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.
Background and Objectives
Identification of antibody characteristics and genetics underlying the development of maternal anti‐A/B linked to inducing haemolytic disease of the foetus and newborn could ...contribute to the development of screening methods predicting pregnancies at risk with high diagnostic accuracy.
Materials and Methods
We examined 73 samples from mothers to 37 newborns with haemolysis (cases) and 36 without (controls). The secretor status was determined by genotyping a single nucleotide polymorphism in FUT2, rs601338 (c.428G>A).
Results
We found a significant association between secretor mothers and newborns developing haemolysis (p = 0.028). However, stratifying by the newborn's blood group, the association was found only in secretor mothers to blood group B newborns (p = 0.032). In fact, only secretor mothers were found in this group. By including antibody data from a previous study, we found higher median semi‐quantitative levels of IgG1 and IgG3 among secretor mothers than non‐secretor mothers to newborns with and without haemolysis.
Conclusion
We found that the maternal secretor status is associated with the production of anti‐A/B, pathogenic to ABO‐incompatible newborns. We suggest that secretors experience hyper‐immunizing events more frequently than non‐secretors, leading to the production of pathogenic ABO antibodies, especially anti‐B.
BACKGROUND
In the past century, blood group determination using serology has been the standard method. Now, molecular methods are gaining traction, which provide additional and easily accessible ...information. Here we designed and validated a high‐throughput extended genotyping setup.
STUDY DESIGN AND METHODS
We developed 35 competitive allele‐specific polymerase chain reaction assays for genotyping of blood donors. Samples from 1034 Danish blood donors were genotyped, and 45,314 red blood cell antigens and 6148 platelet antigens were predicted. Predicted phenotypes were compared with 16,119 serologic phenotypes.
RESULTS
We found 62 discrepancies of which 43 were due to serology. After exclusion of the discrepancies caused by serology, the accuracy of genotyping was 99.9%. Of 17 discrepancies caused by the genotype, three were incorrect antigen‐negative predictions and could potentially, as the solitary analysis, have caused an adverse transfusion reaction.
CONCLUSION
We have established a robust and highly accurate blood group genotyping system with a very high capacity for screening blood donors. The system represents a significant improvement over the former serotyping‐only procedure. Almost all new technology in medicine incurs increased costs, but the presented efficient genotyping system is a rare example of a significant qualitative and quantitative technologic progress that is also more cost‐efficient than previous technologies.
Background and Objectives
Prediction of haemolytic disease of the foetus and newborn (HDFN) caused by maternal anti‐A/‐B enables timely therapy, thereby preventing the development of kernicterus ...spectrum disorder. However, previous efforts to establish accurate prediction methods have been only modestly successful.
Materials and Methods
In a case–control study, we examined 76 samples from mothers and 76 samples from their newborns; 38 with and 38 without haemolysis. The IgG subclass profile of maternal anti‐A and anti‐B was determined by flow cytometry. Samples from newborns were genetically analysed for the A2 subgroup, secretor and FcγRIIa receptor alleles.
Results
Surprisingly, we found a correlation between the newborn secretor allele and haemolysis (p = 0.034). No correlation was found for FcγRIIa alleles. The A2 subgroup was found only in newborns without haemolysis. Unexpectedly, different reaction patterns were found for maternal anti‐A and anti‐B; consequently, the results were treated separately. For the prediction of haemolysis in A‐newborns, the maternal IgG1 subclass determination resulted in an accuracy of 83% at birth. For B‐newborns, an accuracy of 91% was achieved by the maternal IgG2 subclass determination.
Conclusion
We improved the prediction of ABO‐HDFN by characterizing maternal anti‐A and anti‐B by flow cytometry and we presented genetic traits in newborns with correlation to haemolysis. We propose a new understanding of A‐ and B‐substances as immunogens that enhance the maternal immune response and protect the newborn, and we suggest that the development of ABO‐HDFN is different when caused by maternal anti‐A compared to maternal anti‐B.
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.
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.
BACKGROUND: A combination of antenatal and postnatal RhD prophylaxis is more effective in reducing D immunization in pregnancy than postnatal RhD prophylaxis alone. Based on the result from antenatal ...screening for the fetal RHD gene, antenatal RhD prophylaxis in Denmark is given only to those D− women who carry a D+ fetus. We present an evaluation of the first national clinical application of antenatal RHD screening.
STUDY DESIGN AND METHODS: In each of the five Danish health care regions, blood samples were drawn from D− women in Gestational Week 25. DNA was extracted from the maternal plasma and analyzed for the presence of the RHD gene by real‐time polymerase chain reaction targeting two RHD exons. Prediction of the fetal RhD type was compared with serologic typing of the newborn in 2312 pregnancies, which represented the first 6 months of routine analysis.
RESULTS: For the detection of fetal RHD, the sensitivity was 99.9%. The accuracy was 96.5%. The recommendation for unnecessary antenatal RhD prophylaxis for women carrying a D− fetus was correctly avoided in 862 cases (37.3%), while 39 women (1.7%) were recommended for antenatal RhD prophylaxis unnecessarily. Two RHD+ fetuses (0.087%) were not detected, and antenatal RhIG was not given.
CONCLUSION: These data represent the first demonstration of the reliability of routine antenatal fetal RHD screening in D−, pregnant women to ascertain the requirement for antenatal RhD prophylaxis. Our findings should encourage the implementation of such screening programs worldwide, to reduce the unnecessary use of RhIG.
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.
Background and ObjectivesFor 5 years, routine genotyping has been performed for selected blood groups of blood donors in the Copenhagen Capital Region, Denmark. The result is summarized in the ...following.Materials and MethodsGenotyping was carried out by an external service provider using the competitive allele specific PCR (KASP) technology. The genotypes were returned to the blood bank and translated into phenotypes by a proprietary IT application.ResultsIn total, 65 alleles from 16 blood group systems (ABO, MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, Colton, Landsteiner‐Wiener, Cromer, Knops, Vel, secretor status) and the HPA1, HPA5 and HPA15 antigens were interrogated. After translation, phenotypes were imported into the laboratory information management system of the blood bank. The results from 31,538 genotyped blood donors were used to calculate the allele frequencies for a Danish blood donor population. ABO genotyping was done for sample ID purposes. Determination of the 1061delC single nucleotide polymorphism (SNP) (NM_020469.2), most frequently characteristic of ABO*A2, was validated against a series of 1287 samples with Dolichos biflorus lectin determination of the A1 phenotype.ConclusionWe report allele frequencies and phenotype frequencies for 16 blood groups from a total of 31,538 genotyped blood donors. Blood products were supplied from a total of 64,312 active blood donors, and of these active blood donors 25,396 (39.5%) were genotyped. These donors represent a valuable resource for patient treatment. This genotyping has enabled the provision of rare genotyped donor blood for patients with alloantibodies and rare reagent cells for serology.
Abstract
Background and Objectives
For 5 years, routine genotyping has been performed for selected blood groups of blood donors in the Copenhagen Capital Region, Denmark. The result is summarized in ...the following.
Materials and Methods
Genotyping was carried out by an external service provider using the competitive allele specific PCR (KASP) technology. The genotypes were returned to the blood bank and translated into phenotypes by a proprietary IT application.
Results
In total, 65 alleles from 16 blood group systems (ABO, MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Dombrock, Colton, Landsteiner‐Wiener, Cromer, Knops, Vel, secretor status) and the HPA1, HPA5 and HPA15 antigens were interrogated. After translation, phenotypes were imported into the laboratory information management system of the blood bank. The results from 31,538 genotyped blood donors were used to calculate the allele frequencies for a Danish blood donor population.
ABO
genotyping was done for sample ID purposes. Determination of the 1061delC single nucleotide polymorphism (SNP) (NM_020469.2), most frequently characteristic of
ABO*A2
, was validated against a series of 1287 samples with
Dolichos biflorus
lectin determination of the A1 phenotype.
Conclusion
We report allele frequencies and phenotype frequencies for 16 blood groups from a total of 31,538 genotyped blood donors. Blood products were supplied from a total of 64,312 active blood donors, and of these active blood donors 25,396 (39.5%) were genotyped. These donors represent a valuable resource for patient treatment. This genotyping has enabled the provision of rare genotyped donor blood for patients with alloantibodies and rare reagent cells for serology.
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