[Genotyping and its applications, a look to the future]. / Genotipificación y sus aplicaciones, una mirada hacia el futuro.

The detection of the most significant erythrocyte antigens present in each one of the individuals is fundamental when carrying out a transfusion or a transplant. Detection to date is performed by conventional serological methods through the antigen-antibody reaction. But several drawbacks may arise depending on the pathology under study, limiting the availability of blood components. Molecular methods such as genotyping is a tool that complements sensitivity and specificity and has come to revolutionize immunohematology in the blood bank, allowing not only the detection of erythrocyte antigens but also platelet antigens. These methodologies are applicable in patients and in large-scale donors, starting from the allelic variants present in each of the genes that code for the antigens of clinical interest, using microarray systems or systems based on particles labeled with specific probes or their variants that allow an analysis from the immunohematological point of view.

La detección de los antígenos eritrocitarios más significativos presentes en cada uno de los individuos es fundamental cuando se lleva a cabo una transfusión o un trasplante. La detección a la fecha se realiza mediante métodos serológicos convencionales a través de la reacción de antígeno-anticuerpo. Pero se pueden presentar varios inconvenientes dependiendo de la patología en estudio, lo cual limita la disponibilidad de los hemocomponentes. Los métodos moleculares, como la genotipificación, son una herramienta que complementa la sensibilidad y especificidad y que han venido a revolucionar la inmunohematología en el banco de sangre, lo cual permite no solo la detención de antígenos eritrocitarios sino también la de antígenos plaquetarios. Estas metodologías son aplicables en pacientes y en donantes a gran escala, partiendo de las variantes alélicas presentes en cada uno de los genes que codifican para los antígenos de interés clínico, utilizando los sistemas de microarreglos o los sistemas basados en partículas marcadas con sondas específicas o sus variantes que permiten un análisis desde el punto de vista inmunohematológico.

Validating the HPA-1 to -5 and -15 Detection by Homemade PCR-SSP, Real-Time PCR, and PCR-RFLP Methods.

OBJECTIVE: Human platelet antigens (HPAs) are antigenic determinants on platelet membrane glycoproteins that stimulate the host's immune system and cause platelet destruction. In this study, we share our experience with implementing sequence-specific primer-polymerase chain reaction (PCR-SSP), real-time PCR, and PCR-RFLP (restriction fragment length polymorphism) and the validation process used to evaluate the results. METHODS: At the Ardabil Blood Transfusion Center, 10 samples were obtained from blood donors. Validation using PCR-SSP, real-time PCR, and PCR-RFLP methods for genotyping HPAs was done by sequencing. A commercial DNA sample and a commercial kit were also used for validation. RESULTS: The results of PCR-SSP, TaqMan Real-Time PCR, melting curve analysis (HPA-15), and PCR-RFLP (HPA-3) were 100% consistent with sequencing (gold standard) and commercial kit results. CONCLUSIONS: There was a 100% correlation between repeating the methods and the expected results for repeatability, and no false positives and negatives were observed.

Novel method for simultaneously detecting HPA and HLA antibodies using Luminex microbeads.

BACKGROUND: Alloantibodies against human platelet antigens (HPAs) and human leukocyte antigen (HLA) are implicated in several immune-mediated platelet disorders. Detection of these antibodies is crucial in the diagnosis and management of these disorders. The aim of this study was to establish a novel method to simultaneously detect HPA-1, HPA-2, HPA-3, HPA-5 and HLA antibodies with Luminex microbeads technology. METHODS: Monoclonal antibodies specific for platelet glycoproteins and HLA class I molecules were separately coupled to the Luminex microbeads. We validated specificity of the Luminex platform using the following antibodies: anti-HPA-1a, anti-HPA-2b, anti-HPA-3a, anti-HPA-5a, and anti-HLA positive samples. Sensitivity was evaluated by a serial dilution (from neat to 1/1024) using the following antibodies: anti-HPA-1a, anti-HPA-3a standard sera, and anti-HPA-5a positive serum. Serum samples were collected from 36 neonatal alloimmune thrombocytopenia (NAIT) patients suspected of having HPA or HLA antibodies and 8 samples from ISBT platelet workshop were tested using the Luminex assay. RESULTS: The Luminex assay detected all antibodies tested from the known samples. The sensitivities of the Luminex assay detecting anti-HPA-1a, anti-HPA-3a, and anti-HPA-5a were 1:512, 1:64, and 1:128, respectively. The sensitivity of Luminex assay was higher than monoclonal antibody immobilization of platelet antigen method (MAIPA). No cross-reactivity was observed in the samples containing multi-platelet antibodies or mixture antibodies against HPA and HLA. The results of 44 samples with platelet disorders were consistent with those of the same samples processed with the MAIPA assay. CONCLUSION: Luminex microbeads coupled with monoclonal antibodies could be successfully used to detect HPA and HLA antibodies simultaneously, especially with high sensitivity in detecting HPA antibodies.

Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. STUDY DESIGN AND METHODS: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. RESULTS: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. CONCLUSION: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.

Lung fluid biomarkers for acute respiratory distress syndrome: a systematic review and meta-analysis.

BACKGROUND: With the development of new techniques to easily obtain lower respiratory tract specimens, bronchoalveolar lavage fluid and other lung fluids are gaining importance in pulmonary disease diagnosis. We aimed to review and summarize lung fluid biomarkers associated with acute respiratory distress syndrome diagnosis and mortality. METHODS: After searching PubMed, Embase, Web of Science, and the Cochrane Library for articles published prior to January 11, 2018, we performed a meta-analysis on biomarkers for acute respiratory distress syndrome diagnosis in at-risk patients and those related to disease mortality. From the included studies, we then extracted the mean and standard deviation of the biomarker concentrations measured in the lung fluid, acute respiratory distress syndrome etiologies, sample size, demographic variables, diagnostic criteria, mortality, and protocol for obtaining the lung fluid. The effect size was measured by the ratio of means, which was then synthesized by the inverse-variance method using its natural logarithm form and transformed to obtain a pooled ratio and 95% confidence interval. RESULTS: In total, 1156 articles were identified, and 49 studies were included. Increases in total phospholipases A2 activity, total protein, albumin, plasminogen activator inhibitor-1, soluble receptor for advanced glycation end products, and platelet activating factor-acetyl choline were most strongly associated with acute respiratory distress syndrome diagnosis. As for biomarkers associated with acute respiratory distress syndrome mortality, interleukin-1ß, interleukin-6, interleukin-8, Kerbs von Lungren-6, and plasminogen activator inhibitor-1 were significantly increased in the lung fluid of patients who died. Decreased levels of Club cell protein and matrix metalloproteinases-9 were associated with increased odds for acute respiratory distress syndrome diagnosis, whereas decreased levels of Club cell protein and interleukin-2 were associated with increased odds for acute respiratory distress syndrome mortality. CONCLUSIONS: This meta-analysis provides a ranking system for lung fluid biomarkers, according to their association with diagnosis or mortality of acute respiratory distress syndrome. The performance of biomarkers among studies shown in this article may help to improve acute respiratory distress syndrome diagnosis and outcome prediction.

Detection of platelet autoantibodies to identify immune thrombocytopenia: state of the art.

Immune Thrombocytopenia (ITP) is diagnosed by exclusion of other causes for thrombocytopenia. Reliable detection of platelet autoantibodies would support the clinical diagnosis of ITP and prevent misdiagnosis. We optimized our diagnostic algorithm for suspected ITP using the direct monoclonal antibody immobilization of platelet antigens assay (MAIPA), which evaluates the presence of platelet autoantibodies on the glycoproteins (GP) IIb/IIIa, Ib/IX and V bound on the patient platelets. The direct MAIPA was shown to be a valuable technique for the detection of platelet autoantibodies and could possibly become a guide for optimizing therapy towards a more personalized treatment of ITP.

Taking a wider view on fetal/neonatal alloimmune thrombocytopenia.

In fetal/neonatal alloimmune thrombocytopenia (FNAIT), platelets are destroyed by maternal antibodies directed against fetal/neonate antigens. Thrombocytopenia can be severe and lead to intracranial hemorrhage (ICH) in about 10% of cases. Although three types of antigen groups, presented on platelets [ABO blood group antigens, human leukocyte antigens (HLA) and human platelet antigens (HPA)] are known to be implicated in immune platelet destruction, antibodies against HPA are most commonly involved in FNAIT and hence are the target of extensive research. Awareness of FNAIT by physicians as well as the availability of the most sensitive diagnostic methods capable of detecting a wide range of antibodies are crucial for the diagnosis of FNAIT and the prevention of severe thrombocytopenia and its bleeding risks in subsequent pregnancies.

Parallel donor genotyping for 46 selected blood group and 4 human platelet antigens using high-throughput MALDI-TOF mass spectrometry.

Most blood group antigens are defined by single nucleotide polymorphisms (SNPs). Highly accurate MALDI-TOF MS has proven its potential in SNP genotyping and was therefore chosen for blood donor oriented genotyping with high-throughput capability, e.g., 380 samples per day. The Select Module covers a total of 36 SNPs in two single-tube reactions, representative of 46 blood group and 4 human platelet antigens. Using this tool, confirmatory blood group typing for RhD, RhCE, Kell, Kidd, Duffy, MN, Ss, and selected rare antigens is performed on a routine basis.

Prenatal testing for hemolytic disease of the newborn and fetal neonatal alloimmune thrombocytopenia - current status.

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).

Identification of potential platelet alloantigens in the Equidae family by comparison of gene sequences encoding major platelet membrane glycoproteins.

BACKGROUND: Platelet alloantigens in horses may play an important role in the development of neonatal alloimmune thrombocytopenia (NAIT). OBJECTIVE: The objective of this study was to evaluate genes encoding major platelet glycoproteins within the Equidae family in an effort to identify potential alloantigens. METHODS: DNA was isolated from blood samples obtained from Equidae family members, including a Holsteiner-Oldenburg cross, a Quarter horse, a donkey, and a Plains zebra (Equus burchelli). Gene sequences encoding equine platelet membrane glycoproteins IIb, IIIa (integrin subunits αIIb and ß3), Ia (integrin subunit α2), and Ibα were determined using PCR. Gene sequences were compared to the equine genome available on GenBank. Polymorphisms that would be predicted to result in amino acid changes on platelet surfaces were documented and compared with known alloantigenic sites documented on human platelets. RESULTS: Amino acid differences were predicted based on nucleotide sequences for all 4 genes. Nine differences were documented for αIIb, 5 differences were documented for ß3, 7 differences were documented for α2, and 16 differences were documented for Ibα outside the macroglycopeptide region. CONCLUSIONS: This study represents the first effort at identifying potential platelet alloantigens in members of the Equidae Family based on evaluation of gene sequences. The data obtained form the groundwork for identifying potential platelet alloantigens involved in transfusion reactions and neonatal alloimmune thrombocytopenia (NAIT). More work is required to determine whether the predicted amino acid differences documented in this study play a role in alloimmunity, and whether other polymorphisms not detected in this study are present that may result in alloimmunity.

Sensitive detection of idiotypic platelet-reactive alloantibodies by an electrical protein chip.

To prevent and treat immune-mediated platelet disorders (e.g. neonatal allo-immune thrombocytopenia and platelet transfusion refractoriness) the causative idiotypic platelet-reactive antibodies have to be detected with high sensitivity and specificity. The "Monoclonal Antibody Immobilization Platelet Assay" (MAIPA) is the diagnostic gold standard for immunotyping sera with respect to alloantibodies against human platelet antigens (HPA). However, it is labor-intensive and time-consuming. In this work, an automated protein chip assay (enzyme-linked sandwich immunoassay) based on interdigitated gold microelectrodes in combination with an electrical read-out system was developed and optimized. For this purpose, specific capture antibodies were immobilized on the gold electrodes. The binding of the target is detected via an enzyme-labeled detection antibody by a redox-recycling process that corresponds to the amount of bound target molecule. With this electrical chip assay it is possible to detect antibodies against HPA-1a, HPA-5b and HLA with high sensitivity and specificity in less than half the duration of the MAIPA protocol with similar intra- and interassay variance.

Determination of human platelet antigen typing by molecular methods: Importance in diagnosis and early treatment of neonatal alloimmune thrombocytopenia.

Neonatal alloimmune thrombocytopenia (NAIT) is the most common cause of severe thrombocytopenia and intracranial hemorrhage in the perinatal period. While the gold standard for making a diagnosis of NAIT is detection of a human platelet antigen (HPA)-specific antibody in maternal serum, together with identifying an incompatibility between the parents for the cognate HPA antigen, platelet genotyping is the gold standard method for HPA typing. Platelet genotyping is critical in screening at-risk fetuses for the presence ofthe HPA corresponding to the maternal antibody. In addition, platelet genotyping may play a role in population screening to identify women at risk for sensitization, and thus, fetuses at risk for NAIT. The most commonly used methods of platelet genotyping are sequence-specific primer-polymerase chain reaction (PCR-SSP), restriction fragment length polymorphism-PCR (PCR-RFLP), and TaqMan real-time PCR. PCR-SSP and PCR-RFLP are relatively inexpensive and technically simple methods, but they are not easily automated and require expertise for reliable interpretation of results. Newer methods that allow for multiplexing, automation, and easily interpretable results, such as bead arrays, are currently in development and available for research purposes.

Refractory response to platelet transfusion therapy.

Platelet transfusions are commonly used for prophylaxis and treatment of bleeding. After a transfusion, an increment or "boost" is expected to occur. While a number of factors can contribute to a poor posttransfusion increment, refractoriness is typically defined as failure to achieve an appropriate increment after receiving 2 consecutive transfusions with fresh ABO-compatible platelets. The most common cause of refractoriness is contamination of the platelet product with leukocytes, resulting in human leukocyte antigens antibody formation. ABO incompatibility and human platelet antigens are also implicated in the development of refractoriness. Nurses who infuse platelets must understand these causes to assist in prevention and management.

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.

[Genotyping applied to platelet immunology: when? How? Limits]. / Génotypage en immunologie plaquettaire: quand? Comment? Limites.

Platelet alloantigens named Human Platelet Antigens (HPA) are involved in immune conflicts such as post-transfusion purpura, platelet transfusion refractoriness and neonatal alloimmune thrombocytopenia. Biological diagnosis relies on: (1) detection of alloantibodies; (2) identification of the alloantigen involved in the immune conflict. Since the development of methods based on molecular biology, platelet genotyping is preferred to phenotyping. Today, most of the Platelet Immunology Units use PCR-RFLP or PCR-SSP, and few use real-time PCR. An increasing amount of commercial kits based on new technologies is now available, for example microarrays, fluorescent or coloured microbeads, or a combination of both technologies. However, an increasing number of polymorphisms have been discovered that are responsible for erroneous platelet genotypings. Consequently, it would be of interest to develop alternative technologies based on antigen/antibody interaction instead of DNA.

Application of bead array technology to simultaneous detection of human leucocyte antigen and human platelet antigen antibodies.

BACKGROUND: Detection of antibodies against human leucocyte antigens (HLA) and human platelet antigens (HPA) is crucial for patients refractory to platelet transfusion therapy. However, a reliable and high-throughput method for HLA cross-matching and detecting HPA antibodies has not yet been described. STUDY DESIGN AND METHODS: Immunocomplex capture fluorescence analysis (ICFA) was developed for high-throughput, simultaneous detection of HLA and HPA antibodies. Microarray beads were separately coupled with monoclonal antibodies specific for CD36, CD41, CD42b, CD49b, CD61 and HLA class I antigens. Platelets reacting with patient serum were lysed and the lysates were incubated with the bead mixture to specifically capture antigen-antibody complexes via the epitopes on platelet glycoproteins or HLA antigens. The beads capturing immunocomplexes were then subjected to flow cytometric analysis. RESULTS: Immunocomplex capture fluorescence analysis was validated using 50 serum samples containing HLA antibodies and 20 serum samples containing HPA antibodies. The method enabled the detection of all the HLA antibodies with a sensitivity comparable to that of the purified HLA antigen-coated pooled-bead assay (FlowPRA, One Lambda, Canoga Park, CA, USA). The method also enabled the detection of all the HPA antibodies with a sensitivity higher than that of the mixed passive haemagglutination. CONCLUSION: In this study, we developed a rapid, simple and reliable method for the simultaneous analysis of HLA and HPA antibodies. ICFA can also be used as an alternative to the lymphocyte cytotoxicity test for HLA cross-matching.

Allelic frequencies of HPA-1 to 5 human platelet antigens in patients infected with hepatitis C virus.

Studies have suggested that hepatitis C virus (HCV) may infect not only hepatocytes but may also be carried by platelets. Platelets express more than 20 polymorphic antigenic determinants on their surface, which are called human platelet antigens (HPA). To determine the allele frequency of the HPA-1 to -5 in patients infected with HCV, blood samples were collected from 257 blood donors for the control group and from 191 patients infected with HCV. DNA was isolated and amplified for genes HPA-1 to -4 using PCR Sequence Specific Primers (PCR-SSP) and HPA-5 using PCR-Restriction Fragment Length Polymorphism (PCR-RFLP). The allelic and genotypic frequency of HPA-5a in patients infected with HCV was found to be significantly lower (P < 0.05) than in the controls, and HPA-5b from patients infected with HCV was significantly higher (P < 0.05) than in controls. The increase in HPA-5b allelic frequency in HCV infection may indicate a possible association between HCV infection and HPAs.

An International Reference Reagent (minimum sensitivity) for the detection of anti-human platelet antigen 1a.

BACKGROUND AND OBJECTIVES: The platelet-specific alloantibody anti-human platelet antigen (HPA) 1a is involved in feto-maternal alloimmune thrombocytopenia, post-transfusion purpura and platelet refractoriness. The existing minimum potency preparation for the detection of anti-HPA-1a (NIBSC code 93/710) was established by World Health Organization in 1997 and is used by laboratories to validate new assays or to calibrate 'in-house' controls. However, it has been well-used and a replacement is required. This report describes the production and comparative evaluation of a freeze-dried preparation of pooled human plasma, coded 05/106, containing anti-HPA-1a. MATERIALS AND METHODS: Plasma containing anti-HPA-1a was obtained and 2974 1-ml aliquots were prepared and freeze-dried in glass ampoules. In order to characterize the material and compare it to the existing reference material, three collaborative studies were organized, involving a total of 50 different laboratories in 23 countries. RESULTS: As expected only anti-HPA-1a could be detected in the plasma and no additional HPA or human leucocyte antigen antibodies were detected. When tested in titration, there was a wide variation in the sensitivity of antibody detection by different laboratories, irrespective of the technique used. However, there was no significant difference between the two materials when compared using a t-test. CONCLUSIONS: When diluted 1 in 2, most laboratories were able to detect the presence of anti-HPA-1a in both materials and the participants agreed that this was an appropriate level to set as the minimum sensitivity required. In October 2007, the World Health Organization Expert Committee on Biological Standardization approved the material 05/106 as an International Reference Reagent.