A high prevalence of neutrophil-specific antibodies in ELANE-mutated severe congenital neutropenia.

An assay for neutrophil-specific antibodies is frequently used in the workup of chronic severe neutropenia and is suggestive of autoimmune, or sporadically alloimmune neutropenia, rather than severe congenital neutropenia (SCN). We analyzed a neutropenia consortium database for the outcomes of antibody testing initiated before receiving genetic diagnosis in Polish SCN cohort. Test results, performed in a single reference laboratory, were available for 14 patients with ELANE-mutated SCN or cyclic neutropenia, and were frequently positive (36%). We note that the trigger for genetic studies in severe neutropenia should not be affected by antibody-positivity and should be clinically driven.

Noninvasive diagnostics of fetal KEL*01.01 allele from maternal plasma of immunized women using digital PCR protocols.

Allo-antibodies produced by K-negative pregnant women against a fetal K antigen from the Kell blood group system may cause hemolytic disease of the fetus and newborn (HDFN). Predicting the fetal K antigen using noninvasive prenatal testing (NIPT) is important for decisions concerning management of pregnancies. Digital and droplet digital PCR techniques permit the detection of fetal single nucleotide variant with a higher specificity and sensitivity than real-time polymerase chain reaction (PCR). AIM: The aim was to evaluate and compare protocols for fetal KEL*01.01 genotyping using different assays and digital PCR platforms. METHODS: DNA isolated from 59 pregnant women (9-39 weeks of gestation, 49 with anti-K) was tested using home-made and custom-ordered KEL*01.01/KEL*02 assays with Droplet Digital™ and QuantStudio™3D. The results were compared with fetal/neonatal genotypes/phenotypes. RESULTS: Fetal KEL*01.01 results using all tested protocols were concordant with fetal/neonatal KEL*01.01 genotypes/phenotypes. None of the tested combinations of assays or digital PCR platforms gave false KEL*01.01-negative results, but inconclusive KEL*01.01 reads were observed in all tested protocols. For 36 cases compared using two digital PCR platforms and assays, there were not statistically significant differences in a level of fetal KEL*01.01 fraction (p < .72). CONCLUSION: Independent of the applied dPCR and ddPCR platforms and KEL*01.01 assays, prediction of the fetal KEL*01.01 is highly reliable. Before implementation in routine practice further validation of the KEL*01.01 protocol with a larger group of pregnant women should be performed.

Prediction of fetal blood group antigens from maternal plasma using Ion AmpliSeq HD technology.

BACKGROUND: Fetal blood group (BG) and platelet (HPA) antigens may trigger maternal immunization, causing a fetal disease. Noninvasive prenatal diagnostics (NIPT) predicts fetal genotype, identifying pregnancies with no risk. All current techniques detect fetal antigen alleles with unspecific background and without estimation of fetal fraction, thus new protocols for detection of fetal BG/HPA alleles with ultrahigh sensitivity still need to be tested to improve NIPT. AIM: To design NIPT of clinically important antigens using Ion AmpliSeq HD technology. METHODS: Plasma DNA from 36 pregnant women (9-33 week of gestation, 24 immunized with anti-HPA-1a,-3b,-15a, -K, or -D+C+S), with known BG/HPA genotypes of their neonates/partners, was tested on Ion S5 System using the Ion AmpliSeq HD designer custom gene panel. NGS contained 25 rs-targets encoding relevant BG/HPA antigens and 10 markers. RESULTS: Using the NGS protocol, 76 out of 85 differences in fetal/maternal BG/HPA genotypes were determined in concentration above 2% fetal paternally inherited allele chimerism. The level of unspecific reads for BG/HPA alleles was below 0.87%. In 24 immunized women NGS revealed feto-maternal incompatibility in 11 cases (from 2.44% to 7.41%) and excluded in 10 (<0.05%), three cases had inconclusive results (1.79%, 0.19%, 0.11%). The presence of fetal DNA was confirmed in each case by detecting markers with at least 2% chimerism. CONCLUSION: The use of Ion AmpliSeq HD technology improves the prediction of feto-maternal incompatibility, increasing the sensitivity of BG/HPA NIPT and serving confirmation of the fetal DNA at the same workflow.

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

BACKGROUND AND OBJECTIVES: Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. MATERIALS AND METHODS: We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. RESULTS: In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. CONCLUSION: These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

Prediction of fetal blood group and platelet antigens from maternal plasma using next-generation sequencing.

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.

Noninvasive prenatal HPA-1 typing in HPA-1a negative pregnancies selected in the Polish PREVFNAIT screening program.

BACKGROUND: Anti-HPA-1a alloantibodies in HPA-1a negative mothers can lead to fetal/neonatal alloimmune thrombocytopenia (FNAIT). Noninvasive prenatal testing (NIPT) of HPA-1a determines fetuses at risk and the course of maternal antenatal treatment. STUDY DESIGN AND METHODS: The aim was to develop and validate HPA-1a NIPT by real-time polymerase chain reaction (PCR) or next-generation sequencing (NGS) for a high-throughput screening setting. DNA from 328 plasma samples of 299 HPA-1a negative pregnant women was examined for HPA-1a by real-time PCR and in two cases also by NGS (Ion Torrent). The results were compared with neonatal HPA-1a genotyping in 281 cases. RESULTS: HPA-1a NIPT was negative in 44 of 51 HPA-1a negative fetuses, inconclusive in five, and false positive in two. In 228 of 229 HPA-1a positive fetuses, the NIPT results were positive (mean threshold cycle 36.0 ± 1.7) and inconclusive in one. In 22 cases with HPA-1a positive fetuses analyzed twice, the sensitivity of HPA-1a detection was significantly higher at 28 weeks compared with 16 to 20 weeks. NGS efficiently detected the ITGB3 coding HPA-1a/b (1% and 5% fetal HPA-1a reads). CONCLUSION: Real-time PCR is reliable to predict the fetal HPA-1a positive genotype in a screening study, but false-positive results are reported in 4%, with unnecessary prenatal treatment if anti-HPA-1a is detected.

Compound heterozygosity of two novel RHAG alleles leads to a considerable disruption of the Rh complex.

BACKGROUND: The Rhesus (Rh) complex consists of a core comprising the Rh proteins (RhD/RhCE) and the Rh-associated glycoprotein (RhAG) with accessory chains (GPB, LW, CD47). Molecular defects of the RHAG gene may cause a regulator Rhnull phenotype without Rh antigen expression or a Rhmod phenotype with decreased Rh antigen expression. STUDY DESIGN AND METHODS: Blood samples of a donor with strongly diminished Rh antigens and five family members were analyzed by serological phenotyping, flow cytometry, molecular testing, and gene expression analysis of Rh complex candidate genes. RESULTS: RHAG sequencing identified a missense mutation, c.241G>C (p.Gly81Arg) and a splice site mutation, c.640 + 3del14, among the cohort. Compound heterozygosity of these novel alleles identified in the propositus and two siblings gave rise to a strongly diminished expression of RhAG, Rh, and CD47 antigens on the RBC surface. CONCLUSION: The Rhmod phenotype was caused by a novel RHAG splice site mutation in association with a non-functional allele. The primary depression of RhAG is most likely due to posttranslational events that affect the interaction and processing of the RhAG glycoprotein and gave rise to a secondary depression of RhD, RhCE, and CD47, the major members of the Rh complex.

14 Years of Polish Experience in Non-Invasive Prenatal Blood Group Diagnosis.

BACKGROUND: Blood cell antigens may cause maternal alloimmunization leading to fetal/newborn disorders. Non-invasive prenatal diagnostics (NIPD) of the blood group permits the determination of feto-maternal incompatibility. AIM: To evaluate 14 years of blood group NIPD at the Institute of Hematology and Transfusion Medicine (IHTM) in Warsaw. METHODS: Plasma DNA from 536 RhD-negative, 24 Rhc-negative, 26 RhE-negative, 43 K-negative, and 42 HPA-1a-negative pregnant women was examined by real-time PCR to detect RHD, RHCE*c, RHCE*E, RHCE*C, KEL*01 and HPA*1A, respectively. We tested for CCR5, SRY or bi-allelic polymorphisms and quantified the total or fetal DNA. RESULTS: The results of fetal antigen status prediction by NIPD in all but one case (false-positive result of KEL*01) were correct taking neonate serology as a reference. It was confirmed that all negative results of NIPD contained fetal DNA except for four cases where there was no difference between the parents' polymorphisms. CONCLUSIONS: A fetal genotype compatible with the mother was determined in 25% of all pregnancies tested at the IHTM for the fetal blood group. These cases were not at risk of disease, so it was possible to avoid invasive procedures.

[PREVFNAIT prevention of foetal/neonatal alloimmune thrombocytopenia (FNAIT) in Polish foetuses and newborns--the PREVFNAIT program]. / Zapobieganie alloimmunologicznej maloplytkowosci plodów i noworodków (AIMPN) w Polsce--program PREVFNAIT.

The scientific goals related to the grant include 1) estimation of FNAIT prevalence in Poland and 2) search for biomarkers to predict the risk of the antibody production and severity of fetal thrombocytopenia. Fetal/Neonatal Alloimmune Thrombocytopenia (FNAIT) is caused by destruction of fetal blood platelets due to maternal antibodies. This condition, which most commonly results from incompatibility between the mother and the fetus for the Human Platelet Antigen-1a (HPA-1a), may lead to intracranial hemorrhage, damage of the central nervous system (CNS) and even death of the fetus or the newborn. It can be the cause of strokes in term newborns. FNAIT is usually attributed to the presence of anti-HPA-1a antibodies. Its incidence rate is estimated at approximately 1/1000-2000 live births. In the absence of a screening program, it is usually diagnosed after birth of a child with symptoms of thrombocytopenia or CNS hemorrhage. Monitoring of antibody production and thrombocytopenia treatment to effectively minimize the risk of stroke are therefore launched only at the next pregnancy. Testing indications are broader to include fetal ultrasound for symptoms of stroke to the CNS, ventricular enlargement or hydrocephalus, and obstetric failure. Diagnostic process is also recommended prior to the planned cordocentesis, in vitro fertilization and in sisters of mothers with children with FNAIT history. HPA-1a testing remains the best method for diagnosing pregnancies at risk. The detection frequency for FNAIT in Poland remains low. Therefore, the Institute of Hematology and Transfusion Medicine (IHTM) will have performed such HPA-1a antigen testing in 30 000 Polish women within the framework of the PREVFNAIT program by March 2016. HPA-1a negative women (2% of the population) are a risk group for production of anti- HPA-1a antibodies responsible for FNAIT therefore all of them will be monitored for the presence and activity of anti-HPA-1a antibodies. Such testing will be performed free of charge for the women.

RHD variants in Polish blood donors routinely typed as D-.

BACKGROUND: Blood donors exhibiting a weak D or DEL phenotypical expression may be mistyped D- by standard serology hence permitting incompatible transfusion to D- recipients. Molecular methods may overcome these technical limits. Our aim was to estimate the frequency of RHD alleles among the apparently D- 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- were tested by real-time polymerase chain reaction (PCR) for the presence of RHD-specific markers located in Intron 4 and Exons 7 and 10. RHD+ individuals were characterized by PCR or cDNA sequencing and serology. RESULTS: Plasma cross-pool strategy revealed 63 RHD+ 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, and RHD(IVS1-29G>C) and two novel alleles, RHD*(767C>G) (n = 3) and 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) Only 0.2% of D- Polish donors carry some fragments of the RHD gene; all of them were C or E+. 3) Almost 60% of the detected RHD alleles may be potentially immunogenic when transfused to a D- recipient.

What's with the boys? Lower birth weight in boys from HPA-1a alloimmunized pregnancies - New insights from a large prospective screening study in Poland.

Fetomaternal incompatibility in human platelet antigens (HPAs) can cause maternal alloimmunization, which in turn may lead to thrombocytopenia with or without intracranial hemorrhage (ICH) in the fetus or newborn. Retrospective studies suggest that boys from alloimmunized mothers may have higher risk of ICH and lower birth weight than girls. The objective of this study was to assess how maternal HPA-1a alloimmunization, sex of the neonate and birth weight relates in a large prospective cohort. Through a national screening study in Poland (PREVFNAIT) involving HPA-1 typing of 24,259 pregnant women during 2013-2017, 606 HPA-1a negative pregnant women and their offspring were identified and included. Various multivariate models were used to assess if and how maternal HPA-1a alloimmunization status was associated with birth weight and risk of having a small for gestational age (SGA) neonate, and if and how sex of the neonate mattered. Most immunized pregnancies had male fetuses (69 %). Women carrying a male fetus had increased likelihood of having an SGA newborn if they were HPA-1a alloimmunized compared to non-immunized mothers. Increasing maternal anti-HPA-1a antibody levels were significantly associated with reduced birth weight and SGA risk among male-fetus pregnancies, but not if the fetus was female. In conclusion, anti-HPA-1a antibodies in a male fetus pregnancy is associated with increased risk of SGA and lower birth weight, especially if the antibody level is high. Sex of the fetus may therefore be considered as a new clinical predictor of more severe FNAIT neonatal outcome.

[Reticulated platelet count used for differentiation of the type of thrombocytopenia in pregnant women]. / Przydatnosc badania retykuloplytek w róznicowaniu maloplytkowosci wystepujacej u kobiet ciezarnych.

BACKGROUND: Analysis of reticulated platelets (RP), the youngest form of platelets in peripheral blood, is useful for assessment of thrombopoietic response in thrombocytopenic conditions due to intense immunological platelet destruction. AIM: The aim of the study was to assess the value of RP measurement for differentiation between pregnancy-related thrombocytopenia (PT), immunological thrombocytopenia (IM) and hereditary thrombocytopenia (HT) in pregnant women with platelet count below 100 G/L. MATERIAL: The study included 49 pregnant thrombocytopenia women (21 with PT, 22 with IM, 6 with HR) as well as 22 healthy pregnant women (Control). METHODS: The percentage of RP in peripheral blood was measured using double staining with: PE-labeled anti CD41 (Dako) and thiazole orange (Beckton Dickinson). The measurements were performed several times during pregnancy (II and III trimester) and delivery. Anti-platelet antibodies were tested by immunofluorescence and immunoensimatic assays. HPA1a antigen was determined by PCR/SSP. RESULTS: The average platelet count in all groups of thrombocytopenia women was significantly lower than in control group. The mean RP percentage in the control group (5.31%) was within the range of the haematological normal value (0.5-6%), and for thrombocytopenia women it was: 9.19% for PT women, 14.75% for IM women and 14.94% for HT women and was significantly higher than that in the control group. In the group of IM pregnant women the RP percentage was significantly higher in the II trimester than in the PT women. Anti-platelet antibody and HPA1a antigen testing excluded alloimmunological/fetus/neonatal thrombocytopenia in the study material. CONCLUSION: RP analysis has been proved useful for preliminary differentiation of PT and IT in the II trimester of pregnancy. Higher RP percentage informs the physician of the likelihood of immunological thrombocytopenia in the pregnant woman as well as of the delivery of a thrombocytopenia child.

[Noninvasive prenatal diagnosis from maternal plasma in serological conflicts in Poland: current practice and future prospects]. / Nieinwazyjne badania prenatalne z osocza kobiet ciezarnych w konfliktach serologicznych w Polsce.

The principles and results of noninvasive prenatal diagnosis in serological conflicts in Poland were described in the following work. Noninvasive fetal genotyping from plasma of immunized mother identifies women carrying fetus without incompatible antigen and risk of hemolytic disease. The presence of fetal RHD gene or RHCE*c/E alleles in maternal plasma is examined routinely in Institute of Hematology and Blood Transfusion in Warsaw. Preliminary results of successful fetal KEL*1 genotyping from maternal blood were obtained.

[RHD variant in RhD/-/ mother with anti-D makes noninvasive fetal RHD genotyping impossible]. / Obecnosc "niemego" genu RHD u RhD ujemnej kobiety ciezarnej z przeciwcialami anty-RhD uniemozliwia okreslenie genotypu RHD plodu metoda nieinwazyjna.

OBJECTIVES: Noninvasive fetal RHD genotyping from maternal plasma of RhD(/-) pregnant women of Caucasian race may be used for predicting the risk of hemolytic disease because the RHD gene is usually absent in such populations. If detected in plasma of such women, the RHD gene originates from the RhD(+) fetus. The number of fetal copies of the gene in maternal plasma is extremely small. In the presented case of the RhD(/-) pregnant woman with anti-D it was impossible to give a fetal RHD result due to mother's RHD(+) genotype. The fetal RHD was determined from amniocytes. AIM: to present the difficulties related to the interpretation of results of invasive and noninvasive procedures. MATERIAL AND METHODS: whole blood, plasma and amniotic fluid of the RhD(-) woman with anti-D (14 week of pregnancy) as well as whole blood of the newborn. RHD and RHCE*c were genotyped by real-time PCR in DNA isolated from maternal plasma and amniocytes and the RHD and d-genotypes were tested by SSP methods in DNA isolated from whole blood and amniocytes. RESULTS: RHD and RHCE*c were detected in DNA isolated from plasma. The high level of RHD suggested its origin from the mother's DNA therefore it was impossible to determine the fetal RHD. The d-little test identified a RHD(IVS3+ 1G>A) variant in the mother's genome. A weak signal of real-time PCR for the RHD was obtained in amniocytes but the RHD was not detected by SSP. The RHCE*c was detected by both methods. Results were inconclusive; the fetal RHD status remained unknown. The child was RhD(-) with RHD in its DNA undetected by either method. CONCLUSIONS: 1/The RHD(IVS3+ 1G>A) variant in the RhD(-) mother precluded formal noninvasive fetal RHD genotyping. 2/Real-time PCR is too sensitive for amniocyte testing and may lead to false results as it detects trace maternal DNA in amniotic fluid. 3/The frequency of RHD(IVS3+1G>A) occurrence in Poland requires further studies.

[Estimation of diagnostic value of the middle cerebral artery peak systolic velocity in prediction of fetal anemia in pregnancies complicated by alloimmunisation]. / Ocena wartosci diagnostycznej oznaczania wskaznika MoM PSV MCA w przewidywaniu niedokrwistosci u plodu w konflikcie serologicznym.

UNLABELLED: The use of the middle cerebral artery peak systolic velocity (PSV) for the noninvasive diagnosis of fetal anemia in pregnancies complicated by alloimmunisation has the potential to reduce the number of invasive procedures. OBJECTIVES: The study was undertaken to determine the detection of fetal anemia by fetal middle cerebral artery peak systolic velocity (MCA PSV). MATERIAL AND METHODS: 31 fetuses with red cell alloimmunisation were evaluated with Doppler ultrasongraphy. On the basis of ROC (AUC) analysis the cutoff point of MoM=1.215 with the highest sensitivity and specificity was established. We examined the relation between MoM=1.215 and neonatal hemoglobin level and the maternal antibody titre in the indirect antiglobulin test. Sensitivity specificity positive and negative value and statistical significance were calculated. CONCLUSIONS: Data reported to date suggest that a threshold of 1.215 multiples of the median can be used to better diagnostic of fetal anemia.

[Anti-K antibodies in pregnant women and genotyping of K antigen in foetuses]. / Przeciwciala anty-K u kobiet ciezarnych oraz badania genu kodujacego antygen K u plodów.

AIM: 1) We have presented our experiment conducted to detect anti-K antibodies from the Kell-system in pregnant women and their connection with potential destruction of foetal red cells, which may result in haemolytic disease of the foetus and the newborn (HDFN). 2) We have also indicated serological and molecular methods important for a proper diagnosis. MATERIAL AND METHODS: 27 women with anti-K. Serological diagnosis of K antigen in fathers and children. KEL1 gene examination in foetuses from DNA isolated from foetal cells contained in amniotic fluid, using discrimination of alleles--real-time PCR method. RESULTS: Anti-K were detected in most women after blood transfusion, the fathers were usually K negative. Foetomaternal incompatibility was found in 6 out of 27 women. Haemolytic disease was observed in 5 cases: 3-severe, 1-fatal, 1-mild. Foetal genotyping allowed us to avoid cordocentesis in two pregnant women, it appeared that both foetuses have not received KEL1 gene from heterozygous (Kk) fathers--in the previous pregnancies the children had died because of HDFN. CONCLUSIONS: 1) In every pregnant woman with anti-K, the K antigen should be examined in the father. 2) In every K+ father the phenotype should be evaluated and if he is heterozygote (Kk), the fetal KEL1 gene must be examined, to avoid unnecessary cordocentesis. 3) If KEL1 gene is not detected in the foetus, HDFN will not occur. 4/Foetal KEL1 genotyping may be performed in all mothers with anti-K and heterozygous father in our Department, after providing the material from the amniocethesis.

A preliminary evaluation of next-generation sequencing as a screening tool for targeted genotyping of erythrocyte and platelet antigens in blood donors.

BACKGROUND: Matching the compatibility of donor blood with the recipient's antigens prevents alloimmunisation. Next-generation sequencing (NGS) technology is a promising method for extensive blood group and platelet antigen genotyping of blood donors. It circumvents the limitations of detecting known alleles based on predefined polymorphisms and enables targeted sequencing on a massive scale. The aim of this study was to evaluate the NGS AmpliSeq application on the Ion Torrent platform as a screening tool for genotyping blood donors' erythrocyte/platelet antigens. MATERIALS AND METHODS: Primers for regions encoding antigens RhD (exons 5, 7), Rhc, RhE/e, Fya/b, Jka/b, M/N, S/s, HPA-1, 2, 3, 5, 15 were designed with Ion AmpliSeq Designer with manual inclusion of RHCE*C primers. DNA libraries of 57 regular blood donors with determined phenotype/genotype (prepared using the Ion AmpliSeq Library Kit and 14 primer pairs) were sequenced on the Ion Torrent PGM using 316v2 chips and 200 bp chemistry. RESULTS: Sequencing was successful in all but the MN and HPA-5 regions. Mean sequencing coverage in one experiment was 4,606 reads, except for the RHCE*C region (mean 568 reads). NGS results agreed with the known phenotype/genotype of donors except in one phenotypically Fy(a+b-) case in whom FY*A/FY*B alleles were found. Reading rates for homozygotes were 97-100%, while they were around 50% for heterozygotes. NGS of RHD regions led to identification of mutations in two RhD negative donors. DISCUSSION: NGS can be performed as a screening test to determine erythrocyte/platelet antigens in blood donors. This method allowed testing of 48 donors for 14 features (200 bp long) with the depth of a few thousand reads simultaneously, and the estimation of natural chimerism or hemi/homozygotic status. NGS screening can be adjusted to the genetic background of a given tested population.

Identification and follow-up of pregnant women with platelet-type human platelet antigen (HPA)-1bb alloimmunized with fetal HPA-1a.

INTRODUCTION: Pregnant women negative for human platelet antigen 1a (HPA-1a) are at risk of alloimmunization with fetal HPA-1a antigen inherited from the father, and their offspring may develop fetal and neonatal alloimmune thrombocytopenia (FNAIT). The aim of this study was to analyze the frequency of HPA-1a alloimmunization in pregnant Polish women, the feasibility of using maternal platelets for intrauterine transfusions in women subjected to diagnostic fetal blood sampling (FBS) and to discuss potential consequences of alloimmunization. MATERIAL AND METHODS: Fifteen thousand two hundred and four pregnant women were typed for HPA-1a; HPA-1a negative were screened for anti-HPA-1a. Alloimmunized women received specialist perinatology care; some of them were subjected to FBS, followed by transfusion of HPA-1a negative platelet concentrates (PC) prepared from maternal blood. RESULTS: Three hundred seventy-three (2.5%) women were HPA-1a negative, and 32 (8.6%) tested positively for anti-HPA-1a. Antibodies were detected in 22 women during pregnancy. Diagnostic FBS followed by PC transfusion was performed in 14 woman, who were platelet donors for their 16 unborn babies. Blood donations were tolerated well by the patients, and also intrauterine platelet transfusions were uneventful. Pharmacotherapy with intravenous immunoglobulins was implemented in 11/22 patients. CONCLUSIONS: HPA-1a negative women (ca. 2.5% of all pregnant patients) are at risk of alloimmunization with HPA-1a antigen and developing FNAIT. Alloimmunized women can be donors of platelets for their offspring providing removal of antibodies from PC. Owing to potential complications, special care should be taken if an alloimmunized woman was qualified as a blood or stem cell recipient.