Alloimmunization against HPA-1a antigen is the most common cause of fetal/neonatal alloimmune thrombocytopenia (FNAIT) and often occurs during the first pregnancy. Thus, the question is should we introduce an antenatal screening for HPA-1a antigen/antibody? We screened 8013 pregnant women for HPA-1a by ELISA [1] and found 144 (1·8%) women to be HPA-1a-negative; the range reported for Caucasians being 1·6-2·5% [2]. All cases were confirmed by polymerase chain reaction sequence specific primers (PCR SSP) [3] to be HPA-1bb. Anti-HPA-1a was detected by monoclonal antibody immobilization of platelet antigens (MAIPA) [4] in 12 out of 122 HPA-1a-negative women (9·8%) available for further study. The incidence of HPA-1a immunization in 112 cases with a HPA-1a-positive neonate was 10·7%. Our results are comparable to those of Williamson et al . (11·9%) who also used the MAIPA for HPA-1a antibody detection [5]. Half of the HPA-1a immunized mothers were primigravida. Like others, we found that the negative predictive value of

Fetal RHD genotyping from maternal plasma may be used with confidence, although additional polymorphisms for confirmation of fetal DNA should be included for 100 percent predictive value (instead of 99.6%).

Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a relatively rare condition (1/1000–1/2000) that was granted orphan status by the European Medicines Agency in 2011. Clinical consequences of FNAIT, however, may be severe. A thrombocytopenic fetus or new-born is at risk of intracranial hemorrhage that may result in lifelong disability or death. Preventing such bleeding is thus vital and requires a solution. Anti-HPA1a antibodies are the most frequent cause of FNAIT in Caucasians. Its pathogenesis is similar to hemolytic disease of the newborn (HDN) due to anti-RhD antibodies, but is characterized by platelet destruction and is more often observed in the first pregnancy. In 75 % of these women, alloimmunization by HPA-1a antigens, however, occurs at delivery, which enables development of antibody-mediated immune suppression to prevent maternal immunization. As for HDN, the recurrence rate of FNAIT is high. For advancing diagnostic efforts and treatment, it is thereby crucial to understand the pathogenesis of FNAIT, including cellular immunity involvement. This review presents the current knowledge on FNAIT. Also described is a program for HPA-1a screening in identifying HPA-1a negative pregnant women at risk of immunization. This program is now performed at the Institute of Hematology and Transfusion Medicine in cooperation with the Department of Obstetrics and Gynecology of the Medical Centre of Postgraduate Education in Warsaw as well as the UiT The Arctic University of Norway.

The evaluation of immunization by the HNA1a and 1b antigens during pregnancy was based on (i) their genotyping in 1038 unselected mothers and newborns of homozygous mothers, (ii) granulocyte counting in all born infants and (iii) examination of granulocyte antibodies in maternal sera if an HNA1 incompatibile child was born. A total of 548 (52.8%) mothers were heterozygous--thus further examinations were not done. Four hundred and ninety (47.2%) were homozygous, of whom 203 (41.3%) delivered an incompatible child, i.e. 19.6% of all the infants. Among available sera from 195 mothers with feto-maternal incompatibility, the granulocyte-specific antibodies were found in nine (4.5%); six of these (3%) were HNA1 (four anti-1a, two anti-1b), and in three others the specificity was not determined. In the remaining 28 sera, the only antibodies detected were HLA. Hence, six out of 1000 pregnant women can be expected to develop anti-HNA1. In none of the newborns was the cord neutrophil count below 1.5 x 109 L-1 and signs of infection found, thus the incidence of NAIN seems to be lower than 1 per 1000 infants. A comparison with our previous, unpublished data suggests that the incidence of severe NAIN is roughly 1 per 6000 (four cases among 24101 newborns).

BACKGROUND Blood donors exhibiting a weak D or DEL phenotypical expression may be mistyped D negative by standard serology hence permitting incompatible transfusion to D negative recipients. Molecular methods may overcome these technical limits. Our aim was to estimate the frequency of RHD alleles among the apparently D negative Polish donor population and to characterize its molecular background. STUDY DESIGN AND METHODS Plasma pools collected from 31,200 consecutive Polish donors typed as D negative were tested by real-time PCR for the presence of RHD specific markers located in the intron 4, exons 7 and 10. RHD positive individuals were characterized by PCR or cDNA sequencing and serology. RESULTS Plasma cross-pool strategy revealed 63 RHD positive donors harboring RHD*01N.03(n=17), RHD*15(n=12), RHD*11(n=7), RHD*DEL8(n=3), RHD*01W.2(n=3), RHD-CE(10)(n=3), RHD*01W.3, RHD*01W.9, RHD*01N.05, RHD*01N.07, RHD*01N.23, RHD(IVS1-29G>C) and two novel alleles: RHD*(767C>G)(n=3), RHD*(1029C>A). Among 47 cases available for serology, 27 were shown to express the D antigen CONCLUSION 1/ Plasma cross-pool strategy is a reliable and cost-effective tool for RHD screening. 2/ 0.2% of D negative Polish donors carry some fragments of the RHD gene; all of them were C or E positive. 3/ Almost 60% of the detected RHD alleles may be potentially immunogenic when transfused to a D negative recipient.

BACKGROUND Fetuses whose mothers have produced antibodies to red blood cell (RBC) or platelet antigens are at risk of being affected by hemolytic disease or alloimmune thrombocytopenia, respectively, only if they inherit the incompatible antigen. Noninvasive diagnosis of the fetal antigen is employed for management of immunized pregnancies, but the specific detection of SNPs, encoding the majority of antigens, in maternal plasma is still a challenge. We applied targeted next‐generation sequencing (NGS) to predict the fetal antigen based on the detection of fetomaternal chimerism. METHODS AND MATERIALS The DNA of 13 pregnant women (with anti‐K [3] anti‐k [1], anti‐Fya [1], anti‐D + C + Jka [1], anti‐D + E + K [1], anti‐HPA‐1a [1], anti‐HPA‐3b [1], anti‐HPA‐5b [1], and nonimmunized [3]) was sequenced using primers for regions encoding RhD, RhC, Rhc, RhE/e, K/k, Fya/b, Jka/b, MN, Ss, and HPA‐1, 2, 3, 5, 15, 4 X‐polymorphisms on the Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). RESULTS NGS results were in agreement with the phenotype/genotype of women and their neonates (except for the unsuccessful detection of MN and RhC). NGS determined fetal allele chimerism for K, k, Fya, Fyb, Jka, Jkb, S, RhE (from 0.42% to 6.08%); RhD, Rhc (100%); HPA‐1a, −2b, −3a, 3b, −5b, −15a, 15b (from 0.23% to 4.11%). NGS revealed fetal chimerism for incompatible antigens (from 0.7% to 4.8%) in 7 immunized cases, excluded in 3 (with anti‐K, anti‐Fya, anti‐HPA‐3b). CONCLUSION The designed NGS predicts the fetal RBC and platelet antigen status universally in cases with various clinically significant antibodies as well as providing confirmation of the presence of fetal DNA. However, some improvement of the unsuccessful primers is required.

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Anti-HPA-15 antibody detection should be based on carefully selected platelets with high HPA-15 expression level.