Back to base pairs: What is the genetic risk for red bloodcell alloimmunization?

Red blood cell (RBC) alloimmunization is a serious complication of blood transfusions, challenging selection of compatible units for future transfusions. Genetic characteristics may be associated with the risk of RBC alloimmunization and may therefore serve to identify high-risk patients. The aim of this systematic review was to summarize the available evidence on genetic risk factors for RBC alloimmunization. Electronic databases were searched up to April 2020 for studies (Search terms included transfusion, alloimmunization and genetic). A total of 2581 alloimmunized cases and 26,558 controls were derived from 24 studies. The alleles that were most frequently studied and that demonstrated significant associations in a meta-analysis with alloimmunization to the Duffya antigen were HLA-DRB1*04 (Odds Ratio 7.80 (95%CI 4.57-13.33)), HLA-DRB1*15 (OR 3.76 (95%CI 2.14-6.59)), and HLA-DRB1*03 (OR 0.12 (95%CI 0.05-0.29)). Furthermore, significant associations with anti-K formation was found for the alleles HLA-DRB1*10 (OR 2.64 (95%CI 1.41-4.95)), HLA*DRB1*11 (OR 2.11, (95%CI 1.34-3.32)), and HLA-DRB1*13 (OR 1.71 (95%CI 1.26-2.33)). Overall, the available evidence was of moderate to low quality, hampering interpretation of reported results. There is an urgent need for high quality evidence on genetic risk factors for RBC alloimmunization.

RHD genotype and zygosity analysis in the Chinese Southern Han D+, D- and D variant donors using the multiplex ligation-dependent probe amplification assay.

BACKGROUND AND OBJECTIVES: Several comprehensive genotyping platforms for determining red blood cell (RBC) antigens have been established and validated for use in the Caucasian and Black populations, but not for the Chinese. The multiplex ligation-dependent probe amplification (MLPA) assay was validated for RHD genotyping in the Chinese. MATERIALS AND METHODS: The blood samples of 200 D+, 200 D- and 62 D variant Chinese donors were collected. RhD antigen was routinely typed by serological method. D variant phenotype was determined by an anti-D panel (D-Screen), when RBCs were available. The RHD genotype and its zygosity were analysed with the RH-MLPA technique. When the MLPA was unable to identify a RHD variant, direct sequencing of all exons of the RHD gene was performed. RESULTS: In 200 D+ donors, DD (168/200, 84%), D (12/200, 6%), DDD genotype (1/200) and D variant allele carriers (19/200, 9·5%) were found. In 200 D- donors, six reported RHD alleles, RHD*01EL.01, RHD*01N.03, RHD*01N.05, RHD*01N.16, RHD*DFR2 and RHD*weak partial 15 and one novel RHD*1154T allele were identified in 36·5% (73/200) of them. In 62 D variant donors, three novel RHD alleles, RHD*79_81delCTC, RHD*710T and RHD*689A, and twelve reported alleles, RHD*DVI.3, RHD*weak partial 15, RHD*DVI.4, RHD*01EL.01, RHD*01N.03, RHD*DLO, RHD*DV.5, RHD*D-CE(2-10), RHD*730C, RHD*weak D type 25, 33 and 72, were identified, either alone or in combination. CONCLUSION: The RH-MLPA assay correctly identified the common RHD variant alleles in the Chinese population. However, DNA sequencing was required to identify certain alleles; probes to detect these alleles should be added into the assay.

Germline activating TYK2 mutations in pediatric patients with two primary acute lymphoblastic leukemia occurrences.

The contribution of genetic predisposing factors to the development of pediatric acute lymphoblastic leukemia (ALL), the most frequently diagnosed cancer in childhood, has not been fully elucidated. Children presenting with multiple de novo leukemias are more likely to suffer from genetic predisposition. Here, we selected five of these patients and analyzed the mutational spectrum of normal and malignant tissues. In two patients, we identified germline mutations in TYK2, a member of the JAK tyrosine kinase family. These mutations were located in two adjacent codons of the pseudokinase domain (p.Pro760Leu and p.Gly761Val). In silico modeling revealed that both mutations affect the conformation of this autoregulatory domain. Consistent with this notion, both germline mutations promote TYK2 autophosphorylation and activate downstream STAT family members, which could be blocked with the JAK kinase inhibitor I. These data indicate that germline activating TYK2 mutations predispose to the development of ALL.

Prediction of the anti-RhD donor population size for managerial decision-making.

BACKGROUND: Rhesus D (RhD)-negative women pregnant with a RhD-positive child receive prophylactic injections to prevent haemolytic disease of the newborn. Because of the success of the prophylaxis, the number of naturally immunized women has decreased, thereby also decreasing the number of potential donors who provide the plasma from which the prophylaxis is made. As the current donor pool is ageing, the availability of the prophylaxis is threatened. OBJECTIVES: Objectives are to investigate whether the anti-D population and the changes therein can be described by a relatively simple model, to determine the impact of ageing of the anti-D donors on the decline of the population and how many new donors should be recruited to meet future supply demand. METHODS: Data on Dutch anti-D donors in 1994-2013 were used to simulate the donor population size and age composition for various donor recruitment scenarios. RESULTS: With a continuous influx of 27 new donors per year and a donor stopping rate of 10% per year, the population size will stabilize at 195 donors, with 2·3% of donors stopping annually due to reaching the donor age limit. A formula is derived to estimate which donor recruitment and retention efforts are required to maintain a prespecified donor pool. CONCLUSION: A relatively simple model can already describe and predict the size of the anti-D donor population and the impact of ageing accurately.

International society of blood transfusion working party on red cell immunogenetics and terminology: report of the Seoul and London meetings.

The Working Party has met twice since the last report: in Seoul, South Korea 2014, and in London, UK 2015, both in association with the International Society of Blood Transfusion (ISBT) Congress. As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. Eleven new blood group antigens were added to seven blood group systems. This brings the current total of blood group antigens recognized by the ISBT to 346, of which 308 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known blood group system.

Multiplex ligation-dependent probe amplification (MLPA) assay for blood group genotyping, copy number quantification, and analysis of RH variants.

The blood group multiplex ligation-dependent probe amplification (MLPA) is a comprehensive assay, developed for genotyping the majority of clinically relevant blood group antigens in both patients and donors. The MLPA is an easy method to apply and only requires a thermal cycler and capillary electrophoresis equipment. Because the molecular basis of blood group antigens can be a single nucleotide polymorphism, an insertion/deletion polymorphism, or genetic recombination, a single assay such as the MLPA to facilitate these different types of genetic variation is a prerequisite in blood group typing. An MLPA assay allows the simultaneous detection of up to 50 polymorphisms in a single tube. The blood group MLPA currently consists of three separate probe pools targeting 104 different blood group alleles of 18 blood group systems. The assay is performed in a 96-well plate; therefore, a maximum of 32 genomic DNA samples can be processed simultaneously. Results are available within 24 hours,and software for analysis of the MLPA results is available free of charge. In addition to the analysis of genetic variation in blood group genes, a major advantage of the test is the ability to detect aberrations in gene copy numbers, which is especially useful for the determination of homo- or hemizygous status of RHD or other blood group genes and for detection of blood chimerism. A relatively large number of RH wild-type and mutation-specific probes are included in the assay, allowing an extensive analysis of RHD variants. In our reference lab in the Netherlands, the MLPA was validated to detect RH variants in patients, donors, and pregnant women. Furthermore, we have used the MLPA to provide comprehensive typing after blood transfusion of 52 blood group antigens simultaneously, in patients with red cell autoantibodies or patients with rare phenotypes.

Haemolytic disease of the fetus and newborn.

Haemolytic Disease of the Fetus and Newborn (HDFN) is caused by maternal alloimmunization against red blood cell antigens. In severe cases, HDFN may lead to fetal anaemia with a risk for fetal death and to severe forms of neonatal hyperbilirubinaemia with a risk for kernicterus. Most severe cases are caused by anti-D, despite the introduction of antental and postnatal anti-D immunoglobulin prophylaxis. In general, red blood cell antibody screening programmes are aimed to detect maternal alloimmunization early in pregnancy to facilitate the identification of high-risk cases to timely start antenatal and postnatal treatment. In this review, an overview of the clinical relevance of red cell alloantibodies in relation to occurrence of HDFN and recent views on prevention, screening and treatment options of HDFN are provided.

Analysis of false-positive results of fetal RHD typing in a national screening program reveals vanishing twins as potential cause for discrepancy.

OBJECTIVES: We aim to elucidate causes of false-positive fetal RHD screening results obtained with cell-free DNA. METHODS: Fetal RHD screening was performed in 32,222 samples from RhD-negative women by multiplex real-time PCR in triplicate for RHD exons 5 and 7 using cell-free DNA isolated from maternal plasma obtained in the 27th gestational week. PCR results were compared with cord blood serology in 25,789 pregnancies (80.04%). False-positive cases were analyzed. Known biological causes (RHD variant genes), technical causes of discordance, and errors around blood sampling were investigated with leukocyte DNA from maternal and cord blood, and cell-free DNA from stored maternal plasma. RESULTS: Not only RHD but also Y-chromosome (DYS14) sequences were present in four plasma samples from RHD-negative women bearing an RHD-negative girl. Sample mix-up and other sampling errors could be excluded in three samples. CONCLUSIONS: These results indicate that false-positive fetal RHD screening results can be caused by cell-free DNA fragments in maternal plasma derived from a third cell line that is not representative for either the maternal genome or the genome of the vital fetus. We propose that remaining (cyto)trophoblasts of a vanishing twin are the underlying mechanism, and we estimate a frequency of this phenomenon of 0.6%.

Improved flow cytometric detection of minimal residual disease in childhood acute lymphoblastic leukemia.

Most current treatment protocols for acute lymphoblastic leukemia (ALL) include minimal residual disease (MRD) diagnostics, generally based on PCR analysis of rearranged antigen receptor genes. Although flow cytometry (FCM) can be used for MRD detection as well, discordant FCM and PCR results are obtained in 5-20% of samples. We evaluated whether 6-color FCM, including additional markers and new marker combinations, improved the results. Bone marrow samples were obtained from 363 ALL patients at day 15, 33 and 78 and MRD was analyzed using 6-color (218 patients) or 4-color (145 patients) FCM in parallel to routine PCR-based MRD diagnostics. Compared with 4-color FCM, 6-color FCM significantly improved the concordance with PCR-based MRD data (88% versus 96%); particularly the specificity of the MRD analysis improved. However, PCR remained more sensitive at levels <0.01%. MRD-based risk groups were similar between 6-color FCM and PCR in 68% of patients, most discrepancies being medium risk by PCR and standard risk by FCM. Alternative interpretation of the PCR data, aimed at prevention of false-positive MRD results, changed the risk group to standard risk in half (52%) of these discordant cases. In conclusion, 6-color FCM significantly improves MRD analysis in ALL but remains less sensitive than PCR-based MRD-diagnostics.

Report of the fourth International Workshop on molecular blood group genotyping.

The fourth International Society of Blood Transfusion (ISBT) workshop on molecular blood group genotyping was held in 2010, with a feedback meeting at the ISBT Congress in Berlin, Germany. Fifty laboratories participated, 17 more than in 2008. Six samples were distributed. Samples 1-3 were DNA samples for all red cell blood group tests available to the participants. Of the 46 laboratories that tested these samples, 37 obtained completely correct results, although the extent of testing varied considerably. Sample 4, also a DNA sample, was an Rh problem in which RHDΨ and RHCE*ceCF were present, but the participants were only informed that the donor's red cells typed as positive with some monoclonal anti-D. Of the 42 laboratories that participated in this exercise, seven performed the sequencing necessary to obtain the correct result. Samples 5 and 6 were plasma samples from RhD-negative pregnant women, for foetal RhD testing. These were sent to 25 laboratories, and two incorrect results were reported. Overall, the level of accuracy was about equal to that of the previous workshop. The main conclusion for the last two workshops can be reiterated: with greater care and attention to detail, very high standards could be set for molecular blood group genotyping.

Noninvasive fetal genotyping of human platelet antigen-1a.

We describe a reliable noninvasive fetal human platelet antigen (HPA)-1a genotyping assay on a real-time polymerase chain reaction (PCR) platform using cell-free fetal DNA isolated from maternal blood. Nonspecific amplification of maternal cell-free DNA is overcome by pre-PCR digestion of the cell-free DNA with the Msp1 restriction enzyme. Noninvasive fetal HPA-1a genotyping offers a safe method for alloimmunised pregnant women to determine whether their fetus is at risk of fetal or neonatal alloimmune thrombocytopenia (FNAIT) and whether interventions to prevent intracranial haemorrhage are required. The availability of this test is relevant to the ongoing debate on screening pregnancies for HPA-1a-mediated FNAIT.

Noninvasive fetal blood group genotyping of rhesus D, c, E and of K in alloimmunised pregnant women: evaluation of a 7-year clinical experience.

OBJECTIVE: To evaluate the diagnostic performance of noninvasive fetal blood group genotyping. DESIGN: Descriptive analysis. SETTING: Dutch national reference laboratory for pregnancies complicated by alloimmunisation. POPULATION: All consecutive alloimmunised pregnant women for whom fetal blood group genotyping of rhesus D, c, E or of K in maternal plasma was performed from 2003 up to 2010. METHODS: The test results of each individual assay were collected. Real-time polymerase chain reaction was performed for RHD exon 5 and RHD exon 7, or the specific allele of the RHCE or KEL gene. A stringent diagnostic algorithm was applied. In the case of a negative result, the presence of fetal DNA was ascertained by the analysis of the Y chromosome-specific SRY gene or other paternal genetic markers. Results were compared with available serology after birth or genotyping results of amniotic fluid cells. MAIN OUTCOME MEASURES: Percentage of conclusive test results and diagnostic accuracy. RESULTS: A total of 362 tests was performed (D: n = 168; c: n = 49; E: n = 85; K: n = 60). The median gestational age was 17 weeks (range 7-38 weeks). In 351 women (97%), a test result was issued: in seven samples, the presence of fetal DNA could not be confirmed; in two samples, non-specific amplification in the K assay led to an inconclusive result; in two samples, a maternal silent RHD gene prevented fetal RHD genotyping. No false-positive or false-negative results were found among those women for whom cord blood serology or genotyping results of amniotic fluid cells were available (n = 212). CONCLUSIONS: Noninvasive fetal blood group genotyping is accurate and applicable in a clinical diagnostic setting.

The prognostic value of fast molecular response of marrow disease in patients aged over 1 year with stage 4 neuroblastoma.

BACKGROUND: Quantitative real-time (q)PCR for detection of minimal residual disease (MRD) in children with neuroblastoma (NB) can evaluate molecular bone marrow (BM) response to therapy, but the prognostic value of tumour kinetics in the BM during induction treatment remains to be established. The purpose of this study was to analyse at which time points MRD detection by sequential molecular assessment of BM was prognostic for overall survival (OS). METHODS: In this single centre study, qPCR was performed with five NB-specific markers: PHOX2B, TH, DDC, GAP43 and CHRNA3, on 106 retrospectively analysed BM samples of 53 patients >1 year with stage 4 neuroblastoma. The prognostic impact of MRD at diagnosis (n = 39), at 3 months after diagnosis (n = 38) and after completing induction chemotherapy (n = 29) was assessed using univariate and bivariate Cox regression analyses. RESULTS: There was no correlation between tumour load at diagnosis and outcome (p = 0.93). Molecular BM remission was observed in 11/38 (29%) of patients at 3 months after diagnosis and associated with favourable outcome (5-y-OS 62 ± 15.0% versus 19 ± 8%; p = 0.009). After completion of induction chemotherapy, BM of 41% (12/29) of the patients was still MRD positive, which was associated with poor outcome (5-y-OS 0% versus 52 ± 12%; p<0.001). For both time points, the prognostic value of molecular response remained significant in bivariate analysis. CONCLUSIONS: MRD detection measured by a panel of NB specific-PCR targets could identify fast responders, who clear their BM early during treatment. Fast molecular response was a prognostic factor, associated with better outcome. Our data indicate that MRD analysis during induction therapy should be included in prospective MRD studies.

Integrated use of minimal residual disease classification and IKZF1 alteration status accurately predicts 79% of relapses in pediatric acute lymphoblastic leukemia.

Response to therapy as determined by minimal residual disease (MRD) is currently used for stratification in treatment protocols for pediatric acute lymphoblastic leukemia (ALL). However, the large MRD-based medium risk group (MRD-M; 50-60% of the patients) harbors many relapses. We analyzed MRD in 131 uniformly treated precursor-B-ALL patients and evaluated whether combined MRD and IKZF1 (Ikaros zinc finger-1) alteration status can improve risk stratification. We confirmed the strong prognostic significance of MRD classification, which was independent of IKZF1 alterations. Notably, 8 of the 11 relapsed cases in the large MRD-M group (n=81; 62%) harbored an IKZF1 alteration. Integration of both MRD and IKZF1 status resulted in a favorable outcome group (n=104; 5 relapses) and a poor outcome group (n=27; 19 relapses), and showed a stronger prognostic value than each of the established risk factors alone (hazard ratio (95%CI): 24.98 (8.29-75.31)). Importantly, whereas MRD and IKZF1 status alone identified only 46 and 54% of the relapses, respectively, their integrated use allowed prediction of 79% of all the relapses with 93% specificity. Because of the unprecedented sensitivity in upfront relapse prediction, the combined parameters have high potential for future risk stratification, particularly for patients originally classified as non-high risk, such as the large group of MRD-M patients.

Screening in pregnancy for fetal or neonatal alloimmune thrombocytopenia: systematic review.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a potentially devastating disease, which may lead to intracranial haemorrhage (ICH), with neurological damage as a consequence. In the absence of screening, FNAIT is only diagnosed after bleeding symptoms, with preventive options limited to a next pregnancy. OBJECTIVES: To estimate the population incidence of FNAIT and its consequences to prepare for study design of a screening programme. SEARCH STRATEGY: An electronic literature search using MEDLINE, EMBASE and Cochrane database, and references of retrieved articles. No language restrictions were applied. SELECTION CRITERIA: Prospective studies on screening for human platelet antigen 1a (HPA-1a) alloimmunisation in low-risk pregnant women. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed studies for inclusion and extracted data. Main outcome data were prevalence of HPA-1a negativity, HPA-1a immunisation, platelet count at birth and perinatal ICH. We aimed to compare outcome with and without intervention. MAIN RESULTS: HPA-1a alloimmunisation occurred in 294/3028 (9.7%) pregnancies at risk. Severe FNAIT occurred in 71/227 (31%) immunised pregnancies, with perinatal ICH in 7/71 (10%). True natural history data were not found because interventions were performed in most screen-positive women. AUTHORS' CONCLUSIONS: Screening for HPA-1a alloimmunisation detects about two cases in 1000 pregnancies. The calculated risk for perinatal ICH of 10% in pregnancies with severe FNAIT is an underestimation because studies without interventions were lacking. Screening of all pregnancies together with effective antenatal treatment such as intravenous immunoglobulin may reduce the mortality and morbidity associated with FNAIT.

Blood group genotyping: from patient to high-throughput donor screening.

Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.

Risk factors for RhD immunisation despite antenatal and postnatal anti-D prophylaxis.

OBJECTIVE: To identify risk factors for Rhesus D (RhD) immunisation in pregnancy, despite adequate antenatal and postnatal anti-D prophylaxis in the previous pregnancy. To generate evidence for improved primary prevention by extra administration of anti-D Ig in the presence of a risk factor. DESIGN: Case-control study. SETTING: Nation-wide evaluation of the Dutch antenatal anti-D-prophylaxis programme. CASES: 42 RhD-immunised parae-1, recognised by first-trimester routine red cell antibody screening in their current pregnancy, who received antenatal and postnatal anti-D Ig prophylaxis (gifts of 1000 iu) in their first pregnancy. CONTROLS: 339 parae-1 without red cell antibodies. METHODS: Data were collected via obstetric care workers and/or personal interviews with women. MAIN OUTCOME MEASURE: Significant risk factors for RhD immunisation in multivariate analysis. RESULTS: Independent risk factors were non-spontaneous delivery (assisted vaginal delivery or caesarean section) (OR 2.23; 95% CI:1.04-4.74), postmaturity (>or=42 weeks of completed gestation: OR 3.07; 95% CI:1.02-9.02), pregnancy-related red blood cell transfusion (OR 3.51; 95% CI:0.97-12.7 and age (OR 0.89/year; 95% CI:0.80-0.98). In 43% of cases, none of the categorical risk factors was present. CONCLUSIONS: In at least half of the failures of anti-D Ig prophylaxis, a condition related to increased fetomaternal haemorrhage (FMH) and/or insufficient anti-D Ig levels was observed. Hence, RhD immunisation may be further reduced by strict compliance to guidelines concerning determination of FMH and accordingly adjusted anti-D Ig prophylaxis, or by routine administration of extra anti-D Ig after a non-spontaneous delivery and/or a complicated or prolonged third stage of labour.