negative women in gestational weeks 6-37.
Dilution experiments revealed no inhibition of amplification in the multiplex assays. For plasma samples, the duplex assay was significantly more sensitive than the triplex assay (p < 0.0001). For the duplex assay (exon 7/10), accuracy was 99.0%. For the triplex assay (exon 7/10), accuracy was 94.2%. Detection of exon 5 was less reliable.
The duplex assay using exon 7/10 was the most reliable for prenatal prediction of fetal RhD type as a candidate assay for screening of RhD negative women in relation to antenatal RhD prophylaxis. The triplex assay needs further optimization.
Background: Laboratory monitoring of mother, fetus, and newborn in hemolytic disease of fetus and newborn (HDFN) aims to guide clinicians and the immunized women to focus on the most serious problems ...of alloimmunization and thus minimize the consequences of HDFN in general and of anti-D in particular. Here, we present the current approach of laboratory screening and testing for prevention and monitoring of HDFN at the Copenhagen University Hospital in Denmark. Summary: All pregnant women are typed and screened in the 1st trimester. This serves to identify the RhD-negative pregnant women who at gestational age (GA) of 25 weeks are offered a second screen test and a non-invasive fetal RhD prediction. At GA 29 weeks, and again after delivery, non-immunized RhD-negative women carrying an RhD-positive fetus are offered Rh immunoglobulin. If the 1st trimester screen reveals an alloantibody, antenatal investigation is initiated. This also includes RhD-positive women with alloantibodies. Specificity and titer are determined, the fetal phenotype is predicted by non-invasive genotyping based on cell-free DNA (RhD, K, Rhc, RhC, RhE, ABO), and serial monitoring of titer commences. Based on titers and specificity, monitoring with serial peak systolic velocity measurements in the fetal middle cerebral artery to detect anemia will take place. Intrauterine transfusion is given when fetal anemia is suspected. Monitoring of the newborn by titer and survival of fetal red blood cells by flow cytometry will help predict the length of the recovery of the newborn.
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. We report on 2 human IgG3 antibodies with a hinge deletion and a C131S point mutation (IgG3ΔHinge) that eliminate complement activation and binding to all classical Fcγ receptors (FcγRs) and to C1q while binding to FcRn is retained. Additionally, 1 of the antibodies has a single point mutation in the Fc (R435H) at the binding site for FcRn (IgG3ΔHinge:R435H). We compared transplacental transport with wild-type IgG1 and IgG3, and found transport across trophoblast-derived BeWo cells and ex vivo placenta perfusions with hierarchies as follows: IgG3ΔHinge:R435H>wild-type IgG1≥IgG3ΔHinge and IgG3ΔHinge:R435H=wild-type IgG1=wild-type IgG3>>>IgG3ΔHinge, respectively. Collectively, IgG3ΔHinge:R435H was transported efficiently from the maternal to the fetal placental compartment. Thus, IgG3ΔHinge:R435H may be a good candidate for transplacental delivery of a nondestructive antibody to the fetus to combat pathogenic antibodies.
•Retained FcRn binding of an IgG3 antibody devoid of FcgR and C1q binding, cellular cytotoxicity and complement activation.•Inhibition of pathogenic polyclonal anti-D in antibody-dependent cellular toxicity by a hinge region deleted anti-D IgG3 antibody with efficient transplacental transport capacity.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract Immunization against RhD is the major cause of hemolytic disease of the fetus and newborn (HDFN), which causes fetal or neonatal death. The introduction of postnatal immune prophylaxis in ...the 1960s drastically reduced immunization incidents in pregnant, D-negative women. In several countries, antenatal prophylaxis is combined with postnatal prophylaxis to further minimize the immunization risk. Due to lack of knowledge of the fetal RhD type, antenatal prophylaxis is given to all D-negative women. In the European population, approximately 40% of pregnant women carry a D-negative fetus and are thus at no risk of immunization. Noninvasive fetal RhD genotyping enables antenatal prophylaxis to be targeted to only those women carrying a D-positive fetus to avoid unnecessary treatment. Based on an analysis of cell-free fetal DNA from the plasma of pregnant women, this approach has recently undergone technical improvements and rapid clinical implementation. As a screening assay, the sensitivity is >99.3% from a gestational age of approximately 10–11 weeks. In addition, fetal RhD genotyping is widely used to assess the risk of HDFN in anti-D immunized women.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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. The
...ex vivo human placental perfusion system may be used for such validation, here presenting the placental perfusion model in Copenhagen including control substances. The positive control substance antipyrine shows no difference in transport regardless of perfusion media used or of terms of delivery (
n
=
59,
p
<
0.05). Negative control studies with FITC marked dextran correspond with leakage criteria (<3
ml
h
−1 from the fetal reservoir) when adding 2 (
n
=
7) and 20
mg (
n
=
9) FITC-dextran/100
ml fetal perfusion media. Success rate of the Copenhagen placental perfusions is provided in this study, including considerations and quality control parameters. Three checkpoints suggested to determine success rate revealed that 15% of the cannulated placentae received in one year (
n
=
202) were successfully perfused.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
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. Samples that were positive in the screen test were investigated further by adsorption‐elution, antibody consumption, flow cytometry, and sequencing of all RHD exons with intron‐specific primers. Lookback was performed on all recipients of RBCs from RHD+ donors.
RESULTS: We found 13 RHD+ samples (0.26%). No variants or chimeras were found. Characterization of DNA revealed a novel DEL type (IVS2‐2 A>G). In the lookback of the 136 transfusions with subsequent antibody follow‐up, of which 13 were from DEL donors, one recipient developed anti‐D. However, in this case, a competing and more likely cause of immunization was the concurrent transfusion of D+ platelets. Eleven recipients were immunized with 13 antibodies different from anti‐D, of which five were anti‐K.
CONCLUSION: In our laboratory, serologic RhD typing was safe. We detected all D variants and only missed DEL types. In assessing the immunization risk we included a DEL donor, found previous to this study, that did immunize a recipient with anti‐D. We conclude that inadvertent immunization with D antigens in our setting was rare and in the order of 1.4 in 100,000 D− transfusions.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
The aim of this study was to determine whether natural killer T (NKT) cells, including invariant (i) NKT cells, have clinical value in preventing the progression of multiple sclerosis (MS) by ...examining the mechanisms by which a distinct self-peptide induces a novel, protective invariant natural killer T cell (iNKT cell) subset.
We performed a transcriptomic and functional analysis of iNKT cells that were reactive to a human collagen type II self-peptide, hCII707-721, measuring differentially induced genes, cytokines, and suppressive capacity.
We report the first transcriptomic profile of human conventional vs novel hCII707-721-reactive iNKT cells. We determined that hCII707-721 induces protective iNKT cells that are found in the blood of healthy individuals but not progressive patients with MS (PMS). By transcriptomic analysis, we observed that hCII707-721 promotes their development and proliferation, favoring the splicing of full-length AKT serine/threonine kinase 1 (AKT1) and effector function of this unique lineage by upregulating tumor necrosis factor (TNF)-related genes. Furthermore, hCII707-721-reactive iNKT cells did not upregulate interferon (IFN)-γ, interleukin (IL)-4, IL-10, IL-13, or IL-17 by RNA-seq or at the protein level, unlike the response to the glycolipid alpha-galactosylceramide. hCII707-721-reactive iNKT cells increased TNFα only at the protein level and suppressed autologous-activated T cells through FAS-FAS ligand (FAS-FASL) and TNFα-TNF receptor I signaling but not TNF receptor II.
Based on their immunomodulatory properties, NKT cells have a potential value in the treatment of autoimmune diseases, such as MS. These significant findings suggest that endogenous peptide ligands can be used to expand iNKT cells, without causing a cytokine storm, constituting a potential immunotherapy for autoimmune conditions, including PMS.
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