Background and Objectives
Non‐invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental ...consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non‐invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi‐national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations.
Materials and Methods
We reviewed the literature covering validation for in‐vitro diagnostic (IVD) assays in general and for non‐invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co‐authors.
Results
In relation to Annex VIII of the In‐Vitro‐Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non‐invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti‐D prophylaxis for non‐immunized RhD negative women should be placed under risk class C. To ensure high quality of non‐invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre‐analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed.
Conclusion
These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in‐house assays.
Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group ...antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto‐antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)‐expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune‐mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high‐throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high‐frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour‐intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high‐throughput genotyping based on DNA micro‐arrays is a very feasible method to obtain a large pool of well‐typed blood donors. Several systems for high‐throughput blood group genotyping are developed and will be discussed in this review.
Background
Rhesus D (RhD)‐negative women pregnant with a RhD‐positive child receive prophylactic injections to prevent haemolytic disease of the newborn. Because of the success of the prophylaxis, ...the number of naturally immunized women has decreased, thereby also decreasing the number of potential donors who provide the plasma from which the prophylaxis is made. As the current donor pool is ageing, the availability of the prophylaxis is threatened.
Objectives
Objectives are to investigate whether the anti‐D population and the changes therein can be described by a relatively simple model, to determine the impact of ageing of the anti‐D donors on the decline of the population and how many new donors should be recruited to meet future supply demand.
Methods
Data on Dutch anti‐D donors in 1994–2013 were used to simulate the donor population size and age composition for various donor recruitment scenarios.
Results
With a continuous influx of 27 new donors per year and a donor stopping rate of 10% per year, the population size will stabilize at 195 donors, with 2·3% of donors stopping annually due to reaching the donor age limit. A formula is derived to estimate which donor recruitment and retention efforts are required to maintain a prespecified donor pool.
Conclusion
A relatively simple model can already describe and predict the size of the anti‐D donor population and the impact of ageing accurately.
Abstract
STUDY QUESTION
Which clinical and ethical aspects of preimplantation genetic testing for monogenic disorders or structural rearrangements (PGT-M, PGT-SR) should be considered when accepting ...requests and counselling couples for PGT when applied for more than one condition (combination-PGT; cPGT-M/SR)?
SUMMARY ANSWER
cPGT is a feasible extension of the practice of PGT-M/SR that may require adapting the criteria many countries have in place with regard to indications-setting for PGT-M/SR, while leading to complex choices that require timely counselling and information.
WHAT IS KNOWN ALREADY
Although PGT-M/SR is usually performed to prevent transmission of one disorder, requests for PGT-M/SR for more than one condition (cPGT-M/SR) are becoming less exceptional. However, knowledge about implications for a responsible application of such treatments is lacking.
STUDY DESIGN, SIZE, DURATION
Retrospective review of all (40) PGT-M/SR applications concerning more than one genetic condition over the period 1995–2018 in the files of the Dutch national PGT centre. This comprises all relevant national data since the start of PGT in the Netherlands.
PARTICIPANTS/MATERIALS, SETTING AND METHODS
Data regarding cPGT-M/SR cases were collected by means of reviewing medical files of couples applying for cPGT-M/SR. Ethical challenges arising with cPGT-M/SR were explored against the background of PGT-M/SR regulations in several European countries, as well as of relevant ESHRE-guidance regarding both indications-setting and transfer-decisions.
MAIN RESULTS AND THE ROLE OF CHANCE
We report 40 couples applying for cPGT-M/SR of which 16 couples started their IVF treatment. Together they underwent 39 IVF cycles leading to the birth of five healthy children. Of the couples applying for cPGT, 45% differentiated between a primary and secondary condition in terms of perceived severity. In the light of an altered balance of benefits and drawbacks, we argue the ‘high risk of a serious condition’ standard that many countries uphold as governing indications-setting, should be lowered for secondary conditions in couples who already have an indication for PGT-M/SR. As a consequence of cPGT, professionals will more often be confronted with requests for transferring embryos known to be affected with a condition that they were tested for. In line with ESHRE guidance, such transfers may well be acceptable, on the condition of avoiding a high risk of a child with a seriously diminished quality of life.
LIMITATIONS, REASONS FOR CAUTION
We are the first to give an overview of cPGT-M/SR treatments. Retrospective analysis was performed using national data, possibly not reflecting current trends worldwide.
WIDER IMPLICATIONS OF THE FINDINGS
Our observations have led to recommendations for cPGT-M/SR that may add to centre policy making and to the formulation of professional guidelines. Given that the introduction of generic methods for genomic analysis in PGT will regularly yield incidental findings leading to transfer requests with these same challenges, the importance of our discussion exceeds the present discussion of cPGT.
STUDY FUNDING/COMPETING INTEREST(S)
The research for this publication was funded by the Dutch Organization for Health Research and Development (ZonMw), project number: 141111002 (Long term safety, quality and ethics of Preimplantation Genetic Diagnosis). None of the authors has any competing interests to declare.
Objective To identify risk factors for Rhesus D (RhD) immunisation in pregnancy, despite adequate antenatal and postnatal anti‐D prophylaxis in the previous pregnancy. To generate evidence for ...improved primary prevention by extra administration of anti‐D Ig in the presence of a risk factor.
Design Case–control study.
Setting Nation‐wide evaluation of the Dutch antenatal anti‐D‐prophylaxis programme.
Population Cases: 42 RhD‐immunised parae‐1, recognised by first‐trimester routine red cell antibody screening in their current pregnancy, who received antenatal and postnatal anti‐D Ig prophylaxis (gifts of 1000 iu) in their first pregnancy. Controls: 339 parae‐1 without red cell antibodies.
Methods Data were collected via obstetric care workers and/or personal interviews with women.
Main outcome measure Significant risk factors for RhD immunisation in multivariate analysis.
Results Independent risk factors were non‐spontaneous delivery (assisted vaginal delivery or caesarean section) (OR 2.23; 95% CI:1.04–4.74), postmaturity (≥42 weeks of completed gestation: OR 3.07; 95% CI:1.02–9.02), pregnancy‐related red blood cell transfusion (OR 3.51; 95% CI:0.97–12.7 and age (OR 0.89/year; 95% CI:0.80–0.98). In 43% of cases, none of the categorical risk factors was present.
Conclusions In at least half of the failures of anti‐D Ig prophylaxis, a condition related to increased fetomaternal haemorrhage (FMH) and/or insufficient anti‐D Ig levels was observed. Hence, RhD immunisation may be further reduced by strict compliance to guidelines concerning determination of FMH and accordingly adjusted anti‐D Ig prophylaxis, or by routine administration of extra anti‐D Ig after a non‐spontaneous delivery and/or a complicated or prolonged third stage of labour.
Abstract
Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the
NF1
gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA ...diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in
NF1
. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known
NF1
DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in
NF1
.
This study aims to associate the incidence of postoperative vasoplegia and short-term survival to the implantation of various left ventricular assist devices differing in hemocompatibility and flow ...profiles. The overall incidence of vasoplegia was 25.3% (73/289 patients) and 30.3% (37/122), 25.0% (18/72), and 18.9% (18/95) in the axial flow (AXF), centrifugal flow (CF), and centrifugal flow with artificial pulse (CFAP) group, respectively. Vasoplegia was associated with longer intensive care (ICU) and hospital length of stay (LOS) and mortality. ICU and in-hospital LOS and 1-year mortality were the lowest in the CFAP group. Post hoc analysis resulted in a
p
-value of 0.43 between AXF and CF; 0.35 between CF and CFAP; and 0.06 between AXF and CFAP. Although there is a trend in diminished incidence of vasoplegia, pooled logistic regression using flow profile and variables that remained after feature selection showed that flow profile was not an independent predictor for postoperative vasoplegia.
Graphical Abstract
Chemokine-driven migration is accompanied by polarization of the cell body and of the intracellular signaling machinery. The extent to which chemokine receptors polarize during chemotaxis is ...currently unclear. To analyze the distribution of the chemokine receptor CXCR4 during SDF-1 (CXCL12)-induced chemotaxis, we retrovirally expressed a CXCR4-GFP fusion protein in the CXCR4-deficient human hematopoietic progenitor cell line KG1a. This KG1a CXCR4-GFP cell line showed full restoration of SDF-1 responsiveness in assays detecting activation of ERK1/2 phosphorylation, actin polymerization, adhesion to endothelium under conditions of physiological flow, and (transendothelial) chemotaxis. When adhered to cytokine-activated endothelium in the absence of SDF-1, CXCR4 did not localize to the leading edge of the cell but was uniformly distributed over the plasma membrane. In contrast, when SDF-1 was immobilized on cytokine-activated endothelium, the CXCR4-GFP receptors that were present on the cell surface markedly redistributed to the leading edge of migrating cells. In addition, CXCR4-GFP co-localized with lipid rafts in the leading edge of SDF-1-stimulated cells, at the sites of contact with the endothelial surface. Inhibition of lipid raft formation prevents SDF-1-dependent migration, internalization of CXCR4, and polarization to the leading edge of CXCR4, indicating that CXCR4 surface expression and signaling requires lipid rafts. These data show that SDF-1, immobilized on activated human endothelium, induces polarization of CXCR4 to the leading edge of migrating cells, revealing co-operativity between chemokine and substrate in the control of cell migration.
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