Recent advances in non-invasive fetal HPA-1a typing

  • Núria Nogués
    Correspondence
    Correspondence to: Immunohematology Laboratory, Banc de Sang i Teixits, Pg. Taulat 116, 08005 Barcelona, Spain.
    Affiliations
    Immunohematology Laboratory, Banc de Sang i Teixits, Pg. Taulat 116, 08005 Barcelona, Spain

    Department of Medicine, Universitat Autònoma de Barcelona, Passeig Vall d’Hebron 129-139, 08035 Barcelona, Spain
    Search for articles by this author
Open AccessPublished:December 31, 2019DOI:https://doi.org/10.1016/j.transci.2019.102708

      Abstract

      Non-invasive fetal HPA-1a typing is a valuable tool to identify the pregnancies at risk of fetal and neonatal alloimmune thrombocytopenia (FNAIT). At present, prenatal determination of the fetus HPA-1a type is performed for diagnostic purposes in pregnancies of HPA-1 alloimmunized women with history of a previous fetus or child with FNAIT. Different approaches have been used to determine the fetal HPA-1a genotype from cell-free fetal DNA (cffDNA) in the mother’s plasma, mainly based on real-time PCR. Due to the single nucleotide polymorphism (SNP) between the HPA-1a and HPA-1b allelic sequences, a robust and accurate detection of the fetal genotype is challenging, and the sensitivity of most assays is still limited early in pregnancy. Nowadays, the availability of technologies such as next generation sequencing (NGS) or digital PCR offers unprecedented possibilities of analyzing cell-free DNA (cfDNA)-amplified sequences with very high coverage and high sensitivity. In addition, other interesting approaches using variant sequence enrichment strategies have been recently described. In particular, coamplification at lower denaturation temperature PCR (COLD-PCR) offers a simple and sensitive strategy for noninvasive fetal HPA-1 typing. These novel approaches are explained in more detail in this review. Despite no population-based FNAIT screening programs have so far been implemented, the perspectives in terms of treatment and prevention are changing and less costly high-throughput maternal HPA-1a typing methods have been developed. Altogether, this may lead to the implementation of fetal HPA-1a typing with a broader scope in the future, playing a critical role within FNAIT screening programs.

      Keywords

      1. Introduction

      Maternal alloimmunization against the platelet HPA-1a antigen is the most frequent cause of severe fetal and neonatal alloimmune thrombocytopenia (FNAIT) cases in Caucasian population and is involved in more than 80% of reported cases [
      • Davoren A.
      • Curtis B.R.
      • Aster R.H.
      • McFarland J.G.
      Human platelet antigen‐specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia.
      ]. Similar to other fetomaternal blood group incompatibilities, non-invasive determination of the fetal HPA-1a type is a valuable tool to identify the pregnancies at risk of FNAIT. Furthermore, due to a substantial risk of FNAIT recurrence, the management of subsequent pregnancies in HPA-1a-alloimmunized women usually involves weekly treatment with intravenous gammaglobulin (IVIG), to reduce the risk of intracranial hemorrhage (ICH) in the fetus or newborn. In this context, assays for non-invasive fetal HPA-1 genotyping allow us to identify the pregnancies at risk, avoiding unnecessary treatment and procedures if the fetus is HPA-1a negative.
      The implementation of systematic HPA-1a typing of all pregnant women to identify the low percentage of women potentially at risk, has been debated in Norway, Denmark, the UK and the Netherlands. Yet, no countries have so far implemented a screening program to identify pregnancies at risk of FNAIT. However, the current perspective in terms of treatment and prevention options is changing and there have also been significant advances in the development of less costly high-throughput maternal HPA-1a typing methods. Noninvasive determination of the fetal HPA-1a type can also play a critical role within FNAIT screening programs that might be implemented in the future. In this sense, it is important to have available safe, reliable and sensitive fetal HPA-1a genotyping tests, which could ideally be applied early in pregnancy.
      The first methods for noninvasive fetal HPA-1a typing were described less than 10 years ago. At present, technologies such as digital PCR and next generation sequencing (NGS) are promising tools that may offer high sensitivity and the possibility of determining the fetal HPA-1a type early in pregnancy. Recent studies have already provided proof of concept for the application of NGS to the noninvasive antenatal determination of fetal blood groups, with a special focus on clinically relevant blood group single nucleotide polymorphisms [
      • Rieneck K.
      • Banch Clausen F.
      • HanefeldDziegiel M.
      Noninvasive antenatal determination of fetal blood group using next-generation sequencing.
      ]. These novel technologies, as well as early methods for fetal HPA-1a typing and a more recent method based on coamplification at lower denaturation temperature PCR (COLD-PCR), will be presented and discussed in this review. A summary of the different methods is given in Table 1.
      Table 1Overview of currently applied fetal HPA-1a genotyping methods.

      2. Early methods for fetal HPA-1a typing

      Different approaches have been used to determine the fetal HPA-1a genotype from cell-free fetal DNA (cffDNA) in maternal plasma. Due to the single nucleotide polymorphism (SNP) between the HPA-1a and HPA-1b allelic sequences, the robust and accurate detection of the fetal genotype is hampered by the overrepresented maternal HPA-1b alleles. To overcome this technical challenge, several strategies have been designed, including predigestion of the plasma cell-free (cfDNA) with a restriction enzyme (MspI), whose target sequence is only present in the HPA-1b allele. This practical approach, described in 2010 by Scheffer et al. [
      • Scheffer P.
      • Ait Soussan A.
      • Verhagen O.
      • et al.
      Noninvasive fetal genotyping of human platelet antigen‐1a.
      ], was shown to prevent the non-specific amplification of maternal cfDNA, allowing the detection of the intact fetal HPA-1a allele by real-time PCR, when inherited from the father.
      Other fetal HPA-1a genotyping methods, based on real-time PCR as well, have been developed and validated. In one approach, allele-specific primers were used in combination with a TaqMan assay [
      • Freixa L.
      • Nogues N.
      • Ibañez M.
      • Farssac E.
      • Gracia M.
      • Vinyets I.
      • et al.
      Development and validation of a non-invasive approach for fetal HPA-1a genotyping using cell-free fetal DNA present in maternal plasma.
      ], whereas in another method, allele-specific amplification was detected with SYBR Green technology [
      • Le Toriellec E.
      • Chenet C.
      • Kaplan C.
      Safe fetal platelet genotyping: new developments.
      ]. The latter approach was evaluated in parallel with an alternative method based on high-resolution melting (HRM) analysis of an amplicon spanning the HPA-1 SNP. Validation studies carried out in cohorts of 54 [
      • Freixa L.
      • Nogues N.
      • Ibañez M.
      • Farssac E.
      • Gracia M.
      • Vinyets I.
      • et al.
      Development and validation of a non-invasive approach for fetal HPA-1a genotyping using cell-free fetal DNA present in maternal plasma.
      ] and 44 [
      • Le Toriellec E.
      • Chenet C.
      • Kaplan C.
      Safe fetal platelet genotyping: new developments.
      ] samples from HPA-1a negative pregnant women have demonstrated the reliability of these straightforward fetal HPA-1a genotyping assays. However, most of the samples tested were collected in the second or third trimester of gestation. Hence, a negative fetal HPA-1a typing result from a sample collected early in pregnancy should be confirmed by repeating the analysis in a second test later in pregnancy.

      3. COLD-PCR

      COLD-PCR (co-amplification at lower denaturation temperature-PCR) is a modified PCR protocol that enriches variant alleles from a mixture of wild-type and mutation-containing DNA. The ability to preferentially amplify minority alleles in the presence of excess wild-type alleles has been exploited in applications aiming to detect low-level somatic DNA mutations for early cancer detection [
      • Li J.
      • Wang L.
      • Mamon H.
      • Kulke M.H.
      • Berbeco R.
      • Makrigiorgos M.
      Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.
      ].
      The underlying principle is that single nucleotide mismatches will slightly alter the melting temperature (Tm) of the double-stranded DNA.COLD-PCR exploits these melting temperature differences between variant and wild-type sequences. By using a lower denaturation temperature, referred to as "critical denaturation temperature" (Tc), the minority mutated alleles are selectively amplified (Fig. 1).
      Fig. 1
      Fig. 1Outline of Fast COLD-PCR strategy for enrichment of Tm-reducing mutations.
      In the context of noninvasive prenatal diagnosis (NIPD), this approach has been shown to facilitate the detection of the minor fetal component of circulating cfDNA carrying a variant sequence. A recent report by Ferro et al., describes the application of COLD-PCR in combination with HRM analysis to the antenatal determination of the fetal HPA-1 typing [
      • Ferro M.
      • Macher H.C.
      • Fornés G.
      • Martín‐Sánchez J.
      • Jimenez‐Arriscado P.
      • et al.
      Noninvasive prenatal diagnosis by cell‐free DNA screening for fetomaternal HPA‐1a platelet incompatibility.
      ].
      These investigators have devised a strategy in which the mother is initially genotyped for HPA-1 from a plasma cfDNA sample extracted early in gestation. The assay used for this purpose is a conventional HRM PCR and only those samples corresponding to a homozygous HPA-1b1b pregnant women are used for further analysis. In the second analysis, the plasma cfDNA sample is subjected to COLD HRM PCR, to determine whether the fetus is HPA-1a positive or negative.
      The strategy described is easy, straightforward and doesn't require expensive instrumentation. Overall, it would fit very well in a FNAIT screening program. A preliminary validation study has shown accurate results for both maternal and fetal HPA-1 typing and the incompatible fetal HPA-1a allele has been detected as early as 12 weeks of gestation [
      • Ferro M.
      • Macher H.C.
      • Fornés G.
      • Martín‐Sánchez J.
      • Jimenez‐Arriscado P.
      • et al.
      Noninvasive prenatal diagnosis by cell‐free DNA screening for fetomaternal HPA‐1a platelet incompatibility.
      ]. Despite these promising results, only a small number of paired mother and fetus typings have been performed, and extended validation studies involving larger numbers of clinical samples need to be conducted to further assess the sensitivity and accuracy of this fetal HPA-1a genotyping assay.

      4. Targeted massive parallel sequencing

      NGS is a powerful technology with a huge potential in genetic diagnostics as well as in many other fields. As an example, the application of this technology has allowed to ascertain the genetic etiology of some complex hematological and immunological syndromes, providing new means for their accurate diagnosis. Nowadays, besides the numerous research studies using this technology, there are various well-established methodologies based on NGS that have already been incorporated into routine clinical practice, such as high-throughput HLA typing of hemopoietic stem cell donors and NIPD screening for the most common aneuploidies.
      Within the field of NIPD, targeted NGS can also be applied to identify paternally inherited fetal alleles in cfDNA. This strategy has been used for the diagnosis of several inherited diseases but also for noninvasive antenatal determination of fetal blood groups [
      • Rieneck K.
      • Banch Clausen F.
      • HanefeldDziegiel M.
      Noninvasive antenatal determination of fetal blood group using next-generation sequencing.
      ,
      • Rieneck K.
      • Bak M.
      • Jønson L.
      • Clausen F.B.
      • Krog G.R.
      • Tommerup N.
      • et al.
      Next‐generation sequencing: proof of concept for antenatal prediction of the fetal Kell blood group phenotype from cell‐free fetal DNA in maternal plasma.
      ]. In this context, the main advantage of NGS is that it offers the possibility of massively parallel sequencing the targeted amplified fragments spanning the blood-group-determining SNP. Amplicons obtained from maternal plasma cfDNA will comprise both maternal and fetal DNA. NGS-based detection of the paternally inherited allele is the result of sequencing each of the amplified fragments and then counting the number of individual sequences containing the target SNP. Thus, compared to previously described real-time PCR approaches, NGS offers a more in-depth analysis of the sequence of cfDNA amplified products and has, in turn, higher sensitivity.
      Several NGS platforms are available, which differ in terms of throughput, read length and sequencing procedures. The Ion Torrent PGM (Life Technologies) and the MiSeq (Illumina) are the two NGS platforms currently used in most laboratories for noninvasive fetal HPA-1a typing. These two systems have similar pipelines which are summarized in Fig. 2. The sequencing process requires a first step of clonal amplification of the gene-targeted PCR products initially comprising the library. In a second step, the sequencing reaction takes place ‘by synthesis’, which involves a cycle of washing followed by extension with known DNA bases in sequential order. The primary difference is that the Illumina MiSeq detects the nucleotides via fluorescence whereas the Ion Torrent PGM uses semiconductor technology and detects the incorporated nucleotide via pH changes.
      Fig. 2
      Fig. 2Workflow of Ion torrent PGM and Illumina MiSeq NGS platforms.
      Adapted from Silva, Genivaldo (2016). Thesis: “Who Is There and What are They Doing? An Agile and Computationally Efficient Framework for Genome Discovery and Annotation from Metagenomic Big Data”.
      The first study applying NGS for noninvasive fetal HPA-1a typing was published by Wienzek-Lischka et al. in 2015 [
      • Wienzek‐Lischka S.
      • Krautwurst A.
      • Fröhner V.
      • Hackstein H.
      • Gattenlöhner S.
      • Bräuninger A.
      • et al.
      Noninvasive fetal genotyping of human platelet antigen‐1a using targeted massively parallel sequencing.
      ]. In this study, massive parallel sequencing of targeted ITGB3 amplicons was performed in combination with additional polymorphic regions encoding common red cell blood groups as well as other HPA antigens. This panel of customized targeted sequences was amplified from maternal plasma cfDNA in parallel with a set of eight unlinked non-exonic SNP markers (Ion AmpliSeq Sample ID-Panel) that provides an additional internal positive control for the presence of fetal DNA. Massive parallel sequencing was performed on semiconductor chips using the Ion Torrent PGM platform. Correct fetal HPA-1a genotyping results were obtained in this proof of principle study, which included four samples of HPA-1a negative pregnant women with a previous history of FNAIT due to anti-HPA-1a antibodies.
      The same Ion Torrent technology has also been used by Orzinska et al. in a subset of HPA-1a negative pregnant women selected from the Polish PREVFNAIT screening program [
      • Orzińska A.
      • Guz K.
      • Uhrynowska M.
      • Dębska M.
      • Mikula M.
      • Ostrowski J.
      • Ahlen M.T.
      • Husebekk A.
      • Brojer E.
      Noninvasive prenatal HPA‐1 typing in HPA‐1a negative pregnancies selected in the Polish PREVFNAIT screening program.
      ].Their preliminary results have shown a high coverage sequencing of the ITGB3 target SNP position (mean coverage for all examined samples >49,000 total reads), allowing the precise identification of 1 % and 5 % HPA-1a positive reads in two different samples of women carrying an HPA-1a positive fetus.
      Besides these reports, a more extended validation study of a fetal HPA-1a genotyping approach using Illumina NGS technology has been recently presented at the recent cfDNA2019 International Meeting, Copenhagen, May 23–24, 2019. A total of 26 samples from HPA-1a negative pregnant women, collected at different weeks of gestation (range 13–35 WG), were used to validate a novel fetal HPA-1a genotyping approach based on massive parallel sequencing in a MiSeq (Illumina) platform. These samples had been previously tested by allele-specific real-time PCR and the fetal HPA-1a genotype had been confirmed in the newborns [
      • Freixa L.
      • Nogues N.
      • Ibañez M.
      • Farssac E.
      • Gracia M.
      • Vinyets I.
      • et al.
      Development and validation of a non-invasive approach for fetal HPA-1a genotyping using cell-free fetal DNA present in maternal plasma.
      ]. In this validation study, 17 samples corresponding to women carrying an HPA-1a positive fetus, as well as 9 samples of pregnant women carrying homozygous HPA-1b fetuses, were included. An improved high-fidelity DNA polymerase has been used in combination with a paired-end sequencing protocol, which yielded accurate results in all tested samples (unpublished results).
      Despite the current high cost of this technology, the prospects for NGS application in routine diagnostic testing are quite promising, as the obstacles for its further implementation are gradually decreasing. The cost of “benchtop” NGS sequencers and reagents is predicted to drop to more competitive levels in the next years but other factors are also helping, like the availability of low-throughput reagent kits “Nanokits”, which allow deep sequencing of a low number of clinical samples at a reasonable cost. This is particularly suitable for NIPD tests applying to a small number of samples and whose results cannot be delayed until a larger number of samples requiring the same test is collected for parallel processing. Nonetheless, the possibility to combine fetal HPA-1a genotyping tests with other NIPD tests (e.g. fetal K, Rhc, RhE) in reference laboratories enhances the chance for an optimal use of these reagents. This aspect should be taken into consideration when designing new fetal blood group typing NIPD assays to be implemented by NGS technology.

      5. Digital PCR

      Digital PCR (dPCR) is another powerful technology currently available, with a wide variety of applications in clinical diagnostics. Among these, dPCR approaches are in use in applications such as cancer monitoring in liquid biopsies and organ transplant rejection monitoring, both based on the detection of low abundance cell-free DNA in patient samples. Likewise, dPCR methods have also been applied within the field of NIPD, where this technology represents an attractive and sensitive novel tool allowing an accurate analysis of the cell-free fetal DNA circulating in maternal plasma.
      The principle of the dPCR technology (shown in Fig. 3) involves separating the sample with the prepared PCR solution into a large number of partitions (usually nanoliter sized droplets) where the PCR reaction is carried out individually. Every partition contains either zero, one, or several copies of the target DNA, following a Poisson distribution. After an allele-specific PCR reaction in the presence of fluorogenic probes, partitions containing amplified target sequences are detected by fluorescence. Each partition is individually scanned for fluorescence, giving a readout of either “1” or “0” depending on whether copies of the target DNA were present or not. The ratio of positive partitions over the total number allows to determine the concentration of the target sequence within a sample [
      • Nectoux J.
      Current, emerging, and future applications of digital PCR in non-invasive prenatal diagnosis.
      ].
      Fig. 3
      Fig. 3Principle of digital PCR. From Nectoux J. Current, Emerging, and Future Applications of Digital PCR in Non-Invasive Prenatal Diagnosis. Mol Diagn Ther. 2018.
      Several different methods can be used to partition samples. In microchip dPCR methods, reactions are carried out in microchambers, the volume of which is generally in the range of nanoliters (nL). These platforms allow monitoring the fluorescent signal in real-time and display many other advantages, such as being user-friendly and the possibility to automate the system, from injection of the sample to the analysis of the reaction. However, platforms providing a much higher number of digital partitions, such as droplet dPCR (ddPCR) systems, offer even higher precision and improves the detection of low-abundance DNA point mutations. ddPCR consists of aqueous droplets dispersed in oil for the compartmentalization of the PCR reactions. Depending on the platform, the droplets volume can get as low as 5 picoliters (pL) and the number of partitions may range between thousands to even millions.
      Through partitioning of the sample, digital PCR approaches allow a highly reliable and sensitive measurement of the target DNA amount. Indeed, by using digital PCR methodologies it has been shown that the actual fractional concentration of fetal DNA in the maternal plasma in all three trimesters is two times higher than the previously reported amount determined by real-time PCR [
      • Lun F.M.
      • Chiu R.W.
      • Allen Chan K.C.
      • Leung T.Y.
      • Lau T.K.
      • Lo Y.M.D.
      Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma.
      ]. In addition, digital PCR is very versatile and multiple color detection permits the analysis of several SNPs or measurement of a target and a reference sequence in the same reaction.
      Despite its potential, the application of digital PCR approaches to noninvasive fetal blood group genotyping is still limited. The required microfluidic devices are expensive and not yet available in most clinical diagnostic laboratories. However, several dPCR methods have been developed for the NIPD of fetomaternal platelet incompatibilities and validation studies are ongoing on both, chip-based dPCR and ddPCR platforms. Among the latter, the method developed by Mammasse Y et al. simultaneously amplifies four platelet antigen systems (HPA-1,-3,-5 and -15), which altogether covers the specificities implicated in more than 95 % of FNAIT cases. Plasma samples from 46 pregnant women, at risk or with antenatal history of FNAIT, were included in a validation study of this novel approach [
      • Mammasse Y.
      • Chenet C.
      • Drubay D.
      • Martageix C.
      • Cartron J.P.
      • Vainchenker W.
      • et al.
      Implementation of noninvasive prenatal diagnosis of fetomaternal platelet antigen incompatibility in routine clinical testing.
      ]. The methylation status of RASSF1A gene promoter was also tested by ddPCR and was used as an internal control to exclude false-negative results [
      • Mammasse Y.
      • Chenet C.
      • Drubay D.
      • Martageix C.
      • Cartron J.P.
      • Vainchenker W.
      • et al.
      Implementation of noninvasive prenatal diagnosis of fetomaternal platelet antigen incompatibility in routine clinical testing.
      ]. The cohort of pregnant women included nine cases with an HPA-1 fetomaternal incompatibility. Accurate ddPCR results were obtained for all the samples tested within this study, including those corresponding to early stages of pregnancy. Thus, ddPCR seems to be a sensitive and accurate noninvasive method for determination of the fetal HPA genotype [
      • Mammasse Y.
      • Chenet C.
      • Drubay D.
      • Martageix C.
      • Cartron J.P.
      • Vainchenker W.
      • et al.
      Implementation of noninvasive prenatal diagnosis of fetomaternal platelet antigen incompatibility in routine clinical testing.
      ].
      To make it more exciting, further technical developments in microfluidics are expected to occur in the next years, which will likely improve current platforms performance. Together with the growing number of emerging uses of this technology, we can expect that digital PCR will be implemented in a wide range of applications in the near future, including noninvasive fetal genotyping tests in early pregnancy.

      6. Current and future perspectives

      At present, no population-based screening programs are in place to identify the HPA-1a negative pregnant women potentially at risk for HPA-1a alloimmunization, or those already immunized against this platelet antigen. Therefore, at least for now, non-invasive fetal HPA-1a typing is only performed for diagnostic purposes in pregnancies of alloimmunized women who previously have had a fetus or child with FNAIT. Within this context, paternal HPA-1a zygosity is usually examined. If the father is heterozygous HPA-1a1b, there is a 50% chance of the fetus being HPA-1a negative, and then, unnecessary IVIG treatment can be avoided. So, knowing the fetal HPA-1a typing is clinically helpful, as it allows identification of pregnancies at risk and enables the obstetrician to plan the antenatal management accordingly.
      Nowadays, the availability of reliable non-invasive fetal HPA-1a typing methods facilitates the possibility of determining the fetal genotype in all HPA-1a-immunized pregnant women, overcoming the ethical concerns associated with making clinical decisions based on samples obtained from the (presumed) father.
      The rapid technical progress of noninvasive prenatal DNA diagnostics has prompted the implementation of new technologies in fetal blood group typing applications, particularly in those determined by SNPs, which hitherto has been challenging. In this sense, fetal HPA-1a typing from the maternal plasma cfDNA has traditionally been hampered by nonspecific amplification of the overrepresented maternal HPA-1b allele. Different technical approaches have been applied to overcome this problem and have succeeded in detecting the fetal HPA-1a allele, when inherited, with high specificity. However, the sensitivity of most assays currently used is still limited early in pregnancy.
      Recent technologies such as massively parallel sequencing and ddPCR allow the analysis of gene-targeted amplified sequences with an unprecedented high coverage, which in the prenatal testing setting, implies higher sensitivity and accuracy. In addition, these methods can also provide an internal control for fetal DNA, which unfortunately is lacking in assays based on conventional PCR. Hence, NGS and ddPCR may pave the way for more extensive use, not only for fetal HPA-1a typing in alloimmunized pregnant women, but also for future application in FNAIT screening programs that might be implemented in the future.

      Declaration of Competing Interest

      None.

      References

        • Davoren A.
        • Curtis B.R.
        • Aster R.H.
        • McFarland J.G.
        Human platelet antigen‐specific alloantibodies implicated in 1162 cases of neonatal alloimmune thrombocytopenia.
        Transfusion. 2004; 44: 1220-1225https://doi.org/10.1111/j.1537-2995.2004.04026.x
        • Scheffer P.
        • Ait Soussan A.
        • Verhagen O.
        • et al.
        Noninvasive fetal genotyping of human platelet antigen‐1a.
        BJOG. 2011; 118: 1392-1395https://doi.org/10.1111/j.1471-0528.2011.03039.x
        • Freixa L.
        • Nogues N.
        • Ibañez M.
        • Farssac E.
        • Gracia M.
        • Vinyets I.
        • et al.
        Development and validation of a non-invasive approach for fetal HPA-1a genotyping using cell-free fetal DNA present in maternal plasma.
        Vox Sang. 2010; 99: 22
        • Le Toriellec E.
        • Chenet C.
        • Kaplan C.
        Safe fetal platelet genotyping: new developments.
        Transfusion. 2013; 53: 1755-1762https://doi.org/10.1111/j.1537-2995.2012.03954.x
        • Rieneck K.
        • Banch Clausen F.
        • HanefeldDziegiel M.
        Noninvasive antenatal determination of fetal blood group using next-generation sequencing.
        Cold Spring Harb Perspect Med. 2015; 6: a023093https://doi.org/10.1101/cshperspect.a023093
        • Li J.
        • Wang L.
        • Mamon H.
        • Kulke M.H.
        • Berbeco R.
        • Makrigiorgos M.
        Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing.
        Nat Med. 2008; 14: 579-584https://doi.org/10.1038/nm1708
        • Ferro M.
        • Macher H.C.
        • Fornés G.
        • Martín‐Sánchez J.
        • Jimenez‐Arriscado P.
        • et al.
        Noninvasive prenatal diagnosis by cell‐free DNA screening for fetomaternal HPA‐1a platelet incompatibility.
        Transfusion. 2018; 58: 2272-2279https://doi.org/10.1111/trf.14837
        • Rieneck K.
        • Bak M.
        • Jønson L.
        • Clausen F.B.
        • Krog G.R.
        • Tommerup N.
        • et al.
        Next‐generation sequencing: proof of concept for antenatal prediction of the fetal Kell blood group phenotype from cell‐free fetal DNA in maternal plasma.
        Transfusion. 2013; 53: 2892-2898https://doi.org/10.1111/trf.12172
        • Wienzek‐Lischka S.
        • Krautwurst A.
        • Fröhner V.
        • Hackstein H.
        • Gattenlöhner S.
        • Bräuninger A.
        • et al.
        Noninvasive fetal genotyping of human platelet antigen‐1a using targeted massively parallel sequencing.
        Transfusion. 2015; 55: 1538-1544https://doi.org/10.1111/trf.13102
        • Orzińska A.
        • Guz K.
        • Uhrynowska M.
        • Dębska M.
        • Mikula M.
        • Ostrowski J.
        • Ahlen M.T.
        • Husebekk A.
        • Brojer E.
        Noninvasive prenatal HPA‐1 typing in HPA‐1a negative pregnancies selected in the Polish PREVFNAIT screening program.
        Transfusion. 2018; 58: 2705-2711https://doi.org/10.1111/trf.14963
        • Nectoux J.
        Current, emerging, and future applications of digital PCR in non-invasive prenatal diagnosis.
        Mol Diagn Ther. 2018; 22 (Review): 139-148https://doi.org/10.1007/s40291-017-0312-x
        • Lun F.M.
        • Chiu R.W.
        • Allen Chan K.C.
        • Leung T.Y.
        • Lau T.K.
        • Lo Y.M.D.
        Microfluidics digital PCR reveals a higher than expected fraction of fetal DNA in maternal plasma.
        Clin Chem. 2008; 54: 1664-1672https://doi.org/10.1373/clinchem.2008.111385
        • Mammasse Y.
        • Chenet C.
        • Drubay D.
        • Martageix C.
        • Cartron J.P.
        • Vainchenker W.
        • et al.
        Implementation of noninvasive prenatal diagnosis of fetomaternal platelet antigen incompatibility in routine clinical testing.
        Transfusion. 2019; 59https://doi.org/10.1111/trf.15462