Non-invasive fetal HPA-1a typing is a valuable tool to identify the pregnancies at risk of fetal and neonatal alloimmune thrombocytopenia (FNAIT). At present, prenatal determination of the fetus ...HPA-1a type is performed for diagnostic purposes in pregnancies of HPA-1 alloimmunized women with history of a previous fetus or child with FNAIT. Different approaches have been used to determine the fetal HPA-1a genotype from cell-free fetal DNA (cffDNA) in the mother’s plasma, mainly based on real-time PCR. Due to the single nucleotide polymorphism (SNP) between the HPA-1a and HPA-1b allelic sequences, a robust and accurate detection of the fetal genotype is challenging, and the sensitivity of most assays is still limited early in pregnancy. Nowadays, the availability of technologies such as next generation sequencing (NGS) or digital PCR offers unprecedented possibilities of analyzing cell-free DNA (cfDNA)-amplified sequences with very high coverage and high sensitivity. In addition, other interesting approaches using variant sequence enrichment strategies have been recently described. In particular, coamplification at lower denaturation temperature PCR (COLD-PCR) offers a simple and sensitive strategy for noninvasive fetal HPA-1 typing. These novel approaches are explained in more detail in this review. Despite no population-based FNAIT screening programs have so far been implemented, the perspectives in terms of treatment and prevention are changing and less costly high-throughput maternal HPA-1a typing methods have been developed. Altogether, this may lead to the implementation of fetal HPA-1a typing with a broader scope in the future, playing a critical role within FNAIT screening programs.
The analysis of chimerism is crucial to determine the status of patients receiving hematopoietic stem cell transplantation. The variety of relevant techniques available today range from those that ...analyse nucleic acids (i.e. polymerase chain reaction based, next generation sequencing) and cellular phenotype (i.e. flow cytometry) to sophisticated imaging (particularly multimodal imaging using labelling agents). However, current developments of advanced therapies bring chimerism studies into a new dimension in which methods for detection of donor cells in the patient need to adapt to a wider range of cell- and gene-based medicines, routes of administration, target organs and pathologies. Herein we describe and analyze the toolkit of suitable labelling and detection methodologies with actual examples along with a discussion on challenges ahead and potential solutions. Remarkably, existing methods commonly used in chimerism analysis are suitable for use with new cell- and gene-based medicines. Indeed, new developments may facilitate the evolution and combination of such methodologies to the use of non-invasive and highly informative approaches.
The identification of erythrocyte antibodies in the serum of patients rely on panels of human red blood cells (RBCs), which coexpress many antigens and are not easily available for low-incidence ...blood group phenotypes. These problems have been addressed by generating cell lines expressing unique blood group antigens, which may be used as an alternative to human RBCs. However, the use of cell lines implies several drawbacks, like the requirement of cell culture facilities and the high cost of cryopreservation. The application of cell stabilization methods could facilitate their use as reagent cells in clinical laboratories.
We generated stably-transfected cells expressing low-incidence blood group antigens (Dia and Lua). High-expresser clones were used to assess the effect of TransFix® treatment and lyophilization as cell preservation methods. Cells were kept at 4°C and cell morphology, membrane permeability and antigenic properties were evaluated at several time-points after treatment.
TransFix® addition to cell suspensions allows cell stabilization and proper antigen detection for at least 120 days, despite an increase in membrane permeability and a reduction in antigen expression levels. Lyophilized cells showed minor morphological changes and antigen expression levels were rather conserved at days 1, 15 and 120, indicating a high stability of the freeze-dried product. These stabilized cells have been proved to react specifically with human sera containing alloantibodies.
Both stabilization methods allow long-term preservation of the transfected cells antigenic properties and may facilitate their distribution and use as reagent-cells expressing low-incidence antigens, overcoming the limited availability of such rare RBCs.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The limited availability of red cells with extremely rare blood group phenotypes is one of the global challenges in transfusion medicine that has prompted the search for alternative self‐renewable ...pluripotent cell sources for the in vitro generation of red cells with rare blood group types. One such phenotype is the Rhnull, which lacks all the Rh antigens on the red cell membrane and represents one of the rarest blood types in the world with only a few active blood donors available worldwide. Rhnull red cells are critical for the transfusion of immunized patients carrying the same phenotype, besides its utility in the diagnosis of Rh alloimmunization when a high‐prevalence Rh specificity is suspected in a patient or a pregnant woman. In both scenarios, the potential use of human‐induced pluripotent stem cell (hiPSC)‐derived Rhnull red cells is also dependent on ABO compatibility. Here, we present a CRISPR/Cas9‐mediated ABO gene edition strategy for the conversion of blood type A to universal type O, which we have applied to an Rhnull donor‐derived hiPSC line, originally carrying blood group A. This work provides a paradigmatic example of an approach potentially applicable to other hiPSC lines derived from rare blood donors not carrying blood type O.
CRISPR/Cas9 mediated ABO gene edition allows the conversion of blood type A to universal type O in Rhnull donor‐derived hiPSCs
A targeted knock‐in strategy allows to reproduce the c.261delG polymorphism present in the most common inactive ABO*O.01 allele
This work provides a new approach potentially applicable to cell lines established from rare blood donors not carrying blood type O
High-throughput genotyping platforms enable simultaneous analysis of multiple polymorphisms for blood group typing. BLOODchip® ID is a genotyping platform based on Luminex® xMAP technology for ...simultaneous determination of 37 red blood cell (RBC) antigens (ID CORE XT) and 18 human platelet antigens (HPA) (ID HPA XT) using the BIDS XT software.
In this international multicentre study, the performance of ID CORE XT and ID HPA XT, using the centres' current genotyping methods as the reference for comparison, and the usability and practicality of these systems, were evaluated under working laboratory conditions. DNA was extracted from whole blood in EDTA with Qiagen methodologies. Ninety-six previously phenotyped/genotyped samples were processed per assay: 87 testing samples plus five positive controls and four negative controls.
Results were available for 519 samples: 258 with ID CORE XT and 261 with ID HPA XT. There were three "no calls" that were either caused by human error or resolved after repeating the test. Agreement between the tests and reference methods was 99.94% for ID CORE XT (9,540/9,546 antigens determined) and 100% for ID HPA XT (all 4,698 alleles determined). There were six discrepancies in antigen results in five RBC samples, four of which (in VS, N, S and Do(a)) could not be investigated due to lack of sufficient sample to perform additional tests and two of which (in S and C) were resolved in favour of ID CORE XT (100% accuracy). The total hands-on time was 28-41 minutes for a batch of 16 samples. Compared with the reference platforms, ID CORE XT and ID HPA XT were considered simpler to use and had shorter processing times.
ID CORE XT and ID HPA XT genotyping platforms for RBC and platelet systems were accurate and user-friendly in working laboratory settings.
The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping ...platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism.
Background
The D– phenotype is mainly caused by the complete deletion of the RHD gene in Caucasians. However, a plethora of allelic variants have been described among D– individuals from different ...ethnic groups.
Study Design and Methods
A cohort of 1314 routine serologically D– samples from white Argentineans was studied by molecular methods.
Results
Among the 1314 D– samples, 2.1% showed RHD‐specific amplifications. One hybrid Rhesus box was detected in all D–/RHD+ samples, suggesting a hemizygous status. The RHDΨ was found in 0.7% of rr samples while DEL and null variants were detected in 16.7% of the D– samples expressing C and/or E antigens. The variants associated with the C antigen were seven RHD‐CE‐Ds, two RHD(1‐2)‐CE(2‐9)‐D(10), two previously unreported RHD(329T>C)‐CE(3‐9)‐D null alleles, one RHD(M295I), and one new RHCE(1‐2)‐RHD(3361del11‐10) null allele whereas those associated with the E antigen were five RHD(46T>C) and one novel RHD(581insG) null allele responsible for a premature stop codon.
Conclusions
The prevalence of D–/RHD+ samples is higher than that observed in Europeans. More than 50% of the RHD alleles found were represented by RHDψ and RHD‐CE‐Ds showing the African contribution to the genetic pool of the admixed population analyzed. Interestingly, three new alleles were found, two of them being hybrid structures between previously described RHD variants recombined with RHCE sequences. The knowledge of the RHD allele repertoire in our population allowed the implementation of reliable typing and transfusion strategies for a better management of patients and pregnant women.
Platelet transfusions are necessary to prevent and treat haemorrhages in thrombocytopenic patients or those with severe platelet dysfunction. In Latin American countries, including Argentina, blood ...supplies from voluntary non-remunerated blood donors remain dependent on family replacement donors, since altruistic repeat donors are exceptional and platelet donors are very scarce. The aim of this study was to recruit a group of frequent, voluntary, altruistic blood donors and determine their human platelet antigen (HPA)-genotype in order to establish the first registry of HPA-typed voluntary platelet donors in Argentina.
In this study, we invited and recruited voluntary blood donors who attended the Fundación Banco Central de Sangre between July 2016 and July 2017. DNA was extracted from K2EDTA anticoagulated whole blood and genotyping was performed by polymerase chain reaction, using sequence-specific primers to type the HPA-1 to -6, -9 and -15 systems. A subset of samples was also tested using a commercial HPA-TYPE kit. Donors were invited to join the National Register of Haematopoietic Stem Cell Donors of Argentina.
A cohort of 500 platelet donors was recruited and characterised and a database with their personal information, including their genotype for the most relevant HPA alloantigens, was created. Eight of the 500 donors (1.6%) were HPA-1a negative. HPA allelic variants -4b, -6b and -9b were detected for the first time in our population. There was 100% concordance between our in-house assay and the commercial kits in the subset of 150 donor samples assayed in parallel.
The efforts made to recruit, characterise and register voluntary platelet donors will provide the first sustainable source of HPA and human leukocyte antigen-typed platelets for compatible transfusions in the country. Remarkably, we identified a higher percentage of HPA-1a-negative donors than previously detected in the Argentinean population.
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