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Special Report
Prenatal testing for hemolytic disease of the newborn and fetal neonatal alloimmune thrombocytopenia – current status Expert Rev. Hematol. 7(6), 741–745 (2014)
Neil D Avent Plymouth University Peninsula Schools of Medicine and Dentistry, Portland Square Building, Drake Circus, Plymouth, PL4 8AA, UK [email protected]
Incompatibility of red cell and platelet antigens can lead to maternal alloimmunization causing hemolytic disease of the fetus & newborn and fetal neonatal alloimmune thrombocytopenia respectively. As the molecular background of these polymorphisms emerged, prenatal testing using initially fetal DNA obtained from invasively obtained amniotic fluid or chorionic villus was implemented. This evolved into testing using maternal plasma as source of fetal DNA, and this is in routine use as a safe non-invasive diagnostic that has no risk to the fetus of alloimmunization or spontaneous miscarriage. These tests were initially applied to high risk pregnancies, but has been applied on a mass scale, to screen fetuses in D-negative pregnant populations as national screening programs. Fetal neonatal alloimmune thrombocytopenia management has had comparatively small take up in non-invasive testing for causative fetal platelet alleles (e.g., HPA-1A), but mass scale genotyping of mothers to identify at risk HPA-1b1b pregnancies and their treatment with prophylactic anti-HPA-1A is being considered in at least one country (Norway). KEYWORDS: alloimmune diseases of the fetus • blood group genotyping • fetal neonatal alloimmune thrombocytopenia • hemolytic disease of fetus and newborn • non-invasive prenatal diagnosis
Alloimmune diseases of the fetus & newborn
Incompatibility of either red cell or platelet antigens between mother and fetus can lead to alloimmunization, and maternal IgG (thus primarily in secondary immune responses) transfer across the placenta which leads to red cell destruction (hemolytic disease of the newborn and fetus, hemolytic disease of the fetus & newborn [HDFN]) or platelet destruction (fetal neonatal alloimmune thromobocytopenia, fetal neonatal alloimmune thrombocytopenia [FNAIT]). The maternal IgG is transferred via the FcRN transporter that binds IgG at acidic pH (maternal face of placenta) and releases it at neutral/above pH 7.0. FcRN is a MHC class I family member comprising a heavy chain, (Fcgrt) and forms a heterodimer with its light chain, b2 microglobulin [1]. informahealthcare.com
10.1586/17474086.2014.970160
FcRN is involved in maintaining the correct plasma concentrations of IgG (and albumin) in adults, but also facilitates the transport of IgG to the fetus prior to birth and from colostrum and milk in the neonatal period. Thus the FcRN transport system is the cause of placental transfer of the pathogenic IgGs in both HDFN and NAITP. The clinical management of both HDFN and FNAIT involves the detection of blood group or platelet specific antibodies in the mother, followed by escalated clinical management in high-risk pregnancies. For HDFN this largely is focused on D-negative mothers, where antenatal administered prophylactic anti-D has drastically reduced the incidence of fetal anemia [2]. For alloimmunization due to other blood group antigen incompatibility, there is no prophylactic treatment regime currently available, but prenatal diagnosis offers
2014 Informa UK Ltd
ISSN 1747-4086
741
Special Report
Avent
the opportunity to identify antigen-negative fetuses that do not require further treatment [3]. This is especially important as correction of either fetal anemia or thrombocytopenia may involve in utero transfusion of red cells or platelets, which is an inherently risky procedure.
Expert Review of Hematology Downloaded from informahealthcare.com by Nanyang Technological University on 04/29/15 For personal use only.
Hemolytic disease of the fetus & newborn – prenatal testing for blood group antigens
Molecular testing for HDFN can now be broadly categorized into two distinct clinical groups (especially for RhD) [1], the testing of alloimmunized mothers to inform potential further clinical intervention (e.g., in utero transfusion) [2]. To identify mothers not requiring prenatal prophylactic treatment of antiD as they carry D-negative fetuses. Rapidly following the cloning of various blood group active cDNAs and determination of blood group critical single nucleotide polymorphisms, clinically significant testing for fetal blood group status was rapidly applied. The initial tests were performed on amniocyte-derived fetal DNA that was previously discarded during Liley-curve investigations. In the 1990s methods for RhD [4], Kell [5,6], Duffy [7], RhCcEe [8] were initially published. However, there are a substantial number of blood group antigens that can cause HDFN, most of which have been defined at the molecular level. It is therefore relatively straightforward to design PCR-based assays to detect fetal antigens from a source of fetal DNA. Based on the relatively high concentrations of DNA that can be obtained from amniocytes, most of these assays utilized allele-specific primers. The development of these assays has been previously reviewed by others and myself over a decade ago [9,10]. In 1997 Dennis Lo published his classic paper describing the presence of fetal DNA in maternal plasma and serum [11], many subsequent studies applied this knowledge into the clinical management of alloimmunized pregnancies [12,13]. All working in the field immediately recognized the power of testing without the inherent risk of further exacerbating alloimmunization by procedure-related hemorrhage of fetal blood into the maternal circulation, or in the worse case scenario spontaneous procedural-related fetal loss. Initial methods analyzed single regions of the RHD gene, but due to the extreme complexity of RH genetics [14,15] this approach was replaced by analysis of at least two regions of the RHD gene. In particular, the presence of RHDy in large numbers of African D-negative individuals resulted in the development of allele-specific primer methods to identify wild-type RHD and deliberate failure to amplify RHDy [16]. In order to ascertain that fetal DNA was present in samples types as RHD negative, the Y-chromosome gene SRY and marker DYS14 (located on the TSPY1 gene) [17] have been used effectively to type male D-negative fetuses (for review see [18]), whilst short tandem repeats or the fetal hypermethylated promoter of the RASSF1A gene have been used as Universal fetal markers [19,20]. This is regarded critical for RHD diagnosis of pregnancies to alloimmunized mothers, as the risk of scoring a false-negative result in such pregnancies is highly undesirable. 742
Mass-scale RHD testing
Several countries have applied mass scale RHD genotyping of all RhD-negative pregnancies, notably the Netherlands and Denmark. The drive to perform this is based on several criteria, of which some can be considered on economic grounds, others on ethics and safe clinical practice. First, on economic grounds approximately 40% of pregnancies to D-negative mothers will be carrying D-negative fetuses, which will not require the administration of prophylactic anti-D. Whilst the scale of savings has been debated, it still represents a situation where a therapeutic product is being administered unnecessarily. Prophylactic anti-D preparations are inherently safe, but as they are a human derived product have previously have been shown to be responsible for Hepatitis C infections [21], and thus have the potential to unwittingly infect mothers where preparations are contaminated with yet to be described viruses. Secondly, on ethical grounds it is not considered good practice to unnecessarily administer a humanderived product to a vulnerable patient group. Furthermore, the product is prepared from hyperimmunized male D-negative volunteers, and it is considered unethical to waste nearly 40% of this material on pregnancies that do not require it. The mass-scale application RHD analysis in maternal plasma has revealed a number of useful scientific observations regarding the presence of fetal DNA in maternal plasma. For example, a significant range of concentrations of free fetal DNA are found, with most false-negative results in a large scale study coming from a small cohort of mothers with extremely low concentrations of DNA in their plasma. It is these results that cause most concern in diagnostic testing, false positives are less concerning as they will lead to the unnecessary but safe administration of prophylactic anti-D to D-negative fetuses, which is the standard procedure in most countries in any case. In some instances, free fetal DNA concentrations can be used diagnostically for example, prediction of risk of preeclampsia or preterm labor where elevated or depressed levels of fetal DNA in maternal plasma can be used diagnostically [22,23]. New diagnostic approaches to the clinical management of HDFN
Since the late 1990s real-time PCR has been the chosen approach for the diagnostic detection of fetal blood group genotype. The methodology is robust, sensitive and has shown a high degree of accuracy. Most published methods involve the use of allele-specific primers coupled with Taqman probes, and for RHD assessment of at least two regions of the RHD gene. New methods are beginning to emerge, and may provide additional opportunities for more accurate assessment of fetal DNA (especially for RHD). This may include the Multiplex Ligationdependent probe amplification assay for RH [24], digital PCR and possibly next generation sequencing (see below). This is important (whilst noting the widespread abundance of real-time PCR instruments, low cost and significant technical experience) because low levels of fetal DNA found in maternal plasma, especially during the first trimester, and the continued drive for the universal fetal marker for control in prenatal diagnosis. Expert Rev. Hematol. 7(6), (2014)
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Prenatal testing for hemolytic disease and fetal neonatal alloimmune thrombocytopenia
Recent elegant studies by Fan et al., [25] and Yu et al., [26], has shown by paired end massively parallel sequencing of free DNA in plasma samples that free fetal DNA has a predominant 143bp population of molecules, whilst maternal free DNA around 166bp. Thus, by bioinformatic analysis of the sequence reads from samples obtained from maternal plasma samples, preferential analysis of the population of 143bp sequence reads gives comparative fetal-specificity, and a measure of the abundance of fetal DNA in any tested sample. Clearly this analysis can be used in preference to methylated fetal markers, fetal sex or other identified genetic differences, as a control for the quantity and quality of fetal DNA. Whilst massive parallel sequencing (so called next generation sequencing) is now comparatively expensive, the costs have dramatically reduced in the past few years, and may eventually be the first choice for prenatal diagnosis of the fetus. We have recently shown (Sillence et al., in preparation) that digital droplet PCR (ddPCR) can successfully be applied to RHD genotyping maternal plasma samples. We have found that dPCR is inherently more sensitive, more amenable to high throughput methods and is cheaper than real-time PCR. Fetal neonatal alloimmune thrombocytopenia
FNAIT is an analogous disease to HDFN in that it is caused by alloimmunization to platelet antigens carried by fetal platelets but absent from the mother. The condition results in fetal and neonatal thrombocytopenia that can lead to intracranial hemorrhage, and subsequent fetal death or brain damage. The bulk of cases in Europe and North America are caused by antiHPA-1a, but several other antibodies to various HPA antigens have been cited to cause FNAIT in the literature. Notably, in different populations different HPA antigens predominate as the most prevalent cause of FNAIT. For example, in Finland HPA-6bw is the most frequent [27], whilst in Asians antiHPA-21bw [28]. Platelet genotyping has a long heritage [29] for adult platelet transfusion matching, and maternal plasma based testing for these alleles should be straightforward [30,31]. Non-invasive testing for HPA-1A using maternal plasma as a source of fetal DNA has recently been described [32,33]. Notably in the method described by Scheffer et al., the large maternal background of HPA-1b genomic DNA is destroyed by the restriction endonuclease MspI, and this is followed by the allele- specific amplification of HPA-1A using allele-specific primers. Mass – scale HPA-1 genotyping of fetuses carried by HPA-1b1b mothers will be of questionable value as the majority of fetuses will inherit a paternal HPA-1a allele of which the most will be HPA-1a1a. Toward prophylactic anti-HPA-1a?
As almost 10% of HPA-1b1b mothers will make anti-HPA1a when exposed to their fetal platelets [34] prophylactic antiHPA1a treatment may provide viable prevention of this alloimmunization event. For this reason treatment of HPA-1b1b mothers with anti-HPA-1a to eliminate antigen positive platelets from the maternal circulation is being considered for trial in Norway [35]. This approach would require that mothers are informahealthcare.com
Special Report
HPA-1 genotyped at an early stage in pregnancy, and there is a very high probability that these mothers would carry HPA-1a fetuses in all of their pregnancies. Proof of principle studies by NHS Blood and Transplant in the UK has shown that recombinant anti-HPA-1a behaves essentially the same way as maternal IgG1 in removal of platelets by macrophages [36]. This suggests that this reagent (named B2G1Deltanab), could be used as prophylactic treatment of HPA-1b1b mothers, as it will be required in significant amounts. Expert commentary
Noninvasive prenatal diagnosis for blood group and platelet antigens has been the pathfinder for the routine implementation of maternal plasma based testing in molecular diagnostics. NIPD for a host of single gene disorders, Aneuploidy, and fetal sex are now commonplace, and have largely eliminated the usage of invasive testing. Relatively cheap and reliable methods for noninvasive prenatal testing both for fetal blood group and platelet antigen status are now commonplace, and there should be no reason why laboratories are still using amniocyte or chorionic villus samples as the source of fetal DNA. Several countries have applied mass scale RHD NIPD for their entire D-negative pregnant population, to conserve stocks of prophylactic anti-D that is, produced by hyperimmunized male volunteers. There are some arguments [37] that mass-scale NIPD for RHD is not economically worthwhile, but this does not take account of supply issues of a human blood product, which ethically should not be wastefully administered to patients (D-negative mothers) that do not require it –approximately 40% of these mothers will carry D-negative fetuses. Despite over four decades of the highly successful usage of prophylactic anti-D, anti-HPA-1a prophylactic treatment is only just beginning to emerge as a viable approach. Clinical trials are being considered, and may prelude routine treatment of HPA-1b1b mothers (around 2% of the North American and European population). Mass scale non-invasive prenatal diagnosis for HPA-1 status is unlikely to have clinical utility as the majority of fetuses will inherit a paternal HPA-1a allele. Five-year view
Blood group and HPA genotyping are becoming more commonplace, as DNA-based technologies are emerging in transfusion molecular testing laboratories [38–44]. Thus, transfusion medicine has adopted molecular testing into routine use. For economies of scale and for concentration of skill sets and knowledge, specialist centers for molecular blood grouping are the most likely scenario. As National centers for mass-scale RHD prenatal typing have emerged in two European countries, this maybe replicated elsewhere in Europe, whilst this is not standard practice in North America due to limited application of maternal plasma based testing due to licensing issues [45]. Non-invasive prenatal diagnosis for HPA genotyping is less well utilized than RHD, and this may change over the next 5 years. This may be predominantly because of utilization of recombinant anti-HPA1a in prophylactic treatment of HPA-1b1b mothers will be likely be introduced over that time period. 743
Special Report
Avent
Financial & competing interests disclosure
ND Avent is a member of the Transfusion Medicine Advisory Board of Grifols SA. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in
or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Key issues • Free fetal DNA based testing for most blood group alleles that cause hemolytic disease of the fetus & newborn and HPA-1A platelet
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antigens are in widespread use. • The use of amniotic fluid as a source of fetal DNA has largely been eliminated, and should be actively discouraged in preference to maternal plasma testing. • Mass scale application of fetal RHD genotyping has led to the focused and reduced use of prenatally administered prophylactic anti-D. • Whilst the economic value of applying prophylactic anti-D as directed by prenatal diagnosis is debated, there is no doubt that ethically wasteful use of a blood product obtained from male volunteers should be avoided. • A preliminary trial of the efficacy of prophylactic treatment of HPA-1b1b mothers with anti-HPA-1a is being considered in Norway, and has shown to be effective in human volunteers. This may pave the way to the routine prophylaxis in these mothers in the future.
References
9.
Papers of special note have been highlighted as: • of interest •• of considerable interest
Avent ND. Antenatal genotyping of the blood groups of the fetus. Vox Sang 1998; 74(Suppl 2):365-74
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Avent ND, Finning KM, Martin PG, Soothill PW. Prenatal determination of fetal blood group status. Vox Sang 2000; 78(Suppl 2):155-62
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Roopenian DC, Sun VZ. Clinical ramifications of the mhc family fc receptor fcrn. J Clin Immunol 2010;30:790-7
2.
Urbaniak SJ. Consensus conference on anti-d prophylaxis. April 7 & 8, 1997: final consensus statement. Royal College of Physicians of Edinburgh/Royal College of Obstetricians and Gynaecologists. Transfusion 1998;38(1):97-9
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Moise KJ. Hemolytic disease of the fetus and newborn. Clin Adv Hematol Oncol 2013;11:664-6 Bennett PR, Le Van Kim C, Colin Y, et al. Prenatal determination of fetal rhd type by DNA amplification. N Engl J Med 1993;329:607-10
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Lo YM, Corbetta N, Chamberlain PF, et al. Presence of fetal DNA in maternal plasma and serum. Lancet 1997;350:485-7
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Faas BH, Beuling EA, Christiaens GC, et al. Detection of fetal rhd-specific sequences in maternal plasma. Lancet 1998;352:1196
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Finning K, Martin P, Daniels G. A clinical service in the UK to predict fetal rh (rhesus) d blood group using free fetal DNA in maternal plasma. Ann N Y Acad Sci 2004;1022:119-23
19.
Scheffer PG, de Haas M, van der Schoot CE. The controversy about controls for fetal blood group genotyping by cell-free fetal DNA in maternal plasma. Curr Opin Hematol 2011;18:467-73
•
A general assessment of whether fatal identifiers are required during noninvasive prenatal diagnostic investigations.
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Chan KC, Ding C, Gerovassili A, et al. Hypermethylated rassf1a in maternal plasma: a universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006;52: 2211-18
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Kenny-Walsh E. Clinical outcomes after hepatitis c infection from contaminated anti-d immune globulin. Irish hepatology research group. N Engl J Med 1999;340: 1228-33
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Jakobsen TR, Clausen FB, Rode L, et al. High levels of fetal DNA are associated with increased risk of spontaneous preterm delivery. Prenat Diagn 2012;32:840-5
14.
Lee S, Wu X, Reid M, et al. Molecular basis of the kell (k1) phenotype. Blood 1995;85:912-16
Avent ND, Reid ME. The rh blood group system: a review. Blood 2000;95:375-87
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Avent ND, Martin PG. Kell typing by allele-specific pcr (asp). Br J Haematol 1996;93:728-30
Daniels G. Variants of rhd –current testing and clinical consequences. Br J Haematol 2013;161:461-70
16.
Finning KM, Martin PG, Soothill PW, Avent ND. Prediction of fetal d status from maternal plasma: introduction of a new noninvasive fetal rhd genotyping service. Transfusion 2002;42:1079-85
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Jakobsen TR, Clausen FB, Rode L, et al. Identifying mild and severe preeclampsia in asymptomatic pregnant women by levels of cell-free fetal DNA. Transfusion 2013;53: 1956-64
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Vogel T, Schmidtke J. Structure and function of tspy, the y-chromosome gene coding for the "testis-specific protein". Cytogenet Cell Genet 1998;80:209-13
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Avent ND, Chitty LS. Non-invasive diagnosis of fetal sex; utilisation of free fetal DNA in maternal plasma and ultrasound. Prenat Diagn 2006;26:598-603
Haer-Wigman L, Veldhuisen B, Jonkers R, et al. Rhd and rhce variant and zygosity genotyping via multiplex ligation-dependent probe amplification. Transfusion 2013;53: 1559-74
25.
Fan HC, Blumenfeld YJ, Chitkara U, et al. Analysis of the size distributions of fetal and
Olsson ML, Hansson C, Avent ND, et al. A clinically applicable method for determining the three major alleles at the duffy (fy) blood group locus using polymerase chain reaction with allele-specific primers. Transfusion 1998;38:168-73 Le Van Kim C, Mouro I, Brossard Y, et al. Pcr-based determination of rhc and rhe status of fetuses at risk of rhc and rhe haemolytic disease. Br J Haematol 1994;88: 193-5
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Prenatal testing for hemolytic disease and fetal neonatal alloimmune thrombocytopenia
Expert Review of Hematology Downloaded from informahealthcare.com by Nanyang Technological University on 04/29/15 For personal use only.
maternal cell-free DNA by paired-end sequencing. Clin Chem 2010;56:1279-86 ••
First indicator that sizing of DNA fragments can be used to identify fetal DNA.
26.
Yu SC, Chan KC, Zheng YW, et al. Size-based molecular diagnostics using plasma DNA for noninvasive prenatal testing. Proc Natl Acad Sci USA 2014;111: 8583-8
••
27.
28.
Analysis of next generation sequencing data reveals specific fetal fragmentation sizes that can be used for fetal-specific genotyping. Ghevaert C, Rankin A, Huiskes E, et al. Alloantibodies against low-frequency human platelet antigens do not account for a significant proportion of cases of fetomaternal alloimmune thrombocytopenia: evidence from 1054 cases. Transfusion 2009;49:2084-9 Koh Y, Ishii H, Amakishi E, et al. The first two cases of neonatal alloimmune thrombocytopenia associated with the low-frequency platelet antigen hpa-21bw (nos) in Japan. Transfusion 2012;52: 1468-75
29.
Metcalfe P. Platelet antigens and antibody detection. Vox Sang 2004;87(Suppl1):82-6
30.
Bertrand G, Kaplan C. How do we treat fetal and neonatal alloimmune thrombocytopenia? Transfusion 2014;54(7): 1698-703
•
Good general review on fetal neonatal alloimmune thrombocytopenia.
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Gunnink SF, Vlug R, Fijnvandraat K, et al. Neonatal thrombocytopenia: etiology, management and outcome. Expert Rev Hematol 2014;7:387-95
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anti-d prophylaxis in England and Wales. BMC Pregnancy Childbirth 2011;11:5 38.
Avent ND. Large scale blood group genotyping. Transfus Clin Biol 2007;14: 10-15
•
Good review on fetal neonatal alloimmune thrombocytopenia.
39.
32.
Le Toriellec E, Chenet C, Kaplan C. Safe fetal platelet genotyping: new developments. Transfusion 2013;53:1755-62
Avent ND. Large-scale blood group genotyping: clinical implications. Br J Haematol 2009;144:3-13
40.
33.
Scheffer PG, Ait Soussan A, Verhagen OJ, et al. Noninvasive fetal genotyping of human platelet antigen-1a. BJOG 2011;118:1392-5
Daniels G, van der Schoot CE, Olsson ML. Report of the fourth international workshop on molecular blood group genotyping. Vox Sang 2011;101:327-32
41.
•
HPA-1 genotyping using a novel approach to reduce maternal background.
34.
Semple JW. Processed platelet hpa1a peptides au naturel. Blood 2009;114:1724-5
Veldhuisen B, van der Schoot CE, de Haas M. Blood group genotyping: from patient to high-throughput donor screening. Vox Sang 2009;97:198-206
42.
Westhoff CM. The potential of blood group genotyping for transfusion medicine practice. Immunohematol 2008;24:190-5
43.
Flegel WA. Blood group genotyping in Germany. Transfusion 2007;47:47S-53S
44.
Daniels G, Finning K, Martin P, Summers J. Fetal blood group genotyping: present and future. Ann N Y Acad Sci 2006;1075:88-95
45.
Agarwal A, Sayres LC, Cho MK, et al. Commercial landscape of noninvasive prenatal testing in the United States. Prenat Diagn 2013;33:521-31
•
Excellent overview of the IP landscape surrounding non-invasive prenatal diagnosis.
35.
36.
••
37.
Kjeldsen-Kragh J, Ni H, Skogen B. Towards a prophylactic treatment of hpa-related foetal and neonatal alloimmune thrombocytopenia. Curr Opin Hematol 2012;19:469-74 Ghevaert C, Herbert N, Hawkins L, et al. Recombinant hpa-1a antibody therapy for treatment of fetomaternal alloimmune thrombocytopenia: proof of principle in human volunteers. Blood 2013;122:313-20 Pathway to mass scale HPA-1A Ig prophylaxis needs massive production of antibody, this paper describes this potentially. Szczepura A, Osipenko L, Freeman K. A new fetal rhd genotyping test: costs and benefits of mass testing to target antenatal
745
Special Report
Prenatal testing for hemolytic disease of the newborn and fetal neonatal alloimmune thrombocytopenia – current status Expert Rev. Hematol. 7(6), 741–745 (2014)
Neil D Avent Plymouth University Peninsula Schools of Medicine and Dentistry, Portland Square Building, Drake Circus, Plymouth, PL4 8AA, UK [email protected]
Incompatibility of red cell and platelet antigens can lead to maternal alloimmunization causing hemolytic disease of the fetus & newborn and fetal neonatal alloimmune thrombocytopenia respectively. As the molecular background of these polymorphisms emerged, prenatal testing using initially fetal DNA obtained from invasively obtained amniotic fluid or chorionic villus was implemented. This evolved into testing using maternal plasma as source of fetal DNA, and this is in routine use as a safe non-invasive diagnostic that has no risk to the fetus of alloimmunization or spontaneous miscarriage. These tests were initially applied to high risk pregnancies, but has been applied on a mass scale, to screen fetuses in D-negative pregnant populations as national screening programs. Fetal neonatal alloimmune thrombocytopenia management has had comparatively small take up in non-invasive testing for causative fetal platelet alleles (e.g., HPA-1A), but mass scale genotyping of mothers to identify at risk HPA-1b1b pregnancies and their treatment with prophylactic anti-HPA-1A is being considered in at least one country (Norway). KEYWORDS: alloimmune diseases of the fetus • blood group genotyping • fetal neonatal alloimmune thrombocytopenia • hemolytic disease of fetus and newborn • non-invasive prenatal diagnosis
Alloimmune diseases of the fetus & newborn
Incompatibility of either red cell or platelet antigens between mother and fetus can lead to alloimmunization, and maternal IgG (thus primarily in secondary immune responses) transfer across the placenta which leads to red cell destruction (hemolytic disease of the newborn and fetus, hemolytic disease of the fetus & newborn [HDFN]) or platelet destruction (fetal neonatal alloimmune thromobocytopenia, fetal neonatal alloimmune thrombocytopenia [FNAIT]). The maternal IgG is transferred via the FcRN transporter that binds IgG at acidic pH (maternal face of placenta) and releases it at neutral/above pH 7.0. FcRN is a MHC class I family member comprising a heavy chain, (Fcgrt) and forms a heterodimer with its light chain, b2 microglobulin [1]. informahealthcare.com
10.1586/17474086.2014.970160
FcRN is involved in maintaining the correct plasma concentrations of IgG (and albumin) in adults, but also facilitates the transport of IgG to the fetus prior to birth and from colostrum and milk in the neonatal period. Thus the FcRN transport system is the cause of placental transfer of the pathogenic IgGs in both HDFN and NAITP. The clinical management of both HDFN and FNAIT involves the detection of blood group or platelet specific antibodies in the mother, followed by escalated clinical management in high-risk pregnancies. For HDFN this largely is focused on D-negative mothers, where antenatal administered prophylactic anti-D has drastically reduced the incidence of fetal anemia [2]. For alloimmunization due to other blood group antigen incompatibility, there is no prophylactic treatment regime currently available, but prenatal diagnosis offers
2014 Informa UK Ltd
ISSN 1747-4086
741
Special Report
Avent
the opportunity to identify antigen-negative fetuses that do not require further treatment [3]. This is especially important as correction of either fetal anemia or thrombocytopenia may involve in utero transfusion of red cells or platelets, which is an inherently risky procedure.
Expert Review of Hematology Downloaded from informahealthcare.com by Nanyang Technological University on 04/29/15 For personal use only.
Hemolytic disease of the fetus & newborn – prenatal testing for blood group antigens
Molecular testing for HDFN can now be broadly categorized into two distinct clinical groups (especially for RhD) [1], the testing of alloimmunized mothers to inform potential further clinical intervention (e.g., in utero transfusion) [2]. To identify mothers not requiring prenatal prophylactic treatment of antiD as they carry D-negative fetuses. Rapidly following the cloning of various blood group active cDNAs and determination of blood group critical single nucleotide polymorphisms, clinically significant testing for fetal blood group status was rapidly applied. The initial tests were performed on amniocyte-derived fetal DNA that was previously discarded during Liley-curve investigations. In the 1990s methods for RhD [4], Kell [5,6], Duffy [7], RhCcEe [8] were initially published. However, there are a substantial number of blood group antigens that can cause HDFN, most of which have been defined at the molecular level. It is therefore relatively straightforward to design PCR-based assays to detect fetal antigens from a source of fetal DNA. Based on the relatively high concentrations of DNA that can be obtained from amniocytes, most of these assays utilized allele-specific primers. The development of these assays has been previously reviewed by others and myself over a decade ago [9,10]. In 1997 Dennis Lo published his classic paper describing the presence of fetal DNA in maternal plasma and serum [11], many subsequent studies applied this knowledge into the clinical management of alloimmunized pregnancies [12,13]. All working in the field immediately recognized the power of testing without the inherent risk of further exacerbating alloimmunization by procedure-related hemorrhage of fetal blood into the maternal circulation, or in the worse case scenario spontaneous procedural-related fetal loss. Initial methods analyzed single regions of the RHD gene, but due to the extreme complexity of RH genetics [14,15] this approach was replaced by analysis of at least two regions of the RHD gene. In particular, the presence of RHDy in large numbers of African D-negative individuals resulted in the development of allele-specific primer methods to identify wild-type RHD and deliberate failure to amplify RHDy [16]. In order to ascertain that fetal DNA was present in samples types as RHD negative, the Y-chromosome gene SRY and marker DYS14 (located on the TSPY1 gene) [17] have been used effectively to type male D-negative fetuses (for review see [18]), whilst short tandem repeats or the fetal hypermethylated promoter of the RASSF1A gene have been used as Universal fetal markers [19,20]. This is regarded critical for RHD diagnosis of pregnancies to alloimmunized mothers, as the risk of scoring a false-negative result in such pregnancies is highly undesirable. 742
Mass-scale RHD testing
Several countries have applied mass scale RHD genotyping of all RhD-negative pregnancies, notably the Netherlands and Denmark. The drive to perform this is based on several criteria, of which some can be considered on economic grounds, others on ethics and safe clinical practice. First, on economic grounds approximately 40% of pregnancies to D-negative mothers will be carrying D-negative fetuses, which will not require the administration of prophylactic anti-D. Whilst the scale of savings has been debated, it still represents a situation where a therapeutic product is being administered unnecessarily. Prophylactic anti-D preparations are inherently safe, but as they are a human derived product have previously have been shown to be responsible for Hepatitis C infections [21], and thus have the potential to unwittingly infect mothers where preparations are contaminated with yet to be described viruses. Secondly, on ethical grounds it is not considered good practice to unnecessarily administer a humanderived product to a vulnerable patient group. Furthermore, the product is prepared from hyperimmunized male D-negative volunteers, and it is considered unethical to waste nearly 40% of this material on pregnancies that do not require it. The mass-scale application RHD analysis in maternal plasma has revealed a number of useful scientific observations regarding the presence of fetal DNA in maternal plasma. For example, a significant range of concentrations of free fetal DNA are found, with most false-negative results in a large scale study coming from a small cohort of mothers with extremely low concentrations of DNA in their plasma. It is these results that cause most concern in diagnostic testing, false positives are less concerning as they will lead to the unnecessary but safe administration of prophylactic anti-D to D-negative fetuses, which is the standard procedure in most countries in any case. In some instances, free fetal DNA concentrations can be used diagnostically for example, prediction of risk of preeclampsia or preterm labor where elevated or depressed levels of fetal DNA in maternal plasma can be used diagnostically [22,23]. New diagnostic approaches to the clinical management of HDFN
Since the late 1990s real-time PCR has been the chosen approach for the diagnostic detection of fetal blood group genotype. The methodology is robust, sensitive and has shown a high degree of accuracy. Most published methods involve the use of allele-specific primers coupled with Taqman probes, and for RHD assessment of at least two regions of the RHD gene. New methods are beginning to emerge, and may provide additional opportunities for more accurate assessment of fetal DNA (especially for RHD). This may include the Multiplex Ligationdependent probe amplification assay for RH [24], digital PCR and possibly next generation sequencing (see below). This is important (whilst noting the widespread abundance of real-time PCR instruments, low cost and significant technical experience) because low levels of fetal DNA found in maternal plasma, especially during the first trimester, and the continued drive for the universal fetal marker for control in prenatal diagnosis. Expert Rev. Hematol. 7(6), (2014)
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Prenatal testing for hemolytic disease and fetal neonatal alloimmune thrombocytopenia
Recent elegant studies by Fan et al., [25] and Yu et al., [26], has shown by paired end massively parallel sequencing of free DNA in plasma samples that free fetal DNA has a predominant 143bp population of molecules, whilst maternal free DNA around 166bp. Thus, by bioinformatic analysis of the sequence reads from samples obtained from maternal plasma samples, preferential analysis of the population of 143bp sequence reads gives comparative fetal-specificity, and a measure of the abundance of fetal DNA in any tested sample. Clearly this analysis can be used in preference to methylated fetal markers, fetal sex or other identified genetic differences, as a control for the quantity and quality of fetal DNA. Whilst massive parallel sequencing (so called next generation sequencing) is now comparatively expensive, the costs have dramatically reduced in the past few years, and may eventually be the first choice for prenatal diagnosis of the fetus. We have recently shown (Sillence et al., in preparation) that digital droplet PCR (ddPCR) can successfully be applied to RHD genotyping maternal plasma samples. We have found that dPCR is inherently more sensitive, more amenable to high throughput methods and is cheaper than real-time PCR. Fetal neonatal alloimmune thrombocytopenia
FNAIT is an analogous disease to HDFN in that it is caused by alloimmunization to platelet antigens carried by fetal platelets but absent from the mother. The condition results in fetal and neonatal thrombocytopenia that can lead to intracranial hemorrhage, and subsequent fetal death or brain damage. The bulk of cases in Europe and North America are caused by antiHPA-1a, but several other antibodies to various HPA antigens have been cited to cause FNAIT in the literature. Notably, in different populations different HPA antigens predominate as the most prevalent cause of FNAIT. For example, in Finland HPA-6bw is the most frequent [27], whilst in Asians antiHPA-21bw [28]. Platelet genotyping has a long heritage [29] for adult platelet transfusion matching, and maternal plasma based testing for these alleles should be straightforward [30,31]. Non-invasive testing for HPA-1A using maternal plasma as a source of fetal DNA has recently been described [32,33]. Notably in the method described by Scheffer et al., the large maternal background of HPA-1b genomic DNA is destroyed by the restriction endonuclease MspI, and this is followed by the allele- specific amplification of HPA-1A using allele-specific primers. Mass – scale HPA-1 genotyping of fetuses carried by HPA-1b1b mothers will be of questionable value as the majority of fetuses will inherit a paternal HPA-1a allele of which the most will be HPA-1a1a. Toward prophylactic anti-HPA-1a?
As almost 10% of HPA-1b1b mothers will make anti-HPA1a when exposed to their fetal platelets [34] prophylactic antiHPA1a treatment may provide viable prevention of this alloimmunization event. For this reason treatment of HPA-1b1b mothers with anti-HPA-1a to eliminate antigen positive platelets from the maternal circulation is being considered for trial in Norway [35]. This approach would require that mothers are informahealthcare.com
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HPA-1 genotyped at an early stage in pregnancy, and there is a very high probability that these mothers would carry HPA-1a fetuses in all of their pregnancies. Proof of principle studies by NHS Blood and Transplant in the UK has shown that recombinant anti-HPA-1a behaves essentially the same way as maternal IgG1 in removal of platelets by macrophages [36]. This suggests that this reagent (named B2G1Deltanab), could be used as prophylactic treatment of HPA-1b1b mothers, as it will be required in significant amounts. Expert commentary
Noninvasive prenatal diagnosis for blood group and platelet antigens has been the pathfinder for the routine implementation of maternal plasma based testing in molecular diagnostics. NIPD for a host of single gene disorders, Aneuploidy, and fetal sex are now commonplace, and have largely eliminated the usage of invasive testing. Relatively cheap and reliable methods for noninvasive prenatal testing both for fetal blood group and platelet antigen status are now commonplace, and there should be no reason why laboratories are still using amniocyte or chorionic villus samples as the source of fetal DNA. Several countries have applied mass scale RHD NIPD for their entire D-negative pregnant population, to conserve stocks of prophylactic anti-D that is, produced by hyperimmunized male volunteers. There are some arguments [37] that mass-scale NIPD for RHD is not economically worthwhile, but this does not take account of supply issues of a human blood product, which ethically should not be wastefully administered to patients (D-negative mothers) that do not require it –approximately 40% of these mothers will carry D-negative fetuses. Despite over four decades of the highly successful usage of prophylactic anti-D, anti-HPA-1a prophylactic treatment is only just beginning to emerge as a viable approach. Clinical trials are being considered, and may prelude routine treatment of HPA-1b1b mothers (around 2% of the North American and European population). Mass scale non-invasive prenatal diagnosis for HPA-1 status is unlikely to have clinical utility as the majority of fetuses will inherit a paternal HPA-1a allele. Five-year view
Blood group and HPA genotyping are becoming more commonplace, as DNA-based technologies are emerging in transfusion molecular testing laboratories [38–44]. Thus, transfusion medicine has adopted molecular testing into routine use. For economies of scale and for concentration of skill sets and knowledge, specialist centers for molecular blood grouping are the most likely scenario. As National centers for mass-scale RHD prenatal typing have emerged in two European countries, this maybe replicated elsewhere in Europe, whilst this is not standard practice in North America due to limited application of maternal plasma based testing due to licensing issues [45]. Non-invasive prenatal diagnosis for HPA genotyping is less well utilized than RHD, and this may change over the next 5 years. This may be predominantly because of utilization of recombinant anti-HPA1a in prophylactic treatment of HPA-1b1b mothers will be likely be introduced over that time period. 743
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Avent
Financial & competing interests disclosure
ND Avent is a member of the Transfusion Medicine Advisory Board of Grifols SA. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in
or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
Key issues • Free fetal DNA based testing for most blood group alleles that cause hemolytic disease of the fetus & newborn and HPA-1A platelet
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antigens are in widespread use. • The use of amniotic fluid as a source of fetal DNA has largely been eliminated, and should be actively discouraged in preference to maternal plasma testing. • Mass scale application of fetal RHD genotyping has led to the focused and reduced use of prenatally administered prophylactic anti-D. • Whilst the economic value of applying prophylactic anti-D as directed by prenatal diagnosis is debated, there is no doubt that ethically wasteful use of a blood product obtained from male volunteers should be avoided. • A preliminary trial of the efficacy of prophylactic treatment of HPA-1b1b mothers with anti-HPA-1a is being considered in Norway, and has shown to be effective in human volunteers. This may pave the way to the routine prophylaxis in these mothers in the future.
References
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